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ISSN 1743-588910.2217/NNM.12.131 © 2012 Future Medicine Ltd Nanomedicine (2012) 7(10), 1591–1610

Enhancing immunogenicity and cross-reactivity of HIV-1 antigens by in vivo targeting to dendritic cells

BackgroundHighly active antiretroviral treatment (HAART) inhibits the replication of HIV and slows down disease progression, thus keeping viral RNA levels very low [1]. However, alternative treat-ment strategies are required as there are still a number of unresolved problems with HAART. These include high cost, persistent low-grade viral replication, incomplete normalization of T-cell functions, drug-related side effects (lipid metabolism problems: lipodystrophy and athero-sclerosis) and emerging primary HIV infections with multidrug-resistant viruses [2].

During the past two decades, a plethora of immunotherapeutic strategies and HIV vaccines have been developed to treat AIDS. Although these induce a certain level of immune response, they are not associated with significant clinical benefit or with an effective and sustained control of viral replication [3–5]. Nevertheless, a study by Lévy et al. demonstrated that immunization in combination with antiretroviral therapy and IL-2 improves HIV-specific immunity and lowers the viral set-point after withdrawal of HAART [6]. Lu et al. showed that ex vivo pulsed autologous monocyte-derived dendritic cells (DCs) with inactivated autologous virus induce a prolonged reduction of HIV RNA in a substantial num-ber of patients [7]. These results highlighted the potential of immunotherapy strategies targeting HIV antigens to DCs in HIV-infected patients.

The mutation capacity of HIV implies that an infected patient may harbor many genetic variants of the virus. This is undoubtedly a major obstacle for the development of a vaccine

for all variants. However, more than 90% of infections belong to HIV-1 group M [8], which includes at least nine genetically distinct sub-types (or clades). Of these, clade C (HIV-C) strains comprise 56% of all HIV infections worldwide. Therefore, HIV vaccines must include protection against this virus subtype.

While live-inactivated HIV vaccines are strongly immunogenic and can induce both cellular and humoral immune responses, their use remains a major concern because of the risk of HIV mutating back or recombining with a pathogenic virus. Although virus-inactivated vaccines are still being used because of the sim-plicity in obtaining an autologous virus, new HIV vaccine research has focused mainly on the administration of conserved recombinant viral proteins and peptides as single preparations or cocktails. Attention has also been devoted to DNA encoding several HIV antigens, recom-binant vectors and prime–boost strategies, using two vaccination approaches to immunize with the same antigen (Figure 1 shows the genome and virion organization with the potential antigens).

The expression of HIV genes in DCs could induce a response mediated through MHC class I molecules to generate cytotoxic T-lymphocytes (CTLs). In addition, HIV Tat protein stimu-lates the maturation of DCs and increases their antigen-presenting capacity, thereby promot-ing the induction of antigen-specific T-cell responses [9,10]. Recently, several DC-based HIV vaccines have produced encouraging results, thereby indicating that some HIV anti-gens – those more invariant or essential for viral

Current retroviral treatments have reduced AIDS to a chronic disease for most patients. However, given drug-related side effects, the emergence of drug-resistant strains and the persistence of viral replication, the development of alternative treatments is a pressing need. This review focuses on recent developments in HIV immunotherapy treatments, with particular emphasis on current vaccination strategies for optimizing the induction of an effective immune response by the recruitment of dendritic cells. In addition to cell-based therapies, targeted strategies aiming to deliver synthetic HIV peptides to dendritic cell-specific receptors in vivo will be discussed.

Keywords: carrier conjugates n cross-reactivity n delivery vehicle n HIV-1 n immunogenicity n nanoparticle n specific ligands n synthetic peptide vaccines n targeting n V3 loop

Luis J Cruz1,2*, Felix Rueda3, Paul Tacken1, Fernando Albericio2,4, Ruurd Torensma1 & Carl G Figdor1

1Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands 2Institute for Research in Biomedicine and CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain 3Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain 4School of Chemistry, University of KwaZulu-Natal, 4041 Durban, South Africa *Author for correspondence: Endocrinology Research Lab and Molecular Imaging, Leiden University Medical Center, Building 1, C4-R67, Albinusdreef 2, 2333ZA Leiden, The Netherlands [email protected]

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Nanomedicine (2012) 7(10)1592 future science group

Review Cruz, Rueda, Tacken, Albericio, Torensma & Figdor

U3 R U5

LTR

gag

MACANCp6

PR RT IN

pol

vif

vpr rev env

vputat

SU TM

nefLTR

Surface (GP120) SU

Transmembrane(GP41) TM

Nucleocapsid (p24) NCMatrix MA

Integrase IN (p32)

Reverse transcriptase(p66)

p6, vif, vpr, nef

Protease PR(p11)

Transmembrane(GP41) TMSurface (GP120) SU

100 200 300 400 500 600 700

V1 V2 V3 V4 V5

Variable domains (gp120)

Transmembraneanchor

Fusion domainProteolyticcleavageCD4-binding domainV3 loop

1

Nanomedicine © Future Science Group (2012)

Figure 1. HIV-1 genome and virion organization: the potential use of virus proteins for vaccines. The genome of HIV has the characteristics of a lentivirus. It is composed of two LTR regions, which are involved in the replication and integration of the virus. Three regions, gag, pol and env, code for NC and MA, enzymatic proteins (protease, reverse transcriptase and integrase) and envelope proteins, respectively, and a set of accessory proteins (tat, nef, vpr, vif). Gag (p55) polyprotein is processed during maturation to MA (p17), capsid protein (p24), spacer peptide 1 (p2); NC (p7), spacer peptide 2 (p1) and p6. Pol codes for viral enzymes reverse transcriptase, integrase and HIV protease. The most frequently used proteins in HIV vaccine development are Env, envelope glycoproteins and glycoprotein (gp)120 (SU or gp120) in particular. The latter contains variable regions, such as the V3 loop. Consisting of approximately 30 amino acids, this loop is formed by two cysteine residues that form a disulfide bridge, and it has a conserved GPGRAF amino acid sequence at the perimeter. The remainder of the loop is more variable. Gp120 is known to bind with high affinity to the cellular protein CD4. After binding, this glycoprotein binds to cellular chemokine receptors, notably CCR5. The other is a transmembrane glycoprotein (TM or gp41), which contains highly hydrophobic residues in a short segment of the N-terminal (fusion) domain. This region of fusion is crucial because it allows the membrane of the virion to fuse with the cell membrane and thus allows virus entry into the cell. In addition to the fusion region, gp41 has a second hydrophobic region in the middle, which serves to anchor the protein to the lipid bilayer of the viral envelope. The gp120 and gp41 proteins are produced by fragmentation of a large precursor, gp160, and are found as oligomers on the viral particle. LTR: Long terminal repeat; MA: Matrix proteins; NC: Nucleocapsid; TM: Transmembrane glycoprotein.

