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doi: 10.1097/QAD.0000000000000308
Editorial Review

Recent progress in HIV vaccines inducing mucosal immune responses

Pavot, Vincenta,b; Rochereau, Nicolasb; Lawrence, Philipc; Girard, Marc P.d; Genin, Christianb; Verrier, Bernarda; Paul, Stéphaneb

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aInstitut de Biologie et Chimie des Protéines – LBTI, UMR 5305 – CNRS/University of Lyon 1, Lyon, France

bGroupe Immunité des Muqueuses et Agents Pathogènes – INSERM CIE3 Vaccinologie, Faculté de Médecine, Saint-Etienne

cInternational Centre for Research in Infectiology (CIRI), INSERM U1111 – CNRS UMR5308, University of Lyon 1, Lyon

dFrench National Academy of Medicine, Paris, France.

Correspondence to Dr Stéphane Paul, GIMAP - Faculté de Médecine Jacques Lisfranc - 15 rue Ambroise Paré - 42023 Saint-Etienne, France. Tel: +334 77 42 14 67; fax: +334 77 42 14 86; e-mail:

Received 13 February, 2014

Revised 12 April, 2014

Accepted 14 April, 2014

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In spite of several attempts over many years at developing a HIV vaccine based on classical strategies, none has convincingly succeeded to date. As HIV is transmitted primarily by the mucosal route, particularly through sexual intercourse, understanding antiviral immunity at mucosal sites is of major importance. An ideal vaccine should elicit HIV-specific antibodies and mucosal CD8+ cytotoxic T-lymphocyte (CTL) as a first line of defense at a very early stage of HIV infection, before the virus can disseminate into the secondary lymphoid organs in mucosal and systemic tissues. A primary focus of HIV preventive vaccine research is therefore the induction of protective immune responses in these crucial early stages of HIV infection. Numerous approaches are being studied in the field, including building upon the recent RV144 clinical trial. In this article, we will review current strategies and briefly discuss the use of adjuvants in designing HIV vaccines that induce mucosal immune responses.

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Despite the extensive efforts that have been made over almost 30 years, major challenges still exist concerning HIV vaccine design. Most HIV infections by far occur through sexual contact [1]. Women are particularly vulnerable during heterosexual transmission through exposure to contaminated seminal fluids, and indeed, heterosexual women account for more than half of all individuals living with this virus [2]. Mucosal tissues involved in the sexual transmission of HIV include the cervicovaginal and rectal mucosa as well as the foreskin and oral epithelia [3]. Therefore, eliciting a strong preexisting anti-HIV immune response in mucosa-associated lymphoid tissues (MALTs) is probably of vital importance in preventing HIV infection [4].

The development of an effective vaccine is a considerable challenge, especially given the formidable propensity to immune evasion that is intrinsic to HIV. The HIV-1 envelope glycoprotein (Env) that is the target of known HIV-1-directed neutralizing antibodies (NAbs) [5–7] is protected by an evolving shield of glycans, variable immunodominant loops and conformational masking of key viral epitopes [6,8–10]. Although immunization with recombinant Env proteins or vectors encoding Env can induce high levels of HIV-1 specific Abs, vaccine-induced Abs have been unable to neutralize most circulating primary HIV-1 isolates [6,7,11,12]. Indeed, natural infection predominantly induces nonneutralizing or strain-specific Abs during the first months of infection [9,13–15]. However, NAbs are the best correlate of protection for many viral vaccines [16,17]. It was found that approximately 10–20% of HIV-1-infected individuals happen to develop broadly NAbs (bNAbs) after a few years [18–20], which is the type of humoral immune response one would like a vaccine to elicit. These bNAbs are able to neutralize the vast majority of virus strains in a cross-clade manner and have been shown to provide robust protection against mucosal challenges in the macaque model [21–23].

Our understanding of what constitutes a bNAb against HIV has been revolutionized by the isolation of extremely broad and potent neutralizing mAb from a number of HIV-infected individuals [24–27]. These mAbs were identified by dissecting the broad neutralization activity seen in specific patient serum samples and by characterizing mAbs from B-cells [24,28,29].

Some bNAbs, when acting at the earliest steps of viral infection, are able to prevent virus entry into host cells by blocking multiple steps of viral transmission by targeting either the CD4+-binding site or the glycan/V3 loop on HIV-1 gp120 [30]. The incapacity to induce bNAbs to HIV has thus been a major hurdle to HIV vaccine research since the beginning of the epidemic. Substantial obstacles remain in inducing bNAbs by immunization and particularly at the mucosal level. Some studies have shown evidence of the presence of NAbs in mucosal fluids using appropriate immunization vectors and delivery regimens [31–34], but previous attempts to induce bNAbs by vaccination were fairly unsuccessful. Currently, based upon our existing understanding of the mucosal immune system, we would expect that the quality of humoral and cellular immune responses at a given effector site would depend upon the route of vaccination [35]. It is thus essential to choose the suitable immunization route for the desired mucosal site.

