POTENTIAL CLINICAL RELEVANCE

Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 28 38

Review Article

nanomedjournal.com

Nanomedicine applications towards the cure of HIV

Julianna Lisziewicz, PhD a, b,, Enik R. Tke, PhD a
Genetic Immunity Kft., Budapest, Hungary Genetic Immunity Inc., McLean, Virginia Received 10 February 2012; accepted 16 May 2012
b a

Abstract Combination antiretroviral therapy (cART) successfully suppresses HIV replication. However, daily and lifelong treatment is necessary to manage patient illness because cART neither eradicates infected cells from reservoirs nor reconstitutes HIV-specific immunity that could kill infected 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 by boosting 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-cell responses and reduces viral load in comparison with placebo. DermaVir and the nanomedicine pipelines have the potential to improve the health 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 health challenge. In this review, applications of nanomedicine- based technologies are discussed in the context of HIV treatment, including virus elimination by activation of latently infected CD4+ T-cells; infected cell elimination in the reservoirs by boosting HIV-specific T cells, and by 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 identied as the cause of AIDS one year after the virus was isolated. 1-4 Since that time HIV/AIDS has become the leading infectious killer affecting more than 33 million people worldwide. 5 HIV/AIDS is treated with the combination of 25 antiretroviral drugs (ARV) that are divided into six classes according to their interference with HIV life-cycle: fusion/entry inhibitors, integrase inhibitors, protease inhibitors, nonnucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptase inhibitors, and multidrug combination products. HIV/ AIDS treatment using any single class of ARV has not been efcient in controlling infection due to the development of resistant strains of the virus. Hence, three or more ARVs are used in combination (combination antiretroviral therapy, cART) to treat the disease. Currently available cART is potent in suppressing HIV replication and effective in decreasing HIV RNA level below the
Dr. Julianna Lisziewicz holds shares in Genetic Immunity. This work was supported by grants: HIKC05 and DVCLIN01 of the National Ofce for Research 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

limit of detection (50 copies/mL) with only minimum side effects. Long-term cART decreased morbidity and mortality associated with HIV infection. 6 Key products used for the treatment of the majority of patients, such as Atripla (efavirenz/tenofovir/ emtricitabine), Truvada (tenofovir/emtricitabine), Sustiva (efavirenz), Kaletra (lopinavir/ritonavir), Reyataz (atazanavir), and Isentress (raltegravir), satisfy the ARV demand in the market. Successful management of HIV-infected patients is challenging, requiring highly experienced physicians due to resistance and overlapping toxicities of the complex daily ARV regimen that must be taken for the rest of the patient's life. However, even optimal cART, characterized by suppression of viral load to undetectable levels for years, has not provided a cure for the disease. Patients on optimal cART have twelve-year shorter life expectancy than HIV-negative people, 7,8 In addition, increased AIDS-related and non-AIDS-related morbidity and mortality have been described in a signicant proportion of individuals on optimal cART due to the lack of normalization of their CD4 + Tcell counts. 9 Optimal cART failed to decrease the viral reservoirs, especially in the gut mucosa, where the residual low-level viral replication may be the cause of persistent immune activation that facilitates the progression to AIDS and death. 10 One barrier of cure is the stable latent reservoirs of HIV-infected

Please cite this article as: Lisziewicz J., Tke E.R., Nanomedicine applications towards the cure of HIV. Nanomedicine: NBM 2013;9:28-38, http:// dx.doi.org/10.1016/j.nano.2012.05.012