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Enhancing immunogenicity & cross-reactivity of HIV-1 antigens by in vivo targeting to DCs Review

replication – generate specific T lymphocytes in subjects with long-term stable disease [11–13].

direct antigen vaccination using a series of presentation strategies for HIV-1Given that many of the vaccine candidates cur-rently available induce antigen-specific antibody responses and do not cross-react with heterolo-gous strains, improvements in HIV-1 vaccines are needed. To overcome the problem of HIV-1 variability, novel vaccines should be designed to induce an extensive range of reactive antibodies against multiple HIV-1 epitopes. In this regard, the authors and others have focused on achieving a good immunogen on the V3 loop of the HIV-1 envelope. This loop was chosen because it is the most variable region and is responsible for induc-tion. It is also the primary target of many neu-tralizing antibodies [14], and it is involved in vari-ous other aspects that affect infectivity. Thus, sequence changes in V3 affect chemokine recep-tor usage and therefore modulate the cell types infected [15]. Moreover, it appears that antibodies against the principal neutralizing domain of V3 exert control over viral load [16]. For the HIV-1 MN subtype B isolate, a high titer against V3 has been correlated with slow progression rates in seropositive hemophilic Japanese patients [17]. All these findings highlight the immunological relevance of the V3 loop which, in addition, includes both T-helper and CTL epitopes [18], thereby justifying its inclusion in a number of HIV-1 vaccine candidates [19]. However, in a recent report from the RV144 trial, in a vaccine composed of a recombinant canarypox vector expressing HIV-1, gag, pro and membrane-linked gp120 (vCP1521), combined with a bivalent gp120 protein boost (AIDSVAX B/E) (one of the few vaccines that has shown some protection against HIV-1), the T-cell responses were preferentially targeted to epitopes in the V2 region of the HIV-1 envelope, although the neutralizing antibodies were targeted to the V3 region [20,21]. Furthermore, recent results have shown the presence of neutralizing antibodies rec-ognizing quaternary structures in V2 and V3 [22]. These results demonstrate that regions other than V3 could also be important for the generation of new vaccines.

Current HIV vaccine models comprise either recombinant proteins [23] or synthetic HIV-1 multi-epitopic peptide constructs [24,25]; how-ever, naked DNA [26], plasmid DNA [27,28] and mRNA are gaining ground [29–31]. Multi-epitope polypeptide TAB9, containing six V3 epitopes

from isolates LR10, JY1, RF, MN, BRVA and IIIB, were selected for immunological response in animal models (Figure 2), and were subsequently used in a Phase I clinical trial. All immunized volunteers seroconverted, and antibodies were broadly reactive against the V3 peptides included in the protein. However, the immunogenicity of the recombinant protein induced high levels of reactivity directed against the recombinant protein itself, mainly at JY1 and LR150, and only weakly at MN [32]. Naked DNA technology has progressed in the last decade; however, suitable delivery routes continue to be elusive. Recently, Rosenberg et al. [33] demonstrated that the HIV-1 DNA vaccine is safe but poorly immunogenic in subjects treated with antiretrovirals during acute/early HIV-1 infection. However, a combi-nation of polypeptide and a DNA vaccine could boost the immune response as a result of a com-bination with T-helper epitopes [34]. Viral vectors achieve this objective efficiently; however, they present other kinds of problems, including safety issues. The discovery of specific and small anti-gens, such as those of the V3 loop, has allowed the development of synthetic peptides for HIV-1 vaccines (Figure 2) [35,36].

�n Peptides, multiple antigenic peptides & combinatorial libraries, & their conjugation to carrier proteins Given the variability of HIV-1, in a previous study, the authors focused their efforts on several V3-based multiple antigen peptides (MAPs) and peptides. This strategy enhances the immuno-genicity and cross-reactivity of these structures towards other V3 sequences [37]. To this end, several presentation strategies were examined, such as polymers [38], MAPs and peptides cou-pled to dextran microparticles [39]. Combined approaches, such as the coupling of MAPs to carrier proteins [40], were also tested [41].

Cocktails containing several peptide variants were used in an attempt to overcome viral vari-ability. The authors had previously shown that V3 cocktails with MAPs provide an efficient vaccination strategy [28]. In the context of that study, the following points of attention were raised: first, recognition of distinct V3 peptides is closely related to the degree of homology between them; second, conjugation allows the inclusion of more peptide variants in cocktails with a smaller mass of antigen; and third, a combination of con-jugated MAP and peptide significantly enhances cross-reactivity.

In fact, the authors’ results confirmed their hypothesis that the inclusion of V3 peptides,



such as JY1 and RF subtype, in a dendrimeric structure, such as a MAP or MAP coupled to a carrier protein, increases the generation of broadly reactive antibodies [33].

The HBsAg–JY1–MAP conjugates elicited the highest titers against JY1 in comparison with all the other strategies tested. Carriers such as KLH and HBsAg form nanostructures with greater surfaces to incorporate MAP and peptides. Furthermore, these carriers offer more sites for the coupling of synthetic peptides. By contrast, carrier–MAP conjugates are more immunogenic than carrier–peptides [42].

An alternative approach aimed to cover the identification of a wide spectrum of V3 epitopic sequences is the generation of combinatorial

peptide libraries, called mixotopes [43,44]. Mixotopes comprise several peptides that rep-resent variable positions of the V3 loop, and act as model antigens for recognition of the immune system. Promising results using this type of strategy have been reported [45].

In contrast to the findings described by Resnick et al. [46], the authors demonstrated that the mixotope strategy does not generate higher levels of cross-reactivity against several V3 pep-tides than those of the consensus strategy. The mixotope library tends to target mainly the con-served regions of the V3 loop, which are overrep-resented compared with the variant ones. With regard to highly variable antigens, it could be concluded that a rationally designed consensus

=

Recombinant protein vaccine

N-terminus P64K LR150 JY1 RF MN BRVA IIIB

Multiple antigen peptide vaccine:

Loop V3:LR150, JY1,RF, MN, BRVA,IIB

Loop V3 Loop V3

Loop V3

Loop V3

Loop V3

Loop V3

Loop V3

Loop V3

KK

KK

KK

KK

T helper

T helper

T helper

T helper

MAP

MAP

MAPMAP

MAP

MAP MAP

KGC-S—–S-CGK

Complex macromolecules for vaccines

Proteins:Gp120 or gp40,P64k, KLH orHBsAg

COOH

COOHHOOC

HOOC

Conjugation

Peptides

Proteins

Proteins

Proteins


Figure 2. Various strategies for HIV-1 peptide vaccine formulations: recombinant proteins, multiple antigen peptides and protein conjugates. (A) Recombinant protein, a multi-epitope polypeptide including the V3 region from six divergent HIV-1 isolates (LR150, JY1, RF, MN, BRVA and IIIB, in this order) fused to the amino terminus (47 amino acids) of P64K protein from Neisseria meningitidis. (B) The V3-MAP variant includes two monomers linked through a disulfide bond and with a 2:1 epitope ratio. A cathepsin-like enzyme cleavage site (-KK-) was inserted between B- and T-cell epitopes. Six V3-MAP8 constructions bearing divergent V3 HIV-1 sequences LR150, JY1, RF, MN, BRVA and IIIB as B-cell epitopes were synthesized. Monomers were properly assembled for heterodimeric MAP combinations. (C) Complex macromolecule vaccines are conjugates of peptides or MAPs with carrier proteins, such as Gp120, KLH or HBsAg. MAP: Multiple antigen peptide.



sequence, such as the one derived for the V3 loop of gp120, is a valid approach for synthetic vaccine development, providing desirable cross-reactivity and therapeutic advantage.

