POTENTIAL CLINICAL RELEVANCE

Nanomedicine: Nanotechnology, Biology, and Medicine9 (2013) 28–38

Review Article

Nanomedicine applications towards the cure of HIVJulianna Lisziewicz, PhDa,b,⁎, Enikő R. Tőke, PhDa

aGenetic Immunity Kft., Budapest, HungarybGenetic Immunity Inc., McLean, Virginia

Received 10 February 2012; accepted 16 May 2012

nanomedjournal.com

Abstract

Combination antiretroviral therapy (cART) successfully suppresses HIV replication. However, daily and lifelong treatment is necessary tomanage patient illness because cART neither eradicates infected cells from reservoirs nor reconstitutes HIV-specific immunity that could killinfected cells. Toward the cure of HIV, different nanomedicine classes have been developed with the following disease-modifying properties:to eradicate the virus by activation of latently infected CD4+ T-cells and reservoirs flushing; to kill the infected cells in the reservoirs byboosting of HIV-specific T cells; and to prevent infection by the use of microbicides with improved epithelial penetration and drug half-life.Preclinical and clinical trials consistently demonstrated that DermaVir, the most advanced nanomedicine, induces long-lasting memory T-cellresponses and reduces viral load in comparison with placebo. DermaVir and the nanomedicine pipelines have the potential to improve thehealth of HIV-infected people at lower costs, to decrease antiretroviral drug exposure, and to contribute to the cure of HIV/AIDS.

From the Clinical Editor: Despite the leaps and bounds in the development of antiretroviral therapy, HIV remains a significant public healthchallenge. In this review, applications of nanomedicine- based technologies are discussed in the context of HIV treatment, including viruselimination by activation of latently infected CD4+ T-cells; infected cell elimination in the reservoirs by boosting HIV-specific T cells, andby preventing infection by the use of microbicides with improved epithelial penetration and drug half-life.© 2013 Elsevier Inc. All rights reserved.

Key words: HIV vaccine; HIV eradication; Memory T cells; Drug development

In 1984 HIV was identified as the cause of AIDS one year afterthe virus was isolated.1-4 Since that time HIV/AIDS has becomethe leading infectious killer affecting more than 33 million peopleworldwide.5 HIV/AIDS is treated with the combination of 25antiretroviral drugs (ARV) that are divided into six classesaccording to their interference with HIV life-cycle: fusion/entryinhibitors, integrase inhibitors, protease inhibitors, nonnucleosidereverse transcriptase inhibitors, nucleoside analog reverse tran-scriptase inhibitors, and multidrug combination products. HIV/AIDS treatment using any single class of ARV has not beenefficient in controlling infection due to the development ofresistant strains of the virus. Hence, three or more ARVs are usedin combination (combination antiretroviral therapy, cART) to treatthe disease.

Currently available cART is potent in suppressing HIVreplication and effective in decreasing HIV RNA level below the

Dr. Julianna Lisziewicz holds shares in Genetic Immunity. This work wassupported by grants: HIKC05 and DVCLIN01 of the National Office forResearch and Technology (NKTH) in Hungary.

⁎Corresponding author: Genetic Immunity Kft., Berlini utca 47-49,Budapest H-1045, Hungary.

E-mail address: lisziewj@geneticimmunity.com (J. Lisziewicz).

1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.nano.2012.05.012

Please cite this article as: Lisziewicz J., Tőke E.R., Nanomedicine applicatiodx.doi.org/10.1016/j.nano.2012.05.012

limit of detection (50 copies/mL) with onlyminimum side effects.Long-term cART decreased morbidity and mortality associatedwith HIV infection.6 Key products used for the treatment of themajority of patients, such as Atripla (efavirenz/tenofovir/emtricitabine), Truvada (tenofovir/emtricitabine), Sustiva (efa-virenz), Kaletra (lopinavir/ritonavir), Reyataz (atazanavir), andIsentress (raltegravir), satisfy the ARV demand in the market.

Successful management of HIV-infected patients is challeng-ing, requiring highly experienced physicians due to resistanceand overlapping toxicities of the complex daily ARV regimenthat must be taken for the rest of the patient's life. However, evenoptimal cART, characterized by suppression of viral load toundetectable levels for years, has not provided a cure for thedisease. Patients on optimal cART have twelve-year shorter lifeexpectancy than HIV-negative people,7,8 In addition, increasedAIDS-related and non-AIDS-related morbidity and mortalityhave been described in a significant proportion of individuals onoptimal cART due to the lack of normalization of their CD4+ T-cell counts.9 Optimal cART failed to decrease the viralreservoirs, especially in the gut mucosa, where the residuallow-level viral replication may be the cause of persistent immuneactivation that facilitates the progression to AIDS and death.10

One barrier of cure is the stable latent reservoirs of HIV-infected

ns towards the cure of HIV. Nanomedicine: NBM 2013;9:28-38, http://

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resting memory T cells that are able to produce HIV after cellularactivation. HIV-producing cells in the reservoirs that are noteliminated by ARV would be susceptible to immune clearance,but long-term optimal cART diminish HIV-specific T-cellresponses.11 Therefore, the immune systems of successfullytreated HIV-infected people are not prepared to decrease viralreservoirs and control the virus replication.

