Recent progress in HIV vaccines inducing mucosal immune responses.

EDITORI
AL REVIEW
Recent 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-

[email protected] April 2014.

lth | Lippincott Williams & Wilkins 1701

mailto:[email protected]

http://dx.doi.org/10.1097/QAD.0000000000000308

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

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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


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]


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.


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


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


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.




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


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.



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


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


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.


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].


Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibi


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.


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.


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


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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Recent progress in HIV vaccines inducing mucosal immune responses.

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