However, early enthusiasm for exploring pep-tide vaccines against HIV-1 infection was based on the supposition that the V3 loop of HIV-1 was the main neutralizing determinant of the virus and the demonstration that this deter-minant is mimicked antigenically by synthetic peptides [47]. Two Phase III trials showed that bivalent, recombinant gp120 (rgp120) vaccines do not prevent HIV infection [48,49]. Although vaccine efficacy was not demonstrated, a separate analysis revealed an inverse correlation between vaccine-induced antibody activity and HIV infection rate [49]. The authors proposed that the vaccine-induced antibody was a marker of another – presumably innate – immune response that ultimately reduced the rate of infection. Recently, an HIV-1 lipopeptide vaccine tested in healthy adults in a Phase II trial proved immu-nogenic and safe at a range of doses. Moreover, it elicited HIV-specific sustained CD8 and CD4 T-cell responses in healthy adults [50].

However, the limited success of direct pep-tide vaccination strategies in Phase III prompted various research groups to develop cell-based HIV-vaccines using DCs.

strategies to activate HIV-1 antigen presentation in dCsThe functional characteristics of DCs allow them to capture antigens by several pathways. Thus, a number antigen-loading methods for DCs have been described [51]. For instance, synthetic peptides need mature DCs to be more effective, while major antigens, such as proteins and whole virus, are usually loaded into immature DCs.

In the case of HIV-1, where the genetic variation of the virus makes it difficult to fully identify and define the antigen epitopes of the virus infecting each patient, DCs can be loaded with the whole virus. This approach may reduce antigen escape by a loss of epitope variants. If the antigen is processed and presented through both MHC class I and II molecules, a more potent immune reaction is gen-erated because CD4+ T cells play a critical role in inducing and maintaining effective CTL responses [52]. The main disadvantage of this strategy is the potential risk associated with the use of inactivated whole virus (despite the safety of inactivating meth-ods, recombination with integrated HIV DNA may occur and may thereby generate infectious parti-cles). In addition, immune monitoring is hindered because the epitopes involved are unknown.

�n Inactivated HIV-1 virus Several methods to inactivate HIV-1 have been described, with different effects on the integrity of HIV-1 antigens: irradiation, heat, formalin and aldrithiol-2 (AT-2). All these approaches render the virus non-replicative before in vivo administration. Yoshida et al. tested a prophylac-tic vaccine consisting of autologous mature DCs (mDCs) loaded with either AT-2-inactivated HIV-1 or an irrelevant antigen, ovalbumin [53]. Although the study was performed in severe combined immunodeficiency (SCID) mice transplanted with human peripheral blood mononuclear cells, only mice immunized with DC-HIV-1 were protected against HIV-1 chal-lenge in vivo. In another study, 3 days after reconstitution with autologous human peripheral blood lymphocytes (PBLs), human PBL-SCID mice were injected with autologous DCs loaded with AT-2-inactivated HIV-1 [54]. Immunization induced human anti-HIV-1 antibodies and the generation of a human CD8+ T-cell response against HIV-1. In addition, immunized human PBL-SCID mice were more resistant to virus infection than control animals.

In rhesus monkeys infected with simian immunodeficiency virus (SIV), Lu et al. showed that a therapeutic vaccination performed with AT-2-inactivated SIV-pulsed DCs induced effec-tive and sustainable SIV-specific cellular and humoral immunity and a decrease in the viral load induced [55].

In a clinical trial carried out by the same group in 18 patients with chronic HIV-1 infection, the efficacy of a therapeutic AT-2-inactivated HIV-pulsed autologous DC vaccine was assessed [7,56]. A temporary decrease in plasma viral loads of 80% was observed. This reduction correlated with HIV-1-specific IL-2- or IFN-g-expressing CD4+ T cells and with HIV-1 gag-specific per-forin-expressing CD8+ effector cells. These data suggest that a robust virus-specific CD4+ Th1 response is required for inducing and maintain-ing virus-specific CD8+ effectors in order to control HIV-1 replication in vivo. Other clinical trials are currently underway with psoralen and UVB irradiation (312 nm) [57] or heat-inactivated HIV. Recently, results from 24 untreated patients with chronic HIV-1 infection enrolled in a dou-ble-blinded, controlled study of vaccination with three doses of autologous monocyte-derived DCs pulsed with heat-inactivated autologous HIV-1 were reported [58]. The vaccine was safe and well tolerated, and after vaccination, an increase in HIV-specific T-cell responses was observed. This enhancement tended to be inversely associated



with the decrease in plasma viral load in vacci-nated patients, but not in the control group. No positive CD4 lymphoproliferative responses to HIV-1 p24 antigen were observed in any patient. However, as noted above, the use of live-inac-tivated HIV vaccines entails safety concerns. Therefore, the administration of recombinant proteins, peptides, DNA or mRNA encod-ing defined antigens is an attractive alterna-tive (Figure 3). Unfortunately, immunological responses induced by this approach are only low and partial and may allow the virus to escape recognition and neutralization. Therefore, new strategies should be tested with combinations of viral proteins or peptides showing low variability, DNA vaccines composed of several genes that encode HIV subunits, recombinant vectors and prime–boost strategies, which use two ways of administering the same antigen.

�n Protein antigen HIV-1 has several proteins that can be used as antigens for vaccination purposes (Figure 1). However, when administered alone, recombi-nant viral proteins are poor immunogens. An attractive alternative is to combine several HIV proteins into one vaccine. Liposomal prepara-tions containing several HIV-1 structural pro-teins (p24 gag, gp160 env or p55 gag) to load

DCs are delivered to and processed through the HLA class I pathway, and prime CD8+ T cells against HIV-1 in vitro [59]. However, some HIV proteins may induce adverse effects, which represent an important drawback; for instance, HIV-1 gp120 binds to the surface of CD4+ T cells, thus contributing to the immu-nopathogenesis of this virus. This envelope gly-coprotein influences various cell populations of the immune system, including hematopoietic progenitors, T and B lymphocytes, monocytes/macrophages and DCs, as well as neuronal cells [60]. Furthermore, despite displaying a typi-cal phenotypic maturation, immature human monocyte-derived DCs (iDCs) loaded with either AT-2-inactivated HIV-1 or gp120 show an impaired capacity to secrete cytokines or chemokines and to induce T-cell proliferation [61]. This immunosuppressive condition is not reverted by stimulation with lipopolysaccharide or CD40L. However, exogenous addition of IL-12 does restore the allostimulatory capacity of gp120-exposed DCs.

Extracellular Tat has crucial effects on immu-noregulatory mechanisms, driving iDC matu-ration, inducing MHC class I molecules and tipping the balance towards a Th1-inducing phenotype [62]. However, Tat also alters the proteasome function by modifying proteasome composition and enzymatic activity, thereby changing the processing of CTL epitopes. This favors the presentation of subdominant and cryptic epitopes [63]. Furthermore, Tat, directly or indirectly, also induces damaging effects in several organs and host systems, such as the myocardium, kidney, liver and CNS. HIV-1 Nef protein downregulates the expression of HLA class I [64,65] and II molecules [66]. Taken together, these observations indicate that caution should be taken regarding the use of full-length viral proteins for vaccination purposes.

