Post on 26-Jan-2023
EDITORI
AL REVIEWRecent progress in HIV vaccines inducing mucosalimmune responses
Vincent Pavota,b, Nicolas Rochereaub, Philip Lawrencec,
Marc P. Girardd, Christian Geninb, Bernard Verriera
and Stephane Paulb
Copyright © L
aInstitut de BiologiMuqueuses et AgeResearch in InfectiMedicine, Paris, F
Correspondence tEtienne, France.
Tel: +334 77 42 1Received: 13 Febr
DOI:10.1097/QAD
ISSN
In spite of several attempts over many years at developing a HIV vaccine based onclassical strategies, none has convincingly succeeded to date. As HIV is transmittedprimarily by the mucosal route, particularly through sexual intercourse, understandingantiviral immunity at mucosal sites is of major importance. An ideal vaccine shouldelicit HIV-specific antibodies and mucosal CD8þ cytotoxic T-lymphocyte (CTL) as a firstline of defense at a very early stage of HIV infection, before the virus can disseminateinto the secondary lymphoid organs in mucosal and systemic tissues. A primary focus ofHIV preventive vaccine research is therefore the induction of protective immuneresponses in these crucial early stages of HIV infection. Numerous approaches arebeing studied in the field, including building upon the recent RV144 clinical trial. In thisarticle, we will review current strategies and briefly discuss the use of adjuvants indesigning HIV vaccines that induce mucosal immune responses.
� 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
AIDS 2014, 28:1701–1718
Keywords: adjuvants, administration routes, HIV, mucosa, vaccine
Introduction
Despite the extensive efforts that have been made overalmost 30 years, major challenges still exist concerningHIV vaccine design. Most HIV infections by far occurthrough sexual contact [1]. Women are particularlyvulnerable during heterosexual transmission throughexposure to contaminated seminal fluids, and indeed,heterosexual women account for more than half of allindividuals living with this virus [2]. Mucosal tissuesinvolved in the sexual transmission of HIV include thecervicovaginal and rectal mucosa as well as the foreskinand oral epithelia [3]. Therefore, eliciting a strong
ippincott Williams & Wilkins. Unaut
e et Chimie des Proteines – LBTI, UMR 5305 – CNnts Pathogenes – INSERM CIE3 Vaccinologie, Fology (CIRI), INSERM U1111 – CNRS UMR5308,rance.
o Dr Stephane Paul, GIMAP - Faculte de Medec
4 67; fax: +334 77 42 14 86; e-mail: stephane.uary 2014; revised: 12 April 2014; accepted: 1
.0000000000000308
0269-9370 Q 2014 Wolters Kluwer Hea
preexisting anti-HIV immune response in mucosa-associated lymphoid tissues (MALTs) is probably of vitalimportance in preventing HIV infection [4].
The development of an effective vaccine is a considerablechallenge, especially given the formidable propensity toimmune evasion that is intrinsic to HIV. The HIV-1envelope glycoprotein (Env) that is the target of knownHIV-1-directed neutralizing antibodies (NAbs) [5–7] isprotected by an evolving shield of glycans, variableimmunodominant loops and conformational masking ofkey viral epitopes [6,8–10]. Although immunization withrecombinant Env proteins or vectors encoding Env can
horized reproduction of this article is prohibited.
RS/University of Lyon 1, Lyon, France, bGroupe Immunite desaculte de Medecine, Saint-Etienne, cInternational Centre forUniversity of Lyon 1, Lyon, and dFrench National Academy of
ine Jacques Lisfranc - 15 rue Ambroise Pare - 42023 Saint-
paul@chu-st-etienne.fr4 April 2014.
lth | Lippincott Williams & Wilkins 1701
Co
1702 AIDS 2014, Vol 28 No 12
induce high levels of HIV-1 specific Abs, vaccine-inducedAbs have been unable to neutralize most circulatingprimary HIV-1 isolates [6,7,11,12]. Indeed, naturalinfection predominantly induces nonneutralizing orstrain-specific Abs during the first months of infection[9,13–15]. However, NAbs are the best correlate ofprotection for many viral vaccines [16,17]. It was foundthat approximately 10–20% of HIV-1-infected individualshappen to develop broadly NAbs (bNAbs) after a few years[18–20], which is the type of humoral immune responseone would like a vaccine to elicit. These bNAbs are able toneutralize the vast majority of virus strains in a cross-clademanner and have been shown to provide robust protectionagainst mucosal challenges in the macaque model [21–23].
Our understanding of what constitutes a bNAb againstHIV has been revolutionized by the isolation of extremelybroad and potent neutralizing mAb from a number ofHIV-infected individuals [24–27]. These mAbs wereidentified by dissecting the broad neutralization activityseen in specific patient serum samples and by character-izing mAbs from B-cells [24,28,29].
Some bNAbs, when acting at the earliest steps of viralinfection, are able to prevent virus entry into host cells byblocking multiple steps of viral transmission by targetingeither the CD4þ-binding site or the glycan/V3 loop onHIV-1 gp120 [30]. The incapacity to induce bNAbs toHIV has thus been a major hurdle to HIV vaccineresearch since the beginning of the epidemic. Substantialobstacles remain in inducing bNAbs by immunizationand particularly at the mucosal level. Some studies haveshown evidence of the presence of NAbs in mucosal fluidsusing appropriate immunization vectors and deliveryregimens [31–34], but previous attempts to inducebNAbs by vaccination were fairly unsuccessful. Currently,based upon our existing understanding of the mucosalimmune system, we would expect that the quality ofhumoral and cellular immune responses at a given effectorsite would depend upon the route of vaccination [35]. It isthus essential to choose the suitable immunization routefor the desired mucosal site.
Despite the remarkable progress made in understandingthe epitopes that Abs recognize on the Env spikes of thevirion, one of the major goals of HIV vaccine research atthis time is the discovery of immunogens and immuniz-ation strategies that can elicit bNAbs. This challenge ismade even greater by the fact that it is more difficult toinduce high concentrations of NAbs at the mucosal levelthan at a systemic level [36].
It would certainly be beneficial for an HIV-1 vaccine toalso elicit immune responses capable of controlling viralreplication [37]. A wealth of data has shown that cellularimmune responses can mediate the control of viremia inHIV-1-infected humans and simian immunodeficiencyvirus (SIV)-infected rhesus macaques, including CD8þ T
pyright © Lippincott Williams & Wilkins. Unautho
lymphocytes [38–40], natural killer (NK) cells [41] andCD4þ T lymphocytes [42,43]. Moreover, vaccine trials innonhuman primates (NHPs) have shown that sustainedviremic control is achievable after heterologous SIVchallenges. For example, immunizations with an Adeno-virus serotype 26 prime and Modified Vaccinia Ankara(MVA) boost expressing SIV antigens led to a 2.32 logreduction in mean set point viral load following stringentSIVmac251 challenge, which was related to the magnitudeand breadth of the Gag-specific cellular immune responsesmeasured immediately prior to challenge [31].
Even more remarkable was the report that 50% of rhesusmacaques vaccinated with a SIV protein expressingrhesus cytomegalovirus (RhCMV/SIV) vector manifesteddurable, aviraemic control of infection with the highlypathogenic strain SIVmac239 [38]. The RhCMV/SIVvector elicited immune responses that control SIVmac239infection (regardless of the route of challenge) after viraldissemination. Over time, protected rhesus macaques lostsigns of SIV infection, showing a consistent lack ofmeasurable plasma or tissue-associated viral RNA or DNAusing ultrasensitive assays, and a loss of T-cell reactivity toSIV determinants not in the vaccine [44]. Similarly, it wasshown that protection against wild-type SIVmac239challenge by live attenuated SIV vaccines strongly corre-lated with the magnitude and function of SIV-specific,effector-differentiated T cells in the lymph nodes of theanimals. It follows from these observations that SIV-specific T cells can suppress wild-type SIVamplification atan early stage and, if present persistently in sufficient fre-quencies, can completely control and even clear infection[45].
Current assessments aim to evaluate a broad range ofmucosal immune responses and answer key questionssuch as can vaccines delivered parenterally elicit detectablemucosal responses? Whereas systemic immunizationinduces mostly immune responses in peripheral andsystemic sites, mucosal delivery of immunogens isthought to trigger primarily mucosal immune responses[46]. The second question is which mucosal immuneresponses may be associated with protection from HIVinfection? And the third, which mucosal specimens andassays are most relevant for the detection of theseresponses? Answers to these questions will be vital inclarifying which mucosal immune responses are capableof blocking HIV infection, and for developing vaccinesthat can elicit these types of responses.
