Dendritic cell immunotherapy for HIV infection: from theory to reality

1039

Review

ISSN 1750-743XImmunotherapy (2009) 1(6), 1039–105110.2217/IMT.09.68 © 2009 Future Medicine Ltd

Dendritic cell immunotherapy for HIV infection: from theory to reality

A considerable contribution to current knowl-edge regarding vaccines was generated from studies by Edward Jenner (1749–1823) and Louis Pasteur (1822–1895). Since then, vaccines have been used with the objective of artificially inducing what is referred to as immunological memory, a fundamental property of the immune system that permits an individual to defend one-self more rapidly against pathogens to which one has been exposed.

In the composition of a vaccine product, besides the antigen, adjuvants that potenti-ate and prolong the immune response are also important. The nature of such adjuvants defines the type of immune response that vaccines are able to induce (e.g., cytoxicity or antibodies).

Known as a powerful natural adjuvant, den-dritic cells (DCs) were first observed by Paul Langherans in 1868, although the term ‘den-dritic cells’ was only established by Steinman & Cohn in 1973 [1], when more in-depth stud-ies were conducted. Initial reports concerning the physiology of these cells and their ability in amplifying the immune response against anti-gens [2,3] lead to their use in clinical research. Nowadays, as well as their role as adjuvants, DCs are recognized as performing several other func-tions related to the establishment of the innate and adaptive immunity and to the activation or suppression of the immune system. All these functions are regulated by a process known as DC differentiation [4,5].

The beginning of interventional immunology in the field of medicine was marked by observa-tions from William Coley concerning the reduc-tion of the tumor in some patients affected by cancer and simultaneously by erysipelas [6]. Since

then, a parallel can be drawn between immuno-therapy for cancer and the vaccination for infec-tious diseases, especially those caused by viruses, in which the process is located within a host cell. In both cases, transformed and infected cells stimulate a MHC-I-based immune response.

The large number of clinical studies in oncol-ogy led to fundamental knowledge concerning DC-based immunotherapy; however, more pro-found investigation is required to standardize the design and production of this vaccine in diseases other than cancer [7–10].

Prototype antitumoral vaccines with DCs were initially developed from observations that in some types of solid cancer, such as melanomas and metastatic renal tumors, DCs were signifi-cantly reduced or not fully functional [11]. The idea that this phenomenon could also occur dur-ing viral infections drove researchers to study the number and function of DCs in patients with HIV/AIDS [12–14]. Several groups investigated these cells during natural and experimental HIV infection to correlate eventual disturbances in DCs with the well-known dysfunction of the cytotoxic T lymphocyte (CTL) response [15–19].

From a clinical point of view, the control of the HIV/AIDS pandemic with antiretroviral therapy is difficult owing to its high cost, rela-tively low availability, side effects and develop-ment of resistance, among other factors [20–22]. In this context, therapeutic vaccines and/or immunomodulators have been indicated as a way of increasing the capacity of HIV-infected individuals to respond against the virus and to delay the onset of highly active antiretroviral therapy (HAART). Maintaining patients with low circulating viral load and further increasing

Knowledge concerning the immunology of dendritic cells (DCs) accumulated over the last few decades and the development of methodologies to generate and manipulate these cells in vitro has made their therapeutic application a reality. Currently, clinical protocols for DC-based therapeutic vaccine in HIV-infected individuals show that it is a safe and promising approach. Concomitantly, important advances continue to be made in the development of methodologies to optimize DC acquisition, as well as the selection of safe, immunogenic HIV antigens and the evaluation of immune response in treated individuals.

KEYWORDS: dendritic cell n HIV infection n therapeutic vaccine Telma Miyuki Oshiro1, Alexandre de Almeida1,2 & Alberto José da Silva Duarte1,3,†

†Author for correspondence: 1Laboratório de Investigação em Dermatologia e Imunodeficiências – LIM 56, Faculdade de Medicina da Universidade de São Paulo, Instituto de Medicina Tropical – prédio II, Av. Dr. Enéas de Carvalho Aguiar, 470 – 3o andar, CEP 05403-05000, São Paulo, Brazil Tel.: +55 11 30617499 Fax: +55 11 30817190 [email protected] [email protected]@usp.br

For reprint orders, please contact: [email protected]

Immunotherapy (2009) 1(6)1040 future science group

Review Oshiro, de Almeida & da Silva Duarte

patient’s quality of life, reducing virus transmis-sion and effectively diminishing the use of drugs are all important considerations.

DCs in HIV immunotherapyAs sentinels and regulators of immune response, DCs present expressive plasticity in their prop-erties and functions and constitute a very h eterogeneous population [4].

Although all DCs share common features, including dendritic morphology, high expression of MHC-II and potent ability to stimulate T cells, they are divided into several subtypes according to characteristics such as origin, location, expres-sion of markers, migratory capacity and immune function [23,24]. The most commonly used divi-sion, which will also be used in this review, based on their origin, classifies them as myeloid or p lasmacytoid DCs (pDCs; lymphoid origin).

Myeloid DCs are potent antigen-presenting cells because of their unique property of initiat-ing the immune response by naive T-lymphocyte activation [4]. They constitute less than 1% of peripheral blood mononuclear cells [25] and show great versatility, because they can process pro-teins within MHC-I and MHC-II pathways and stimulate both CD8+ and CD4+ T lymphocytes. Moreover, they are capable of cross-presentation, a phenomenon by which exogenous antigens, classically presented by MHC-II, gain access to the cytoplasm and are presented by MHC-I, eventually activating CTLs [26].

Plasmacytoid DCs, on the other hand, are potent IFN-a producers in response to viral infection and their capacity to regulate the function of natural killer (NK) cells, NKT cells, B lymphocytes, monocytes and myeloid DCs has been well described [27–30]. Although it was initially believed that pDCs were not capable of capturing and processing antigens [31], these properties were discovered [32], together with their ability to cross-present antigens to specific CD8+ T lymphocytes [33].

In the immature stage, DCs possess a strong capacity to capture antigens. Initial recognition of pathogens by DCs occurs by pathogen-recog-nition receptors (PRRs), principally belonging to the mannose receptor family of C-type lec-tins endocytic receptors, and Toll-like receptor (TLR) family.

Among C-type lectin receptors, DC-SIGN (CD209) and DEC-205 (CD205) receptors have been widely investigated in the context of HIV infection and development of immunotherapy. DC-SIGN is highly expressed in DCs residing in mucosal and lymphoid tissues and is able to

recognize and bind to the HIV protein gp120, mediating the internalization of the virus into the intracellular lysosomal compartment [34]. DEC-205 mediates the processing and presen-tation of antigens through MHC-I and MHC-II [35]. This receptor is highly expressed on DCs in the T cell areas of lymph nodes, suggesting that CD205 could play a key role in the selection of specific T cell clones [36]. Moreover, it has been demonstrated that CD205 mediates the presen-tation of HIV Gag-derived peptides to CD8+ T lymphocytes in the context of MHC-I. For this reason, CD205 is currently under investiga-tion as a way of delivering HIV antigens to DCs for more effective activation of CD8+ cells [37].

On the other hand, it is well known that TLRs on antigen-presenting cells are critically involved in the generation of effector T response [38,39]. The profile of TLR expression depends on the DC subtype. pDCs express TLR7 and TLR9, while myeloid dendritic cells express TLR2, 3, 4, 5, 6 and 8, but not TLR7 and 9 [40,41]. Each TLR specifically binds to a type of pathogen-associ-ated molecular pattern (PAMP) and is capable of generating an appropriate response. Adjuvants derived from microbial components, such as TLR agonists, augment the expression of PRRs in DCs. For these reasons, research on the role of TLRs in antigen presentation is a promising field for the development of new vaccines [42].