Currently, there are two major unmet needs in HIV treatment.They are (i) simplification of the daily treatment and (ii) disease-modifying therapies. Large pharmaceutical companies focus onthe treatment simplification approach with single-tablet cARTregimen using conventional drug formulations. Nanomaterials areexploited for the development of innovative disease modifyingtherapies aiming to prevent infections, stop disease progressionand cure HIV/AIDS.

We found it curious that the two different treatment strategies forthe cure ofHIV/AIDSwerefirst implemented inBerlin, Germany bythe publication of two exceptional case reports. Eradication of thevirus was demonstrated after bone marrow transplantation withdonor cells resistant to HIV infection.12 We have described immunecontrol of HIV in the case of the first Berlin patient, whose immunesystem was boosted by his own virus emerging during shortinterruptions of cART.13 This work led to the identification of “elitecontrollers” representing a model for remission as cure. Theseindividuals have large numbers of cells containing replication-competent HIV controlled by the cellular arm of the immunesystem.14 Along these lines, Deeks and coworkers recently haveshown an inverse correlation between the frequency of HIV-specificT cells in the gut and the size of the reservoir, suggesting thatboosting of T-cell responses might contribute to the clearance oflatent HIV.15 Siliciano and his team have recently demonstrated thatboosting of HIV-specific T-cell responses prior to reactivating latentHIV will be essential for eradication of the virus.16

Here we review novel disease-modifying treatment ap-proaches that exploit nanomaterials to cure HIV/AIDS. Thefirst section discusses the experiments that showed hownanomedicines activate resting cells to flush HIV and improvepharmacokinetic features of ARV. The second section describesnanomedicines application as microbicides to be used forprevention of HIV infection. In the third section we providedetailed description on the mechanism of action and clinicalresults of immune-modulating nanomedicines, including Der-maVir therapeutic vaccine. In the final chapter we discuss hownanomedicines could contribute to the cure of HIV/AIDS.

Nanomedicine drug formulations

Application of nanotechnology to antiretroviral drug deliveryholds promise in the cure of HIV, because it could modify tissuedistribution by targeting drugs to HIV reservoirs and byincreasing the half-lives of drugs. The use of nano-deliverysystems has been extensively reviewed previously.17-19 There-fore we only highlight some of the recent advances in the fieldthat could play a role in achieving a cure.

Optimal cART cannot eradicate HIV. A portion of the virusof patients successfully treated with cART resides in “latentreservoirs” within memory CD4+ T cells and macrophages

concentrated mainly in lymphoid tissues, testes, the gut, and thecentral nervous system. A new eradication approach is to targetdrugs to the viral reservoirs and force the activation of latentlyinfected cells to virus production. Consequently, these produc-tively infected cells can be recognized and eliminated by theimmune system. To target and activate primary human CD4+

T cells, a nanomedicine formulation of a protein kinase Cactivator, bryostatin-2 (LNP-Bry), was studied in a humanizedmouse model. LNP-Bry was also loaded with the proteaseinhibitor nelfinavir producing a nanomedicine capable of bothactivating latent virus and inhibiting viral spread.20

To target antiretroviral drugs to the lymphoid organs, a pH-dependent nanomedicine formulation of indinavir was investi-gated in macaques.21 This nanomedicine formulation increasedindinavir concentration in the lymph nodes.22 These 50 – 80 nmnanoparticles (NPs) were trapped in lymph nodes as theycirculated through the lymphatic system. The authors concludedthat the targeting effect of NPs to the lymphoid tissues wasmainly particle-size dependent.