Mucosal transmission of HIV-1: a rationalefor the role of HIV-specific mucosalimmune responses
Natural transmission of HIV-1 occurs through vaginalmucosa, the male genital tract, that is penile mucosa
rized reproduction of this article is prohibited.
Progress in HIV vaccines inducing mucosal responses Pavot et al. 1703
(inner foreskin, penile urethra), gastrointestinal mucosaand via breastmilk (vertical transmission). Although thereare challenges in quantifying risk by sex act, all studiesconsistently report that anal intercourse is a higher risk actthan vaginal intercourse and the probability of infectionby the vaginal route has been estimated to be one in 200or less [3]. Considering this route, HIV-1 can infect thevaginal, ectocervical and endocervical mucosa, but therelative contribution of each site to the establishment ofthe initial infection is unknown.
Both free and cell-associated HIV and SIV virions canestablish mucosal infection [1,47]. This has been showndirectly in vivo in female macaques [48–50], or ex vivousing human cervical explants [49,51], and indirectly inhumans through genetic sequence comparisons of viralisolates from acutely infected women with those fromseminal leukocytes (cell-associated virions) and plasmafrom their infected source partners [52–54]. Ex-vivostudies using human cervical explants and reconstructedvaginal mucosa have confirmed transmission of cell-freeand cell-associated HIV-1 [55–58]. Cervical mucus cantrap infected seminal cells or free virions [59,60].Conceivably, this could facilitate viral transmission byprolonging the time of contact of the virions with themucosa. However, although immobilized, the virionsmay also become more susceptible to innate antiviralsubstances or to Abs.
Several reports have shown that HIV virions bind to andenter epithelial cells in the female genital tract [61–63].Virions that are initially free, or those that are releasedfrom infected donor seminal T cells, interact withepithelial cells and traverse the epithelium by severalpathways, including transcytosis, endocytosis and sub-sequent exocytosis, by causing productive infection, ormerely by penetrating through the gaps betweenepithelial cells in the vaginal multilayered epithelium[64].
Transcytosis, which occurs across single layeredepithelia, has been shown to occur in cell lines andalso in primary cells, but has not been definitivelydemonstrated in intact mucosal tissues. Interestingly,cell-associated virions secreted from infected seminalleukocytes appear markedly more efficient at transcy-tosis than cell-free virions [65,66]. It is actually likelythat HIV is not transmitted as a naked particle, butrather as an immune complex with Env-binding IgGsthat are abundant in the semen of HIV-positive men.The low pH of the vagina and urethra also plays animportant role in transcytosis, as the neonatal FcRnreceptor that is expressed on epithelial cells of thepenile urethra and endocervix binds the IgGs at low pHand releases them at neutral pH, thus favouring thecapture of the immune virus complexes on the acidicapical (vaginal) side of the epithelium and their releasein the neutral basolateral environment [67].
Copyright © Lippincott Williams & Wilkins. Unaut
Upon release from epithelial cells, the virions can readilyinfect susceptible leukocytes [63]. It has been reportedthat virions can also productively infect the cervicalepithelial cells themselves [63,68], although this pointremains contested [69,70]. Conceivably, HIV-1 can alsobe transported through the cervicovaginal epithelium tothe draining lymph nodes by infected lymphocytes,macrophages, monocytes and dendritic cells, as has beensuggested in both in-vitro systems and mouse studies[68,71–75]. The rationale for the role of specific mucosalimmune responses in protection from HIV-1 transmissionhas been highlighted by numerous studies in human andNHPs (Table 1) [76–89].
Anatomic sites of HIV-1 persistence
Another rationale for eliciting HIV-specific mucosalimmune responses is linked to their potential to preventthe establishment of viral reservoirs within a newlyinfected host. A viral reservoir can be defined as cell typesor anatomical sites in which replication-competent formsof the virus can and do persist throughout infection and inthe presence of otherwise efficient antiretroviral therapy(ART) [90]. Gastrointestinal and vaginal mucosal tissuesare major reservoirs for initial HIV replication andamplification, and the sites of rapid CD4þ T-celldepletion [91]. Such viral reservoirs are currently thoughtto be a key factor explaining our difficulty in successfullyeradicating HIV-1 from an infected host via the currentlyavailable treatments regimes. A successful vaccine wouldneed to prevent the establishment of these reservoirs at avery early step of viral infection. This is especiallyimportant considering the fact that aggressive ART invery early acute infection can substantially decrease thesize of the viral reservoir in terms of integrated DNA andRNA concentrations [92]. Although most HIV pro-viralDNA is found in CD4þ T lymphocytes in lymphoidtissue, blood viral reservoirs may also be maintained incentral and transitional memory T cells that persistthrough mechanisms of homeostatic proliferation andrenewal. Other potential sources may include monocytesand macrophages, astrocytes and microglial cells [92].
A related aspect is also the notion of viral compartmentswithin an infected individual. A viral compartment maybe defined as a cell or tissue replication site wherein apopulation of viral variants is at least partially restricted inits ability to enter, leave and replicate and therefore displaya limited exchange of viral genetic information withother sites [90]. It is unclear as to whether all sites of viralcompartmentalization represent viral reservoirs in thestrictest sense. This anatomical compartmentalization ofHIV-1 variants has been well described for the centralnervous system (CNS), the gut-associated lymphoidtissue (GALT) [93,94] and the genital tract, althoughthere are also data for viral compartmentalization within
horized reproduction of this article is prohibited.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1704 AIDS 2014, Vol 28 No 12
Tab
le1.
Exam
ple
sof
studie
sdem
onst
rati
ng
corr
elat
esof
muco
sal
imm
une
pro
tect
ion
inH
IV-1
infe
ctio
n.
Model
Study
des
ign
Outc
om
eR
efer
ence
s
Hum
anH
IV-1
spec
ific
muco
sal
IgA
ina
cohort
of
HIV
-1re
sist
ant
Ken
yan
fem
ale
sex
work
ers.
HIV
-1-s
pec
ific
IgA
ispre
senti
nth
ege
nit
altr
acto
fmost
HIV
-1re
sist
antK
enya
nse
xw
ork
ers.
[76,7
7]
Hum
anA
bil
ity
aten
rolm
ent
of
genit
alIg
Ato
neu
tral
ize
pri
mar
yH
IVis
ola
tes
asw
ell
assy
stem
icH
IV-s
pec
ific
cell
ula
rre
sponse
s.G
enit
alH
IV-n
eutr
aliz
ing
IgA
and
syst
emic
HIV
-spec
ific
pro
life
rati
vere
sponse
sw
ere
pro
spec
tive
lyas
soci
ated
wit
hH
IVnonac
quis
itio
n.
[78]
Hum
anH
IV-1
sero
dis
cord
antc
ouple
sin
Nai
robiw
ere
foll
ow
edquar
terl
yup
to2
year
s.C
ervi
cova
ginal
IgA
was
asse
ssed
forH
IV-1
neu
tral
izin
gac
tivi
tyby
aper
ipher
alblo
od
mononucl
earce
llbas
edas
say
usi
ng
anH
IV-1
clad
eA
pri
mar
yis
ola
te.
HES
Nw
om
enw
ere
five
tim
esm
ore
like
lyto
hav
eneu
tral
izin
gIg
Ain
cerv
icova
ginal
secr
etio
ns
than
low
-ris
kco
ntr
ol
wom
en.
Res
ponse
sw
ere
inve
rsel
yas
soci
ated
wit
hpar
tner
vira
llo
ad.
Thes
eobse
rvat
ions
support
the
exis
tence
ofan
tivi
ralac
tivi
tyin
the
muco
sal
IgA
frac
tion
foll
ow
ing
sexu
alH
IV-1
exposu
re.
[79]
Hum
anSu
b-p
reputial
swab
san
dfo
resk
inti
ssue
from
HES
Nan
dunex
pose
dco
ntr
ol
men
wer
eco
llec
ted.
Adec
reas
ein
the
rela
tive
abundan
ceof
susc
epti
ble
CD
4þ
Tce
lls,
inco
mbin
atio
nw
ith
HIV
-neu
tral
izin
gIg
Aan
da
-def
ensi
ns
may
repre
sent
apro
tect
ive
imm
une
mil
ieu
ata
site
of
HIV
exposu
re.