Despite the remarkable progress made in understanding the epitopes that Abs recognize on the Env spikes of the virion, one of the major goals of HIV vaccine research at this time is the discovery of immunogens and immunization strategies that can elicit bNAbs. This challenge is made even greater by the fact that it is more difficult to induce high concentrations of NAbs at the mucosal level than at a systemic level [36].

It would certainly be beneficial for an HIV-1 vaccine to also elicit immune responses capable of controlling viral replication [37]. A wealth of data has shown that cellular immune responses can mediate the control of viremia in HIV-1-infected humans and simian immunodeficiency virus (SIV)-infected rhesus macaques, including CD8+ T lymphocytes [38–40], natural killer (NK) cells [41] and CD4+ T lymphocytes [42,43]. Moreover, vaccine trials in nonhuman primates (NHPs) have shown that sustained viremic control is achievable after heterologous SIV challenges. For example, immunizations with an Adenovirus serotype 26 prime and Modified Vaccinia Ankara (MVA) boost expressing SIV antigens led to a 2.32 log reduction in mean set point viral load following stringent SIVmac251 challenge, which was related to the magnitude and breadth of the Gag-specific cellular immune responses measured immediately prior to challenge [31].

Even more remarkable was the report that 50% of rhesus macaques vaccinated with a SIV protein expressing rhesus cytomegalovirus (RhCMV/SIV) vector manifested durable, aviraemic control of infection with the highly pathogenic strain SIVmac239 [38]. The RhCMV/SIV vector elicited immune responses that control SIVmac239 infection (regardless of the route of challenge) after viral dissemination. Over time, protected rhesus macaques lost signs of SIV infection, showing a consistent lack of measurable plasma or tissue-associated viral RNA or DNA using ultrasensitive assays, and a loss of T-cell reactivity to SIV determinants not in the vaccine [44]. Similarly, it was shown that protection against wild-type SIVmac239 challenge by live attenuated SIV vaccines strongly correlated with the magnitude and function of SIV-specific, effector-differentiated T cells in the lymph nodes of the animals. It follows from these observations that SIV-specific T cells can suppress wild-type SIV amplification at an early stage and, if present persistently in sufficient frequencies, can completely control and even clear infection [45].

Current assessments aim to evaluate a broad range of mucosal immune responses and answer key questions such as can vaccines delivered parenterally elicit detectable mucosal responses? Whereas systemic immunization induces mostly immune responses in peripheral and systemic sites, mucosal delivery of immunogens is thought to trigger primarily mucosal immune responses [46]. The second question is which mucosal immune responses may be associated with protection from HIV infection? And the third, which mucosal specimens and assays are most relevant for the detection of these responses? Answers to these questions will be vital in clarifying which mucosal immune responses are capable of blocking HIV infection, and for developing vaccines that can elicit these types of responses.

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Mucosal transmission of HIV-1: a rationale for the role of HIV-specific mucosal immune responses

Natural transmission of HIV-1 occurs through vaginal mucosa, the male genital tract, that is penile mucosa (inner foreskin, penile urethra), gastrointestinal mucosa and via breastmilk (vertical transmission). Although there are challenges in quantifying risk by sex act, all studies consistently report that anal intercourse is a higher risk act than vaginal intercourse and the probability of infection by the vaginal route has been estimated to be one in 200 or less [3]. Considering this route, HIV-1 can infect the vaginal, ectocervical and endocervical mucosa, but the relative contribution of each site to the establishment of the initial infection is unknown.

Both free and cell-associated HIV and SIV virions can establish mucosal infection [1,47]. This has been shown directly in vivo in female macaques [48–50], or ex vivo using human cervical explants [49,51], and indirectly in humans through genetic sequence comparisons of viral isolates from acutely infected women with those from seminal leukocytes (cell-associated virions) and plasma from their infected source partners [52–54]. Ex-vivo studies using human cervical explants and reconstructed vaginal mucosa have confirmed transmission of cell-free and cell-associated HIV-1 [55–58]. Cervical mucus can trap infected seminal cells or free virions [59,60]. Conceivably, this could facilitate viral transmission by prolonging the time of contact of the virions with the mucosa. However, although immobilized, the virions may also become more susceptible to innate antiviral substances or to Abs.

Several reports have shown that HIV virions bind to and enter epithelial cells in the female genital tract [61–63]. Virions that are initially free, or those that are released from infected donor seminal T cells, interact with epithelial cells and traverse the epithelium by several pathways, including transcytosis, endocytosis and subsequent exocytosis, by causing productive infection, or merely by penetrating through the gaps between epithelial cells in the vaginal multilayered epithelium [64].

Transcytosis, which occurs across single layered epithelia, has been shown to occur in cell lines and also in primary cells, but has not been definitively demonstrated in intact mucosal tissues. Interestingly, cell-associated virions secreted from infected seminal leukocytes appear markedly more efficient at transcytosis than cell-free virions [65,66]. It is actually likely that HIV is not transmitted as a naked particle, but rather as an immune complex with Env-binding IgGs that are abundant in the semen of HIV-positive men. The low pH of the vagina and urethra also plays an important role in transcytosis, as the neonatal FcRn receptor that is expressed on epithelial cells of the penile urethra and endocervix binds the IgGs at low pH and releases them at neutral pH, thus favouring the capture of the immune virus complexes on the acidic apical (vaginal) side of the epithelium and their release in the neutral basolateral environment [67].