Bioavailability is a major challenge in drug development. Forexample, the protease inhibitors indinavir, ritonavir, andnevirapine have different, but still acceptable, oral bioavailabilityof 39%, 60% –70%, and 92%, respectively. In contrast,saquinavir possesses only 4% of bioavailability that influencedthe efficacy and toxicity of this drug, and the company decided towithdraw the drug from the market after obtaining marketauthorization.23 To improve bioavailability nanomedicine for-mulation of efavirenz (EFV) was investigated. The drug wasincorporated into the core of linear and branched poly(ethyleneoxide)–poly(propylene oxide) block copolymer micelles. In rats,EFV-nanomedicine had up to 88% higher plasma concentrationsin comparison with EFV suspensions.24

ARV resistance occurs often in the case of non-adherence orwhen the viral load is insufficiently suppressed. The presence ofresistant HIV then limits the treatment options of patients. Toimprove adherence with longer dosing intervals, the nanomedi-cine formulation of rilpivirine was investigated in rats and dogs.This nanomedicine demonstrated a sustained and dose-propor-tional release over 2 months and a significant half-lifeenhancement in comparison with the 38 hours of the free drug.25

Preventive nanomedicines

Topical microbicides represent a promising strategy to preventvaginal and rectal HIV transmission. Clinical proof of concept hasbeen achieved recently with a vaginal gel containing one ARV,tenofovir. This hallmark study demonstrated partial protection,suggesting that achieving sustainable concentrations of an ARV atthe genital mucosal tissue is a crucial step toward efficacy.26

However, a later clinical trial (VOICE) in a different patientpopulation and administration schedule did not confirm the efficacyof protection.27 These results present the opportunity for nanotech-nology to improve the mucosal penetration and half-life of ARV.

To increase the epithelial penetration of ARV-based micro-bicides, specific mucoadhesive and non-mucoadhesive nanome-dicine formulations were investigated.28,29 The interaction of thenanomedicine with the mucus fluids covering the vaginal

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mucosal epithelium can work either as docking point or as barrierfor diffusion. The surface chemistry of the nanomedicinedetermines attraction/repulsion with mucin fibers, whereas thediameter controls their ability to “fit” within the mucin meshpores. In particular, positively charged polymers, such aschitosan, could increase mucoadhesion by ionic interactionwith negatively charged mucin chains. Furthermore, thiolmodification of chitosan enhanced mucoadhesion of thenanomedicine in comparison with chitosan.30 However, theuse of chitosan in a microbicide formulation is a concern,because it stimulates the mucosal translocation of HIV and otherviruses.28 Moreover, recent findings suggest that mucoadhesiveNPs can substantially alter the microstructure of mucus,highlighting the potential of mucoadhesive environmental orengineered NPs to disrupt mucus barriers and cause greaterexposure to foreign particles, including pathogens and otherpotentially toxic nanomaterials.31

Certain nanomedicines have an intrinsic activity to inhibit viralentry. For example, either empty or drug-loaded (2-RANTES,MC1220) liposomes showed partial protection against infectionin a macaque model after vaginal instillation.32,33 Liposomes arelipid vesicles with an aqueous core used to encapsulatehydrophilic drugs whereas hydrophobic and amphiphilic drugscan be solubilized within the phospholipid bilayers. Liposomesare either phagocytosed by macrophages or enter into the cells bymembrane–membrane fusion. The free-circulating liposomes arequickly cleared from after uptake by the reticuloendothelialsystem.34 Both polyvinyl pyrrolidone-coated silver NPs andmannose-coated gold NPs inhibited the entry of HIV into the hostcell.35,36 Nanomedicine applied as topical microbicide consistingof L-lysine dendrimers formulated as a carbomer gel (VivaGel®,Starpharma) showed dose-dependent resistance to viral challengein macaques.37 The mechanism of action is based on the surfacechemistry of the nanomedicine: The polylysine branches of thedendrimer are terminally derivatizedwith naphthalene disulfonategroups that are responsible for the direct interaction with HIVenvelope glycoproteins.38 The safety of Vivagel (SPL7013) hasbeen demonstrated in human subjects (Phase I).38

A vaccine that produces strong HIV-specific humoral andcellular immune responses might be desirable for the preventionof HIV infection. Intramuscular injection with gag and envpDNA adsorbed to the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substan-tially more potent in the induction of immune responses thancorresponding naked pDNA vaccines in mice, guinea pigs, andrhesus macaques.39 PLG microparticles were generally welltolerated in HIV-negative volunteers and env-specific CD4+ T-cell responses were detected after protein boosting.40 Strongneutralizing antibody responses against the homologous HIVwere present in the majority of vaccine recipients. However,unfortunately, neutralization breadth against heterologous HIVwas minimal.41