In response to signals triggered by PRRs, DCs initiate a profound phenotypic and functional transformation called activation/maturation, which involves alterations in their morphology, acquisition of mobility, reduction of phago-cytic capacity, expression of specific chemokine receptors, upregulation of costimulatory (CD80, CD86 and CD40) and presentation (MHC-I and MHC-II) molecules and production of c ytokines, such as IL-12 and IL-23.

Dendritic cell migration is mediated by the expression of specific surface receptors. As such, they are capable of migrating from periphery to secondary lymphoid organs. Immature DCs are characterized by the expression of chemokine receptors CCR2 and CCR6 and are generally localized in peripheral tissues [43], where they can encounter pathogens [44]. Migration from the periphery to secondary lymphoid organs occurs after DC maturation, due to CCR7 expression in these cells.

Recently, it has been shown in an animal model that CD74 (the MHC-II-associated invariant chain) plays an important role in DC migration. CD74 induces low-motility phases that, coupled with its known role of antigen

www.futuremedicine.com 1041future science group

Dendritic cell immunotherapy for HIV infection: from theory to reality Review

uptake and processing, facilitates the sampling of the microenvironment. In this way, DCs may coordinate both functions of antigen detection and processing at the same time [45].

The process of activation/maturation can make DCs immunogenic, capable of antigen presentation to T lymphocytes and, eventually, capable of producing an appropriate immune response [4,39,46], depending on three signals. The first signal provides information on the pathogen identity and is mediated by bind-ing of MHC–peptide complex to its specific T-cell receptor. The second, mediated by the interaction between costimulatory molecules (e.g., CD40, CD80 and CD86) on DCs and their respective ligands on T cells, provides information regarding the pathogenic potential of the presented antigen. Finally, the third signal reflects the nature of the pathogen and the type of infected tissue and leads to the selection of the most appropriate effector mechanism. This sig-nal is mediated by soluble factors or membrane receptors, such as certain interleukins (IL-12, IL-10 and type I interferon) that promote the polarization of T helper (Th) lymphocytes [47].

In the context of an anti-HIV therapeutic vaccine, it is desirable to induce a specific and e fficient immune response against the virus while regulating the chronic activation of the immune system.

Much effort have been made to develop a DC-based vaccine capable of fully stimulat-ing an anti-HIV specific response. Given this requirement, certain critical issues must be considered, including the subtype of inoculated DCs, the kind of stimulus for ex vivo DC matu-ration, the ability of DCs to produce cytokines and their migratory potential. All together, these factors determine the effector function of the DCs used in immunotherapy.

It has been recently demonstrated in mice that an additional lymph node DC type, the blood-derived inflammatory DC, plays a key role in the induction of acute T-cell response. These DCs are capable of antigen uptake, production of large quantities of IL-12p70 and inducing IFN-g secre-tion by CD4+ T lymphocytes much more potently than other lymph node DC subtypes [48]. This subpopulation of DCs could represent an ideal cell type for the vaccine design, although they have not yet been described in humans.

The protocol for a DC-based vaccine begins from a large quantity of precursor cells cultivated in the presence of cytokines. The DCs obtained are then treated with the antigen of interest and re-injected into the patient (Figure 1).

Currently, protocols for obtaining DCs dedi-cated to immunotherapy use myeloid precur-sors. Although pDCs play an important role in controlling viral replication, they have a major limitation for using in vaccines, owing to the d ifficulty in obtaining large quantities of precursor cells.

The most widely used protocol is based on differentiation of CD14+ monocytes in the pres-ence of IL-4 and GM-CSF for 5–7 days [49,50]. These DCs, termed monocyte-derived DCs (MoDCs) present an immature phenotype, while the stage of maturity is achieved through the cultivation for 1 or 2 days in the presence of different stimuli.

Several protocols for DC maturation have been used and the most common is the treatment of cells with a pool of proinflammatory cytokines: TNF-a, IL-1b, IL-6 and prostaglandin E2 alone or in various combinations [51–54]. In this model, cytokines act as PAMPs, inducing a danger signal that activates DCs. Although these protocols are commonly used, there is evidence that the signal induced by cytokines may not be sufficient to gen-erate an adaptive immune response, in particular owing to the deficiency in the induced produc-tion of IL-12 [55]. It had been shown that DCs activated only by a cocktail of cytokines led to the expansion of populations of noncommitted T lymphocytes, potentially capable of inducing tolerance rather than activation [56].

The introduction of CD40L into the cocktail to improve DC maturation has also been pro-posed, because of its ability to induce IL-12p70 production, a crucial cytokine to the induction of a Th1 response. A commonly used PAMP, the bacterial lipopolysaccharide, provides good results in stimulating DC activation when used in in vitro studies, but its application in clinical protocols could be restricted owing to the toxic nature of this component.

The use of IFN-a for the generation of DCs from monocytes has also been proposed. The exposure of monocytes to this cytokine would be a more physiological stimulus, given that IFN-a is secreted by pDCs during viral infection and plays a critical role in linking innate and adaptive immunity [57].

Alternative approaches, such as the use of antigenic combinations capable of promoting the activation of DCs, while avoiding the use of other stimuli, have also been proposed, as it will be discussed later.

Current work is aimed at reducing the period of culture of DCs to 3 days, in contrast to the conventionally used 6–7 days. Tawab et al.



demonstrated that cells cultivated for less time present more homogenous expression of sur-face molecules, aside from producing more IL-12 and less IL-10; these are desirable charac-teristics for an efficient cellular response [58]. Landi et al. demonstrated that the generation of DCs in 3 days promoted greater viability of immature DCs for transfection [59]. Since these works were performed in vitro, further stud-ies should be conducted to consider its use in clinical protocols.

Regarding the immunogenic capacity of MoDCs from HIV-infected patients, inconsis-tent results have been obtained in in vitro studies. While some works demonstrate the competence of these cells to present antigens and promote a potent cellular response [60,61], a recent work by Buisson et al. demonstrated that, in compari-son with healthy subjects, MoDCs from infected patients presented diminished capacity to stimu-late T cells and produce IL-12, principally in

patients treated with HAART [62]. More studies are required concerning the factors that could interfere in the differentiation, maturation and antigenic presentation processes in HIV-infected individuals.

In the context of immunotherapy, it is important to underline that aside from stimu-lating a specific immune response, DCs may be able to promote peripheral tolerance in CD4+ and CD8+ T cells by inducing deletion, anergy or through the expansion of CD4+ regulatory T cells (Tregs) [4,63,64].

The role of Tregs in HIV infection is not yet fully understood mainly because the lack of information on the dynamic of these cells during the course of infection. Although some studies have shown that Tregs are involved in impaired immunity and persistence of viral infections [65–67], others underlined the beneficial effect of a high rate of Tregs in HIV infection, due to their ability in reducing immune activation [68,69].

8. Antigen-loaded DC expected features:- CCR7- CD80high

- CD86high

- CD83high

- CD40- IL-12 production

9. Vaccination

1. Leukapheresis

2. PBMC collection3. Monocyteisolation

4. Culture with IL-4 + GM-CSF

5. iDC:- CD80low

- CD86low

6. DC loading withHIV antigen:- Whole inactivated viruses- Apoptotic infected cells- Recombinant protein- Synthetic peptide- RNA- DNA

7. Maturation stimulus:- Cytokines- TLR ligands- CD40L

Immunotherapy © Future Science Group (2009)

Figure 1. Treatment of HIV-infected patients with monocyte-derived dendritic cells. PBMCs are obtained by leukapheresis and monocytes are separated and cultured in the presence of IL-4 and GM-CSF to obtain immature DCs (iDCs). iDCs are loaded with the antigen of interest and are activated by different stimuli for maturation. Mature DCs, potentially able to migrate and present antigens, are reinoculated into the patient. DC: Dendritic cell; PBMC: Peripheral blood mononuclear cell; TLR: Toll-like receptor.