�n Antigen-encoding DNA & RNA Among the methods available for HIV-1 anti-gen loading on DCs, genetic techniques for the introduction of RNA or DNA are gaining popularity owing to their flexibility and simplic-ity. The use of RNA isolated from pathogens or infected cells has a number of advantages. First, sufficient RNA can be generated from a small amount of sample by amplification tech-niques [67]. Second, target-restricted RNA can be enriched before DC loading by subtractive hybridization with RNA from uninfected cells. Pathogen-specific immune responses are thus augmented and the risk of autoimmunity is

DC vaccination strategy

IL-4GM-CSF

ImmatureDC Maturation

stimuli HIV-1 peptides,RNA, DNA,

recombinant protein

Injection in patient

Monocyte

Isolationfrom blood

Disadvantages:- Time consuming- High costs- Preparation of individual vaccine

Figure 3. HIV vaccination strategy with dendritic cells. DCs can be loaded with the whole protein when a pathogenic antigen has been defined but the peptide epitope has not yet been identified. This approach not only circumvents the need to identify peptide epitopes, but also expands the clinical application to patients who are excluded because of MHC restriction, a problem associated with the use of peptide epitopes. Adapted with permission from [170].



reduced. However, the major drawback of the use of RNA is the instability of its products and the greater technical demands required [68]. Similar to RNA, it is possible to generate DNA encoding a target antigen that can be expressed in the antigen-presenting cell (APC). Thus, the antigen can be processed and presented in a way that closely resembles the processing of endog-enous proteins or virus, where protein-derived peptides are loaded onto MHC class I molecules to activate CD8+ T cells [69].

Because DNA is less prone to degradation than RNA, antigen presentation may last lon-ger. However, the generation of immunocom-petent DCs with target DNA continues to be difficult because of the limitations of DNA delivery systems. The most common method for introducing RNA into DCs is electropora-tion [70]. This approach allows the expression of exogenous antigens by DCs, without the risk of viral vectors. Furthermore, genetically modified recombinant vectors have proved to be the most suitable vehicle for DNA delivery into DCs. Recombinant vector vaccines use attenuated virus or bacteria as carriers of modified target antigens into the host. The target genetic infor-mation is incorporated into the vector genome and target proteins are produced. DCs loaded ex vivo with recombinant vectors, such as rep-lication-defective herpes simplex virus type 1 amplicons that express HIV gp120 (herpes sim-plex virus gp120MN/LAI), replication-defective adenovirus serotype 5 vectors expressing SIV Gag antigens p17 and p45 [71] and recombinant Lactococcus lactis vector expressing the V2-V4 loops of HIV Env on its cell surface (IL1403-pHIV) [72], induce HIV-specif ic immune responses in vivo. However, vector-specific neu-tralizing antibodies may limit the effectiveness of repeated booster injections. Therefore, the use of these vectors in clinical vaccination trials is still limited [73]. Furthermore, although the new generation of recombinant viruses offers greater safety, these viruses pose a certain degree of risk for immunocompromised patients. Despite this risk, some clinical trials have been performed with recombinant viral vectors. Autologous DCs from healthy volunteers were transfected ex vivo with ALVAC-HIV (vCP205), a canarypox-based vaccine containing env, gag and pol genes from HIV. Although only modest cytotoxic CD8 T-cell responses were presented, volunteers who received the DC autologous vaccine loaded in mDCs (which showed lower expression of the target genes than those loaded with iDCs) developed a broader and more durable immune

response than those who received ALVAC-HIV by intramuscular, intradermal or subcutaneous injection. Another possibility is the use of viral structural proteins that resemble virus particles or virus-like particles (VLPs), particularly HPV capsid proteins, which show the same capacity to induce an immune response [74] as the viral vector. Moreover, the affinity of the VLP for heterologous DNA allows its use in gene trans-fer of antigens [75], but this approach shows less efficiency than that achieved by a viral vector.

�n Exosomes as a tool for HIV-1 vaccinationIn 1998, DC-derived exosomes were shown to have immunogenic properties leading to tumor eradication in mice [76]. Exosomes are small membrane vesicles of approximately 30–100 nm in diameter. Exosome secretion is a process in which multivesicular bodies fuse with the cell membrane. Exosomes derived from tumor or APCs are released into the extracellular environ-ment and fuse with the membranes of neighbor-ing cells, thus delivering membrane and cyto-plasmic proteins from one cell to another. Such proteins, for example, MHC molecules, costim-ulatory molecules and heat-shock proteins, play key roles in immune responses [77]. DCs could be primed with antigens and then the DC-derived exosomes could be used as cell-free vaccines [78].

A lentiviral vector encoding HIV gag–pol without env would form viral cores in transduced DCs, but would release noninfectious particles. It was shown that accumulation of viral cores inside transduced DCs and the exosomes from these cells produce a second-round transduction of iDCs. Mice were immunized with transduced DCs to induce specific lymphocyte activation, and they produced antigen-specific T-cell responses and specific antibodies after immunization. This approach could provide a novel strategy for HIV

Table 1. receptors used to target antigens to dendritic cells.

Targeted receptor ref.

Mannose receptor [98,99]

Dec-205 [100–102]

LOX-1 [103,104]

Langerin [105]

DC-SIGN [107–111]

Clec9A [112,113]

CD40 [117–119]

FcR family [120–132]



vaccination [79]. However, exosomes produced by T cells and DCs from HIV-1-infected patients have been associated with HIV-1 dissemina-tion, although a budding pathway is not shared by exosomes and HIV-1 [80]. Some studies have shown a previously undescribed mechanism of DC-mediated HIV-1 transmission and suggest that virus particle trafficking to multivesicu-lar endosomal bodies and subsequent exocyto-sis can provide an avenue for immune escape for HIV-1 particles captured by DCs [81], thus allowing viral internalization and final transin-fection of CD4+ T cells [82]. Furthermore, exo-somes released from HIV-1-infected cells harbor regulatory molecules that affect cell apoptosis (ANXA5 and LDHB) and proliferation (CD38) [83], and export Nef, which facilitates the depletion of CD4+ T cells, a hallmark of AIDS [84]. Given all these observations, although exosomes are a prom-ising tool for vaccination for tumors and infectious pathogens, caution is needed when considering them in the setting of HIV-1 infection.

Targeting of antigens to dCs in vivo�n Ligands to deliver protein or

peptides to DC receptorsDespite the promising results of vaccines using antigen loading on DCs ex vivo, the cost and technical demand of ex vivo generation of DCs makes this approach unsuitable for large-scale immunization programs. Ex vivo procedures require the isolation of rare naturally occur-ring DCs or the ex vivo generation of DCs from monocytes isolated from the patient. Both procedures are labor-intensive and require spe-cial equipment to collect the cells from blood. Moreover, it is costly to perform ex vivo culture of cells following good manufacturing proce-dures. The cell collection procedure takes hours and is an extra burden for the patient. These drawbacks of cellular therapies could be pre-vented by targeting antigens to DCs in vivo via specific surface receptors. These constructs are cost effective and can be used from the shelf [85].

Triggering the appropriate receptors might even lead to the activation and maturation of DCs and to the boosting of immune responses. The antigen delivery system will determine the route of entry into the cell and the pathways, mecha-nisms and efficiency of antigen processing and presentation via MHC class I and II molecules. Receptor ligands can be directly attached to pro-tein and peptide antigens for targeting aims or, alternatively, proteins, peptides or DNA encod-ing the antigen could be incorporated on other suitable drug delivery systems, such as liposomes,

nanoparticles and dendrimers [86]. Two categories of receptor ligands target antigens to DC recep-tors: first, ‘natural’ receptor ligands, including bacterial toxins, heat-shock proteins and sugar residues; and second, receptor-specific antibodies.