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resting memory T cells that are able to produce HIV after cellular activation. HIV-producing cells in the reservoirs that are not eliminated by ARV would be susceptible to immune clearance, but long-term optimal cART diminish HIV-specic T-cell responses. 11 Therefore, the immune systems of successfully treated HIV-infected people are not prepared to decrease viral reservoirs and control the virus replication. Currently, there are two major unmet needs in HIV treatment. They are (i) simplication of the daily treatment and (ii) diseasemodifying therapies. Large pharmaceutical companies focus on the treatment simplication approach with single-tablet cART regimen using conventional drug formulations. Nanomaterials are exploited for the development of innovative disease modifying therapies aiming to prevent infections, stop disease progression and cure HIV/AIDS. We found it curious that the two different treatment strategies for the cure of HIV/AIDS were rst implemented in Berlin, Germany by the publication of two exceptional case reports. Eradication of the virus was demonstrated after bone marrow transplantation with donor cells resistant to HIV infection. 12 We have described immune control of HIV in the case of the rst Berlin patient, whose immune system was boosted by his own virus emerging during short interruptions of cART. 13 This work led to the identication of elite controllers representing a model for remission as cure. These individuals have large numbers of cells containing replicationcompetent HIV controlled by the cellular arm of the immune system. 14 Along these lines, Deeks and coworkers recently have shown an inverse correlation between the frequency of HIV-specic T cells in the gut and the size of the reservoir, suggesting that boosting of T-cell responses might contribute to the clearance of latent HIV. 15 Siliciano and his team have recently demonstrated that boosting of HIV-specic T-cell responses prior to reactivating latent HIV will be essential for eradication of the virus. 16 Here we review novel disease-modifying treatment approaches that exploit nanomaterials to cure HIV/AIDS. The rst section discusses the experiments that showed how nanomedicines activate resting cells to ush HIV and improve pharmacokinetic features of ARV. The second section describes nanomedicines application as microbicides to be used for prevention of HIV infection. In the third section we provide detailed description on the mechanism of action and clinical results of immune-modulating nanomedicines, including DermaVir therapeutic vaccine. In the nal chapter we discuss how nanomedicines could contribute to the cure of HIV/AIDS.

concentrated mainly in lymphoid tissues, testes, the gut, and the central nervous system. A new eradication approach is to target drugs to the viral reservoirs and force the activation of latently infected cells to virus production. Consequently, these productively infected cells can be recognized and eliminated by the immune system. To target and activate primary human CD4 + T cells, a nanomedicine formulation of a protein kinase C activator, bryostatin-2 (LNP-Bry), was studied in a humanized mouse model. LNP-Bry was also loaded with the protease inhibitor nelnavir producing a nanomedicine capable of both activating latent virus and inhibiting viral spread. 20 To target antiretroviral drugs to the lymphoid organs, a pHdependent nanomedicine formulation of indinavir was investigated in macaques. 21 This nanomedicine formulation increased indinavir concentration in the lymph nodes. 22 These 50 80 nm nanoparticles (NPs) were trapped in lymph nodes as they circulated through the lymphatic system. The authors concluded that the targeting effect of NPs to the lymphoid tissues was mainly particle-size dependent. Bioavailability is a major challenge in drug development. For example, the protease inhibitors indinavir, ritonavir, and nevirapine have different, but still acceptable, oral bioavailability of 39%, 60% 70%, and 92%, respectively. In contrast, saquinavir possesses only 4% of bioavailability that inuenced the efcacy and toxicity of this drug, and the company decided to withdraw the drug from the market after obtaining market authorization. 23 To improve bioavailability nanomedicine formulation of efavirenz (EFV) was investigated. The drug was incorporated into the core of linear and branched poly(ethylene oxide)poly(propylene oxide) block copolymer micelles. In rats, EFV-nanomedicine had up to 88% higher plasma concentrations in comparison with EFV suspensions. 24 ARV resistance occurs often in the case of non-adherence or when the viral load is insufciently suppressed. The presence of resistant HIV then limits the treatment options of patients. To improve adherence with longer dosing intervals, the nanomedicine formulation of rilpivirine was investigated in rats and dogs. This nanomedicine demonstrated a sustained and dose-proportional release over 2 months and a signicant half-life enhancement in comparison with the 38 hours of the free drug. 25

Preventive nanomedicines Topical microbicides represent a promising strategy to prevent vaginal and rectal HIV transmission. Clinical proof of concept has been achieved recently with a vaginal gel containing one ARV, tenofovir. This hallmark study demonstrated partial protection, suggesting that achieving sustainable concentrations of an ARV at the genital mucosal tissue is a crucial step toward efcacy. 26 However, a later clinical trial (VOICE) in a different patient population and administration schedule did not conrm the efcacy of protection. 27 These results present the opportunity for nanotechnology to improve the mucosal penetration and half-life of ARV. To increase the epithelial penetration of ARV-based microbicides, specic mucoadhesive and non-mucoadhesive nanomedicine formulations were investigated. 28,29 The interaction of the nanomedicine with the mucus uids covering the vaginal