[80]
Inve
stig
ators
assa
yed
swab
sfo
rH
IV-n
eutr
aliz
ing
IgA
.H
um
anG
ener
atio
nof
aC
D4þ
-induce
dhum
anm
Ab,
F425A
1g8
.The
IgA
1va
rian
tdis
pla
yed
sign
ifica
nt
indep
enden
tneu
tral
izat
ion
acti
vity
agai
nst
ara
nge
of
HIV
clad
eB
isola
tes.
[81]
Char
acte
riza
tion
of
the
impac
tof
its
isoty
pe
vari
ants
on
HIV
neu
tral
izin
gac
tivi
ty.
Hum
anA
bil
ity
of
IgA
isola
ted
from
HEP
Sin
div
idual
sto
inhib
ittr
ansc
ytosi
sac
ross
ati
ght
epit
hel
ial
cell
laye
rin
vitr
o.
Inhib
itio
nw
asse
enin
thre
eof
six
cerv
icova
ginal
fluid
sam
ple
s,5/1
0sa
liva
sam
ple
san
dth
ree
of
six
pla
sma
sam
ple
sag
ainst
pri
mar
yH
IV-1
isola
tes
test
ed.
[82]
Hum
anM
uco
sal
Fab
IgA
libra
ryfr
om
HEP
San
din
-vit
roch
arac
teri
zati
on
of
seri
esof
HIV
-1Ig
As
spec
ific
for
gp41.
IgA
are
tran
scyt
osi
s-blo
ckin
gan
din
fect
ion-n
eutr
aliz
ing.
[83]
Hum
anN
inet
een
indiv
idual
sat
risk
for
HIV
infe
ctio
nw
ere
scre
ened
for
viru
s-sp
ecifi
cm
emory
CTL.
HIV
-spec
ific
CTL
wer
edet
ecte
din
seve
nof1
7ex
pose
dunin
fect
edin
div
idual
s.[8
4]
Rec
ogn
itio
nof
viru
sby
CTL
inex
pose
dunin
fect
edin
div
idual
sw
asM
HC
-Ire
stri
cted
.H
um
an37
HIV
-1-u
nin
fect
edper
sons
wit
hre
pea
ted
hig
h-r
isk
sexu
alac
tivi
tyw
ith
anH
IV-1
-infe
cted
par
tner
wer
epro
spec
tive
lyst
udie
dto
det
erm
ine
pro
tect
ing
fact
ors
agai
nst
HIV
-1tr
ansm
issi
on.
Thir
teen
of
36
dem
onst
rate
dH
IV-1
spec
ific
cyto
toxi
city
.[8
5]
Hum
anA
sses
smen
tof
HIV
-spec
ific
epit
ope
reco
gnitio
n,
pla
sma
vira
llo
adan
dex
pre
ssio
nofH
LAcl
ass
Iall
eles
ina
cohort
ofH
IV-s
eroposi
tive
bar
work
ers.
CD
8T-c
ell
reco
gnit
ion
of
mult
iple
epit
opes
wit
hin
spec
ific
Gag
regi
ons
isas
soci
ated
wit
hm
ainte
nan
ceof
alo
wst
eady-
stat
evi
rem
iain
HIV
-1se
roposi
tive
pat
ients
.
[86]
Hum
anR
ecen
tw
ork
has
focu
sed
grea
ter
atte
nti
on
on
qual
itat
ive
feat
ure
sof
the
HIV
-spec
ific
CD
8þ
T-c
ell
resp
onse
.In
vest
igat
ion
from
mult
iple
groups
has
now
focu
sed
upon
HIV
-spec
ific
CD
8þ
T-c
ell
gran
ule
-exo
cyto
sis
med
iate
dcy
toto
xici
tyas
aco
rrel
ate
of
imm
unolo
gic
contr
ol
of
HIV
.
[39]
Hum
anD
etai
led
study
of
the
inte
rpla
ybet
wee
nT-c
ell
funct
ional
attr
ibute
susi
ng
aban
kof
HIV
-spec
ific
CD
8þ
T-c
ell
clones
.H
ighly
sensi
tive
CD
8þ
Tce
lls
dis
pla
ypoly
funct
ional
pro
file
san
dpote
nt
HIV
-suppre
ssiv
eac
tivi
ty.
[87]
NH
PdIg
A1,dIg
A2
and
IgG
1ve
rsio
ns
ofnm
Ab
HG
N194
wer
eap
plied
intr
arec
tall
yin
thre
erh
esus
mac
aque
groups
bef
ore
intr
arec
tal
SHIV
chal
lenge
.dIg
A1-m
edia
ted
captu
ring
of
viri
ons
inm
uco
sal
secr
etio
ns
and
inhib
itio
noftr
ansc
ytosi
sca
npro
vide
sign
ifica
ntpre
venti
on
ofle
nti
vira
lac
quis
itio
n.
[88]
Inhib
itio
nof
tran
scyt
osi
sof
cell
-fre
evi
rus
acro
ssan
epit
hel
ial
cell
laye
rin
vitr
o.
NH
PThey
asse
ssed
whet
her
hum
anM
Absb12
and
b6
agai
nst
the
CD
4þ
-bin
din
gsi
teon
HIV
-1gp
120
and
F240
agai
nst
anim
mundom
inan
tep
itope
on
gp41
could
pre
vent
vagi
nal
tran
smis
sion
of
SHIV
.
Appli
edva
ginal
ly,
the
stro
ngl
yneu
tral
izin
gM
Ab
b12
pro
vided
ster
iliz
ing
imm
unit
yin
seve
nof
seve
nan
imal
s.[8
9]
Progress in HIV vaccines inducing mucosal responses Pavot et al. 1705
the lung, liver, kidney and breast milk (for a recent reviewsee [95]).
Env-specific antibodies to protect againstHIV-1 acquisition at mucosal surfaces
The design of immunogens able to elicit NAb remains amajor goal of HIV-1 vaccine development [96]. Manystudies in NHPs have shown that passive infusion of HIVNAbs, especially bNAbs, can prevent rectal or vaginalinfection by a chimeric simian-HIV (SHIV) containingthe env gene of HIV-1. This was initially shown using asingle oral or vaginal inoculation sufficient to infect 100%of control animals [97–100]. In this setting, protectionagainst SHIV infection was most directly associated withthe neutralization potency of the infused Abs [101,102].However, recent passive transfer studies have employedlow-dose multiple mucosal challenges to infect all controlanimals [103,104]. This model may be more physiologi-cally relevant to the relatively low probability of sexualinfection seen with HIV-1 in humans. In the low-doseNHP model, approximately 10-fold fewer Abs wererequired to mediate protection against infection thanprior studies with high-dose virus challenge: serum Abtitres sufficient to mediate 90% virus neutralization at 1 : 5serum dilution were associated with protection.
It has been suggested that Fc-mediated Ab effectorfunctions might also play an important role in conferringprotection. Indeed, although direct antibody-mediatedneutralization is highly effective against cell-free virus,increasing evidence suggests an important role for IgGFcg receptor (FcgR)-mediated inhibition of HIVreplication. Thus, bNAb IgG1 b12 showed a diminishedprotective potency after its Fc region was altered to knockout complement binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity without decreas-ing its in-vitro neutralizing activity [105]. A recent studyscreened a panel of bNAbs and nonneutralizing Abs(NoNAbs) for their ability to block HIV acquisitionand replication in vitro in either an independent orFcgR-dependent manner. In the NHP model, vaginalapplication of a gel containing the selected bNAbs2G12, 2F5 and 4E10 prevented SHIV transmission in 10out of 15 macaques after vaginal challenge, whereas theNoNAbs 246-D and 4B3 had no impact on SHIVacquisition but reduced plasma viral load [22]. Theseresults highlight that distinct neutralization and inhibitoryactivity of anti-HIV Abs affect in-vivo HIV acquisitionand replication in different ways and demonstrate thepotential interest of NAbs for microbicide and vaccinedevelopment. It follows that vaccines may not need toachieve extraordinarily high levels of HIV-1 NAbs toelicit protection at mucosal surfaces, but the Ab responsewill likely need to be durable, and NAbs will have tocross-react with a genetically diverse spectrum of HIV-1strains.