Immunotherapeutic nanomedicines

Immunotherapeutic nanomedicines are new, complex, multi-modular vaccines that provide superior therapeutic effects in

comparison with all previous approaches. Their physical size isusually over 50 nm, which is the approximate threshold ofimmune recognition.42 Soluble antigens, less than 50 nm in size,are generally not recognized by the immune system as particlesand are not immunogenic. In fact, the size ranges of immuno-therapeutic nanomedicines correspond to the size range ofviruses. Nature developed an effective and specific immunesurveillance against viruses. Consequently, triggering theimmune system with nanomedicines provides exceptionalimmunogenicity because the body considers nanomedicine as aharmful virus that needs to be eliminated.43 The best examples forthe superior immune recognition of nanomedicine are the humanPapilloma virus (HPV) vaccines, Gardasil, and Cervarix. Thesevaccines composed are from one surface protein (L1) of the HPVthat self-assemble to virus-like particles (VLP). These VLPs,morphologically similar to the wild type HPV, induce potentimmune responses in the absence of adjuvants. In contrast, the L1protein purified from bacteria remains soluble, does not assembleto VLPs, and does not induce immune responses.44 These VLPvaccines are safe and protect young uninfected people fromcancer. However, none of these VLPs was effective for thetreatment of HPV-associated cancer, because they unsuccessfullyinduce therapeutically beneficial T-cell responses.45

Creating “particulate vaccines” has recently been recognizedin the HIV field to improve the immunogenicity of small solubleantigens. This approach involves increasing the physical size ofthe antigen to the size of pathogens. There are so-called “natural”particulate vaccines, based on VLPs in the 40-nm range thatinduce both humoral and cellular immune responses againstHIV.46,47 HIV VLPs are essentially non-infective virusesconsisting of self-assembled viral envelope proteins withoutthe accompanying genetic material. A different approach is touse an adjuvant that increases the size of the antigen. One of theseveral proposed mechanisms of aluminum salts, the adjuvantapproved in the United States and the European Union, isattributed to their particulate nature; however, recently concernsare raised regarding their safety.47

An alternative approach is the use of a plasmid DNA (pDNA)that can express one or more antigens in the body. For example,pDNA is attractive for immunotherapeutic nanomedicinedevelopment because (i) its excellent safety profile, (ii)intracellularly expressed antigens are processed and presentedon the host MHC molecules, and (iii) recently improved large-scale manufacturing capabilities enable cost-effective produc-tion. Unfortunately, the very promising animal studies demon-strating the induction of immune responses with naked pDNAinjected intramuscularly or intradermally were not reproduced inhuman subjects. Possible reasons of the weak immunogenicity isthat the naked pDNA do not enter the cell and reach the nucleus,and/or the expressed soluble antigens are not recognized by theimmune system, similarly to the previously described soluble L1protein of the HPV.

Various biodegradable and nonbiodegradable polymeric andliposomal delivery systems have been explored for transform-ing HIV-antigens to synthetic NPs to increase their immuno-genicity and to protect them against extra- and intracellulardegradation.23,48,49 Targeting dendritic cells (DC) that areessential for initiating immune responses, can be achieved by

Table 1Immunotherapeutic nanomedicines developed for HIVAIDS

Features DermaVir NP77 Gold/DNA microparticle78 PLA/p24 NP79 Naked DNA80

Potential indications Therapeutic Prophylactic andtherapeutic

Prophylactic andtherapeutic

Prophylactic andtherapeutic

Dev. stage Phase II Preclinical Preclinical Phase IIAPI pDNA pDNA Protein pDNAImmune response Th1 Th1 and Th2 Th1 and Th2 Th1 and Th2Size 70-300 nm (virus size) 1-3 μm (bacteria size) ~500 nm (virus size) SolubleTargeting DC Mannose residues No No NoCellular entry Endocytosis by LC-s “Forced” entry Endocytosis by DCs NoPost-entry Targets API to the nucleus N/A N/A N/ADose 0.4 mg Up to 0.01 mg 0.01 mg Up to 8 mgAPI/Carrier ratio 1:1 1:1000 1:10 No carrierAPI content Exactly determined “Theoretical maximum” Exactly determined Exactly determinedAdmin. Topical, DermaPrep

(targets LC)Gene gun to keratinocytes(bystander LC)

Injection ofautologous DC

Needle andelectroporation

Repeated administration Yes Yes 1-3 times YesScalable Yes Yes No Yes

API: active pharmaceutical ingredient; PEIm: mannosylated polyethyleneimine; PLA: Poly(D,L-lactic acid); DC: Dendritic cells; LC: Langerhans cells,precursors of DCs.