�n Targeting DC surface receptorsMany receptors on the surface of DCs induce intracellular signaling pathways upon interaction with the ligand. The general characteristics and qualities that make receptors amenable as vac-cine targets include specific and high expression on DCs, endocytic activity, capacity to induce antigen processing and presentation via MHC class I and II pathways, as well as a potential to trigger signaling pathways to stimulate an immune response. Receptors expressed by DCs displaying at least several of these properties include CD40, Fc-receptors (FcRs), Toll-like receptors (TLRs), C-type lectin receptors, Mac-1 (CD11b/CD18) and globotriaosylceramide (Gb3/CD77). Table 1

presents a summary of DC surface receptors that have been used to target antigens to DCs.

�n Targeting antigens to the MHC class I & II pathwaysAn efficient strategy for HIV vaccination should induce both humoral and cellular immune responses. To this end, antigens should be pro-cessed and presented by both MHC class I and II pathways. For antibodies, the B cell must also be reached, and this is difficult when the packed antigen is targeted to a DC, where it is unzipped inside this cell. In general, strategies targeting antigens to be released directly into the cytosol, or DCs transfected with DNA-encoding anti-gens will be efficiently presented via the MHC class I pathway. As cellular immune responses are considered crucial in reducing viral load, routing antigens into the MHC class I loading pathway appears to be an essential requisite for HIV-1 vaccines. However, targeting protein antigen to receptors on DCs will essentially result in MHC class II presentation; never-theless, antigens captured this way are able to escape from endosomes and can be presented by MHC class I by means of a cross-presentation mechanism (Figure 4) [87]. Although particulate antigens loaded by phagocytosis are more effi-ciently cross-presented than soluble antigens taken up via endocytosis, both soluble and par-ticulate antigens can be targeted to DC surface receptors for class I presentation [88].

Several bacterial toxins have been used to deliver antigens to be presented by MHC class I molecules. These toxins bind to DC receptors



and can be directly translocated into the cyto-sol or cross the endosomal membrane to reach the cytosol, or the endoplasmic reticulum (ER) via a retrograde endosome–Golgi–ER pathway [89]. In this way, antigens coupled to toxins that target the cytosol and ER gain direct access to the classical MHC class I pathway and induce CTL responses in vivo [90].

Regarding the recognition of structurally conserved molecules derived from infectious agents, several TLRs, which are among the most potent activators of the immune response, are present on DCs [91]. TLR engagement alerts the immune system and leads to the activation of innate and adaptive immune responses. TLRs trigger DC maturation, stimulate the prolifera-tion of CD4+ and CD8+ T cells and modulate the suppressive function of Tregs [92]. These

immune-stimulating properties have resulted in the use of TLR agonists in cancer treatment, both as monotherapy to induce innate responses and as adjuvants for vaccines [93]. It is possible to target viral antigens to TLRs, either by conjuga-tion of the antigen to an appropriate TLR lig-and, or by using HIV-1 itself as a TLR agonist, as HIV ssRNA is a potent immune activator triggering TLR 7/8. However, TLR triggering in HIV-infected individuals is not without risks as the activation of certain TLRs, including TLR-2 and -9, induces HIV-1 replication and thereby disease progression [94]. Thus, while flagellin (TLR5 agonist) treatment enhances the repli-cation of CC chemokine receptor 5-tropic and CXC chemokine receptor-tropic HIV-1, treat-ment with oligodeoxynucleotide M362 (TLR9 agonist) suppresses these two viral variants [95].

Targeting antigens to MHC-I and -II

Target receptor

Direct entrance

MHC-I

Proteicantigens

MHC-II

Phagosome

MHC-I

Endosome

MHC-II

Virus orDNA

Proteosome

Peptides

Endoplasmic reticulum


Figure 4. Targeting antigens to the MHC class I and II pathways. In order to transport and present exogenous antigens into the MHC class II pathway, endocytotic and phagocytotic endosomes fuse with vesicles that transport MHC class II molecules from the endoplasmic reticulum compartment. There are several vaccination strategies to target antigens across the cytoplasmic membrane to reach the MHC class I pathway. Furthermore, there is evidence that antigens escape from the phagosome and endosome compartments into the cytosol. The same happens with DNA vaccines encoding antigen that enter DCs via the endosomal or phagosomal compartment, where they can escape, thus resulting in the expression of the antigen in the cytosol. Finally, certain bacterial toxins linked to antigens target them via the endosome and Golgi to the ER through the retrograde pathway. In addition, endocytosed proteins can be transferred from the endosome to the ER, where they can reach the cytosol.



Another strategy to target antigens to DCs comprises the attachment of sugar residues to the antigen. This procedure allows recognition by C-type lectin receptors on DCs, thereby resulting in presentation via the MHC class I and II pathways. C-type lectins belong to a family of lectins that share primary structural homology in their carbohydrate-recognition domain, which binds to sugar residues in a cal-cium-dependent manner. The choice of sugar residue determines the receptors targeted and thus the immunological outcome. For example, antigens coupled to oxidized mannan induce mostly cellular responses, whereas those cou-pled to reduced mannan produce humoral responses [96]. Expressed by DCs, these recep-tors are implicated in immunoregulatory pro-cesses, such as antigen capture, DC trafficking and DC–T-cell interactions [97]. Of the C-type lectin receptors used for antigen targeting for vaccination purposes, mannose receptor (MR), DEC-205 and DC-SIGN have received the most attention.

Certain MR ligands and antibodies induce an immunosuppressive response in DCs [98]. However, recent studies involving monoclonal antibodies specifically targeting antigens to MR conclusively demonstrate enhanced antigen uptake and presentation via the MHC class I and II pathways [99].

The characteristics associated with C-type lectin receptors have led many researchers to assay them in vaccine designs. Thus, DEC-205 is a type I C-type lectin receptor that is highly expressed on mDCs and to a much lesser extent on certain leukocyte subpopulations [100]. DCs cross-present HIV gag to CD8+ T cells, but when the antigen is delivered within an antibody to DEC-205 receptor, cross-presentation becomes 100-fold more efficient than nontargeted anti-gens [101]. Antibody–antigen conjugates target-ing tumor antigens to MR and HIV gag p24 to DEC-205 are currently being evaluated in clinical trials [102].

DC LOX-1 is a type II C-type lectin receptor expressed on several cell types, including macro-phages and peripheral blood myeloid DCs [103]. LOX-1 is a receptor for Hsp70, and targeting DCs with protein antigen conjugated to Hsp70 results in cross-presentation in vitro and in vivo [104]. Targeting vaccine antigen to Langerin+ DCs favors the induction of T-cell responses [105], whereas targeting LOX-1+ DCs enhances the induction of antibody responses [106].

DC-SIGN is a type II C-type lectin with a more restricted expression to professional APCs than that shown by DEC-205 and MRs. DC-SIGN is highly expressed on DCs residing in lymphoid tissues, mucosal surfaces and der-mal tissue, and on specialized macrophages in the placenta and lung. This expression profile repre-sents a major advantage of targeting this lectin over other C-type lectin receptors [107]. Although DC-SIGN has been associated mainly with the spread of HIV-1 in humans and most of the work reported concerns blocking HIV-1–DC-SIGN interactions, in vitro studies show efficient target-ing of liposomes [108], adenovirus [109] and pro-tein antigens [110] to DCs via DC-SIGN, which induces both naive and recall T-cell responses via MHC classes I and II molecules. In primary DCs, DC-SIGN-captured HIV virions are rapidly degraded, possibly in a lysosomal compartment. Thus, a fraction of the incoming viral material is processed by the proteasome, leading to activation of anti-HIV-specific cytotoxic T-lymphocytes by DC-SIGN-expressing cells [111].