Upon release from epithelial cells, the virions can readily infect susceptible leukocytes [63]. It has been reported that virions can also productively infect the cervical epithelial cells themselves [63,68], although this point remains contested [69,70]. Conceivably, HIV-1 can also be transported through the cervicovaginal epithelium to the draining lymph nodes by infected lymphocytes, macrophages, monocytes and dendritic cells, as has been suggested in both in-vitro systems and mouse studies [68,71–75]. The rationale for the role of specific mucosal immune responses in protection from HIV-1 transmission has been highlighted by numerous studies in human and NHPs (Table 1) [76–89].

Table 1
Table 1
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Anatomic sites of HIV-1 persistence

Another rationale for eliciting HIV-specific mucosal immune responses is linked to their potential to prevent the establishment of viral reservoirs within a newly infected host. A viral reservoir can be defined as cell types or anatomical sites in which replication-competent forms of the virus can and do persist throughout infection and in the presence of otherwise efficient antiretroviral therapy (ART) [90]. Gastrointestinal and vaginal mucosal tissues are major reservoirs for initial HIV replication and amplification, and the sites of rapid CD4+ T-cell depletion [91]. Such viral reservoirs are currently thought to be a key factor explaining our difficulty in successfully eradicating HIV-1 from an infected host via the currently available treatments regimes. A successful vaccine would need to prevent the establishment of these reservoirs at a very early step of viral infection. This is especially important considering the fact that aggressive ART in very early acute infection can substantially decrease the size of the viral reservoir in terms of integrated DNA and RNA concentrations [92]. Although most HIV pro-viral DNA is found in CD4+ T lymphocytes in lymphoid tissue, blood viral reservoirs may also be maintained in central and transitional memory T cells that persist through mechanisms of homeostatic proliferation and renewal. Other potential sources may include monocytes and macrophages, astrocytes and microglial cells [92].

A related aspect is also the notion of viral compartments within an infected individual. A viral compartment may be defined as a cell or tissue replication site wherein a population of viral variants is at least partially restricted in its ability to enter, leave and replicate and therefore display a limited exchange of viral genetic information with other sites [90]. It is unclear as to whether all sites of viral compartmentalization represent viral reservoirs in the strictest sense. This anatomical compartmentalization of HIV-1 variants has been well described for the central nervous system (CNS), the gut-associated lymphoid tissue (GALT) [93,94] and the genital tract, although there are also data for viral compartmentalization within the lung, liver, kidney and breast milk (for a recent review see [95]).

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Env-specific antibodies to protect against HIV-1 acquisition at mucosal surfaces

The design of immunogens able to elicit NAb remains a major goal of HIV-1 vaccine development [96]. Many studies in NHPs have shown that passive infusion of HIV NAbs, especially bNAbs, can prevent rectal or vaginal infection by a chimeric simian-HIV (SHIV) containing the env gene of HIV-1. This was initially shown using a single oral or vaginal inoculation sufficient to infect 100% of control animals [97–100]. In this setting, protection against SHIV infection was most directly associated with the neutralization potency of the infused Abs [101,102]. However, recent passive transfer studies have employed low-dose multiple mucosal challenges to infect all control animals [103,104]. This model may be more physiologically relevant to the relatively low probability of sexual infection seen with HIV-1 in humans. In the low-dose NHP model, approximately 10-fold fewer Abs were required to mediate protection against infection than prior studies with high-dose virus challenge: serum Ab titres sufficient to mediate 90% virus neutralization at 1 : 5 serum dilution were associated with protection.

It has been suggested that Fc-mediated Ab effector functions might also play an important role in conferring protection. Indeed, although direct antibody-mediated neutralization is highly effective against cell-free virus, increasing evidence suggests an important role for IgG Fcγ receptor (FcγR)-mediated inhibition of HIV replication. Thus, bNAb IgG1 b12 showed a diminished protective potency after its Fc region was altered to knock out complement binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity without decreasing its in-vitro neutralizing activity [105]. A recent study screened a panel of bNAbs and nonneutralizing Abs (NoNAbs) for their ability to block HIV acquisition and replication in vitro in either an independent or FcγR-dependent manner. In the NHP model, vaginal application of a gel containing the selected bNAbs 2G12, 2F5 and 4E10 prevented SHIV transmission in 10 out of 15 macaques after vaginal challenge, whereas the NoNAbs 246-D and 4B3 had no impact on SHIV acquisition but reduced plasma viral load [22]. These results highlight that distinct neutralization and inhibitory activity of anti-HIV Abs affect in-vivo HIV acquisition and replication in different ways and demonstrate the potential interest of NAbs for microbicide and vaccine development. It follows that vaccines may not need to achieve extraordinarily high levels of HIV-1 NAbs to elicit protection at mucosal surfaces, but the Ab response will likely need to be durable, and NAbs will have to cross-react with a genetically diverse spectrum of HIV-1 strains.