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different nanomedicine size; N 100-nm nanomedicine target theperipheral immature DCs, and the smaller size ~20-nm – 50-nmnanomedicine drain to the lymph node resident DCs.50

Modification of the surface of the nanomedicine with DC-specific receptor ligands has shown to increase the targetingspecificity.51 However, several challenges, including crossingphysical barriers like the cell and nuclear membranes or adhesionto nontarget tissues still need to be overcome during thedevelopment of a synthetic delivery system. Table 1 comparesthe most advanced HIV-specific nanomedicines developed toimprove the immunogenicity of soluble antigens by formulationinto nano- or microparticles. These formulations generally havethree objectives: (i) increase the size of the antigens to either virusor bacteria size; (ii) protect the antigens from degradation; and(iii) target the antigens to DC. Most of the formulations achievethe first two objectives resulting improved immunogenicity.Targeting of DC are approached by adding ligands onto thesurface of the particles,51 using either very sophisticatedtechnologies including administering the NPs directly toautologous DC or developing new medical devices like genegun, electroporation instrument, microneedles and topical patches(Table 1). The main deficiency is the lack of “active targeting” ofthe vaccines to the DC and most importantly delivering theantigens to the compartment of the DC responsible for antigenpresentation. DermaVir nanomedicine differs from others byapplying multiple targeting elements to ensure active targeting ofDC and potent antigen presentation: (i) the polymer and thepDNA together forms a pathogen-like NP. The surface of theDermaVir NPs contains sugar residues that are important for theuptake by antigen-presenting cells, including DC and Langerhanscells; (ii) inside the cells the polymer protects the pDNA fromendosomal degradation and facilitate the delivery of the pDNA tothe nucleus. These steps are essential for potent expression ofDNA-encoded antigens. (iii) DermaVir nanomedicine is topicallyadministered with a new medical device (DermaPrep) thatsupports the loading of the NP into the epidermis, the proximityto activated Langerhans cells (the precursors of DC) (Table 1).

As nanomedicine products approach a pharmaceutical realitya number of issues need to be comprehensively addressedbeyond their clinical efficacy and safety to make them suitablefor the global market. These include (i) the selection of theresponding patient population by linking the mechanism ofaction of the nanomedicine with clinical efficacy; (ii) thedevelopment of commercial manufacturing technologies withrelevant quality-control assays; and (iii) cost of a dose andlogistics of treatment. Therefore, during the development ofnanomedicines we need to demonstrate the mechanism of action,develop scalable manufacturing technologies, optimize theclinical dose, minimize the API (active pharmaceutical ingredi-ent) content and the amount of “carriers” near the API, anddevelop methods for targeted and safe administration.

DermaVir is the first nanomedicine developed for thetreatment of HIV/AIDS that has demonstrated encouragingPhase II clinical safety, immunogenicity, and efficacy results.52

We provide here a detailed overview of the development of thetechnology of DermaVir, because it represents a promisingimmunotherapeutic nanomedicine platform for the cure ofHIV/AIDS.

DermaVir immunotherapeutic nanomedicine

DermaVir features three soft-particle nanoelements accord-ing to the classification proposed by Tomalia53 (Figure 1). TheAPI is a pDNA (S-6) that expresses 15 HIV proteins. Theseproteins assemble to a complex virus-like particle (S-5).54 Todeliver the pDNA to DC and achieve effective proteinexpression the pDNA is condensed in a “core” and packagedinto a mannosylated polyethylenimine “envelope” (S-3)formingNPs of 70-300 nm in a buffered solution.55,56 The immunizationprocedure is performed topically with DermaPrep device. Herewe present a rational, target product profile-oriented design ofDermaVir nanomedicine including the importance of detailed

Figure 1. Nano-elements in DermaVir. Soft particle nano-element categories53: S-6: plasmid DNA; S-5: viruses; S-3: polymeric micelles.

Figure 2. Key features of DermaVir nanomedicine. (A) One single pDNA encoding 15 HIV antigens serves as the API54; (B) Expression of VLP+ (visualizedby transmission electron microscopy); (C) Effect of the degree of association between the pDNA and PEIm on the DermaVir nanomedicine.56 Optimalassociation led to a stable “pathogen-like” NP (visualized by atomic force microscopy) that escaped from endosomal degradation, released the pDNA in thecytosol near the nucleus, where the pDNA-encoded antigens are expressed.

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physicochemical analysis of the components and their effect onthe product quality.

Nanoelements in the API

DermaVir's active pharmaceutical ingredient (API) providesantigens for the induction of HIV-specific immune responses. Theobjective of the antigen design was to preserve the structure and the

broad epitope content of the wild-type HIV and to create a safeimmunogen.54 We constructed a single pDNA to drive theexpression of 15 HIV proteins in a cell (Figure 2, A). Theseproteins self assemble to replication-, reverse transcription- andintegration-defective complex virus-like particles (VLP+)(Figure 2, B). The pDNA is inherently safe because irreversiblemolecular modifications prevent the replication and integrationof the VLP+. The expression of 15 HIV proteins from the single