Alternatively, a dual role has been proposed for Tregs, depending on the stage of the dis-ease. In the acute phase of HIV infection, the increase in the number of Tregs could contribute to suppressing the anti-HIV specific response, promoting acute viremia and persistent infec-tion. During the course of the infection, because of CD4 expression, Tregs become targets for the virus, which would lead to their gradual deple-tion and, consequently, to immune activation. In this context, the remaining Tregs could play a critical role in controlling cell activation and preserving the individual’s immune system [70].

Another important point that should be con-sidered for further investigation concerning the role of Tregs in HIV infection is the kind of tech-nical approach that would be required to study Treg phenotype. For example, the expression of fork-head box P3 (Foxp3), which was initially considered a good marker for Tregs within the CD4+CD25+ T lymphocyte population, seems to be transitorily expressed in all proliferating T lymphocytes [71,72]. As such, Foxp3 expres-sion may not to be very useful as a Treg marker in the context of the chronic immune activa-tion that characterizes HIV infection. More recently, latency-associated peptide (LAP) mol-ecules, and IL-1 receptor type I (CD121a) and II (CD121b) were identified as three unique cell surface markers that distinguish activated Tregs from a ctivated Foxp3- and Foxp3+ non-Tregs [73].

Studies should be conducted to elucidate the role of Tregs in infection by HIV, considering the central role of DCs in directing the immune response to activation and/or regulation.

HIV antigens for potential use in immunotherapyIn immunotherapy protocols, one of the greatest challenges is the introduction of relevant anti-gens into DCs to stimulate the presentation of high-efficiency peptide–MHC complexes.

The obtention of an immunogenic antigen capable of eliciting a specific anti-HIV response constitutes another fundamental aspect of a vac-cine design. The HIV virus is known for its abil-ity to exploit numerous genetic and evolutionary mechanisms to ensure its proliferation, such as high replication, mutation and recombination rates. This leads to considerable virus variability within the infected population and also within each individual [74–77].

Taking into account this high virus variabil-ity, the use of autologous HIV for the develop-ment of a vaccine could be proposed to induce a specific individual immune response [53,78].

Indeed, it has been demonstrated that cellular response to peptides derived from autologous viral sequences is significantly greater compared with the response to a consensus sequence [79]. This suggests that a more accurate host response could depend on very specific viral epitopes that infected each individual.

Moreover, efforts have been made in develop-ing strategies to improve the design of immuno-gens using prediction programs based on the frequency of variants in the population level, for example the polyvalent mosaic protein strat-egy [80] and toggled peptides approach [81]. Using this strategy, it is possible to create more effective immunogens by the exclusion of rare epitopes and the incorporation of variants potentially related to escape mutants.

This strategy is especially interesting in the case of a therapeutic vaccine, in which, despite amplifying the memory response in vaccines, the induction of naive T cells directly against any possible virus variants is highly desirable.

The ability of DCs pulsed with inactivated autologous virus to induce a HIV-specific response has been evaluated both in in vitro studies [15] and in clinical trials [53,78]. However, the obtention of autologous viruses for the pro-duction of vaccines is laborious and complex. Initially the virus is isolated from the patient’s peripheral blood, expanded in autologous CD4+

T cells, inactivated and then purified. This pro-cedure could present difficulties, first owing to low viral load in some individuals, and second, because of the requirement of large quantities of autologous CD4+ cells.

The most common method for HIV i nactivation is the use of aldrithiol-2, which prevents viral replication, but maintains the conformational structure of the virus intact [82].

Potential alternatives to the use of autologous viruses have been widely proposed, including apoptotic CD4+ T lymphocytes infected with HIV, HIV peptides, HIV recombinant proteins, mRNA vectors or even DNA that encodes HIV proteins. These products offer the possibil-ity of generating a specific immune response against previously selected epitopes. Methods to introduce these materials into DCs are also in full development, including electroporation, n ucleofection and viral vector transduction.

Synthetic peptides are relatively easy to pro-duce in comparison with virus isolation and expansion. Also, owing to their noninfectious nature, they offer less risk compared with whole viral fragments and provide the possi-bility of monitoring a peptide-specific cellular



response. Thus, it has been demonstrated that DCs loaded with HIV-derived peptides were capable of stimulating IFN-g production by T cells in both in vitro assays and in a Phase I clinical trial [83,84].

Another strategy currently being explored is the use of HIV proteins encoded by synthetic mRNA. Combined with adequate methodolo-gies for DC transfection, such as electropora-tion, mRNA offers high transfection efficiency and reproducibility and constitutes a promising alternative for a DC-based therapeutic vaccine. This technology offers a safe protocol with no risk of infection and low toxicity. MoDCs from HIV-infected individuals electroporated with mRNA encoding consensus or autologous sequences of HIV proteins or with chimeric mRNA encoding the DC lysosome-associated membrane glycoprotein (DC-LAMP) sequence are capable of inducing a specif ic cellular response against the virus [85–87].

Dendritic cell transduction with vectors car-rying specific HIV genes has also been proposed in several studies, showing different results in relation to transduction and activation efficiency. The expression of target genes, alone or in associ-ation, can result in the endogenous processing of peptides in the context of MHCs that promotes their expression over a long period. Viral vectors offer the advantage of transporting target genes inside the cell, through conventional viral infec-tion procedure, without the need for physical or chemical methods. Moreover, in certain cases, the vector itself induces DC maturation, without requiring the addition of proinflammatory cyto-kines. Known vectors include lentivirus, adeno-virus and poxvirus, among others. In an in vitro study using leukocytes from HIV patients, DCs infected with canarypox virus carrying HIV-1 genes induced a strong anti-HIV response by CD4+ and CD8+ T cells [88].

The use of virus-like particles (VLPs) has also been described. VLPs from HIV Gag, obtained by sprouting yeast spheroplasts, were incorporated into DCs using macropinocytosis and endocytosis and were capable of inducing their maturation. Once matured, the DCs were capable of stimulating a Gag-specific response in CD8+ T cells by cross-presentation [89].

Apoptotic CD4+ T lymphocytes infected with HIV have been used to load DCs. In fact, immature DCs have the ability to capture apop-totic cells and to efficiently cross-present antigen derivatives from these cells through MHC class I to to CD8+ T lymphocytes. On their surface, apoptotic cells expose altered molecules that

facilitate their rapid recognition and internal-ization by DCs, offering a complete repertoire of antigenic information [90]. In the case of HIV that predominantly infects CD4+ T lympho-cytes, it has been demonstrated that the cross-presentation of small amounts of HIV proteins derived from apoptotic CD4+ T lymphocytes by DCs to CD8+ T lymphocytes was very effi-cient in comparison with DC presentation of infectious or defective viruses [91].

Another promising approach under develop-ment is the use of cell surface antireceptor antibodies to increase the immune response by targeting antigens to DCs. In an in vitro study, DCs targeted with monoclonal antibody anti-DEC-205 carrying a p24 Gag protein from HIV were capable of inducing a CD8+ T cell response by cells of HIV-infected patients [37]. Current research is aimed at targeting anti-gens to DCs in situ, which would eliminate the ex vivo differentiation stage, reduce the quantity of antigen required to simulate DCs and increase vaccine efficacy.