We also do not know which type of immunoglobulins are the best at blocking the virus at mucosal surfaces. IgG1 Abs certainly can play a role, as passive infusion of such anti-HIV bNAbs into the blood can protect animals from mucosal challenge [106,107]. However, it is known that IgA is the predominant Ab in the majority of mucosal secretions [108]. Mucosal IgA Ab is generated primarily in the mucosal epithelial compartment and transported across the epithelial cell boundary into external secretions by interacting with the polymeric immune globulin receptor (pIgR) [109]. It was recently reported that rectally applied dimeric IgA Abs derived from bNAb HGN194 could not only protect NHPs from rectal challenge with SHIV-1157ipEL-p, but also that they did it more effectively than corresponding IgGs [88]. Comparison of the IgG1 version of bNAb HGN194 with its dimeric IgA versions, dIgA1 and dIgA2, showed that the dIgA1 version protected the animals better than the dIgA2 and IgG1. Thus, five out of the six animals treated with the dIgA1 version remained uninfected, whereas only one of the six dIgA2-treated animals and two of the six IgG1-treated animals remained virus free. All 11 untreated animals got infected.

The increased protection observed in the dIgA1-treated animals was initially puzzling, as all three HGN194 versions neutralized the challenge virus equally well. The explanation could be that the dIgA1 version can bind twice as many virus particles as the dIgA2 version. As a result, a dIgA1 molecule can accommodate four virus particles between its antigen-binding sites, while dIgA2 can only accommodate two. This also might explain why only dIgA1 and not dIgA2 nor IgG1 were able to prevent most HIV particles from crossing a cultured epithelial cell layer in an in-vitro transcytosis assay [88].

Altogether, these results suggest that one should try to develop vaccines that can elicit dIgA1s at mucosal surfaces. These Abs do not necessarily have to be neutralizing, because it is the ability of dIgA1s to be able to bind more HIV particles, and not necessarily better virus neutralization, which seems to be responsible for their higher level of protective efficacy.

Studies in humans have also revealed a correlation between a high level of secretory IgA (SIgA) and protection in high-risk individuals who remain seronegative (highly exposed persistently seronegative persons) [76,78,110,111]. These studies concluded that protection could be mediated by the interaction between these SIgA and HIV-1 on mucosal surfaces or within epithelial cells capable of internalizing IgA-bound HIV-1. This conclusion is however disputed, as there is no evidence for IgA-mediated intraepithelial HIV-1 neutralization [112–114].

However, in addition to its ability to neutralize virus, it is thought that IgA may contribute to the elimination of virus in the form of exocrine immune complexes via the lamina propria. In this manner, the mechanism of IgA protection may be wider than that provided by IgG-mediated neutralization. It also follows that assays based on neutralizing IgG Abs may not be suitable for assessing the activity of mucosal IgA [115].

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Vaccination strategies that elicit mucosal neutralizing antibodies

In clinical trials that show the efficacy of a vaccine, the identification of immune responses that are predictive of trial outcomes generates hypotheses about which of those responses are responsible for protection [116,117]. The RV144 phase 3 trial in Thailand was an opportunity to perform such a hypothesis-generating analysis for an HIV-1 vaccine. This trial of the canarypox vector vaccine (ALVAC-HIV [vCP1521]) as well as the gp120 AIDSVAX B/E vaccine showed an estimated vaccine efficacy of 31.2% for the prevention of HIV-1 infection over a period of 42 months after the first of four planned vaccinations [118]. This result enabled a systematic search by Haynes et al.[119] who performed a case–control analysis to identify Ab and cellular immune correlates of infection risk. This immune-correlates study generated the hypotheses that levels of V1V2 Abs correlated inversely with the risk of infection, whereas high levels of Env-specific IgA may have mitigated the effects of protective Abs. However, any protective role of mucosal Abs in the context of HIV-1 vaccination could not be evaluated in the RV144 trial, because mucosal samples were not collected.

Several studies have shown that immunization by the nasal route (i.n.) can be most effective at eliciting Abs and cellular immunity in the female genital tract [35]. Thus, the use of live replicating recombinant Ad5hr-vectored vaccine, in the rhesus macaques model, administered first by i.n. and oral routes then intratracheally followed by Env protein boosts resulted in systemic and mucosal Ab responses, including NAb, ADCC and transcytosis inhibition, together with potent cell immune responses [120]. Mucosal IgA immunity correlated with delayed acquisition following a repeated low-dose rectal SIV(mac251) challenge. The replicating Ad5 vector was shown to disseminate across multiple mucosal sites irrespective of delivery route [121]. These results suggest that initial mucosal vaccination with a replicating vector inducing NAbs in combination with a potent protein boost may significantly reinforce protective immunity against SIV mucosal transmission.

As another example, four of five rhesus macaques vaccinated first by intramuscular route (i.m.) and then i.n. with gp41-subunit antigens presented on virosomes were protected against 13 consecutive vaginal challenges with SHIV-SF162P3, and the fifth specimen showed only transient infection. All of the animals displayed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or ADCC activities [32].