Figure 3. Mechanism of action of DermaVir immunotherapy: 1. Targeted delivery of the “pathogen-like” NPs to Langerhans cells using DermaPrep52; 2.Cellular uptake; 3. Intracellular processing of NPs; 4. Antigen expression and processing; 5. Antigen presentation in the lymph nodes and priming of naïve CD4+

and CD8+ T cells66; 6. HIV-specific central memory T cells with high proliferation capacity.69

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pDNA supports the presentation of the highest number of HIVepitopes and the induction of HIV-specific T-cell responses withthe broadest specificity. The expressed VLP+ is structurallyauthentic to the wild-type HIV. Beyond inducing T-cellresponses, this antigen might be suitable for inducing neutral-izing antibodies against viral and cellular antigens naturallyoccurring on the surface of HIV or structures present only duringbudding or entry.57 Such a multifaceted immune response is aunique feature of pDNA-based vaccines expressing an authentic-looking HIV.57

Synthetic nanomedicine formulation

The objective of DermaVir nanomedicine formulation was toexpress the API in DCs, because DC-mediated antigenpresentation is essential to boost T-cell immunity in HIV-infected people. pDNA delivery to DCs is a complex challengeinvolving DC binding, antigen uptake, expression, processing,and presentation to naïve T cells.58 We designed a “pathogen-like” nanomedicine to encapsulate the pDNA within thepositively charged linear mannobiosylated polyethylenimine(PEIm). DermaVir nanomedicine is similar in size, appearance,and DNA-delivery features to viruses that naturally evolved todeliver genetic materials to cells. DermaVir's PEIm “envelope”protects the condensed pDNA “core” from extra- and intracel-lular degradations. Its particle size (70 nm – 300 nm) is optimalfor receptor-mediated endocytosis into cells, and it has sufficientstability to support the release of the pDNA from the endosomalcompartment and the delivery of the pDNA to the nucleus. Thesefeatures, unique for DermaVir nanomedicine, are essential forpotent expression of antigens.,54,59 Consequently, the biological

activity of DermaVir is dependent on its inherent structure andbinding,55,60,61 (Figure 2, C).

Langerhans cell-targeting nanomedicine administration

The objective was to deliver the nanomedicine in vivo to thelymph-node DCs, the location where T-cell responses areoriginated in the body. We developed DermaPrep, the first LC-targeting nanomedicine administration device. DermaPrepemploys a skin preparation method that interrupts the stratumcorneum facilitating nanomedicine penetration and providing theessential “danger” signal to the LCs residing just below thisprotective layer.62,63 Once activated, LCs are naturally lookingfor pathogens and capturing the pathogen-like DermaVirnanomedicine applied to the prepared skin surface under asemi-occlusive patch. The main advantage of DermaPrep is thenatural targeting of a large number of LCs (8 million) that form ahorizontal 900 to 1,800 cells/mm2 network under the skinsurface.64 After DermaVir has been captured, LCs mature toDCs and migrate to the local lymph nodes. Here DCs expresspDNA-encoded antigens and present most HIV epitopes to thepassing naïve T cells. HIV-specific precursor/memory T cellsprimed by DCs further differentiate into HIV-specific effector Tcells circulating out of the lymph node to seek virus-infectedtargets. Each killer effector cell can destroy several HIV-infectedcells (Figure 3).

The human proof of concept

Prior to human clinical evaluation, the novel mechanism ofaction and the antiviral efficacy of DermaVir was demon-strated in macaques, some of them with AIDS. DermaVir

Figure 4. Single DermaVir immunization boosts long-lasting HIV-specific central memory T cells in HIV-infected individuals.69 Patients on cART received 0.1,0.4, and 0.8 mg pDNA doses in DermaVir nanomedicine. Increase of HIV-specific central memory T cells from baseline (net PHPC counts/106 PBMC) isshown. Gag, Tat and Rev represents three of 15 DermaVir-expressed antigens.

Table 2Features of nanomedicines developed toward the cure of HIV in comparisonwith the current state of art treatment of HIV/AIDS

Features/Approach Immunotherapy Activators cART

Therapeutic target HIV-expressingcells

Resting cells HIV life cycle

Treatment benefit Slow & Durable Rapid Rapid & TransientSafety Transient, skin Unknown Cumulative,

systemicAdministration

scheduleYearly (4x) Short period Daily

HIV RNA Slow decrease Increase Rapid decreaseHIV immunity Boosting No effect DecreasingHIV-infected cells Killing No effect No effectCure Remission Eradication No

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immunization targeted and expressed the pDNA-encodedantigens into the DCs of the lymph nodes (Figure 3). TheseDermaVir-expressing DCs primed naïve T cells and inducedboth HIV-specific helper and cytotoxic T cells.65 Importantly,immune responses following topical DermaVir immunizationwere similar to ex vivo immunization with cultured DCs.66

DermaVir immunotherapy suppressed viral load and providedsurvival benefit for chronically SIV251-infected macaques.67

Repeated DermaVir immunizations in the absence of cARTtransiently suppressed virus replication that led to improve-ment of median survival time from 18 to 38 weeks incomparison with no treatment.67 In other trials, DermaViradministered in combination with cART boosted SIV-specificT cells that possessed significant antiretroviral activity in bothchronically infected and macaques with AIDS.67 Theseprimate experiments provided the rationale to investigateDermaVir immunizations in HIV-infected human subjects.