Nanomedicine drug formulations Application of nanotechnology to antiretroviral drug delivery holds promise in the cure of HIV, because it could modify tissue distribution by targeting drugs to HIV reservoirs and by increasing the half-lives of drugs. The use of nano-delivery systems has been extensively reviewed previously. 17-19 Therefore we only highlight some of the recent advances in the eld that could play a role in achieving a cure. Optimal cART cannot eradicate HIV. A portion of the virus of patients successfully treated with cART resides in latent reservoirs within memory CD4 + T cells and macrophages

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mucosal epithelium can work either as docking point or as barrier for diffusion. The surface chemistry of the nanomedicine determines attraction/repulsion with mucin bers, whereas the diameter controls their ability to t within the mucin mesh pores. In particular, positively charged polymers, such as chitosan, could increase mucoadhesion by ionic interaction with negatively charged mucin chains. Furthermore, thiol modication of chitosan enhanced mucoadhesion of the nanomedicine in comparison with chitosan. 30 However, the use of chitosan in a microbicide formulation is a concern, because it stimulates the mucosal translocation of HIV and other viruses. 28 Moreover, recent ndings suggest that mucoadhesive NPs can substantially alter the microstructure of mucus, highlighting the potential of mucoadhesive environmental or engineered NPs to disrupt mucus barriers and cause greater exposure to foreign particles, including pathogens and other potentially toxic nanomaterials. 31 Certain nanomedicines have an intrinsic activity to inhibit viral entry. For example, either empty or drug-loaded (2-RANTES, MC1220) liposomes showed partial protection against infection in a macaque model after vaginal instillation. 32,33 Liposomes are lipid vesicles with an aqueous core used to encapsulate hydrophilic drugs whereas hydrophobic and amphiphilic drugs can be solubilized within the phospholipid bilayers. Liposomes are either phagocytosed by macrophages or enter into the cells by membranemembrane fusion. The free-circulating liposomes are quickly cleared from after uptake by the reticuloendothelial system. 34 Both polyvinyl pyrrolidone-coated silver NPs and mannose-coated gold NPs inhibited the entry of HIV into the host cell. 35,36 Nanomedicine applied as topical microbicide consisting of L-lysine dendrimers formulated as a carbomer gel (VivaGel, Starpharma) showed dose-dependent resistance to viral challenge in macaques. 37 The mechanism of action is based on the surface chemistry of the nanomedicine: The polylysine branches of the dendrimer are terminally derivatized with naphthalene disulfonate groups that are responsible for the direct interaction with HIV envelope glycoproteins. 38 The safety of Vivagel (SPL7013) has been demonstrated in human subjects (Phase I). 38 A vaccine that produces strong HIV-specic humoral and cellular immune responses might be desirable for the prevention of HIV infection. Intramuscular injection with gag and env pDNA adsorbed to the surface of cationic poly(lactidecoglycolide) (PLG) microparticles were shown to be substantially more potent in the induction of immune responses than corresponding naked pDNA vaccines in mice, guinea pigs, and rhesus macaques. 39 PLG microparticles were generally well tolerated in HIV-negative volunteers and env-specic CD4 + Tcell responses were detected after protein boosting. 40 Strong neutralizing antibody responses against the homologous HIV were present in the majority of vaccine recipients. However, unfortunately, neutralization breadth against heterologous HIV was minimal. 41

Immunotherapeutic nanomedicines Immunotherapeutic nanomedicines are new, complex, multimodular vaccines that provide superior therapeutic effects in