The immunogenicity of virosomes spiked with a gp41 MPER peptide (P1) was tested in a phase I, double-blind, randomized, placebo-controlled trial in 24 healthy HIV-uninfected young women [122]. Antigen-specific serum IgGs and IgAs were elicited in all high-dose recipients after the first i.m. injection, but vaginal and rectal gp41-specific IgGs could be detected only after boosting via the i.n. route.

Although these data speak highly in favour of the nasal route of immunization to elicit mucosal anti-HIV Abs in the female genital tract, numerous studies have also shown that parenteral immunization is able to induce protective mucosal immune responses, notably with viral vectors (Table 2) [123–129]. Indeed, studies have demonstrated the capacity of adenovirus/poxvirus and adenovirus/adenovirus vector based vaccines expressing HIV-1 mosaic Env, Gag and Pol administered by i.m. route to protect rhesus macaques against acquisition of infection following repetitive intrarectal inoculations of the difficult-to-neutralize SHIV-SF162P3 or SIVmac251 [31,123].

Table 2
Table 2
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Cellular immune responses mediate control of viremia

Whereas Env-specific Abs appear necessary to block HIV-1 acquisition, Gag-specific cellular immune responses appear important for the control of virus replication and viral load after infection. Gag-specific CD8+ T cells, but not Env- nor Pol-specific CD8+ T cells, correlate with in-vivo viral load control following SIV challenge in vaccinated monkeys [130]. This result is consistent with studies demonstrating the association of Gag-specific cellular immune responses with viremia control in HIV-1 infected individuals [131–133] and SIV-infected rhesus macaques [31,134–136]. Vif and Nef may also contribute to viral load control in monkeys [137].

As conservation of polyfunctional HIV-specific CD8+ T-cells appears to correlate with the control of viremia in infected people [138], the polyfunctionality of the T-cell response is perceived as one of the best correlates of T-cell immunity [87]. Thus, Ferre et al.[139] showed that mucosal CD8+ cytotoxic T-lymphocyte (CTL) responses in controllers are more complex and significantly stronger than in antiretroviral-suppressed persons: HIV-controllers show long-lasting, high avidity, polyfunctional Gag-specific CD8+ T-cell responses in mucosal compartments as compared with noncontrollers.

Another critical aspect of HIV cellular immune responses is the location of the HIV-specific immune cells elicited by immunization. Thus, the degree of protection mediated by a live attenuated SIV vaccine strongly correlates with the location of SIV-specific effector CD8+ T cells, in lymph nodes [45]. The maintenance of this protective T-cell response seems to be associated with persistent replication of the live attenuated virus vaccine in follicular helper T (Tfh) cells.

Surprisingly, none of the candidate HIV vaccines tested so far in human volunteers has been able to elicit viral load control. Neither the Step trial, based on the use of a recombinant Ad5 vector, nor the HVTN 505 trial, which used a DNA prime followed by a recombinant Ad5 boost, nor the RV144 trial, using a recombinant Canarypox prime followed by gp120 boosts, showed any significant impact on viral load in vaccine recipients who became infected with HIV-1 [118,140,141]. There actually was some evidence for immune selection pressure on breakthrough HIV-1 sequences in the Step study, suggesting that, although too weak to be efficient, vaccine-elicited cellular immune responses did exert immunologically relevant biological effects in humans [142]. The disappointing results of the Step and HVTN 505 vaccine trials highlight the likely importance of inducing mucosal immune responses that could significantly decrease virus replication in the mucosa and subsequent viral dissemination to peripheral lymphoid tissues and blood. Another, but different example is the SIV protein encoding RhCMV, which is able to maintain differentiated effector memory T-cell responses at viral entry sites that show high efficacy at impairing SIV replication at its earliest stage. This strategy can maintain robust SIV-specific CD4+ and CD8+ effector memory T-cell (TEM) responses that provide protection against repeated limiting-dose intrarectal challenge with SIVmac239 [124].

Studies of the early kinetics of T-cell responses in previously vaccinated, acutely SIV-infected NHPs will allow the determination of whether an initial influx of virus-specific CD4+ T cells precedes robust CTL responses and correlates with early containment [143]. Alternatively, CD4+ CTL may directly contribute to containment of HIV infection [43].

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Vaccination strategies that elicit cellular mucosal immune responses

Hansen et al.[38] reported that RhCMV/SIV vectors used by subcutaneous route (s.c.) in the rhesus macaques model are able to induce immune-mediated control of highly pathogenic SIVmac239 after repeated intrarectal challenges and prior to irreversible establishment of infection. An early complete control of SIV was observed in 13 of 24 rhesus macaques receiving either RhCMV alone or RhCMV (s.c. prime)/Ad5 (i.m. boost) vectors, and a long-term protection (≥1 year) was observed in 12 of these 13 animals [124]. The immunologic assays performed in mononuclear cell preparations from blood and tissues suggest that this control is related to the high frequency of SIV-specific T-cell responses (CD8+, and possibly CD4+). These responses are located both in mucosal portals of entry and at potential sites of distant viral spread and are indefinitely maintained by the persistent RhCMV vectors, and can protect without anamnestic expansion.