Clinical trials‘Rapid results’ is not a term that corresponds to the reality of vaccine development. Although we live in an era of technological and medi-cal advances with no precedent, the history of modern vaccinology reveals that it almost always takes decades from the discovery of a pathogenic agent to the licensing of an efficient vaccine against the same [92].

In the initial attempts of DC-based immu-notherapy with recombinant proteins/synthetic peptides, a small number of HIV+ individuals (n = 5–6) were analyzed without reporting encouraging results. Although the products were well tolerated, no impact was observed on plasma viral levels and the characteristics of the induced immune response (antibody produc-tion and CTL response) were not homogeneous. These differences were related to factors such as CD4+ T-cell blood counts, human leucocyte antigen (HLA) polymorphisms and the survival of DCs after infusion [93,94].

Further studies, performed with a slightly larger number of patients (n = 18) and using peptides or inactivated viral particles, revealed an increase in the specific immune response and in the control of viral replication [53,84], although a clear relationship between the two parameters was not always established [78]. Currently, six studies based on DC immuno-therapy against HIV infection are in progress worldwide (Table 1).



Although Phase I and II clinical trials using DCs showed that these products were well tolerated and safe, their design are still very heterogeneous [53,78,84,93,95,96]. For this reason, basic aspects of the production and application of these vaccines require more extensive inves-tigation: DC generation and activation, load-ing of DCs with antigens, DC subpopulations, quantity of DCs per dose, interval between applications, vaccine administration route and postvaccination laboratorial monitoring.

Undoubtedly, the technology for generat-ing DCs in vitro from bone marrow precursors (CD34+) or peripheral blood (monocytes) con-stituted a major advance in the field of DC-based immunotherapy [97,49]. Several differentiation protocols have been developed using diverse com-binations of cytokines, along with proposals for implementing quality control criteria (e.g., viabil-ity, purity and DC activation/maturation), which enable the reproduction of results [98,99].

The fact that immunogenicity could be related to the administration route suggests a crucial role for the homing of DCs postvaccina-tion. The capacity of DCs to migrate represents a fundamental aspect of their immunological function and many researchers have been study-ing this phenomenon [100,101]. The administra-tion route of DCs is a controversial subject in the literature and several routes have already been described [99]. Intradermal and subcutane-ous inoculation appears to be better, since these routes could act as selective filters for mature cells with greater migratory capacity and the capability of developing stimulation, rather than tolerance, of the immune system [102]. However, such concepts have been challenged by studies that demonstrate that mature DCs are also capable of inducing tolerance, notably through the generation of Tregs [103,104]. Furthermore, Barratt-Boyes et al. [105] and Thomas et al. [106]

demonstrated that immature DCs adminis-tered into the skin possess migratory capacity. Alternatively, DC injection directly into the lymphatic system appears to be a good option, although intralymphonodal administration has the disadvantage of potentially destroying the architecture of this lymphoid structure [99]. Thus, the best choice of an administration route remains to be demonstrated.

In HIV infection, the availability of func-tional antigen-presenting cells is considered a limiting factor for the development of an ade-quate immune response. Nevertheless, defining the ideal dose of DCs to be used in clinical tri-als remains a difficult task since the correlation between the number of inoculated DCs, the immune response elicited and the real clinical response has not been clearly established.

Moreover, the interval between vaccine injec-tions must also be taken into account. Studies evaluating the kinetics of the immune response suggest that short intervals may cause apop-tosis of antigen-specific lymphocytes, induced by cellular activation (activation-induced cell death) [107,108].

In the context of postvaccination monitor-ing, it is worth highlighting that the concept of clinical–laboratorial stability should be applied to HIV-infected patients recruited for clinical trials using DC-based vaccines. Such stability permits better evaluation of the impact of the vaccine product in relation to its immunogenicity and immunovirological endpoints.

Regarding the evaluation of immunogenicity, trials have focused on the detection of acquired cellular properties after the activation of memory lymphocyte clones. Similar to other vaccines, the production of neutralizing antibodies, lympho-proliferative capacity, cytokine production and cytotoxicity have been evaluated, as described in Table 2.

Table 1. Ongoing clinical trials involving anti-HIV DC vaccines.

Title Site Phase Patients (N)

DC vaccine in HIV-1 infection Hospital Clinic of Barcelona, Spain I/II 60

Autologous DC vaccine in HIV-1 infection University of Pittsburgh, PA, USA I/II 24

A pilot study of a DC vaccine in HIV-1-infected subjects (PARC002) Massachusetts General Hospital, MA, USA I/II 21

Safety and tolerability of a therapeutic DNA DC vaccine in HIV-infected children, adolescents, and young adults

International Maternal PediatricAdolescent AIDS ClinicalTrials Group (NIAID)

I/II 32

Vaccination of HIV-1 infected patients with dendritic cells in addition to antiretroviral treatment (DALIA trial)

Baylor Research Institute, TX, USA I 19

Safety/immunogenicity of immunizations of ALVAC-DC-SC vs ALVAC-SC

Massachusetts General Hospital, and New York University School of Medicine, NY, USA

I 29

DALIA: Distribution-analyzing latex immunoassay; DC: Dendritic cell; NIAID: National Institute of Allergy and Infectious Diseases. Adapted from [201].



It is noteworthy that in HIV infection, an effective protection against the virus appears to be related to the presence of polyfunctional lym-phocytes combining different characteristics, such as proliferative and cytotoxic capacity, and simultaneous production of specific cytokines (e.g., IL-2, TNF-a and IFN-g) [109–111].

Difficulties inherent to HIV infection, such as the lack of correlates of well-defined immu-nity, make the analysis of immunogenicity more complex than in other pathologies in which DC-based immunotherapy is used. Another peculiar characteristic to this disease is the chronic activation of the immune system [112], making the determination of specific activated subpopulations much more difficult. For that reason, analysis by HLA tetramers appears to be the most precise technique for evaluating spe-cific response. Besides, DCs have the property to regulate the immune response and may reduce this hyperactivation, which could be extremely promising in the field of an anti-HIV vaccine. Either way, definitive determination of surrogate markers in anti-HIV vaccine response must be defined in future studies.

Although DC-based vaccine products have been shown to be safe up until now, the pos-sibility of developing autoimmunity [113] and neo plasias should be considered, since these cells play a central role in the regulation of the immune response. As such, these aspects require rigorous monitoring during clinical trials.

Our group is currently leading a study aimed at characterizing aspects of the in vitro anti-HIV immune response using different models of infection/transfection. We then intend to relate these to changes in the genetic expression profile

of inactivated HIV-loaded MoDCs obtained from HIV-infected patients. Understanding the immune mechanisms related to the induction of a protective response mediated by DCs may con-tribute to the develop ment of approaches capable of improving the therapeutic vaccine according to the protocol proposed by Lu et al. [46].

Conclusion & future perspectiveKnowledge generated over the last few years in the field of DC immunobiology, such as their role in the regulation of immune response, the descrip-tion of different subtypes, the signaling pathways and mechanisms of antigen presentation, has con-tributed enormously to the advancement of thera-peutic vaccine research. Moreover, DC-vaccine models developed in the field of oncology are also applicable in the treatment of infectious diseases, particularly in HIV infection.