comparison with all previous approaches. Their physical size is usually over 50 nm, which is the approximate threshold of immune recognition. 42 Soluble antigens, less than 50 nm in size, are generally not recognized by the immune system as particles and are not immunogenic. In fact, the size ranges of immunotherapeutic nanomedicines correspond to the size range of viruses. Nature developed an effective and specic immune surveillance against viruses. Consequently, triggering the immune system with nanomedicines provides exceptional immunogenicity because the body considers nanomedicine as a harmful virus that needs to be eliminated. 43 The best examples for the superior immune recognition of nanomedicine are the human Papilloma virus (HPV) vaccines, Gardasil, and Cervarix. These vaccines composed are from one surface protein (L1) of the HPV that self-assemble to virus-like particles (VLP). These VLPs, morphologically similar to the wild type HPV, induce potent immune responses in the absence of adjuvants. In contrast, the L1 protein puried from bacteria remains soluble, does not assemble to VLPs, and does not induce immune responses. 44 These VLP vaccines are safe and protect young uninfected people from cancer. However, none of these VLPs was effective for the treatment of HPV-associated cancer, because they unsuccessfully induce therapeutically benecial T-cell responses. 45 Creating particulate vaccines has recently been recognized in the HIV eld to improve the immunogenicity of small soluble antigens. This approach involves increasing the physical size of the antigen to the size of pathogens. There are so-called natural particulate vaccines, based on VLPs in the 40-nm range that induce both humoral and cellular immune responses against HIV. 46,47 HIV VLPs are essentially non-infective viruses consisting of self-assembled viral envelope proteins without the accompanying genetic material. A different approach is to use an adjuvant that increases the size of the antigen. One of the several proposed mechanisms of aluminum salts, the adjuvant approved in the United States and the European Union, is attributed to their particulate nature; however, recently concerns are 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 nanomedicine development because (i) its excellent safety prole, (ii) intracellularly expressed antigens are processed and presented on the host MHC molecules, and (iii) recently improved largescale manufacturing capabilities enable cost-effective production. Unfortunately, the very promising animal studies demonstrating the induction of immune responses with naked pDNA injected intramuscularly or intradermally were not reproduced in human subjects. Possible reasons of the weak immunogenicity is that the naked pDNA do not enter the cell and reach the nucleus, and/or the expressed soluble antigens are not recognized by the immune system, similarly to the previously described soluble L1 protein of the HPV. Various biodegradable and nonbiodegradable polymeric and liposomal delivery systems have been explored for transforming HIV-antigens to synthetic NPs to increase their immunogenicity and to protect them against extra- and intracellular degradation. 23,48,49 Targeting dendritic cells (DC) that are essential for initiating immune responses, can be achieved by

J. Lisziewicz, E.R. Tke / Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 2838 Table 1 Immunotherapeutic nanomedicines developed for HIVAIDS Features Potential indications Dev. stage API Immune response Size Targeting DC Cellular entry Post-entry Dose API/Carrier ratio API content Admin. Repeated administration Scalable DermaVir NP 77 Therapeutic Phase II pDNA Th1 70-300 nm (virus size) Mannose residues Endocytosis by LC-s Targets API to the nucleus 0.4 mg 1:1 Exactly determined Topical, DermaPrep (targets LC) Yes Yes Gold/DNA microparticle 78 Prophylactic and therapeutic Preclinical pDNA Th1 and Th2 1-3 m (bacteria size) No Forced entry N/A Up to 0.01 mg 1:1000 Theoretical maximum Gene gun to keratinocytes (bystander LC) Yes Yes PLA/p24 NP 79 Prophylactic and therapeutic Preclinical Protein Th1 and Th2 ~500 nm (virus size) No Endocytosis by DCs N/A 0.01 mg 1:10 Exactly determined Injection of autologous DC 1-3 times No Naked DNA 80

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Prophylactic and therapeutic Phase II pDNA Th1 and Th2 Soluble No No N/A Up to 8 mg No carrier Exactly determined Needle and electroporation Yes Yes

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

different nanomedicine size; N 100-nm nanomedicine target the peripheral immature DCs, and the smaller size ~20-nm 50-nm nanomedicine drain to the lymph node resident DCs. 50 Modication of the surface of the nanomedicine with DCspecic receptor ligands has shown to increase the targeting specicity. 51 However, several challenges, including crossing physical barriers like the cell and nuclear membranes or adhesion to nontarget tissues still need to be overcome during the development of a synthetic delivery system. Table 1 compares the most advanced HIV-specic nanomedicines developed to improve the immunogenicity of soluble antigens by formulation into nano- or microparticles. These formulations generally have three objectives: (i) increase the size of the antigens to either virus or bacteria size; (ii) protect the antigens from degradation; and (iii) target the antigens to DC. Most of the formulations achieve the rst two objectives resulting improved immunogenicity. Targeting of DC are approached by adding ligands onto the surface of the particles, 51 using either very sophisticated technologies including administering the NPs directly to autologous DC or developing new medical devices like gene gun, electroporation instrument, microneedles and topical patches (Table 1). The main deciency is the lack of active targeting of the vaccines to the DC and most importantly delivering the antigens to the compartment of the DC responsible for antigen presentation. DermaVir nanomedicine differs from others by applying multiple targeting elements to ensure active targeting of DC and potent antigen presentation: (i) the polymer and the pDNA together forms a pathogen-like NP. The surface of the DermaVir NPs contains sugar residues that are important for the uptake by antigen-presenting cells, including DC and Langerhans cells; (ii) inside the cells the polymer protects the pDNA from endosomal degradation and facilitate the delivery of the pDNA to the nucleus. These steps are essential for potent expression of DNA-encoded antigens. (iii) DermaVir nanomedicine is topically administered with a new medical device (DermaPrep) that supports the loading of the NP into the epidermis, the proximity to activated Langerhans cells (the precursors of DC) (Table 1).