Copyright © Lippincott Williams & Wilkins. Unaut
We also do not know which type of immunoglobulins arethe best at blocking the virus at mucosal surfaces. IgG1Abs certainly can play a role, as passive infusion of suchanti-HIV bNAbs into the blood can protect animals frommucosal challenge [106,107]. However, it is known thatIgA is the predominant Ab in the majority of mucosalsecretions [108]. Mucosal IgA Ab is generated primarilyin the mucosal epithelial compartment and transportedacross the epithelial cell boundary into external secretionsby interacting with the polymeric immune globulinreceptor (pIgR) [109]. It was recently reported thatrectally applied dimeric IgA Abs derived from bNAbHGN194 could not only protect NHPs from rectalchallenge with SHIV-1157ipEL-p, but also that they didit more effectively than corresponding IgGs [88].Comparison of the IgG1 version of bNAb HGN194with its dimeric IgA versions, dIgA1 and dIgA2, showedthat the dIgA1 version protected the animals better thanthe dIgA2 and IgG1. Thus, five out of the six animalstreated with the dIgA1 version remained uninfected,whereas only one of the six dIgA2-treated animals andtwo of the six IgG1-treated animals remained virus free.All 11 untreated animals got infected.
The increased protection observed in the dIgA1-treatedanimals was initially puzzling, as all three HGN194versions neutralized the challenge virus equally well. Theexplanation could be that the dIgA1 version can bindtwice as many virus particles as the dIgA2 version. As aresult, a dIgA1 molecule can accommodate four virusparticles between its antigen-binding sites, while dIgA2can only accommodate two. This also might explain whyonly dIgA1 and not dIgA2 nor IgG1 were able to preventmost HIV particles from crossing a cultured epithelial celllayer in an in-vitro transcytosis assay [88].
Altogether, these results suggest that one should try todevelop vaccines that can elicit dIgA1s at mucosalsurfaces. These Abs do not necessarily have to beneutralizing, because it is the ability of dIgA1s to be ableto bind more HIV particles, and not necessarily bettervirus neutralization, which seems to be responsible fortheir higher level of protective efficacy.
Studies in humans have also revealed a correlation betweena high level of secretory IgA (SIgA) and protection in high-risk individuals who remain seronegative (highly exposedpersistently seronegative persons) [76,78,110,111]. Thesestudies concluded that protection could be mediated by theinteraction between these SIgA and HIV-1 on mucosalsurfaces or within epithelial cells capable of internalizingIgA-boundHIV-1. This conclusion is howeverdisputed, asthere is no evidence for IgA-mediated intraepithelial HIV-1 neutralization [112–114].
However, in addition to its ability to neutralize virus, it isthought that IgA may contribute to the elimination ofvirus in the form of exocrine immune complexes via the
horized reproduction of this article is prohibited.
Co
1706 AIDS 2014, Vol 28 No 12
lamina propria. In this manner, the mechanism of IgAprotection may be wider than that provided by IgG-mediated neutralization. It also follows that assays basedon neutralizing IgG Abs may not be suitable for assessingthe activity of mucosal IgA [115].
Vaccination strategies that elicit mucosalneutralizing antibodies
In clinical trials that show the efficacy of a vaccine, theidentification of immune responses that are predictive oftrial outcomes generates hypotheses about which of thoseresponses are responsible for protection [116,117]. TheRV144 phase 3 trial in Thailand was an opportunity toperform such a hypothesis-generating analysis for anHIV-1 vaccine. This trial of the canarypox vector vaccine(ALVAC-HIV [vCP1521]) as well as the gp120 AIDS-VAX B/E vaccine showed an estimated vaccine efficacyof 31.2% for the prevention of HIV-1 infection over aperiod of 42 months after the first of four plannedvaccinations [118]. This result enabled a systematic searchby Haynes et al. [119] who performed a case–controlanalysis to identify Ab and cellular immune correlates ofinfection risk. This immune-correlates study generatedthe hypotheses that levels of V1V2 Abs correlatedinversely with the risk of infection, whereas high levels ofEnv-specific IgA may have mitigated the effects ofprotective Abs. However, any protective role of mucosalAbs in the context of HIV-1 vaccination could not beevaluated in the RV144 trial, because mucosal sampleswere not collected.
Several studies have shown that immunization by the nasalroute (i.n.) can be most effective at eliciting Abs andcellular immunity in the female genital tract [35]. Thus,the use of live replicating recombinant Ad5hr-vectoredvaccine, in the rhesus macaques model, administered firstby i.n. and oral routes then intratracheally followed byEnv protein boosts resulted in systemic and mucosal Abresponses, including NAb, ADCC and transcytosisinhibition, together with potent cell immune responses[120]. Mucosal IgA immunity correlated with delayedacquisition following a repeated low-dose rectal SIV(mac251) challenge. The replicating Ad5 vector wasshown to disseminate across multiple mucosal sitesirrespective of delivery route [121]. These results suggestthat initial mucosal vaccination with a replicating vectorinducing NAbs in combination with a potent proteinboost may significantly reinforce protective immunityagainst SIV mucosal transmission.
As another example, four of five rhesus macaquesvaccinated first by intramuscular route (i.m.) and theni.n. with gp41-subunit antigens presented on virosomeswere protected against 13 consecutive vaginal challengeswith SHIV-SF162P3, and the fifth specimen showed only
pyright © Lippincott Williams & Wilkins. Unautho
transient infection. All of the animals displayed gp41-specific vaginal IgAs with HIV-1 transcytosis-blockingproperties and vaginal IgGs with neutralizing and/orADCC activities [32].
The immunogenicity of virosomes spiked with a gp41MPER peptide (P1) was tested in a phase I, double-blind,randomized, placebo-controlled trial in 24 healthy HIV-uninfected young women [122]. Antigen-specific serumIgGs and IgAs were elicited in all high-dose recipientsafter the first i.m. injection, but vaginal and rectal gp41-specific IgGs could be detected only after boosting via thei.n. route.
Although these data speak highly in favour of the nasalroute of immunization to elicit mucosal anti-HIV Abs inthe female genital tract, numerous studies have also shownthat parenteral immunization is able to induce protectivemucosal immune responses, notably with viral vectors(Table 2) [123–129]. Indeed, studies have demonstratedthe capacity of adenovirus/poxvirus and adenovirus/adenovirus vector based vaccines expressing HIV-1mosaic Env, Gag and Pol administered by i.m. route toprotect rhesus macaques against acquisition of infectionfollowing repetitive intrarectal inoculations of thedifficult-to-neutralize SHIV-SF162P3 or SIVmac251[31,123].
Cellular immune responses mediatecontrol of viremia
Whereas Env-specific Abs appear necessary to blockHIV-1 acquisition, Gag-specific cellular immuneresponses appear important for the control of virusreplication and viral load after infection. Gag-specificCD8þ T cells, but not Env- nor Pol-specific CD8þ Tcells, correlate with in-vivo viral load control followingSIV challenge in vaccinated monkeys [130]. This result isconsistent with studies demonstrating the association ofGag-specific cellular immune responses with viremiacontrol in HIV-1 infected individuals [131–133] andSIV-infected rhesus macaques [31,134–136]. Vif and Nefmay also contribute to viral load control in monkeys[137].
As conservation of polyfunctional HIV-specific CD8þ
T-cells appears to correlate with the control of viremia ininfected people [138], the polyfunctionality of the T-cellresponse is perceived as one of the best correlates of T-cellimmunity [87]. Thus, Ferre et al. [139] showed thatmucosal CD8þ cytotoxic T-lymphocyte (CTL) responsesin controllers are more complex and significantly strongerthan in antiretroviral-suppressed persons: HIV-controllersshow long-lasting, high avidity, polyfunctional Gag-specific CD8þ T-cell responses in mucosal compartmentsas compared with noncontrollers.
rized reproduction of this article is prohibited.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Progress in HIV vaccines inducing mucosal responses Pavot et al. 1707
Tab
le2.
Exam
ple
sof
vacc
ines
elic
itin
gpro
tect
ive
resp
onse
sag
ainst
muco
sal
chal
lenge
sin
the
NH
Pm
odel
.
Del
iver
yst
rate
gies
Route
of
imm
uniz
atio
nM
uco
sal
resp
onse
Ref
eren
ces
Ad26/A
d35/M
VA
vect
or-
bas
edva
ccin
esex
pre
ssin
gH
IV-1
mosa
icEn
v,G
ag,
and
Pol
Intr
amusc
ula
rSu
bst
anti
alpar
tial
pro
tect
ion
(�45%
)ag
ainst
repet
itiv
e,in
trar
ecta
l,het
erolo
gous
SHIV
-SF1
62P3
chal
lenge
s.[1
23]
Env-
spec
ific
bin
din
gA
bs
corr
elat
edm
ost
stro
ngl
yw
ith
pro
tect
ion.