Finally, the type II C-type lectin receptor CLEC9A was recently proposed as an interest-ing target for vaccination purposes. In humans and mice, CLEC9A expression is relatively restricted to a specific DC subset, which is desig-nated BDCA3+ in humans and CD8a+ in mice. Moreover, in mice, this subset is specialized at cross-presenting antigens to CD8+ T cells [112,113].

Antibodies, and not the natural receptor ligands, appear to be the most efficient system to target antigens to DCs, and they are more potent than ex vivo antigen-loading DCs [114]. The production of humanized and human anti-bodies has improved the clinical efficiency of these tools and has led to the approval of many antibody-based therapies for clinical use [115]. Numerous antibodies targeted to cell-surface receptors induce receptor-mediated endocytosis, a relatively inefficient process for the activation of cross-presentation. However, antibody-mediated targeting of protein and peptide antigens to DC surface receptors induces strong CTL responses in vivo [116].

Table 2. delivery systems to target dendritic cells.

Group delivery system ref.

Live vector Live-attenuated virusLive recombinant virus (including HIV-1) or bacteria (gene transfer)

[133][134,135]

Micro/nanoparticles Exosomes Virus-like particlesVirosomesLiposomesPolymer micro/nanoparticles

[76–78,81–84][136][137]

[138,139][140–143,146,147]



A paradigmatic case is the targeting of anti-bodies to CD40 in DCs. This protein is expressed in many cell types, including macrophages, DCs and endothelial cells [117].

CD40 is an excellent candidate for HIV vac-cines based on DC targeting, as its activation not only causes maturation of APCs but also allows efficient presentation and cross-presentation of HIV epitopes, thereby stimulating strong recall T-cell responses [118]. In addition, studies with recombinant monoclonal antibodies (rAbs) for multiple internalizing human DC recep-tors, engineered by fusion via the heavy chain C-terminus to a string of five 19- to 32-amino acid long sequences from HIV-1 gag, Nef and Pol proteins (called rAb.HIV5pep), showed that anti-CD40.HIV5pep was strikingly more effec-tive. CD4+ T cells were found to produce multiple cytokines, while the CD8+ T cells produced cyto-kines, in addition to perforin, granzyme B and surface CD107a, and had CTL characteristics and proliferative capacity [119].

As an alternative to antibodies that recognize target receptors, those recognizing antigens could also be used for vaccination purposes. These antibodies form immune complexes with the antigen, which are captured by FcRs recog-nizing the constant fragment of the antibody. Antigens targeted to FcRs are presented via the MHC class I pathway, thereby simultane-ously inducing DC maturation and increasing the efficiency of T-cell activation by a factor of 1000–10,000 [120].

FcRs comprise a family of membrane pro-teins that are important mediators of immune responses, such as phagocytosis, antibody-dependent cell-mediated cytotoxicity, regula-tion of lymphocyte proliferation and antigen presentation [121]. DCs express receptors for IgG (FcgR), IgA (FcaR) and IgE (FceR).

Immune complexes of IgG and antigen induce DC maturation and the priming of peptide-spe-cific CD8+ T cells in vivo [122]. However, the bal-ance between activating and inhibitory signaling after engagement of the various FcRs on the DC dictates whether the uptake of the immune com-plexes results in efficient naive T-cell activation and protective immunity [123]. This can be over-come by targeting vectors containing antibodies that recognize a specific activating FcR. These vectors should be carefully designed as FcRs need to be cross-linked in order to induce inter-nalization. Therefore, single-chain Fv constructs are not suitable for FcR targeting unless they are themselves cross-linked. Moreover, single-chain Fv constructs might inhibit immune responses,

as continuous stimulation of FcaRI with single-chain Fv has been described to block activating responses of heterologous FcgRI and FceRI [124]. Targeting antigens specifically to FcgRI results in antigen presentation via the MHC class I and II pathway in vitro [125].

At least 1% of human antibodies recognize a-gal residues and the expression of a-gal epit-opes in HIV gp120 and influenza virus vaccines increases immunogenicity by approximately 100-fold. The immunogenicity of a microbial vaccine can be markedly increased by linked a-gal epitopes as a result of in vivo formation of immune complexes with anti-Gal and the effec-tive internalization of these complexes by APCs via Fc/FcgR interaction. The increased transport to lymph nodes and processing of anti-Gal com-plexed vaccines internalized by APCs results in effective activation of vaccine-specific CD4+ and CD8+ T cells and potent cellular and humoral immune responses [126]. In addition, rgp120 vac-cination can elicit antibodies with antiviral activity against clinical strains of HIV-1. However, this activity requires the presence of FcR-bearing effec-tor cells. This result provides further evidence that antibody-dependent cell-mediated virus inhibition can be harnessed to prevent HIV infection [127].

An example of the relevance of FcRs in HIV vaccines is that Fc/FcgR interactions play a criti-cal role in the biological function of an antibody. Moreover, these receptors may be essential in preventing or modulating lentiviral infection. Exploiting antibody responses that depend on Fc/FcgR interactions may help overcome some of the difficulties associated with HIV vaccine development by increasing the potency of the antibody response. Although the importance of generating optimal Fab–antigen interactions can-not be overestimated, improving Fc/FcgR interac-tions through adjuvants, by directly altering the Fc segment of monoclonal antibodies or by other strategies, provides another option for ameliorat-ing HIV vaccines and immunotherapies [128–130].

The effect of neutralizing antibodies that are specific for HIV-1 gp41 membrane-proximal external region were moderately stronger for IgG1 than for IgG3 subtypes, and were ineffective when using fab fragments. It was concluded that FcgRI and FcgRIIb facilitate antibody-mediated neutral-ization of HIV-1 by a mechanism that is depen-dent on the Fc region, IgG subclass and epitope specificity of the antibody [131].

The importance of FcgRs in AIDS pathogen-esis and the critical role of interactions between FcgRs and immune complexes in disease pro-gression are reflected by the observation that



patients with IgG2 anti-p24 and who express the ‘high-affinity’ polymorphism of FcgRIIa exhibit lower HIV replication than those without the polymorphism [132].

�n Drug delivery systems to target DCs Drug delivery systems to target DCs can be subdivided into live vectors, micro- or nano-particles and receptor ligands (Table 2). Live infectious vectors and certain microparticles are particularly well suited to deliver DNA to DCs. For delivery of protein and peptide antigens to these cells, both microparticles/nanoparticles, liposomes and receptor ligands are used.

Live vectors to deliver DNA to DCsLive-attenuated viruses and bacteria potently induce cellular immune responses in HIV vac-cination therapy in animal models [133]. The live vectors drive expression of antigen encoded by DNA inside the cells they invade and the antigen then enters the classical MHC class I pathway.

Viruses in particular show promise as vehicles for targeted gene delivery. The incorporation of DC surface receptor ligands, mostly antibodies, into viruses allows them to use these receptors for cell entry and to specifically infect DCs [134]. A DC-targeted recombinant lentiviral vector was reported to effectively transfect DCs in vivo and to induce strong antigen-specific humoral and cellular responses. DC-specific targeting of the vector was accomplished by introducing an engineered gly-coprotein derived from Sindbis virus, which serves as a ligand for the DC-specific receptor DC-SIGN [135]. Several issues, including some related to safety, remain to be addressed regarding the use of live vectors as vaccines. Reversion of attenuated live vectors to virulent strains by mutation or genetic recombination cannot be excluded and may be especially problematic in immunocompromised individuals such as AIDS patients.