As nanomedicine products approach a pharmaceutical reality a number of issues need to be comprehensively addressed beyond their clinical efcacy and safety to make them suitable for the global market. These include (i) the selection of the responding patient population by linking the mechanism of action of the nanomedicine with clinical efcacy; (ii) the development of commercial manufacturing technologies with relevant quality-control assays; and (iii) cost of a dose and logistics of treatment. Therefore, during the development of nanomedicines we need to demonstrate the mechanism of action, develop scalable manufacturing technologies, optimize the clinical dose, minimize the API (active pharmaceutical ingredient) content and the amount of carriers near the API, and develop methods for targeted and safe administration. DermaVir is the rst nanomedicine developed for the treatment of HIV/AIDS that has demonstrated encouraging Phase II clinical safety, immunogenicity, and efcacy results. 52 We provide here a detailed overview of the development of the technology of DermaVir, because it represents a promising immunotherapeutic nanomedicine platform for the cure of HIV/AIDS.

DermaVir immunotherapeutic nanomedicine DermaVir features three soft-particle nanoelements according to the classication proposed by Tomalia 53 (Figure 1). The API is a pDNA (S-6) that expresses 15 HIV proteins. These proteins assemble to a complex virus-like particle (S-5). 54 To deliver the pDNA to DC and achieve effective protein expression the pDNA is condensed in a core and packaged into a mannosylated polyethylenimine envelope (S-3)forming NPs of 70-300 nm in a buffered solution. 55,56 The immunization procedure is performed topically with DermaPrep device. Here we present a rational, target product prole-oriented design of DermaVir nanomedicine including the importance of detailed

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Figure 1. Nano-elements in DermaVir. Soft particle nano-element categories 53: 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 API 54; (B) Expression of VLP + (visualized by transmission electron microscopy); (C) Effect of the degree of association between the pDNA and PEIm on the DermaVir nanomedicine. 56 Optimal association led to a stable pathogen-like NP (visualized by atomic force microscopy) that escaped from endosomal degradation, released the pDNA in the cytosol near the nucleus, where the pDNA-encoded antigens are expressed.

physicochemical analysis of the components and their effect on the product quality. Nanoelements in the API DermaVir's active pharmaceutical ingredient (API) provides antigens for the induction of HIV-specic immune responses. The objective of the antigen design was to preserve the structure and the

broad epitope content of the wild-type HIV and to create a safe immunogen. 54 We constructed a single pDNA to drive the expression of 15 HIV proteins in a cell (Figure 2, A). These proteins self assemble to replication-, reverse transcription- and integration-defective complex virus-like particles (VLP + ) (Figure 2, B). The pDNA is inherently safe because irreversible molecular modications prevent the replication and integration of the VLP +. The expression of 15 HIV proteins from the single