The Phase I dose-escalation study was designed to evaluatethe safety and immunogenicity of a single DermaVir immuni-zation in nine HIV-infected subjects on fully suppressivecART.68 Increasing DermaVir doses were administered byDermaPrep simultaneously on two, four, and eight skin siteslocated on the back and the tight. The LCs of these skin sitesdrain into four different lymph nodes. Low-dose DermaVircontained 0.1mg DNA targeted to two lymph nodes. Medium-and high-dose DermaVir contained 0.4 mg and 0.8 mg DNAtargeted to four lymph nodes. DermaVir-associated side effectswere limited to the skin, mild, transient and not dose dependent.Boosting of HIV-specific effector CD4+ and CD8+ T cellsexpressing IFN-gamma and IL2 was detected against severalHIV antigens in every subject of the medium dose cohort. Thestriking result was the dose-dependent expansion of HIV-specific precursor/memory T cells with high proliferationcapacity,68,69 (Figure 4). The findings suggest that DermaVircould boost robust and long-lasting memory T-cell responses to15 HIV antigens. We concluded that for durable immunereactivity repeated DermaVir immunizations might be required

because the frequency of DermaVir-boosted HIV-specific T cellsdecreased during the 48-week follow-up period (Figure 4).

A Phase I/II clinical trial conducted in several USA clinicalcenters was designed to investigate repeated administrations(three times) of escalating DermaVir doses (0.2, 0.4, and 2 × 0.4mg pDNA) or placebo on 24 HIV-infected adults receiving fullysuppressive cART. The incidence of adverse events was similaracross groups, suggesting that DermaVir was as safe as aplacebo.70 Immunogenicity data demonstrated the boosting ofHIV-specific precursor/memory T cells with high proliferativecapacity.69 The highest frequency of HIV-specific memory Tcells was induced in the 0.4 mg dose group.70 The results of thistrial were consistent with the macaque and Phase I studies andconfirmed the excellent safety and immunogenicity features ofDermaVir immunizations in patients on cART.

The Phase II randomized, multicenter, and placebo-con-trolled clinical trial was designed to evaluate the safety and totest the immunogenicity and antiviral efficacy of repeatedDermaVir immunizations in the absence of cART. Thirty-sixHIV-infected, treatment naïve adults were randomized toreceive one of three DermaVir doses (0.2, 0.4, or 0.8 mg

Figure 5. Nanomedicine applications toward the cure of HIV/AIDS: (A) Untreated HIV infection is characterized by high viral load, high amount of HIV-infected cells in the reservoirs and the disease is partially controlled by the immune system alone for approximately 15 years. (B) cART is effective in potentand durable suppression of HIV RNA to b50 copies/mL that is required to avoid the development of drug-resistant mutants. However, cART does noteliminate latently infected HIV-infected cells. In addition, HIV-specific T-cell responses diminish in patients treated optimally with fully suppressive cART.Consequently, HIV rebounds even after short interruption of therapy. (C) DermaVir immune intensification of patients treated with optimal cARTdemonstrated the maintenance of undetectable load and induction of long-lasting and broadly specific HIV-specific T-cell responses.68 These T-cell responses,prior to reactivation of the latently infected cells, are essential for the clearance of latent HIV from the reservoirs and eradication of HIV.15,16 (D) At one point,at least theoretically, cART could be safely interrupted because the HIV-specific T cells are fully reconstituted. This stage of the disease the immune systemalone will control the virus (remission) in a manner similar to that of the Berlin patient and the elite controllers.13,14 Eradication of the virus cannot be achievedin the absence of reactivation of latent HIV, because the immune system cannot recognize the latently infected cells where the integrated provirus istranscriptionally silent.

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pDNA) or placebo at StudyWeeks 0, 6, 12, and 18. The primaryendpoint was safety at Week 24 and secondary endpoints wereHIV RNA and immunogenicity.71 Only one Grade 2 adverseevent occurred in the low-dose cohort judged to be possiblyrelated to DermaVir treatment, confirming the excellent safetyfeatures of DermaVir immunizations in a different patientpopulation. Based on induction of HIV-specific memory/precursor T cell, the 0.4 mg DermaVir dose was superior tothe others. In this group the medium HIV RNA significantlydecreased by 70% in comparison with the placebo. Viral loadsuppression occurred slowly, as predicted by DermaVirmechanism of action, similarly to cancer vaccines.71 Theseresults were consistent with the macaque and previous clinicalstudies and confirmed the safety and immunogenicity andantiviral activity of repeated DermaVir immunizations.