Given the difficulty in obtaining a prophylac-tic anti-HIV vaccine, and considering the adju-vant nature of DCs, immunotherapy for HIV-infected patients offers a unique opportunity to study the mechanisms of immune response against the virus and to contribute to defining correlates of protection in HIV infection. Thus, it is expected that the knowledge derived from studies of DC-based immunotherapy will aid the development of p rophylactic vaccines.

Currently, the production of DC-based vac-cine therapy is laborious and expensive, mainly because the focus on individual features makes large-scale production a limiting factor. New technologies capable of optimizing the produc-tion process are currently under development. Proposals of new methodologies to generate DCs for use in immunotherapy have been

Table 2. Parameters used in the post-vaccine immune response assessment.

Authors (year) Patients (N) Type of antigen Immunogenicity assessment Ref.

Kundu et al., (1998)

6 Recombinant HIV-1 MN gp160 or HLA-A2-restricted synthetic peptides of Env, Gag and Pol

Envelope-specific CTL- and lymphocyte-proliferative responses, IFN-g and IL-2 production, peptide-specific lymphocyte-proliferative responses

[93]

Lu W et al., (2004)

18 Autologous aldrithiol-2-inactivated HIV-1

Serum neutralizing antibody titers, HIV-1-specific IFN-g-expressing CD4+ and CD8+ cells, HIV-1-specific IL-2- expressing CD4+ T cells, HIV-1 Gag-specific CD8+ T cells, HIV-1 Gag-specific CD8+ T cells expressing perforin

[53]

Garcia F et al., (2005)

12 Heat-inactivated autologous HIV-1

Lymphoproliferation, Th1 cell levels, CTL levels, serum neutralizing antibody titers and changes in lymphoid tissue

[78]

Ide F et al., (2006) 4 HIV-1-derived CTL peptides IFN-g production in CD8 lymphocytes [95]

Connolly NC et al., (2008)

18 Gag, Env and Pol peptides IFN-g-producing cells (PBMCs) [84]

Gandhi RT et al., (2009)

29 Viral vector (canarypox)expressing HIV-1 Env and Gag and a synthetic polypeptide encompassing epitopes from Nef and Pol

IFN-g-producing cells (PBMCs), lymphocyte-proliferative responses

[96]

CTL: Cytotoxic T lymphocyte; HLA: Human leukocyte antigen; PBMC: Peripheral blood mononuclear cell.



Executive summary

Introduction � In addition to being a powerful natural adjuvant, dendritic cells (DCs) play a central role in regulating the immune system. � In HIV infection, the gradual depletion of DCs correlates to increased viral load. � The control of the HIV/AIDS pandemic with only the use of antiretroviral drugs is limited, stimulating the development of new

therapeutic approaches. � Therapeutic vaccines and/or immunomodulators have been identified as a condition for increasing the body’s ability to respond against

the virus.DCs in HIV immunotherapy � The recognition of pathogens by DCs occurs by means of pathogen-recognition receptors. � DCs are able to capture, process and present antigens in the context of MHC-I and -II, and to perform cross-presentation, triggering an

effector-cell response. � The effectiveness of an effector cell response is dependent on three signals: peptides in the context of MHC and their specific T-cell

receptors, costimulatory molecules and related ligands, and the action of soluble factors associated with the cell membrane. � Critical factors for vaccine therapy: DC subtypes, how to stimulate DC maturation, DC cytokine production capacity and DC

migratory potential. � Conflicting results are observed in studies assessing the ability of immunogenic monocytic DCs of HIV-infected patients.

HIV antigens for potential use in immunotherapy � The high genetic variability of HIV makes the achievement of an immunogenic antigen a major challenge. � A desirable anti-HIV vaccine would be able to amplify the memory response and induce a primary response against a mutating virus to

prevent viral escape mechanisms. � Antigens derived from autologous virus have been used in protocols for the induction of HIV-specific response. � Potential alternatives to the use of autologous virus include: HIV-derived peptides; recombinant HIV proteins; HIV RNA or DNA;

HIV-infected apoptotic cells; and in situ target DCs.Clinical trials � Clinical trials of Phase I/II using DCs for anti-HIV have shown that the product is safe and well tolerated. � The elucidation of basic aspects of production and application of this vaccine are required, including generation and activation of DCs;

supply of DCs with relevant antigens; choice of subpopulations of DCs ideal for vaccination; quantity of DCs in each dose; interval between applications; route of administration of the vaccine; and postvaccination laboratorial monitoring.

Conclusion & future perspective � Knowledge of DCs and models of immunobiological vaccine in the field of oncology has contributed to the advancement of

anti-HIV immunotherapy. � In view of the heterogeneity of the studies conducted to date, a greater number of preclinical studies are still necessary.

developed to overcome certain limitations of conventional protocols. In addition, antigenic formulations directed at the induction of an adequate immune response have been devel-oped, which can be allied with the availabil-ity of advanced biotechnological methods to i ntroduce these components into DCs.

Moreover, the exciting possibility of in situ, rather than ex vivo, production of the vac-cine has been proposed. This would make its use on a larger scale viable. Results obtained from in vitro studies are promising, creating the prospect of a new generation of vaccine therapy [37].

Information provided by clinical trials f inalized up-to-date shows that DC-based i mmunotherapy in HIV-infected patients appears to be safe and presents no significant toxicity. On the other hand, given the expres-sive diversity between vaccine products and the experimental designs used, the standardization of protocols is required so that the studies can be reproduced and directly compared. In this

context, the realization of a greater number of preclinical studies that would permit the imple-mentation of large Phase III clinical trails is highly recommended. These studies may assist in the selection of candidates potentially responsive to vaccine therapy, since it is easier to evaluate in vitro how DCs might promote a specific cellular immune response.

Although the unprecedented magnitude of studies in biomedical research have resulted in extraordinary advances in this field, the collaboration between academic research c enters and biotechnology industry is e ssential for the future development of a DC-based immunotherapy.

The development of an anti-HIV vaccine, even of partial efficacy, would have a major impact on the pandemic. DC-based immuno-therapy represents the possibility of a safe and effective therapeutic strategy, capable of l imiting viral replication and T-cell activation through specific response to strategic HIV epitopes.



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

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

BibliographyPapers of special note have been highlighted as:n of interestnn of considerable interest

1 Steinman RM, Cohn ZA: Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J. Exp. Med. 137(5), 1142–1162 (1973).

2 Inaba K, Metlay JP, Crowley MT, Steinman RM: Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ. J. Exp. Med. 172(2), 631–640 (1990).

n Initial evidence of immunogenicity induced by dendritic cells (DCs).

3 Inaba K, Inaba M, Naito M, Steinman RM: Dendritic cell progenitors phagocytose particulates, including bacillus Calmette–Guerin organisms, and sensitize mice to mycobacterial antigens in vivo. J. Exp. Med. 178(2), 479–488 (1993).

4 Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature 392(6673), 245–252 (1998).

nn Review on aspects of the DCs immunobiology.

5 Fajardo-Moser M, Berzel S, Moll H: Mechanisms of dendritic cell-based vaccination against infection. Int. J. Med. Microbiol. 298(1–2), 11–20 (2008).

6 Coley WB: The treatment of malignant tumors by repeated inoculations of erysipelas: with a report of ten original cases, 1893. Clin. Orthop. Relat. 262, 3–11 (1991).

7 Jefford M, Maraskovsky E, Cebon J, Davis ID: The use of dendritic cells in cancer therapy. Lancet Oncol. 2, 343–353 (2001).

8 Steinman RM, Dhodapkar M: Active immunization against cancer with dendritic cells: the near future. Int. J. Cancer 94, 459–473 (2001).