Ad26/M
VA
or
Ad35/A
d26
vect
or-
bas
edva
ccin
esex
pre
ssin
gSI
Vsm
E543
Gag
,Pol
and
Env
anti
gens
Intr
amusc
ula
rPro
tect
ion
agai
nst
acquis
itio
nof
SIV
(mac
251)
isco
rrel
ated
wit
hEn
v-sp
ecifi
cA
bre
sponse
s.[3
1]
Vir
olo
gic
contr
ol
may
be
corr
elat
edw
ith
both
Tly
mphocy
tean
dA
bre
sponse
s(r
ecta
lse
cret
ions
and
colo
rect
albio
psi
es).
rAd26
enco
din
gSI
Vm
ac239
Gag
Intr
amusc
ula
rD
ura
ble
Gag
-spec
ific
cell
ula
rim
mune
resp
onse
sin
mult
iple
muco
sali
mm
une
com
par
tmen
ts,
incl
udin
gin
the
ora
lan
dga
stro
inte
stin
alm
uco
sa.
[128]
Pla
smid
DN
A(p
rim
e)rA
d5
(boost
)In
tram
usc
ula
rLo
wle
vels
ofN
Abs
and
anEn
v-sp
ecifi
cC
D4þ
T-c
ellr
esponse
wer
eas
soci
ated
with
vacc
ine
pro
tect
ion
agai
nst
muco
sali
nfe
ctio
nby
SIV
smE6
60
(�50%
).[1
25]
Nonre
pli
cati
ng
rAd
vect
ors
from
vari
ous
sero
types
expre
ssin
gSI
Vm
ac239
Gag
Intr
amusc
ula
rSI
V-s
pec
ific
Tly
mphocy
tere
sponse
sth
atper
sist
edfo
rove
r2
year
sin
both
per
ipher
alblo
od
and
multip
lem
uco
sal
tiss
ues
(colo
rect
al,
duoden
alan
dva
ginal
bio
psy
spec
imen
s)an
dbro
nch
oal
veola
rla
vage
fluid
.
[126]
GM
-CSF
DN
Aw
ith
DN
Apri
me
for
aSH
IV-8
9.6
vacc
ine
Intr
amusc
ula
ror
intr
ader
mal
Avi
dity
mat
ura
tion
of
anti
-Env
IgG
inblo
od.
[34]
Pre
sence
of
long-
last
ing
anti
vira
lIg
Ain
rect
alse
cret
ions.
DN
Aw
ith
afu
llSI
Vm
ac239
genom
e/rM
VA
expre
ssin
gSI
VG
ag-P
ol
and
Env
pro
tein
sIn
tram
usc
ula
ror
intr
anas
alSI
V-s
pec
ific
rect
alIg
Are
sponse
sw
ere
more
sign
ifica
ntly
per
sist
ent
ini.n.
vacc
inat
edth
anin
i.m
.va
ccin
ated
rhes
us
mac
aques
.[3
3]
I.n.
imm
uniz
atio
nin
duce
dsi
gnifi
cant
anti
-SIV
T-c
ell
resp
onse
sin
the
colo
rect
alm
uco
sa.
gp41-s
ubunit
anti
gens
graf
ted
on
viro
som
esIn
tram
usc
ula
ran
din
tran
asal
gp41-s
pec
ific
vagi
nal
IgA
s.[3
2]
HIV
-1tr
ansc
ytosi
s-blo
ckin
gpro
per
ties
.V
agin
alIg
Gs
with
neu
tral
izin
gan
d/o
rA
DC
Cac
tivi
ties
.R
ecom
bin
ant
repli
cati
ng
Vac
cinia
viru
sTia
nta
n/S
IVgp
e(p
rim
e)–
Ad5SI
Vgp
e(b
oost
)In
trao
ral
and
intr
anas
al(p
rim
e)an
din
tram
usc
ula
r(b
oost
)SI
V-s
pec
ific
CD
8þ
T-c
ellm
edia
ted
imm
unit
yag
ainst
Gag
and
Poli
sas
soci
ated
with
pro
tect
ion
agai
nst
hig
h-d
ose
intr
arec
talin
ocu
lati
on
ofSI
V(m
ac239).
[129]
RhC
MV
/SIV
Subcu
taneo
us
Induct
ion
of
SIV
-spec
ific
TEM
resp
onse
sat
pote
nti
alsi
tes
of
SIV
replica
tion.
[38]
RhC
MV
vect
ors
expre
ssin
gSI
VG
ag,R
ev/N
ef/T
at/E
nv
Subcu
taneo
us
Mai
nta
indif
fere
nti
ated
effe
ctor
mem
ory
T-c
ell
resp
onse
s.[1
24]
Mai
nta
inro
bust
SIV
-spec
ific
CD
4þ
and
CD
8þ
effe
ctor
mem
ory
T-c
ell
resp
onse
s.
Incr
ease
dre
sist
ance
toac
quis
itio
nof
SIV
mac
239
infe
ctio
n(i
ntr
arec
tal
chal
lenge
).Pri
me:
repli
cation-c
om
pet
ent
Ad5hr-
SIV
smH
4en
v/re
vm
uta
nt
and
Ad5hr-
SIV
239ga
gB
oost
:SI
Vm
ac251
gp120
pro
tein
ina
MPLA
-sta
ble
emuls
ion
Sublingu
alIn
tran
asal
/intr
atra
chea
lIn
trav
agin
alIn
trar
ecta
l
All
imm
uniz
atio
nro
ute
sel
icit
edsI
gAre
sponse
sat
mult
iple
muco
sal
site
s.Si
gnifi
cant
corr
elat
ion
of
vacc
ine-
induce
dsI
gAti
ters
inre
ctal
secr
etio
ns
with
del
ayed
acquis
itio
n.
[120]
Lact
ob
acill
us
pla
nta
rum
and
inac
tiva
ted
SIV
mac
239
Ora
lThe
tole
roge
nic
vacc
ine
induce
dM
HC
-Ib/E
-res
tric
ted
CD
8þ
Tre
gsth
atsu
ppre
ssed
SIV
-har
bori
ng
CD
4þ
T-c
ell
acti
vati
on.
[127]
Ex-v
ivo
SIV
repli
cati
on
in15/1
6an
imal
sw
ithout
induci
ng
SIV
-spec
ific
Abs
or
cyto
toxi
cT
lym
phocy
tes.
15/1
6an
imal
sw
ere
ster
ilel
ypro
tect
edaf
ter
intr
arec
tally
chal
lenge
with
SIV
mac
239
or
het
erolo
gous
stra
inSI
VB
670.
Co
1708 AIDS 2014, Vol 28 No 12
Another critical aspect of HIV cellular immune responsesis the location of the HIV-specific immune cells elicitedby immunization. Thus, the degree of protectionmediated by a live attenuated SIV vaccine stronglycorrelates with the location of SIV-specific effectorCD8þ T cells, in lymph nodes [45]. The maintenance ofthis protective T-cell response seems to be associated withpersistent replication of the live attenuated virus vaccinein follicular helper T (Tfh) cells.
Surprisingly, none of the candidate HIV vaccines tested sofar in human volunteers has been able to elicit viral loadcontrol. Neither the Step trial, based on the use of arecombinant Ad5 vector, nor the HVTN 505 trial, whichused a DNA prime followed by a recombinant Ad5 boost,nor the RV144 trial, using a recombinant Canarypoxprime followed by gp120 boosts, showed any significantimpact on viral load in vaccine recipients who becameinfected with HIV-1 [118,140,141]. There actually wassome evidence for immune selection pressure onbreakthrough HIV-1 sequences in the Step study,suggesting that, although too weak to be efficient,vaccine-elicited cellular immune responses did exertimmunologically relevant biological effects in humans[142]. The disappointing results of the Step and HVTN505 vaccine trials highlight the likely importance ofinducing mucosal immune responses that could signifi-cantly decrease virus replication in the mucosa andsubsequent viral dissemination to peripheral lymphoidtissues and blood. Another, but different example is theSIV protein encoding RhCMV, which is able to maintaindifferentiated effector memory T-cell responses at viralentry sites that show high efficacy at impairing SIVreplication at its earliest stage. This strategy can maintainrobust SIV-specific CD4þ and CD8þ effector memoryT-cell (TEM) responses that provide protection againstrepeated limiting-dose intrarectal challenge with SIV-mac239 [124].
Studies of the early kinetics of T-cell responses inpreviously vaccinated, acutely SIV-infected NHPs willallow the determination of whether an initial influx ofvirus-specific CD4þ T cells precedes robust CTLresponses and correlates with early containment [143].Alternatively, CD4þ CTL may directly contribute tocontainment of HIV infection [43].