The finding that RhCMV/SIV vector-protected rhesus macaques are able to control haematogenous SIV dissemination after both intrarectal and intravaginal challenge suggested that the immune responses elicited by these vectors might provide protection even when mucosal surfaces are bypassed [44]. Thus, persistent vectors such as CMV and their associated TEM responses might significantly contribute to an efficacious HIV/AIDS vaccine.

The NHP model was also used to test the vaccination approach using a plasmid DNA prime/rAd5 boost vaccine developed to induce both CD8+ T lymphocyte responses and Env-specific Ab responses [125]. After repeated intrarectal challenges, the vaccine failed to protect against SIVmac251, but 50% of vaccinated monkeys were protected from infection with SIVsmE660. Although the exploration of immune correlates suggests that a NAb may be responsible for the conferred protection against mucosal acquisition of SIVsmE660, the reduction in peak plasma virus RNA implicates CTL in the control of SIV replication once infection is established.

Intramuscular vaccination of rhesus macaques with Ad/MVA or Ad/Ad vector expressing SIV Gag, Pol and Env antigens, were also investigated for their capacity to induce CD8+ T lymphocytes and to test whether these responses predict virologic control following SIV mucosal challenge [130].

They observed that CD8+ cell mediated SIV inhibition was significantly associated with Gag-specific cellular immunity but not Pol or Env-specific cellular immunity and that CD8+ lymphocytes from 23 vaccinated rhesus macaques inhibited replication of the virus in vitro. Moreover, the level of inhibition prior to challenge was inversely correlated with set point SIV plasma viral load after intrarectal challenge. These findings demonstrate that in-vitro viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with in-vivo control of viremia following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which control is desired.

Rhesus macaques immunized by the i.n. route with a SIV DNA/MVA prime-boost regimen also demonstrated significant anti-SIV CTL responses in the colorectal mucosa and a better control of rectal SIVmac251 infection when compared with macaques given the same vaccine by the i.m. route [33]. However, it was reported that an i.m. injection of nonreplicating recombinant Adenovirus vectors into rhesus macaques is able to significantly induce SIV-specific CTL responses that persist for over 2 years in multiple mucosal tissues, such as colorectal, duodenal and vaginal biopsy specimens [126].

Despite the fact that mucosal vaccination often elicits lower magnitude HIV-specific T-cell responses when compared with systemic vaccination, mucosal immunization can elicit better protection against HIV challenge mediated by higher avidity CTLs in macaques, as assessed in systemic fluids [144]. Interestingly, interleukin (IL)-13 seems to be detrimental to the efficient avidity of these T-cells in the mouse model [145]. Recombinant HIV-1 vaccines that coexpress the soluble or membrane-bound forms of the IL-13 receptor α2 (IL-13Rα2), and which can block IL-13 activity at the immunization site, were used to make wild-type mice comparable to IL-13 knock-out animals [146]. After an i.n./i.m. prime-boost vaccination, these vaccines adjuvanted with IL-13Rα2 were shown to induce multifunctional mucosal CD8+ T-cell responses in the lung, genito-rectal nodes and Peyer's patches with greatly enhanced functional avidity and broader cytokine/chemokine profiles that provided greater protection against a surrogate mucosal HIV-1 challenge [146].

As mucosal prime-boost immunizations elicit significant numbers of high avidity effector memory CD8+ CTL in mucosal and systemic compartments, they appear to be an essential component of any immunization approach that aims at establishing protective frontline defenses against HIV-1 infection.

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Exploring mucosal routes of immunization

Systemically delivered viral vectors can induce mucosal immune responses against HIV-1 or SIV, most notably in the gut, rectal and genital mucosa [147,148]. However, the strength of these responses is generally poor. For example, Ad-vectored vaccines have been shown to induce low levels of mucosal immune responses after systemic inoculation, which are approximately 10 times lower than the immune responses induced at systemic sites. Low-level mucosal immune responses have also been seen in individuals inoculated i.m. with a recombinant pox virus vector [149]. However, at this time, there are very few validated mucosal vaccines against any infectious disease [35] and the mucosal vaccines already available provide protection only via induction of Ab responses. There are no current mucosal vaccines that are known to induce strong protective cellular immune responses at the systemic or mucosal level. Therefore, understanding the biology of the mucosal immune system in order to develop better mucosal vaccines that can induce both humoral and cellular immunity is needed.

Mucosal vaccines using oral or nasal routes have the great advantages of being painless, easy to administer on a large scale and easier to store and to deliver than current systemic vaccines [35]. Vaccination at a mucosal site stimulates local immunity as well as immunity in other mucosal sites and usually also induces systemic immune responses detectable in the blood, spleen and peripheral lymph nodes. This is in contrast to systemically delivered vaccines, which are usually limited in their ability to stimulate an immune response in mucosal tissues [150,151].

Vaccines that target the nasal, oral, rectal or urogenital mucosa have been under investigation for some time, using attenuated virus, inactivated virus, recombinant virus, DNA, dendritic cells or peptides [152–155]. Oral immunization strategies have been shown to induce HIV/SIV-specific immune responses in the gastrointestinal tract [156–158], whereas nasal immunization strategies have been reported to induce robust immune responses in the colorectal mucosa and genitourinary tracts in the NHP model [32,33,120]. Therefore, a mucosal immunization strategy using both the oral and nasal routes should be able to induce potent immune responses at the mucosal surfaces potentially involved in HIV entry.