Perspectives of nanomedicines towards the cure ofHIV/AIDS

More than 30 ARV and drug combinations are currentlyavailable to achieve long-term suppression of HIV RNA to N 50copies/mL. Additional potency, accomplished by ARV inten-sification, did not provide additional treatment benefits15,72-76

Nanomedicines developed for the cure of HIV might overcomethe following limitations of cART: (i) “Activators” targetlatently infected cells to reactivate HIV and flush the reservoirs;and (ii) DermaVir reconstitutes HIV-specific immune responsesthat decreased during optimal cART and deplete HIV-infectedcells. In contrast, current ARVs reduce viral load by inhibitingone step in HIV life cycle (Table 2). In comparison with ARV,

the antiviral activity of immunotherapies are slower and lesspotent, because recognizing and killing of infected cells byHIV-specific T cells takes more time than blocking HIVreplication by drugs. The effectiveness of killing of infectedcells is revealed by their capacity to manage the infection forapproximately 15 years in the absence of cART. Therefore, 0.5log reduction of HIV RNA in 24 weeks, demonstrated withDermaVir, should be sufficient to decrease the amount of HIV-infected cells that are not eliminated by cART. In comparisonwith ARV “Activators” might be taken after immune intensi-fication for a short period of time to flush the reservoirs.Consequently, the activated HIV producing cells will beeliminated by cytotoxic T cells and reservoirs will decrease orbe eliminated (eradication).

To demonstrate the eradication of all HIV-infected cells of apatient seems not to be feasible. The other approach is to achieveremission (functional cure) of HIV/AIDS characterized by theabsence of HIV rebound after cART interruption (Figure 5). Thethree different mechanisms of action of ARV, Activators, andDermaVir are complementary, suitable to achieve remission: Therapid and potent viral load reduction with cART is essential toblock HIV replication and reach undetectable viral load. Afterthat DermaVir immune intensification could address what drugintensification could not achieve: (i) killing of HIV-infected cellsthat remained in reservoirs after optimal cART; and (ii) boostingHIV-specific T cells to reconstitute immune responses thatsuppressed during cART. After immune intensification, patientsmight be treated with “Activators” to achieve rapid reactivationof the virus. That point DermaVir-induced HIV-specific T cellswould kill the latently infected cells and decrease the reservoirs.Because the immune system is slow to kill infected cells, it will

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take time to substantially decrease the infected cells from thereservoirs and fully reconstitute HIV-specific immune responses.We envision that repeated DermaVir immune intensification incombination with “Activators” could eliminate significantamount of infected cells from the reservoirs. Consequently,patients could decrease ARV exposure, and their immune systemcould maintain the HIV RNA level under the detection limit.During remission, demonstrated by undetectable HIV RNA afterinterruption of cART, maintenance of high-level T-cell re-sponses might require the repeated administration of DermaVirimmunotherapy (e.g., 4 times yearly).

Potential advantages of immunotherapy in comparison withcART include its excellent safety profile, potentially higherspecificity, the longevity of an immune response, and likely costsavings as well as at least theoretically the chance to achieve acure (remission). However, despite decades of research, notherapeutic vaccine has reached the market. Challenges include(i) the disease mechanisms and interaction with the immunesystem; (ii) immune escape from T-cell recognition based on thehigh genetic diversity of the virus and the HLA diversity of thehost; and (iii) the shortage of funding in comparison withprophylactic vaccine development.

Any treatment that can eradicate the virus from infected patientsor cure the disease (remission) would have a huge commercialopportunity. Nanotechnology offers opportunities to develop newtreatment approaches that could contribute to the cure of HIV/AIDS. To affect public health the new approaches mustsignificantly improve the health of HIV-infected patients atlower cost than the that of the current cART. One concernregarding to the systemic nanomedicine administrations is toxicity,because a new immunotoxicity was observed during the treatmentof cancer with liposomal nanomedicine formulations.43 Would theimprovement in half-life or the increase of bioavailability of thepresently used ARV justify a new potential toxicity? Anotherconcern is an increase in price due to the need of sophistication inmanufacturing and quality control that is usually accompanied bymore difficult scale-up and higher production costs. We envisionthat disease modifying topical nanomedicines developed forimmunotherapy and microbicides might have the safety, efficacy,and cost features to contribute to the cure of HIV/AIDS andsignificantly improve public health.

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