Micro/nanoparticles to deliver DNA, protein or peptides to DCsMicroparticles include lipid particles such as liposomes, polymer microparticles, VLPs [136] and virosomes [137]. Particulate delivery of anti-gens often increases their stability and bioavail-ability. Furthermore, microparticles are effi-ciently taken up by APCs through phagocytosis, thus resulting in antigen presentation.

Targeting of liposomes containing peptide antigen or DNA to professional APCs effectively enhances their capacity to induce CTL responses in vivo [138]. Several methods to target DCs with liposomes have been described [139].

As occurs with liposomes, polymer micro/nanoparticles allow the protection of antigenic proteins, peptides and DNA from immedi-ate degradation. Entrapment of HIV protein antigen in poly(lactide-co-glycolide) (PLGA) microparticles results in an effective vaccine that induces specific MHC class I- and class II-mediated responses in mice [140].

Some data provide proof of principle that both the antigen and DC maturation signal can be delivered as a complex with polyelectro-lyte capsules to stimulate virus-specific T cells both in vitro and in vivo. Other nanomaterials could be useful for in vivo immunization against HIV-1 and other infections [141,142].

Targeting nanoparticles to human DCs as an effective strategy to deliver HIV-1 antigenSpecific antigens for CTL response (such as gag, pol, env and nef antigens) and TLR ligands (such as Poly I:C, PAM and R848) can be incorpo-rated into biodegradable polymers, such as PLGA (Figure 5). This polymer is safely used in clinical practice. Combining TLR ligands (as adjuvant) and antigen in PLGA particles enhances the induction of CTL responses [143]. For optimal antigen presentation, it has been shown that the TLR ligands and antigen should be in the same compartment [144]. Recently, PLGA nanopar-ticles carrying a combination of TLR ligands were reported to protect mice and nonhuman primates against a variety of influenza strains and to induce long-lasting humoral memory [145]. Importantly, such PLGA nanoparticles can be modified to target DC-specific recep-tors on human DCs by introducing humanized antibodies recognizing the DC-specific recep-tor DC-SIGN [146,147]. Alongside antibodies, the HIV-1 gp120 protein might be used for targeting purposes. This glycoprotein binds to DC-SIGN and could thereby simultaneously serve as an antigen and the targeting moiety [148].

Immunological regulation in the setting of HIV-1 vaccination with dCsHIV-1 infection is associated with elevated immunological activation, which may cause T-cell exhaustion. Depending on the dura-tion and magnitude of antigenic activation of T cells during HIV-1 infection, some intrinsic and extrinsic factors are produced that influ-ence the severity of T-cell exhaustion. Recent observations have detailed the upregulation of inhibitory receptors, which are the major intrinsic factors of T-cell exhaustion. Among the



extrinsic factors are the production of suppres-sive cytokines, T-cell priming by either nonpro-fessional APCs or myeloid-derived suppressor cells, a kind of tolerogenic DC, and alteration of Tregs [149]. In the setting of HIV infection, most immunological studies have shown an increase of Tregs within the T-cell pool, with respect to healthy individuals, but a decrease in the absolute number of T cells in all HIV-infected individuals. On the basis of evidence that Tregs suppress virus-specific immune responses, it has been proposed that these cells are deleterious during HIV infection. Conversely, some authors suggested that Tregs could be beneficial, par-ticularly during early HIV infection when effec-tor T cells are not yet activated, as these cells limit immune activation, thus controlling the availability of HIV targets as well as prevent-ing immune-based pathologies [150,151]. The fre-quency of Tregs in peripheral blood mononuclear cells and gut-associated lymphoid tissue using CD4+Foxp3+ and CD4+Foxp3+CD127Low/- as markers was found to be higher in HIV patients than in controls [152]. Conversely, other authors reported that while HIV ‘elite controllers’ and uninfected individuals had similar Treg num-bers and frequencies, the absolute numbers of Tregs declined in blood and gut-associated lym-phoid tissue in patients with chronic progressive HIV-1 infection, but was largely normalized by HAART. However, HIV-1 infection was not associated with an impairment of ex vivo sup-pressive function of flow-sorted Tregs in either HIV controllers or untreated chronic progres-sors [153,154]. It is possible that Tregs contribute to the low number of repopulating CD4 T cells in patients under HAART, since a higher ratio of Tregs to naive CD4 T cells was observed in these patients. The role of Tregs seems to be

related rather to the control of homeostasis and cell proliferation of naive CD4 T cells, than to immune activation [155]. Alterations in Treg fre-quency in HIV/AIDS are more directly related to the degree of CD4 depletion than to viremia [156]. The relative frequency of Tregs directly correlate with HIV viral load and inversely with CD4+ counts. However, the absolute number of these cells was reduced in HIV-infected patients compared with healthy controls (p < 0.0001), with the exception of elite controllers (p > 0.05). The loss of absolute Treg numbers coincided with increased markers of immune activation (p < 0.0006). The initiation of antiviral therapy significantly increased absolute Treg numbers (p < 0.0031). The authors found that the expres-sion of CD39, a newly defined ectonucleotidase with immunomodulatory functions in Tregs, correlated with progressive HIV disease, HIV viral load and immune activation. Of note, when Tregs were tested in peripheral blood mononu-clear cells of healthy volunteers, their in vitro capacity to suppress T-cell proliferation was limited to CD4+CD25highCD39+ T cells [157]. The sustained increase of the peripheral Treg pool in IL-2-treated HIV patients may account for the unexpected clinical observation that patients with the greatest expansion of CD4+ T cells had the highest relative risk of clinical progression to AIDS [158]. Although most HIV-specific CD8+ T cells lose proliferative capacity during chronic infection, T cells restricted by HLA-B*27 or HLA-B*57 allele groups do not. CD8+ T cells restricted by ‘protective’ HLA allele groups are not suppressed by Treg cells. HLA-B*27- and HLA-B*57-restricted effec-tors also evade Treg-cell-mediated suppression by directly killing Treg cells [159]. In monkeys, tissue myeloid DCs from SIV-infected animals

Target HIV-1 antigen to DC via DC-SIGN receptor

gag, pol, envand nefantigens

TLR ligands

Surface modification:PEGylated lipid layer

gp120

Targeting moietiesanti-DC-SIGN:

gp120 or Abs anti-DC-SIGN

DC-SIGN receptor

Adaptive immuneresponse

Antigen-presentingcell

T cell


Figure 5. Targeting HIV-1 antigen to dendritic cells via dC-sIGN receptor. Schematic representation of a new strategy in the design of next-generation anti-HIV-1 vaccines.



exhibit an enhanced capability to induce Tregs and may contribute to the accumulation of these cells in lymphoid tissues during progres-sive infection [160]. Furthermore, Nef protein is a potent factor for increasing Treg numbers in vitro [161]. In HIV-1-infected progressors and AIDS patients, the T-cell costimulatory mol-ecule programmed death-1 (PD-1) ligand-1 (B7-H1) is significantly upregulated on peripheral mDCs, but is maintained at a relatively low level in long-term nonprogressors. When HAART achieved complete immune reconstitution by full suppression of HIV-1 replication and sub-stantial increases of CD4 T-cell counts, a cor-relation with a decrease in B7-H1 expression was found. Interestingly, X4 HIV-1 isolates directly induce B7-H1 expression on mDCs in vitro, while the addition of antiviral agents hamper this B7-H1 upregulation [162]. Myeloid-derived suppressor cells (a kind of inhibitory DC) pres-ent elevated levels in chronic uncontrolled HIV infection. Such levels potentially contribute to the impaired T-cell responses characteristic in this progressive stage of the disease [163]. HAART seems to enhance the DC population, increasing counts at week 60 post-therapy and achieving similar levels to healthy controls, but is still lower than those of long-term nonprogres-sors. During HAART treatment, DC counts correlate directly with CD4 counts [164]. Taken together, all these results indicate that interac-tion of B7-H1–PD-1 with APCs and T cells correlates with the impairment of CD4+ T cells and CTL responses in vivo. This is associated with the progression of the disease in infected individuals. Blockade of this pathway may have therapeutic implications for HIV-infected patients [165].