9 Koido S, Hara E, Homma S, Ohkusa T, Gong J, Tajiri H: Cancer immunotherapy by fusions of dendritic cells and tumor cells. Immunotherapy 1(1), 49–62 (2009).

10 Thomas-Kaskel AK, Veelken H: Dendritic-cell vaccination for prostate cancer. Immunotherapy 1(1), 63–72 (2009).

11 Gabrilovich D: Mechanisms and functional significance of tumour-induced dendritic-cell defects. Nat. Rev. Immunol. 4(12), 941–952 (2004).

12 Grassi F, Hosmalin A, McIlroy D, Calvez V, Debré P, Autran B: Depletion in blood CD11c-positive dendritic cells from HIV-infected patients. AIDS 13(7), 759–766 (1999).

13 Donaghy H, Pozniak A, Gazzard B et al.: Loss of blood CD11c+ myeloid and CD11c- plasmacytoid dendritic cells in patients with HIV-1 infection correlates with HIV-1 RNA virus load. Blood 98(8), 2574–2576 (2001).

14 Jones GJ, Watera C, Patterson S et al.: Comparative loss and maturation of peripheral blood dendritic cell subpopulations in African and non-African HIV-1-infected patients. AIDS 15(13), 1657–1663 (2001).

15 Lu W, Andrieu JM: In vitro human immunodeficiency virus eradication by autologous CD8+ T cells expanded with inactivated-virus-pulsed dendritic cells. J. Virol. 75(19), 8949–8956 (2001).

16 Lu W, Wu X, Lu Y, Guo W, Andrieu JM: Therapeutic dendritic-cell vaccine for simian AIDS. Nat. Med. 9(1), 27–32 (2003).

17 Carbonneil C, Aouba A, Burgard M et al.: Dendritic cells generated in the presence of granulocyte–macrophage colony-stimulating factor and IFN-a are potent inducers of HIV-specific CD8 T cells. AIDS 17(12), 1731–1740 (2003).

18 Almeida M, Cordero M, Almeida J, Orfao A: Different subsets of peripheral blood dendritic cells show distinct phenotypic and functional abnormalities in HIV-1 infection. AIDS 19(3), 261–271 (2005).

19 Nehete PN, Gambhira R, Nehete BP, Sastry KJ: Dendritic cells enhance detection of antigen-specific cellular immune responses by lymphocytes from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine. J. Med. Primatol. 32(2), 67–73 (2003).

20 WHO, UNAIDS & UNICEF: Towards universal access: scaling up priority HIV/AIDS Interventions in the health sector 1–92. WHO, Geneva, Switzerland (2007).

21 Carr A: HIV protease inhibitor-related lipodystrophy syndrome. Clin. Infect. Dis. 30(Suppl. 2), S135–S142 (2000).

22 Feinberg M: Hidden dangers of incompletely suppressive antiretroviral therapy. Lancet 349, 1408 –1409 (1997).

23 Shortman K, Naik SH: Steady-state and inflammatory dendritic-cell development. Nat. Rev. Immunol. 7(1), 19–30 (2007).

24 Steinman RM: Dendritic cells in vivo: a key target for a new vaccine science. Immunity 29, 319–324 (2008).

25 Freudenthal PS, Steinman RM: The distinct surface of human blood dendritic cells as observed after an improved isolation method. Proc. Natl Acad. Sci. USA 87(19), 7698–7702 (1990).

26 Albert ML, Sauter B, Bhardwaj N: Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392(6671), 86–89 (1998).

27 Siegal FP, Kadowaki N, Shodell M et al.: The nature of the principal type 1 interferon-producing cells in human blood. Science 284(5421), 1835–1837 (1999).

28 Marschner A, Rothenfusser S, Hornung V et al.: CpG ODN enhance antigen-specific NKT cell activation via plasmacytoid dendritic cells. Eur. J. Immunol. 35(8), 2347–2357 (2005).

29 Jego G, Palucka AK, Blanck JP, Chalouni C, Pascual V, Banchereau J: Plasmacytoid dendritic cells induce plasma cell differentiation through type I interferon and interleukin 6. Immunity 19(2), 225–234 (2003).

30 Fonteneau JF, Larsson M, Beignon AS et al.: Human immunodeficiency virus type 1 activates plasmacytoid dendritic cells and concomitantly induces the bystander maturation of myeloid dendritic cells. J. Virol. 78(10), 5223–5232 (2004).

31 Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ: The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J. Exp. Med. 185(6), 1101–1111 (1997).

32 Fonteneau JF, Gilliet M, Larsson M et al.: Activation of influenza virus-specific CD4+ and CD8+ T cells: a new role for plasmacytoid dendritic cells in adaptive immunity. Blood 101(9), 3520–3526 (2003).

33 Hoeffel G, Ripoche AC, Matheoud D et al.: Antigen crosspresentation by human plasmacytoid dendritic cells. Immunity 27, 481–492 (2007).

34 Kwon D, Gregorio G, Bitton N, Hendrickson W, Littman D: DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cell infection. Immunity 16(1), 135–144 (2002).

35 Jiang W, Swiggard WJ, Heufler C: The receptor DEC-205 expressed by dendritic cell and thymic epithelial cells is involved in antigen processing. Nature 375(6527), 151–155 (1995).

future science group 1049www.futuremedicine.com


36 Granelli-Piperno A, Pritsker A, Pack M et al.: Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin/CD209 is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction. J. Immunol. 175(7), 4265–4273 (2005).

37 Bozzacco L, Trumpfheller C, Siegal FP et al.: DEC-205 receptor on dendritic cells mediates presentation of HIV-Gag protein to CD8+ T cells in a spectrum of human MHC I haplotypes. Proc. Natl Acad. Sci. USA 104(4), 1289–1294 (2007).

n View to increase the effectiveness of DC vaccine.

38 Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R: Toll-like receptors control activation of adaptive immune responses. Nat. Immunol. 2(10), 947–950 (2001).

39 Medzhitov R: Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1(2), 135–145 (2001).

40 Hornung V, Rothenfusser S, Britsch S et al.: Quantitative expression of Toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168(9), 4531–4537 (2002).

41 Jarrossay D, Napolitani G, Collona M, Sallusto F, Lanzavecchia A : Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. Eur. J. Immunol. 31(11), 3388–3393 (2001).

42 De Gregorio E, D’Oro U, Wack A: Immunology of TLR-independent vaccine adjuvants. Curr. Opin. Immunol. 21(3), 339–345 (2009).

43 Vanbervliet B, Homey B, Durand I et al.: Sequential involvement of CCR2 and CCR6 ligands for immature dendritic cell recruitment: possible role at inflamed epithelial surfaces. Eur. J. Immunol. 32(1), 231–242 (2002).

44 McWilliam AS, Nelson D, Thomas JA, Holt PG: Rapid dendritic cell recruitment is a hallmark of the acute inflammatory response at mucosal surfaces. J. Exp. Med. 179(4), 1331–1336 (1994).

45 Faure-André G, Vargas P, Yuseff MI et al.: Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain. Science 322(5908), 1705–1710 (2008).

46 Iwasaki A, Medzhitov R: Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5(10), 987–995 (2004).

nn First clinical report showing the impact of vaccine on viral load in HIV infection.

47 Kalinski P, Hilkens CMU, Wierenga EA, Kapsenberg ML: T-cell priming by type-1 and type-2 polarized dendritic cells: the concept of a third signal. Immunol. Today 20(12), 561–567 (1999).

48 Nakano H, Lin KL, Yanagita M et al.: Blood-derived inflammatory dendritic cells in lymph nodes stimulate acute T helper type 1 immune responses. Nat. Immunol. 10(4), 394–402 (2009).