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Figure 3. Mechanism of action of DermaVir immunotherapy: 1. Targeted delivery of the pathogen-like NPs to Langerhans cells using DermaPrep 52; 2. Cellular uptake; 3. Intracellular processing of NPs; 4. Antigen expression and processing; 5. Antigen presentation in the lymph nodes and priming of nave CD4 + and CD8 + T cells 66; 6. HIV-specic central memory T cells with high proliferation capacity. 69

pDNA supports the presentation of the highest number of HIV epitopes and the induction of HIV-specic T-cell responses with the broadest specicity. The expressed VLP + is structurally authentic to the wild-type HIV. Beyond inducing T-cell responses, this antigen might be suitable for inducing neutralizing antibodies against viral and cellular antigens naturally occurring on the surface of HIV or structures present only during budding or entry. 57 Such a multifaceted immune response is a unique feature of pDNA-based vaccines expressing an authenticlooking HIV. 57 Synthetic nanomedicine formulation The objective of DermaVir nanomedicine formulation was to express the API in DCs, because DC-mediated antigen presentation is essential to boost T-cell immunity in HIVinfected people. pDNA delivery to DCs is a complex challenge involving DC binding, antigen uptake, expression, processing, and presentation to nave T cells. 58 We designed a pathogenlike nanomedicine to encapsulate the pDNA within the positively charged linear mannobiosylated polyethylenimine (PEIm). DermaVir nanomedicine is similar in size, appearance, and DNA-delivery features to viruses that naturally evolved to deliver genetic materials to cells. DermaVir's PEIm envelope protects the condensed pDNA core from extra- and intracellular degradations. Its particle size (70 nm 300 nm) is optimal for receptor-mediated endocytosis into cells, and it has sufcient stability to support the release of the pDNA from the endosomal compartment and the delivery of the pDNA to the nucleus. These features, unique for DermaVir nanomedicine, are essential for potent expression of antigens., 54,59 Consequently, the biological

activity of DermaVir is dependent on its inherent structure and binding, 55,60,61 (Figure 2, C). Langerhans cell-targeting nanomedicine administration The objective was to deliver the nanomedicine in vivo to the lymph-node DCs, the location where T-cell responses are originated in the body. We developed DermaPrep, the rst LCtargeting nanomedicine administration device. DermaPrep employs a skin preparation method that interrupts the stratum corneum facilitating nanomedicine penetration and providing the essential danger signal to the LCs residing just below this protective layer. 62,63 Once activated, LCs are naturally looking for pathogens and capturing the pathogen-like DermaVir nanomedicine applied to the prepared skin surface under a semi-occlusive patch. The main advantage of DermaPrep is the natural targeting of a large number of LCs (8 million) that form a horizontal 900 to 1,800 cells/mm 2 network under the skin surface. 64 After DermaVir has been captured, LCs mature to DCs and migrate to the local lymph nodes. Here DCs express pDNA-encoded antigens and present most HIV epitopes to the passing nave T cells. HIV-specic precursor/memory T cells primed by DCs further differentiate into HIV-specic effector T cells circulating out of the lymph node to seek virus-infected targets. Each killer effector cell can destroy several HIV-infected cells (Figure 3). The human proof of concept Prior to human clinical evaluation, the novel mechanism of action and the antiviral efcacy of DermaVir was demonstrated in macaques, some of them with AIDS. DermaVir

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Figure 4. Single DermaVir immunization boosts long-lasting HIV-specic 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-specic central memory T cells from baseline (net PHPC counts/10 6 PBMC) is shown. Gag, Tat and Rev represents three of 15 DermaVir-expressed antigens.

immunization targeted and expressed the pDNA-encoded antigens into the DCs of the lymph nodes (Figure 3). These DermaVir-expressing DCs primed nave T cells and induced both HIV-specic helper and cytotoxic T cells. 65 Importantly, immune responses following topical DermaVir immunization were similar to ex vivo immunization with cultured DCs. 66 DermaVir immunotherapy suppressed viral load and provided survival benet for chronically SIV251-infected macaques. 67 Repeated DermaVir immunizations in the absence of cART transiently suppressed virus replication that led to improvement of median survival time from 18 to 38 weeks in comparison with no treatment. 67 In other trials, DermaVir administered in combination with cART boosted SIV-specic T cells that possessed signicant antiretroviral activity in both chronically infected and macaques with AIDS. 67 These primate experiments provided the rationale to investigate DermaVir immunizations in HIV-infected human subjects. The Phase I dose-escalation study was designed to evaluate the safety and immunogenicity of a single DermaVir immunization in nine HIV-infected subjects on fully suppressive cART. 68 Increasing DermaVir doses were administered by DermaPrep simultaneously on two, four, and eight skin sites located on the back and the tight. The LCs of these skin sites drain into four different lymph nodes. Low-dose DermaVir contained 0.1mg DNA targeted to two lymph nodes. Mediumand high-dose DermaVir contained 0.4 mg and 0.8 mg DNA targeted to four lymph nodes. DermaVir-associated side effects were limited to the skin, mild, transient and not dose dependent. Boosting of HIV-specic effector CD4 + and CD8 + T cells expressing IFN-gamma and IL2 was detected against several HIV antigens in every subject of the medium dose cohort. The striking result was the dose-dependent expansion of HIVspecic precursor/memory T cells with high proliferation capacity, 68,69 (Figure 4). The ndings suggest that DermaVir could boost robust and long-lasting memory T-cell responses to 15 HIV antigens. We concluded that for durable immune reactivity repeated DermaVir immunizations might be required