Vaccination strategies that elicit cellularmucosal immune responses
Hansen et al. [38] reported that RhCMV/SIV vectorsused by subcutaneous route (s.c.) in the rhesus macaquesmodel are able to induce immune-mediated control ofhighly pathogenic SIVmac239 after repeated intrarectalchallenges and prior to irreversible establishment ofinfection. An early complete control of SIV was observed
pyright © Lippincott Williams & Wilkins. Unautho
in 13 of 24 rhesus macaques receiving either RhCMValone or RhCMV (s.c. prime)/Ad5 (i.m. boost) vectors,and a long-term protection (�1 year) was observed in 12of these 13 animals [124]. The immunologic assaysperformed in mononuclear cell preparations from bloodand tissues suggest that this control is related to the highfrequency of SIV-specific T-cell responses (CD8þ, andpossibly CD4þ). These responses are located both inmucosal portals of entry and at potential sites of distantviral spread and are indefinitely maintained by thepersistent RhCMV vectors, and can protect withoutanamnestic expansion.
The finding that RhCMV/SIV vector-protected rhesusmacaques are able to control haematogenous SIVdissemination after both intrarectal and intravaginalchallenge suggested that the immune responses elicitedby these vectors might provide protection even whenmucosal surfaces are bypassed [44]. Thus, persistentvectors such as CMV and their associated TEM responsesmight significantly contribute to an efficacious HIV/AIDS vaccine.
The NHP model was also used to test the vaccinationapproach using a plasmid DNA prime/rAd5 boostvaccine developed to induce both CD8þ T lymphocyteresponses and Env-specific Ab responses [125]. Afterrepeated intrarectal challenges, the vaccine failed toprotect against SIVmac251, but 50% of vaccinatedmonkeys were protected from infection withSIVsmE660. Although the exploration of immunecorrelates suggests that a NAb may be responsible forthe conferred protection against mucosal acquisition ofSIVsmE660, the reduction in peak plasma virus RNAimplicates CTL in the control of SIV replication onceinfection is established.
Intramuscular vaccination of rhesus macaques withAd/MVA or Ad/Ad vector expressing SIV Gag, Poland Env antigens, were also investigated for their capacityto induce CD8þT lymphocytes and to test whether theseresponses predict virologic control following SIV mucosalchallenge [130].
They observed that CD8þ cell mediated SIV inhibitionwas significantly associated with Gag-specific cellularimmunity but not Pol or Env-specific cellular immunityand that CD8þ lymphocytes from 23 vaccinated rhesusmacaques inhibited replication of the virus in vitro.Moreover, the level of inhibition prior to challenge wasinversely correlated with set point SIV plasma viral loadafter intrarectal challenge. These findings demonstratethat in-vitro viral inhibition following vaccinationlargely reflects Gag-specific cellular immune responsesand correlates with in-vivo control of viremia followinginfection. These data suggest the importance ofincluding Gag in an HIV-1 vaccine in which controlis desired.
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Progress in HIV vaccines inducing mucosal responses Pavot et al. 1709
Rhesus macaques immunized by the i.n. route with a SIVDNA/MVA prime-boost regimen also demonstratedsignificant anti-SIV CTL responses in the colorectalmucosa and a better control of rectal SIVmac251infection when compared with macaques given the samevaccine by the i.m. route [33]. However, it was reportedthat an i.m. injection of nonreplicating recombinantAdenovirus vectors into rhesus macaques is able tosignificantly induce SIV-specific CTL responses thatpersist for over 2 years in multiple mucosal tissues, such ascolorectal, duodenal and vaginal biopsy specimens [126].
Despite the fact that mucosal vaccination often elicitslower magnitude HIV-specific T-cell responses whencompared with systemic vaccination, mucosal immuniz-ation can elicit better protection against HIV challengemediated by higher avidity CTLs in macaques, as assessedin systemic fluids [144]. Interestingly, interleukin (IL)-13seems to be detrimental to the efficient avidity of theseT-cells in the mouse model [145]. Recombinant HIV-1vaccines that coexpress the soluble or membrane-boundforms of the IL-13 receptor a2 (IL-13Ra2), and whichcan block IL-13 activity at the immunization site, wereused to make wild-type mice comparable to IL-13knock-out animals [146]. After an i.n./i.m. prime-boostvaccination, these vaccines adjuvanted with IL-13Ra2were shown to induce multifunctional mucosal CD8þ
T-cell responses in the lung, genito-rectal nodes andPeyer’s patches with greatly enhanced functional avidityand broader cytokine/chemokine profiles that providedgreater protection against a surrogate mucosal HIV-1challenge [146].
As mucosal prime-boost immunizations elicit significantnumbers of high avidity effector memory CD8þ CTL inmucosal and systemic compartments, they appear to be anessential component of any immunization approach thataims at establishing protective frontline defenses againstHIV-1 infection.
Exploring mucosal routes of immunization
Systemically delivered viral vectors can induce mucosalimmune responses against HIV-1 or SIV, most notably inthe gut, rectal and genital mucosa [147,148]. However,the strength of these responses is generally poor. Forexample, Ad-vectored vaccines have been shown toinduce low levels of mucosal immune responses aftersystemic inoculation, which are approximately 10 timeslower than the immune responses induced at systemicsites. Low-level mucosal immune responses have alsobeen seen in individuals inoculated i.m. with arecombinant pox virus vector [149]. However, at thistime, there are very few validated mucosal vaccinesagainst any infectious disease [35] and the mucosalvaccines already available provide protection only via
Copyright © Lippincott Williams & Wilkins. Unaut
induction of Ab responses. There are no current mucosalvaccines that are known to induce strong protectivecellular immune responses at the systemic or mucosallevel. Therefore, understanding the biology of themucosal immune system in order to develop bettermucosal vaccines that can induce both humoral andcellular immunity is needed.
Mucosal vaccines using oral or nasal routes have the greatadvantages of being painless, easy to administer on a largescale and easier to store and to deliver than currentsystemic vaccines [35]. Vaccination at a mucosal sitestimulates local immunity as well as immunity in othermucosal sites and usually also induces systemic immuneresponses detectable in the blood, spleen and peripherallymph nodes. This is in contrast to systemically deliveredvaccines, which are usually limited in their ability tostimulate an immune response in mucosal tissues[150,151].
Vaccines that target the nasal, oral, rectal or urogenitalmucosa have been under investigation for some time,using attenuated virus, inactivated virus, recombinantvirus, DNA, dendritic cells or peptides [152–155]. Oralimmunization strategies have been shown to induceHIV/SIV-specific immune responses in the gastrointes-tinal tract [156–158], whereas nasal immunizationstrategies have been reported to induce robust immuneresponses in the colorectal mucosa and genitourinarytracts in the NHP model [32,33,120]. Therefore, amucosal immunization strategy using both the oral andnasal routes should be able to induce potent immuneresponses at the mucosal surfaces potentially involved inHIV entry.
A promising approach to mucosal vaccination has beenthe use of virus-like particle (VLP) vaccines. VLPs aregenomeless viral particles (pseudovirions), obtained byspontaneous assembly of viral capsid proteins. They aresimilar in size and conformation to intact virions but arenonreplicating and nonpathogenic. These immunogenscan be administered as purified particles or as DNAplasmids expressing the viral proteins necessary to formVLPs in vivo [159,160]. Several successful VLP vaccineshave been developed against the sexually transmittedHPVs and tested in human trials (influenza) attesting tothe potential efficacy of VLPs as HIV-1 vaccine candidates[161,162]. VLPs can be used as potent mucosal HIV-1vaccine candidates (HIV-VLPs). Their administration byi.n. (prime) and i.m. (boost) has been shown to elicitvaginal and systemic humoral immune responses in therhesus macaques model [163]. Despite the fact that i.n.vaccines delivered into the nostrils are an attractive modeof immunization, one should be cautious of the risks ofpassage into the brain through olfactory nerves that couldbe the source of important adverse effects. As an example,the i.n. vaccine NasalFlu (Berna Biotech, Switzerland),containing an enzymatically active Escherichia coli labile
horized reproduction of this article is prohibited.
Co
1710 AIDS 2014, Vol 28 No 12
toxin adjuvant, was recalled after the establishment of anassociation with facial nerve paralysis (Bell’s palsy) [164].