A promising approach to mucosal vaccination has been the use of virus-like particle (VLP) vaccines. VLPs are genomeless viral particles (pseudovirions), obtained by spontaneous assembly of viral capsid proteins. They are similar in size and conformation to intact virions but are nonreplicating and nonpathogenic. These immunogens can be administered as purified particles or as DNA plasmids expressing the viral proteins necessary to form VLPs in vivo[159,160]. Several successful VLP vaccines have been developed against the sexually transmitted HPVs and tested in human trials (influenza) attesting to the potential efficacy of VLPs as HIV-1 vaccine candidates [161,162]. VLPs can be used as potent mucosal HIV-1 vaccine candidates (HIV-VLPs). Their administration by i.n. (prime) and i.m. (boost) has been shown to elicit vaginal and systemic humoral immune responses in the rhesus macaques model [163]. Despite the fact that i.n. vaccines delivered into the nostrils are an attractive mode of immunization, one should be cautious of the risks of passage into the brain through olfactory nerves that could be the source of important adverse effects. As an example, the i.n. vaccine NasalFlu (Berna Biotech, Switzerland), containing an enzymatically active Escherichia coli labile toxin adjuvant, was recalled after the establishment of an association with facial nerve paralysis (Bell's palsy) [164].

Another concept that has been recently assayed in the rhesus macaques model demonstrated that induction of immunological tolerance with a tolerogenic vaccine by mucosal route can prevent SIV infection [127]. The oral administration of iSIVmac239 and Lactobacillus plantarum, a commensal bacterium of the digestive tract that is known to induce immunologic tolerance, stimulated macaques to develop a thus far unrecognized type of SIV-specific tolerance. This tolerance was characterized by the suppression of SIV-specific Ab and CTL responses, and activation of a subset of CD8+ T cells that are SIV-specific, noncytolytic and MHC-Ib/E restricted. These cells apparently have the ability to suppress CD4+ T cells activated by SIV and thereby prevent the establishment of productive SIV infection both in vivo and in vitro.

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Adjuvants as tools to orientate mucosal immune responses

Adjuvants can be defined as substances that enhance the immune response to the antigen(s) with which they are coadministered. Despite their potentially critical role in the efficacy of vaccines, relatively few adjuvants are currently used in commercial vaccines. Both the choice of the adjuvant and the route of administration can greatly affect the type and potency of the immune response elicited. To date, a number of approaches have been developed in an effort to increase the immunogenicity of HIV vaccines, including the use of molecular adjuvants and cytokine adjuvants for protein antigens (Table 3 ) [165–177].

Table 3
Table 3
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Table 3
Table 3
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The addition of toxins or nontoxic derivatives of cholera toxin or mutant E. coli labile toxin to mucosal immunization regimens has been shown to enhance systemic immune responses [178]. The adjuvant activity of cholera toxin or labile toxin (and derivatives) can be explained by their ability to affect several steps involved in the induction of the immune response such as an increased permeability of intestinal epithelium resulting in increased antigen uptake, enhancing antigen presentation, the promotion of IgA formation via B-cell isotype differentiation as well as effects on T-cell proliferation and cytokine production [179]. However, these adjuvants are not devoid of a possible risk of severe adverse effects, as seen with the NasalFlu labile toxin adjuvanted vaccine [164].

Regarding the potential role of cytokines as adjuvants in mucosal HIV vaccine development, early clinical studies using 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 IgGs but also vaginal IgAs [180]. Many of the cytokine approaches that have been tested in HIV vaccine development 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 granulocyte-macrophage colony-stimulating factor (GM-CSF) have yielded mixed results, although, in combination with a DNA/MVA prime boost regimen, GM-CSF was shown to effectively induce protective mucosal IgG and IgA production [34]. Of note, promising results have been observed in rhesus macaques using an IL-2 adjuvanted DNA vaccine, which allowed control of viremia and prevention of AIDS in an NHP model [181].