In a murine model of AIDS, which may not fully mimic the disease in humans, following in vivo depletion of CD4 Tregs and/or selec-tive interruption of PD-1-negative signaling in the CD8 T-cell compartment, retroviral pathogenesis was significantly decreased upon the combined treatment of CD4 Treg deple-tion and PD-1 blockade, which worked in a synergistic fashion to substantially reduce the induction of murine AIDS [166]. A combina-tion of 4-1BBL and CD40L overexpression on DCs dramatically enhances CD4+ and CD8+ T-cell responses. Signaling through 4-1BB, but not through CD40, can alleviate the suppres-sive effect of Tregs on CD8+ T-cell proliferation. Thus, the combination of 4-1BBL and CD40L enhances HIV-specific CD8+ T-cell responses in a synergistic way [167]. However, the complexity

of immune regulation in HIV-1 infection, as shown above, demonstrates that applying Treg immunotherapeutic strategies tested in dis-eases other than HIV could involve the risk of inducing further progression of HIV infection. Therefore, extreme care must be taken when designing such studies [168].

ConclusionThe current developments in DC-based vaccines against HIV-1 infection or to treat AIDS patients hold promise for improving the control of HIV infection. The ex vivo production and activation of DCs is expensive and technically complex. Therefore, the use of this technique in broad-scale immunization programs is not viable. Vaccines that target DCs in vivo provide an interesting alternative with a dramatically reduced cost with respect to the in vitro obtention of DCs and activa-tion systems, thereby facilitating their application across the wide geographical and socioeconomic range of people infected by the virus.

As outlined in this review, the huge efforts devoted over recent decades to the design of effec-tive immunotherapies for HIV infection have failed to deliver effective vaccines. However, promising results have been obtained. In fact, the experiences from DC therapy studies in cancer show that there could be many setbacks before advances are made because of the numerous factors that potentially affect DC immunotherapy. Nevertheless, we should learn from experience in developing a DC-based immunotherapy for the fearsome enemy of cancer when pursuing a DC immunotherapeutic vaccine against the equally formidable foe, HIV [169].

It is to be hoped that a solution, although com-plex, will be found that takes into account virus variability, antigen design, the immunological state of the patients and the appropriate receptor targets that ensure DC activation, maturation and MHC class I and II presentation, possibly by several pathways, together with strong adju-vants that help the immunological response and maintenance. The administration route and the delivery systems of the vaccine are two addi-tional key factors that will determine whether an effective immunotherapy for HIV infection is achieved. Given the intense dedication of the hundreds of research groups cited in this review and many others, the authors hope the promise of DC-targeted therapy for HIV infection will become a reality in the not too distant future.

Future perspectiveThe history of modern medicine and possi-bly of humankind has changed thanks to the



executive summary

� Dendritic cell (DC)-based vaccines for HIV have been shown to be the most effective strategy to induce an effective and prolonged immunological response; however, this response is still insufficient. Furthermore, the generation of DCs ex vivo is not suitable for broad vaccination programs owing to cost and technical difficulties.

� A series of presentation strategies for HIV-1 vaccinations, both with respect to antigens as well as to more appropriate vehicles, are presented.

� Several strategies to activate HIV-1 antigen presentation to DCs, by means of whole inactivated virus, proteins and peptides or by DNA or mRNA, are also presented.

� Appropriate ligands to target antigens in vivo to DCs and the best routes to gain access to MHC class-I and -II pathways, as well as the current delivery systems used are reviewed.

� Negative regulatory mechanisms associated with HIV-1 infection and that could impair the effectivity of the vaccines in development are described.

development of vaccines against multiple patho-gens. However, the variability of some viruses, such as HIV, has made the generation of effec-tive vaccines elusive. With current knowledge, it is easier to envisage a curative vaccine than a preventive one, because it is possible to charac-terize HIV variants in an infected patient, but very difficult to develop a vaccine covering all the HIV-1 subtypes or clades. However, these dif-ficulties were overcome in other highly variable viruses, such as influenza virus subtypes.

HIV-1 infection defines a complex and evolv-ing situation with marked differences between each stage of the disease and genetic background of the individuals, but future directions in vac-cine development should uncover several crucial aspects that are still poorly understood.

Depending on the duration and magnitude of antigenic activation, chronic HIV infection involves T-cell exhaustion, which is character-ized by a progressive loss of T-cell function. It is therefore important to identify/study the factors that are implicated in the maintenance of this situation and to control them for a better vac-cination schedule. Furthermore, as T-cell acti-vation is associated with an increase in viremia and disease progression, it would be appropri-ate to direct vaccines that specifically activate CD8 T cells, thus controlling CD4 activation by maintaining Tregs (since CD4 activation or a decreased number of Tregs have been associated with HIV-1 replication).

DCs have been shown to be the most suitable tools for the development of efficient immune responses against HIV-1; however, ex vivo gen-eration and manipulation of these cells for their use as vaccines is expensive, time-consuming and technically difficult. Therefore, new vac-cines should be designed to target DCs in vivo. This strategy should not imply a lower response, since targeted antigens in nanoparticles, nano-liposomes or polymers to DC receptors allows the use of several kinds of antigens and protects

them from degradation and dispersion in blood. In addition, some receptors on DCs trigger acti-vation and maturation signals to improve antigen presentation by the desired route. However, HIV infection has been associated with elevated levels of myeloid-derived suppressor cells, a type of DC that induces Treg development and potentially contributes to impaired T-cell responses. The expression of B7-H1 by DCs (a PD-1 ligand that induces apoptosis in CD8 T cells) and CTLA-4 by T cells (an inhibitory receptor that binds costimulatory molecules CD80 and CD86 but inhibits T-cell responses) could impair vaccine responses. Therefore, an appropriate choice of adjuvant could help to reverse this inhibitory sit-uation, and blockade of this molecule with anti-bodies may improve vaccine immune activation.

Finally, we recommend that efforts be focused on finding the vaccine administration route that, in addition to CD8 T-cell-specific activation, pro-duces high levels of neutralizing antibodies, since the combination of cellular and humoral activity is essential to achieve satisfactory results. Neutralizing antibodies generate immune complexes that activate DCs and reinforce memory T cells.

Although our understanding of the com-plex regulation associated with HIV-1 infec-tion is still in its infancy, our knowledge of the mechanisms involved advances daily. Thus, it is expected that the near future will bring about successful manipulation of natural antiviral activity and effective vaccination strategies.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.



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