49 Romani N, Gruner S, Brang D et al.: Proliferating dendritic cell progenitors in human blood. J. Exp. Med. 180(1), 83–93 (1994).

50 Sallusto F, Lanzavecchia A: Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor a. J. Exp. Med. 179(4), 1109–1118 (1994).

51 Jonuleit H, Kühn U, Müller G et al.: Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions. Eur. J. Immunol. 27(12), 3135–3142 (1997).

52 Weissman D, Ni H, Scales D et al.: HIV-Gag mRNA transfection of dendritic cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response. J. Immunol. 165, 4710–4717 (2000).

53 Lu W, Arraes LC, Ferreira WT, Andrieu JM: Therapeutic dendritic-cell vaccine for chronic HIV-1 infection. Nat. Med. 10, 1359–1365 (2004).

54 Allard SD, Pletinckx K, Breckpot K et al.: Functional T-cell responses generated by dendritic-cells expressing the early HIV-1 proteins Tat, Rev and Nef. Vaccine 26(29–30), 3735–3741 (2008).

55 Ahlers JD, Belyakov IM: Strategies for recruiting and targeting dendritic cells for optimizing HIV vaccines. Trends Mol. Med. 15(6), 263–274 (2009).

56 Banerjee DK, Dhodapkar MV, Matayeva E, Steinman RM, Dhodapkar KM: Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients. Blood 108(8), 2655–2661 (2006).

57 Jacobs B, Wuttke M, Papewalis C et al.: Characterization of monocyte-derived IFN-a-generated dendritic cells. Horm. Metab. Res. 40(2), 117–121 (2008).

58 Tawab A, Fan Y, Read EJ, Kurlander RJ: Effect of ex vivo culture duration on phenotype and cytokine production by mature dendritic cells derived from peripheral blood monocytes. Transfusion 49(3), 536–547 (2009).

59 Landi A, Babiuk LA, van Drunen Littel-van den Hurk S: High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer. J. Leukoc. Biol. 82(4), 849–860 (2007).

60 Sapp M, Engelmayer J, Larsson M, Granelli-Piperno A, Steinman R, Bhardwaj N: Dendritic cells generated from blood monocytes of HIV-1 patients are not infected and act as competent antigen presenting cells eliciting potent T-cell responses. Immunol. Lett. 66(1–3), 121–128 (1999).

61 Chougnet C, Cohen SS, Kawamura T et al.: Normal immune function of monocyte-derived dendritic cells from HIV-infected individuals: implications for immunotherapy. J. Immunol. 163(3), 1666–1673 (1999).

62 Buisson S, Benlahrech A, Gazzard B, Gotch F, Kelleher P, Patterson S: Monocyte-derived dendritic cells from HIV type 1-infected individuals show reduced ability to stimulate T cells and have altered production of interleukin (IL)-12 and IL-10. J. Infect. Dis. 199(12), 1862–1871 (2009)

63 Reis e Sousa C: Dendritic cells in a mature age. Nat. Rev. 6, 476–483 (2006).

64 Yamazaki S, Steinman RM: Dendritic cells as controllers of antigen-specific Foxp3+ regulatory T cells. J. Dermatol. Sci. 54(2), 69–75 (2009).

65 Weiss L, Donkova-Petrini V, Caccavelli L, Balbo M, Carbonneil C, Levy Y: Human immunodeficiency virus-driven expansion of CD4+CD25+ regulatory T cells, which suppress HIV-specific CD4 T-cell responses in HIV-infected patients. Blood 104(10), 3249–3256 (2004).

66 Nilsson J, Boasso A, Velilla PA et al.: HIV-1-driven regulatory T-cell accumulation in lymphoid tissues is associated with disease progression in HIV/AIDS. Blood 108(12), 3808–3817 (2006).

67 Kinter A, McNally J, Riggin L, Jackson R, Roby G, Fauci AS: Suppression of HIV-specific T cell activity by lymph node CD25+ regulatory T cells from HIV-infected individuals. Proc. Natl Acad. Sci. USA 104(9), 3390–3395 (2007).

68 Eggena MP, Barugahare B, Jones N et al.: Depletion of regulatory T cells in HIV infection is associated with immune activation. J. Immunol. 174(7), 4407–4414 (2005).

69 Card CM, McLaren PJ, Wachihi C, Kimani J, Plummer FA, Fowke KR: Decreased immune activation in resistance to HIV-1 infection is associated with an elevated frequency of CD4+CD25+FOXP3+ regulatory T cells. J. Infect. Dis. 199(9), 1318–1322 (2009).



70 Holmes D, Jiang Q, Zhang L, Su L: Foxp3 and Treg cells in HIV-1 infection and immuno-pathogenesis. Immunol. Res. 41(3), 248–266 (2008).

71 Sereti I, Imamichi H, Natarajan V et al.: In vivo expansion of CD4CD45RO-CD25 T cells expressing FoxP3 in IL-2-treated HIV-infected patients. J. Clin. Invest. 115(7), 1839–1847 (2005).

72 Ziegler SF: FoxP3: not just for regulatory T cells anymore. Eur. J. Immunol. 37(1), 21–23 (2007).

73 Tran DQ, Andersson J, Hardwick D, Bebris L, Illei GG, Shevach EM: Selective expression of latency-associated peptide (LAP) and IL-1 receptor type I/II (CD121a/CD121b) on activated human FoxP3+ regulatory T cells allows for their purification from expansion cultures. Blood 113(21), 5125–5133 (2009).

74 Loussert-Ajaka I, Chaix ML, Korber B et al.: Variability of human immunodeficiency virus type 1 group O strains isolated from Cameroonian patients living in France. J. Virol. 69(9), 5640–5649 (1995).

75 Korber B, Muldoon M, Theiler J et al.: Timing the ancestor of the HIV-1 pandemic strains. Science 288(5472), 1789–1796 (2000).

76 Taylor BS, Hammer SM: The challenge of HIV-1 subtype diversity. N. Engl. J. Med. 359(18), 1965–1966 (2008).

77 Malim MH, Emerman M: HIV-1 sequence variation: drift, shift, and attenuation. Cell 104(4), 469–472 (2001).

78 Garcia F, Lejeune M, Climent N et al.: Therapeutic immunization with dendritic cells loaded with heat-inactivated autologous HIV-1 in patients with chronic HIV-1 infection. J. Infect. Dis. 191, 1680–1685 (2005).

79 Altfeld M, Addo MM, Shankarappa R et al.: Enhanced detection of human immunodeficiency virus type 1-specific T-cell responses to highly variable regions by using peptides based on autologous virus sequence. J. Virol. 77(13), 7330–7340 (2003).

80 Fischer W, Perkins S, Theiler J et al.: Polyvalent vaccines for optimal coverage of potential T-cell epitopes in global HIV-1 variants. Nat. Med. 13(1), 100–106 (2007).

81 Frahm N, Kaufmann DE, Yusim K et al.: Increased sequence diversity coverage improves detection of HIV-specific T cell responses. J. Immunol. 179(10), 6638–6650 (2007).

82 Rossio JL, Esser MT, Suryanarayana K et al.: Inactivation of human immunodeficiency virus type I infectivity with preservation of conformational and functional integrity of virion surface proteins. J. Virol. 72, 7992–8001 (1998).

83 Larsson M, Wilkens DT, Fonteneau JF et al.: Amplification of low-frequency antiviral CD8 T cell responses using autologous dendritic cells. AIDS 16, 171–180 (2002).