Table 2 Features of nanomedicines developed toward the cure of HIV in comparison with the current state of art treatment of HIV/AIDS Features/Approach Therapeutic target Treatment benet Safety Administration schedule HIV RNA HIV immunity HIV-infected cells Cure Immunotherapy HIV-expressing cells Slow & Durable Transient, skin Yearly (4x) Slow decrease Boosting Killing Remission Activators Resting cells Rapid Unknown Short period Increase No effect No effect Eradication cART HIV life cycle Rapid & Transient Cumulative, systemic Daily Rapid decrease Decreasing No effect No

because the frequency of DermaVir-boosted HIV-specic T cells decreased during the 48-week follow-up period (Figure 4). A Phase I/II clinical trial conducted in several USA clinical centers was designed to investigate repeated administrations (three times) of escalating DermaVir doses (0.2, 0.4, and 2 0.4 mg pDNA) or placebo on 24 HIV-infected adults receiving fully suppressive cART. The incidence of adverse events was similar across groups, suggesting that DermaVir was as safe as a placebo. 70 Immunogenicity data demonstrated the boosting of HIV-specic precursor/memory T cells with high proliferative capacity. 69 The highest frequency of HIV-specic memory T cells was induced in the 0.4 mg dose group. 70 The results of this trial were consistent with the macaque and Phase I studies and conrmed the excellent safety and immunogenicity features of DermaVir immunizations in patients on cART. The Phase II randomized, multicenter, and placebo-controlled clinical trial was designed to evaluate the safety and to test the immunogenicity and antiviral efcacy of repeated DermaVir immunizations in the absence of cART. Thirty-six HIV-infected, treatment nave adults were randomized to receive one of three DermaVir doses (0.2, 0.4, or 0.8 mg

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Figure 5. Nanomedicine applications toward the cure of HIV/AIDS: (A) Untreated HIV infection is characterized by high viral load, high amount of HIVinfected cells in the reservoirs and the disease is partially controlled by the immune system alone for approximately 15 years. (B) cART is effective in potent and durable suppression of HIV RNA to b50 copies/mL that is required to avoid the development of drug-resistant mutants. However, cART does not eliminate latently infected HIV-infected cells. In addition, HIV-specic T-cell responses diminish in patients treated optimally with fully suppressive cART. Consequently, HIV rebounds even after short interruption of therapy. (C) DermaVir immune intensication of patients treated with optimal cART demonstrated the maintenance of undetectable load and induction of long-lasting and broadly specic HIV-specic 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-specic T cells are fully reconstituted. This stage of the disease the immune system alone 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 achieved in the absence of reactivation of latent HIV, because the immune system cannot recognize the latently infected cells where the integrated provirus is transcriptionally silent.

pDNA) or placebo at Study Weeks 0, 6, 12, and 18. The primary endpoint was safety at Week 24 and secondary endpoints were HIV RNA and immunogenicity. 71 Only one Grade 2 adverse event occurred in the low-dose cohort judged to be possibly related to DermaVir treatment, conrming the excellent safety features of DermaVir immunizations in a different patient population. Based on induction of HIV-specic memory/ precursor T cell, the 0.4 mg DermaVir dose was superior to the others. In this group the medium HIV RNA signicantly decreased by 70% in comparison with the placebo. Viral load suppression occurred slowly, as predicted by DermaVir mechanism of action, similarly to cancer vaccines. 71 These results were consistent with the macaque and previous clinical studies and conrmed the safety and immunogenicity and antiviral activity of repeated DermaVir immunizations.