Another concept that has been recently assayed in therhesus macaques model demonstrated that induction ofimmunological tolerance with a tolerogenic vaccine bymucosal route can prevent SIV infection [127]. The oraladministration of iSIVmac239 and Lactobacillus plantarum,a commensal bacterium of the digestive tract that isknown to induce immunologic tolerance, stimulatedmacaques to develop a thus far unrecognized type ofSIV-specific tolerance. This tolerance was characterizedby the suppression of SIV-specific Ab and CTL responses,and activation of a subset of CD8þ T cells that areSIV-specific, noncytolytic and MHC-Ib/E restricted.These cells apparently have the ability to suppress CD4þ
T cells activated by SIV and thereby prevent theestablishment of productive SIV infection both in vivoand in vitro.
Adjuvants as tools to orientate mucosalimmune responses
Adjuvants can be defined as substances that enhance theimmune response to the antigen(s) with which they arecoadministered. Despite their potentially critical role inthe efficacy of vaccines, relatively few adjuvants arecurrently used in commercial vaccines. Both the choiceof the adjuvant and the route of administration can greatlyaffect the type and potency of the immune responseelicited. To date, a number of approaches have beendeveloped in an effort to increase the immunogenicity ofHIV vaccines, including the use of molecular adjuvantsand cytokine adjuvants for protein antigens (Table 3)[165–177].
The addition of toxins or nontoxic derivatives of choleratoxin or mutant E. coli labile toxin to mucosalimmunization regimens has been shown to enhancesystemic immune responses [178]. The adjuvant activityof cholera toxin or labile toxin (and derivatives) can beexplained by their ability to affect several steps involved inthe induction of the immune response such as anincreased permeability of intestinal epithelium resultingin increased antigen uptake, enhancing antigen presen-tation, the promotion of IgA formation via B-cell isotypedifferentiation as well as effects on T-cell proliferation andcytokine production [179]. However, these adjuvants arenot devoid of a possible risk of severe adverse effects, asseen with the NasalFlu labile toxin adjuvanted vaccine[164].
Regarding the potential role of cytokines as adjuvants inmucosal HIV vaccine development, early clinical studiesusing protein antigens have shown that using pro-inflammatory cytokine adjuvants such as IL-1 by the i.n.
pyright © Lippincott Williams & Wilkins. Unautho
route effectively induced not only serum and vaginal IgGsbut also vaginal IgAs [180]. Many of the cytokineapproaches that have been tested in HIV vaccinedevelopment have been covered in a recent review[177] and will not be addressed here in further detail.Various combinations of IL-12, IL-15 and/or granulo-cyte-macrophage colony-stimulating factor (GM-CSF)have yielded mixed results, although, in combinationwith a DNA/MVA prime boost regimen, GM-CSF wasshown to effectively induce protective mucosal IgG andIgA production [34]. Of note, promising results havebeen observed in rhesus macaques using an IL-2adjuvanted DNA vaccine, which allowed control ofviremia and prevention of AIDS in an NHP model [181].
Over the past 10 years, there have been considerableadvances in both our understanding of the signallingpathways and receptors involved in recognition ofpathogens by the innate immune system and in theimportance of this system in then influencing an adaptiveimmune response. Detection of microbes by the innateimmune system is largely driven by pattern recognitionreceptors, including the toll-like receptors (TLRs) thatrecognize common molecular structures found on thosemicrobial agents that represent a potential danger for thedefending host organism. For HIV, it has been shown thatpolymorphisms in TLR4, 7, 8 and 9 can play a role inboth disease progression and viral load. This improvedunderstanding is now leading to the development ofnovel HIV vaccine adjuvants. TLR3 shows promisingresults when used with vaccine Ags and selective DEC-205/CD205 Ab delivery to dendritic cells. Similarly,TLR7/8 and TLR9 vaccine conjugates have been shownto enhance immune responses. Used together, IL-15 andagonists for TLR2/6, 3 and 9 synergistically upregulatedvaccine responses to recombinant MVA virus expressingviral proteins from SIVmac239 [182]. The activation ofTLR9 via unmethylated CpG motifs or related syntheticoligodeoxynucleotides (CpG ODN) mimicking bacterialDNA and thus acting as a danger signal of bacterialinvasion has also been shown to rapidly activate a varietyof innate immune cells through the Toll/IL-1 pathway toproduce Th1 cytokines and activation of APCs andB-cells. Vaginal administration of CpG ODN can inducethe rapid production of Th1 cytokines such as interferon-gamma (IFN-g), IL-12 or IL-18 in the female genitaltract [183]. These studies highlight the potential of theTLRs as agonists for HIV vaccines. Similarly, severalvaccine approaches using the TLR agonist Poly I:C andderivatives have revealed their capacity to stimulate HIVspecific immune responses in specific cell types and at sitesof mucosal exposure to pathogens [184–187]. Likewise,monophosphoryl lipid A (MPLA) from lipopolysacchar-ide (LPS) of Salmonella minnesota used as an adjuvant inparenterally administered vaccines has been shown toinduce antigen-specific mucosal and systemic cellularimmunity and Ab responses following oral or i.n. delivery,probably through activation of TLR2 and 4 [188,189].
rized reproduction of this article is prohibited.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibi
Progress in HIV vaccines inducing mucosal responses Pavot et al. 1711
Tab
le3.
Exam
ple
sof
vacc
inat
ion
stra
tegi
esto
induce
spec
ific
muco
sal
imm
une
resp
onse
sag
ainst
HIV
anti
gens.
Adju
vant
Del
iver
yst
rate
gyR
oute
of
imm
uniz
atio
nM
odel
Muco
sal
imm
une
resp
onse
Ref
eren
ces
GM
-CSF
DN
AD
NA
/SH
IV.8
9.6
.VLP
and
MV
A/S
HIV
-89.6
Intr
ader
mal
or
intr
amusc
ula
rR
hes
us
mac
aque
GM
-CSF
contr
ibute
sto
pro
tect
ion
by
enhan
cing
the
avid
ity
mat
ura
tion
of
anti
-Env
IgG
inblo
od
and
the
pre
sence
of
long-
last
ing
antivi
ral
IgA
inre
ctal
secr
etio
ns.
[34]
Pla
smid
CC
L19
or
CC
L28
HIV
-1gp
140
Intr
anas
alor
intr
amusc
ula
rM
ouse
pC
CL1
9en
han
ced
vagi
nal
-spec
ific
IgG
resp
onse
sw
hen
adm
inis
tere
di.
m.
or
i.n.
[165]
pC
CL2
8en
han
ced
vagi
nal
IgG
resp
onse
sfo
llow
ing
i.n.
but
not
i.m
.pC
CL1
9an
dpC
CL2
8en
han
ced
vagi
nal
IgA
by
i.m
.pC
CL1
9an
dpC
CL2
8en
han
ced
IgA
leve
lsw
ith
abal
ance
dIg
G:IgA
resp
onse
by
i.n.
CC
L19
and
CC
L28
also
incr
ease
dIg
Aþ
cell
sin
colo
rect
alti
ssue.
CC
L19
and
CC
L28
pro
mote
den
han
ced
neu
tral
izin
gre
sponse
sin
sera
and
vagi
nal
secr
etio
ns.
Chole
rato
xin
(CT)
HIV
-1pep
tide
Intr
arec
tal,
intr
anas
ally
or
intr
agas
tric
ally
Mouse
Intr
arec
talim
muniz
atio
nw
ith
CT
induce
dlo
ng-
last
ing,
antige
n-s
pec
ific
CTL
mem
ory
inPey
er’s
pat
ches
,la
min
ap
rop
ria
and
sple
en.
[166]
Syst
emic
imm
uniz
atio
nin
duce
dsp
ecifi
cC
TL
only
inth
esp
leen
.C
TA
1-D
DM
onom
eric
or
trim
eric
HIV
-1En
vIn
tran
asal
or
intr
aper
itonea
lM
ouse
CTA
1-D
Dst
imula
tes
HIV
-1an
ti-E
nv
seru
mIg
Gan
dm
uco
sal
IgA
foll
ow
ing
i.n.
adm
inis
trat
ion.
[167]
Muta
nt
E.co
lila
bile
toxi
nLT
(R192G
)H
IV-1
pep
tide
Intr
arec
tal
Mouse
LT(R
192G
)w
asas
effe
ctiv
eas
or
more
effe
ctiv
eth
anC
Tat
induci
ng
am
uco
sal
CTL
resp
onse
.