84 Connolly NC, Whiteside TL, Wilson C, Kondragunta V, Rinaldo CR, Riddler SA: Therapeutic immunization with human immunodeficiency virus type 1 (HIV-1) peptide-loaded dendritic cells is safe and induces immunogenicity in HIV-1-infected individuals. Clin. Vac. Immunol. 15(2), 284–292 (2008).

n Clinical trial using vaccine made of DCs pulsed with viral peptides.

85 Van Gulck ERA, Ponsaerts P, Heyndrickx L et al.: Efficient stimulation of HIV-1-specific T cells using dendritic cells electroporated with mRNA encoding autologous HIV-1 Gag and Env proteins. Blood 107(5), 1818–1827 (2006).

86 Kavanagh DG, Kaufmann DE, Sunderji S et al.: Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysossome-targeted Nef. Blood 107(5), 1963–1969 (2006).

87 Van Gulck ER, Vanham G, Heyndrickx L et al.: Efficient in vitro expansion of human immunodeficiency virus (HIV)-specific T-cell responses by gag mRNA-electroporated dendritic cells from treated and untreated HIV type 1-infected individuals. J. Virol. 82(7), 3561–3573 (2008).

88 Engelmayer J, Larsson M, Lee A et al.: Mature dendritic cells infected with canarypox virus elicit strong anti-human immunodeficiency virus CD8+ and CD4+ T-cell responses from chronically infected individuals. J. Virol. 75(5), 2142–2153 (2000).

89 Tsunetsugu-Yokota Y, Morikawa Y, Isogai M et al.: Yeast-derived human immunodeficiency virus type 1 p55gag virus-like particles activate dendritic cells (DCs) and induce perforin expression in gag-specific CD8+ T cells by cross-presentation of DCs. J. Virol. 77(19), 10250–10259 (2003).

90 Albert ML: Death-defying immunity: do apoptotic cells influence antigen processing and presentation? Nat. Rev. Immunol. 4(3), 223–231 (2004).

91 Marañón C, Desoutter JF, Hoeffel G, Cohen W, Hanau D, Hosmalin A: Dendritic cells cross-present HIV antigens from live as well as apoptotic infected CD4+ T lymphocytes. Proc. Natl Acad. Sci. USA 101(16), 6092–6097 (2004).

92 Heyward WL, MacQueen KM, Goldenthal KL: HIV vaccine development and evaluation: realistic expectations. AIDS Res. Hum. Retrovir. (Suppl. 3) S205–S210 (1998).

93 Kundu SK, Engleman E, Benike C et al.: A pilot clinical trial of HIV antigen-pulsed allogeneic and autologous dendritic cell therapy in HIV infected patients. AIDS Res. Hum. Retrovir. 14, 551–560 (1998).

94 Shapero MH, Kundu SK, Engleman E, Laus R, van Schooten WC, Merigan TC: In vivo persistence of donor cells following adoptive transfer of allogeneic dendritic cells in HIV-infected patients. Cell Transplant 9(3), 307–317 (2000).

95 Ide F, Nakamura T, Tomizawa M et al.: Peptide-loaded dendritic-cell vaccination followed by treatment interruption for chronic HIV-1 infection: a Phase 1 trial. J. Med. Virol. 78(6), 711–718 (2006).

96 Gandhi RT, O’Neill D, Bosch RJ et al.: A randomized therapeutic vaccine trial of canarypox-HIV-pulsed dendritic cells vs. canarypox-HIV alone in HIV-1-infected patients on antiretroviral therapy. Vaccine doi:10.1016/j.vaccine.2009.05.016 (2009).

97 Caux C, Dezutter-Dambuyant D, Banchereau J: GM-CSF and TNF-a cooperate in the generation of dendritic Langerhans cells. Nature 360, 258–261 (1992).

98 Nestle FO, Banchereau J, Hart D: Dendritic cells: On the move from bench to bedside. Nat. Med. 7(7), 761–765 (2001).

nn Discusses various aspects of standardization in the protocols of DC-based vaccine.

99 Figdor CG, de Vries IJ, Lesterhuis WJ, Melief CJ: Dendritic cell immunotherapy: mapping the way. Nat. Med. 10(5), 475–480 (2004).

nn Focuses on the difficulties and discusses aspects of standardization in clinical trials using DCs.

100 Ruiz A, Nomdedeu M, Ortega M et al.: Assessment of migration of HIV-1-loaded dendritic cells labeled with 111In-oxine used as a therapeutic vaccine in HIV-1-infected patients. Immunotherapy 1(3), 347–354 (2009).

101 Alvarez D, Vollmann EH, von Andrian UH: Mechanisms and consequences of dendritic cell migration. Immunity 29(3), 325–342 (2008).

102 De Vries IJ, Krooshoop DJ, Scharenborg NM et al.: Effective migration of antigen-pulsed dendritic cells to lymph nodes in melanoma patients is determined by their maturation state. Cancer Res. 63(1), 12–17 (2003).

103 Luo X, Tarbell KV, Yang H: Dendritic cells with TGF-b1 differentiate naive CD4+CD25- T cells into islet-protective Foxp3+ regulatory T cells. Proc. Natl Acad. Sci. USA 104(8), 2821–2826 (2007).

future science group 1051www.futuremedicine.com


104 Belkaid Y, Oldenhove G: Tuning microenvironments: induction of regulatory T cells by dendritic cells. Immunity 29(3), 362–371 (2008).

105 Barratt-Boyes SM, Zimmer MI, Harshyne LA et al.: Maturation and trafficking of monocyte-derived dendritic cells in monkeys: implications for dendritic cell-based vaccines. J. Immunol. 164(5), 2487–2495 (2000).

106 Thomas R, Chambers M, Boytar R et al.: Immature human monocyte-derived dendritic cells migrate rapidly to draining lymph nodes after intradermal injection for melanoma immunotherapy. Melanoma Res. 9, 474–481 (1999).

107 Dhodapkar MV, Krasovsky J, Steinman RM, Bhardwaj N: Mature dendritic cells boost functionally superior CD8+ T-cell in humans without foreign helper epitopes. J. Clin. Invest. 105(6), R9–R14 (2000).

108 Serody JS, Collins EJ, Tisch RM, Kuhns JJ, Frelinger JA: T cell activity after dendritic cell vaccination is dependent on both the type of antigen and the mode of delivery. J. Immunol. 164, 4961–4967 (2000).

109 De Rosa SC, Lu FX, Yu J et al.: Vaccination in humans generates broad T cell cytokine responses. J. Immunol. 173(9), 5372–5380 (2004).

110 Makedonas G, Betts MR: Polyfunctional analysis of human T cell responses: importance in vaccine immunogenicity and natural infection. Springer Semin. Immunopathol. 28(3), 209–219 (2006).

nn Reports the presence of multiple cytokine-producing lymphocytes in response to infection and immunization.

111 Betts MR, Nason MC, West SM et al.: HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood 107(12), 4781–4789 (2006).

112 Brenchley JM, Price DA, Schacker TW et al.: Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat. Med. 12(12), 1365–1371 (2006).

nn Evidence of the role of microbial translocation as vital in HIV infection.

113 Ludewig B, Ochsenbein AF, Odermatt B, Paulin D, Hengartner H, Zinkernagel RM: Immunotherapy with dendritic cells directed against tumor antigens shared with normal host T cells results in severe autoimmune disease. J. Exp. Med. 191, 795–803 (2000).

Website201 Clinicaltrials.gov. A service of the U.S.

National Institutes of Health www.clinicaltrials.gov

Documents

Dendritic cell immunotherapy for HIV infection: from theory to reality