Over the past 10 years, there have been considerable advances in both our understanding of the signalling pathways and receptors involved in recognition of pathogens by the innate immune system and in the importance of this system in then influencing an adaptive immune response. Detection of microbes by the innate immune system is largely driven by pattern recognition receptors, including the toll-like receptors (TLRs) that recognize common molecular structures found on those microbial agents that represent a potential danger for the defending host organism. For HIV, it has been shown that polymorphisms in TLR4, 7, 8 and 9 can play a role in both disease progression and viral load. This improved understanding is now leading to the development of novel HIV vaccine adjuvants. TLR3 shows promising results when used with vaccine Ags and selective DEC-205/CD205 Ab delivery to dendritic cells. Similarly, TLR7/8 and TLR9 vaccine conjugates have been shown to enhance immune responses. Used together, IL-15 and agonists for TLR2/6, 3 and 9 synergistically upregulated vaccine responses to recombinant MVA virus expressing viral proteins from SIVmac239 [182]. The activation of TLR9 via unmethylated CpG motifs or related synthetic oligodeoxynucleotides (CpG ODN) mimicking bacterial DNA and thus acting as a danger signal of bacterial invasion has also been shown to rapidly activate a variety of innate immune cells through the Toll/IL-1 pathway to produce Th1 cytokines and activation of APCs and B-cells. Vaginal administration of CpG ODN can induce the rapid production of Th1 cytokines such as interferon-gamma (IFN-γ), IL-12 or IL-18 in the female genital tract [183]. These studies highlight the potential of the TLRs as agonists for HIV vaccines. Similarly, several vaccine approaches using the TLR agonist Poly I:C and derivatives have revealed their capacity to stimulate HIV specific immune responses in specific cell types and at sites of mucosal exposure to pathogens [184–187]. Likewise, monophosphoryl lipid A (MPLA) from lipopolysaccharide (LPS) of Salmonella minnesota used as an adjuvant in parenterally administered vaccines has been shown to induce antigen-specific mucosal and systemic cellular immunity and Ab responses following oral or i.n. delivery, probably through activation of TLR2 and 4 [188,189].

Other studies have shown that liposomes containing lipid A and HIV-1 proteins or peptide antigens could induce neutralizing ‘multispecific’ Abs in which the antigen-binding site of the Ab simultaneously binds both to the immunizing lipid and protein epitope [190,191].

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The challenges involved in the development of a HIV prophylactic vaccine are unprecedented in the history of vaccinology. Three decades after the discovery of the virus, the quest for a vaccine is still actively ongoing. The major obstacles met in the development of an efficacious vaccine are the mutational variability and global diversity of the virus, which allow its easy escape from both the cellular and humoral responses of the host. Moreover, HIV-1 mainly infects the organism through the mucosal sites of the genital and intestinal tracts and rapidly integrates into memory T cells that become latent viral reservoirs. An efficient vaccine will therefore need to not only induce potent and functional virus-specific Abs able to block virus entry at the site of initial infection but also CD8+ T cells for virological control in lymphoid tissues and lymph nodes.

As illustrated by the example of the human papillomavirus (HPV) vaccine, the parenteral route of immunization could be an efficient way to elicit protective mucosal immunity, and indeed, numerous studies on HIV/SIV vaccines have shown that it is possible to induce protection against rectal or vaginal challenges in NHP models by systemic active or passive immunization. However, systemic immunization usually generates only low humoral responses at mucosal sites that stem from the transudation of IgGs from the blood into the genitourinary tract, and can also induce the secretion of immunoglobulins that act through interaction with the neonatal Fc receptor. It is nevertheless possible that mucosal immune responses induced by parenteral immunization will be improved in the future by the development of specific adjuvants that would amplify and favourize such a response.

Immunization by the mucosal route preferentially induces IgA responses at the site of antigen delivery, as well as in secretions from anatomically remote mucosal sites. Thus, an effective mucosal route of immunization able to elicit specific IgAs and CTL immunity in the genital mucosa appears to be the nasal mucosa and the aerodigestive tract. Data collected so far show that MALT-targeted adjuvanted vaccine design could be universally applied to any form of HIV vaccine candidate, including peptides, subunit vaccines, VLPs, DNA or live recombinant vaccines. However, initial mucosal HIV-1 immunization of immunologically naive individuals may induce a state of mucosal tolerance. Systemic priming followed by mucosal boosting is likely to prevent this undesirable outcome. Furthermore, such a sequence of immunizations should elicit humoral immune responses in both the systemic and the mucosal compartments.

Unfortunately, at this time, mucosal immunization has been tested in only a limited number of studies, mainly because of the relatively inefficient uptake of antigens by mucosal surfaces and the unavailability of mucosal adjuvants approved for human use. Also, whereas systemic immunity can readily be assessed from peripheral blood samples, systemic responses do not necessarily reflect responses in mucosal compartments. Thus, in their NHP vaccination study, Bomsel et al.[32] observed high protection after intravaginal challenge that was correlated with HIV-1 blocking Abs developed in the mucosal compartment, but not in serum. Thus, antiviral mucosal immune responses may be missed in peripheral blood. Numerous studies have assayed HIV-specific mucosal responses in preclinical and clinical research, but a number of difficulties have slowed progress in incorporating such measurements. Indeed, processing mucosal samples is more challenging and sampling procedures provide lower amounts of fluid or cells than blood sampling. Moreover, mucosal sampling is more invasive than blood sampling and takes more time and training of clinical personnel.

If we are to ever fully realize the potential benefits of mucosal vaccines to control HIV/AIDS, current research should be extended to the development of innovative immunological tools such as safe adjuvants, targeting molecules and delivery vectors.

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Authors would like to thank the ANRS (Agence Nationale de la Recherche sur le SIDA) to S.P. and N.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 (grant ANR PECSDELLI and Euronanomed iNanoDCs; support to V.P., S.P. and B.V.) and the grant from the Fondation pour la Recherche Médicale to V.P.

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Conflicts of interest

The authors declare that there are no conflicts of interest.

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adjuvants; administration routes; HIV; mucosa; vaccine

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