[168]
GM
-CSF
syner
gize
dw
ith
LT(R
192G
)Poly
I:C
(TLR
3)
HIV
pep
tides
and
anti
bodie
sto
DEC
-205/C
D205
Subcu
taneo
us
or
intr
aper
itonea
lM
ouse
DEC
-tar
gete
d,
HIV
Gag
p24
along
wit
hpoly
I:C
induce
pro
tect
ive
CD
4þ
Tce
llre
sponse
sat
airw
aym
uco
sal
surf
aces
.
[169]
MPLA
(TLR
4)
HIV
-1gp
140
Subli
ngu
al,
intr
anas
al,
intr
avag
inal
or
subcu
taneo
us
Mouse
MPLA
enhan
ced
resp
onse
saf
ter
i.n.
or
s.c.
[170]
Vag
inal
spec
ific
IgA
:N
onad
juva
nte
dgp
140:
s.l.>
i.n.¼
s.c.
Adju
vante
dgp
140:
i.n.>
s.l.>
s.c.
I.V
agfa
iled
toin
duce
any
det
ecta
ble
muco
sal
resp
onse
sev
enin
the
pre
sence
of
MPLA
.
ted.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
1712 AIDS 2014, Vol 28 No 12
Tab
le3
(continued
)
Adju
vant
Del
iver
yst
rate
gyR
oute
of
imm
uniz
atio
nM
odel
Muco
sal
imm
une
resp
onse
Ref
eren
ces
Glu
copyr
anosy
lLi
pid
Adju
vant
(GLA
,TLR
4)
DN
A-M
VA
-Pro
tein
vacc
ine
Intr
amusc
ula
rM
ouse
and
rabbit
GLA
impro
ved
spec
ific
IgG
vagi
nal
resp
onse
s.[1
71]
GLA
(TLR
4)
HIV
-1gp
140
Intr
anas
alM
ouse
GLA
induce
dhig
hIg
Gan
dIg
Atitr
esin
nas
al,
vagi
nal
lava
ges
and
faec
es.
[172]
Hig
hnum
ber
sof
gp140-s
pec
ific
Ab
secr
etin
gce
lls
inth
ege
nit
altr
act.
Bac
teri
alflag
elli
n(F
liC
)or
trunca
ted
FliC
(tFl
iC)
(TLR
5)
HIV
VLP
sIn
tram
usc
ula
ror
intr
anas
alG
uin
eapig
Flag
elli
n-c
onta
inin
gV
LPs
elic
ithig
hle
vels
of
syst
emic
Ab
resp
onse
sby
eith
eri.
m.
or
i.n.,
asw
ell
asboth
syst
emic
and
muco
sal
imm
unit
yby
i.n.
[173]
VLP
s-Fl
iCw
ere
more
effe
ctiv
ein
induci
ng
syst
emic
resp
onse
s,w
hil
eV
LPs-
tFli
Cw
ere
more
effe
ctiv
ein
induci
ng
vagi
nal
IgA
resp
onse
sby
i.n.
CpG
OD
N(T
LR9)
Inac
tiva
ted
gp120-d
eple
ted
HIV
-1vi
rions
Intr
anas
alM
ouse
Lym
phocy
tes
isola
ted
from
genit
altr
acts
of
mic
eim
muniz
edw
ith
Ag
and
CpG
show
edsi
gnifi
cant
spec
ific
pro
life
rati
on
and
pro
duce
dsi
gnifi
cantl
yhig
her
leve
lsof
IFN
-g.
[174]
CD
8þ
lym
phocy
tes
wer
ein
crea
sed
inth
ege
nital
trac
tsof
mic
eim
muniz
edw
ith
HIV
-1A
gan
dC
pG
TLR
2,
3,
9ag
onis
ts/I
L-15
Pep
tide
(pri
me)
and
MV
A(b
oost
)In
trar
ecta
lR
hes
us
mac
aque
Com
bin
atio
nof
IL-1
5an
dTLR
agonis
tsm
edia
ted
par
tial
pro
tect
ion
agai
nst
SIV
rect
alch
alle
nge
.
[175]
Pre
serv
atio
nof
CD
4þ
Tce
llnum
ber
sin
the
colo
nm
uco
sa.
Hig
her
leve
lof
SIV
-spec
ific
tetr
amerþ
CD
8þ
resp
onse
sin
the
colo
ns
of
adju
vante
dan
imal
s.
Progress in HIV vaccines inducing mucosal responses Pavot et al. 1713
Other studies have shown that liposomes containing lipidA and HIV-1 proteins or peptide antigens could induceneutralizing ‘multispecific’ Abs in which the antigen-binding site of the Ab simultaneously binds both to theimmunizing lipid and protein epitope [190,191].
Conclusion
The challenges involved in the development of a HIVprophylactic vaccine are unprecedented in the history ofvaccinology. Three decades after the discovery of thevirus, the quest for a vaccine is still actively ongoing. Themajor obstacles met in the development of an efficaciousvaccine are the mutational variability and global diversityof the virus, which allow its easy escape from both thecellular and humoral responses of the host. Moreover,HIV-1 mainly infects the organism through the mucosalsites of the genital and intestinal tracts and rapidlyintegrates into memory T cells that become latent viralreservoirs. An efficient vaccine will therefore need to notonly induce potent and functional virus-specific Abs ableto block virus entry at the site of initial infection but alsoCD8þ T cells for virological control in lymphoid tissuesand lymph nodes.
As illustrated by the example of the human papillomavirus(HPV) vaccine, the parenteral route of immunizationcould be an efficient way to elicit protective mucosalimmunity, and indeed, numerous studies on HIV/SIVvaccines have shown that it is possible to induceprotection against rectal or vaginal challenges in NHPmodels by systemic active or passive immunization.However, systemic immunization usually generates onlylow humoral responses at mucosal sites that stem from thetransudation of IgGs from the blood into the genitour-inary tract, and can also induce the secretion ofimmunoglobulins that act through interaction with theneonatal Fc receptor. It is nevertheless possible thatmucosal immune responses induced by parenteralimmunization will be improved in the future by thedevelopment of specific adjuvants that would amplify andfavourize such a response.
Immunization by the mucosal route preferentially inducesIgA responses at the site of antigen delivery, as well as insecretions from anatomically remote mucosal sites. Thus,an effective mucosal route of immunization able to elicitspecific IgAs and CTL immunity in the genital mucosaappears to be the nasal mucosa and the aerodigestive tract.Data collected so far show that MALT-targeted adju-vanted vaccine design could be universally applied to anyform of HIV vaccine candidate, including peptides,subunit vaccines, VLPs, DNA or live recombinantvaccines. However, initial mucosal HIV-1 immunizationof immunologically naive individuals may induce a stateof mucosal tolerance. Systemic priming followed by
Copyright © Lippincott Williams & Wilkins. Unaut
mucosal boosting is likely to prevent this undesirableoutcome. Furthermore, such a sequence of immuniz-ations should elicit humoral immune responses in boththe systemic and the mucosal compartments.
Unfortunately, at this time, mucosal immunization hasbeen tested in only a limited number of studies, mainlybecause of the relatively inefficient uptake of antigens bymucosal surfaces and the unavailability of mucosaladjuvants approved for human use. Also, whereas systemicimmunity can readily be assessed from peripheral bloodsamples, systemic responses do not necessarily reflectresponses in mucosal compartments. Thus, in their NHPvaccination study, Bomsel et al. [32] observed highprotection after intravaginal challenge that was correlatedwith HIV-1 blocking Abs developed in the mucosalcompartment, but not in serum. Thus, antiviral mucosalimmune responses may be missed in peripheral blood.Numerous studies have assayed HIV-specific mucosalresponses in preclinical and clinical research, but anumber of difficulties have slowed progress in incorpor-ating such measurements. Indeed, processing mucosalsamples is more challenging and sampling proceduresprovide lower amounts of fluid or cells than bloodsampling. Moreover, mucosal sampling is more invasivethan blood sampling and takes more time and training ofclinical personnel.
If we are to ever fully realize the potential benefits ofmucosal vaccines to control HIV/AIDS, current researchshould be extended to the development of innovativeimmunological tools such as safe adjuvants, targetingmolecules and delivery vectors.
Acknowledgements
Authors would like to thank the ANRS (AgenceNationale de la Recherche sur le SIDA) to S.P. andN.R., the European Commission FP7 ADITEC program(HEALTH-F4-2011-280873) and FP7 Cut’hivac(HEALTH- 241904) to V.P. and B.V., the ANR (grantANR PECSDELLI and Euronanomed iNanoDCs;support to V.P., S.P. and B.V.) and the grant from theFondation pour la Recherche Medicale to V.P.
Conflicts of interestThe authors declare that there are no conflicts of interest.
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