Perspectives of nanomedicines towards the cure of HIV/AIDS More than 30 ARV and drug combinations are currently available to achieve long-term suppression of HIV RNA to N 50 copies/mL. Additional potency, accomplished by ARV intensication, did not provide additional treatment benets 15,72-76 Nanomedicines developed for the cure of HIV might overcome the following limitations of cART: (i) Activators target latently infected cells to reactivate HIV and ush the reservoirs; and (ii) DermaVir reconstitutes HIV-specic immune responses that decreased during optimal cART and deplete HIV-infected cells. In contrast, current ARVs reduce viral load by inhibiting one step in HIV life cycle (Table 2). In comparison with ARV,

the antiviral activity of immunotherapies are slower and less potent, because recognizing and killing of infected cells by HIV-specic T cells takes more time than blocking HIV replication by drugs. The effectiveness of killing of infected cells is revealed by their capacity to manage the infection for approximately 15 years in the absence of cART. Therefore, 0.5 log reduction of HIV RNA in 24 weeks, demonstrated with DermaVir, should be sufcient to decrease the amount of HIVinfected cells that are not eliminated by cART. In comparison with ARV Activators might be taken after immune intensication for a short period of time to ush the reservoirs. Consequently, the activated HIV producing cells will be eliminated by cytotoxic T cells and reservoirs will decrease or be eliminated (eradication). To demonstrate the eradication of all HIV-infected cells of a patient seems not to be feasible. The other approach is to achieve remission (functional cure) of HIV/AIDS characterized by the absence of HIV rebound after cART interruption (Figure 5). The three different mechanisms of action of ARV, Activators, and DermaVir are complementary, suitable to achieve remission: The rapid and potent viral load reduction with cART is essential to block HIV replication and reach undetectable viral load. After that DermaVir immune intensication could address what drug intensication could not achieve: (i) killing of HIV-infected cells that remained in reservoirs after optimal cART; and (ii) boosting HIV-specic T cells to reconstitute immune responses that suppressed during cART. After immune intensication, patients might be treated with Activators to achieve rapid reactivation of the virus. That point DermaVir-induced HIV-specic T cells would 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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J. Lisziewicz, E.R. Tke / Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 2838 7. Holtgrave DR. Causes of the decline in AIDS deaths, United States, 1995-2002: Prevention, treatment or both? Int J STD AIDS 2005;16: 777-81. 8. Losina E, Schackman BR, Sadownik SN, Gebo KA, Walensky RP, et al. Racial and sex disparities in life expectancy losses among HIVinfected persons in the United States: impact of risk behavior, late initiation, and early discontinuation of antiretroviral therapy. Clin Infect Dis 2009;49:1570-8. 9. Baker JV, Peng G, Rapkin J, Abrams DI, Silverberg MJ, et al. CD4+ count and risk of NON-AIDS diseases following initial treatment for HIV infection. AIDS 2008;22:841-8. 10. Mavigner M, Delobel P, Cazabat M, Dubois M, L'faqihi-Olive FE, et al. HIV-1 residual viremia correlates with persisitent T-cell activation in poor immunological responders to combination antiretroviral therapy. PLoS One 2009;4:e7658. 11. Casazza JP, Betts MR. Picker LJ and Koup RA Decay kinetics of Human Immunodeficiency Virus-specific CD8+ T cells in peripheral blood after initiation of highly active antiretroviral therapy. J Virol 2001;75: 6508-16. 12. Allers K, Htter G, Hofmann J, Loddenkemper C, Rieger K, et al. Evidence for the cure of HIV infection by CCR5D32/D32 stem cell transplantation. Blood 2011;117:2791-9. 13. Lisziewicz J, Rosenberg E, Lieberman J, Jessen H, Lopalco L, et al. Control of HIV despite the discontinuation of antiretroviral therapy. New Engl J Med 1999;340:1683-4. 14. Deeks SG, Walker BD. Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy. Immunity 2007;27:406-16. 15. Hatano H, Hayes TL, Dahl V, Sinclair E, Lee TH, et al. A randomized, controlled trial of raltegravir intensification in antiretroviral-treated, HIV-infected patients with a suboptimal CD4+ T cell response. J Infect Dis 2011;203:894-7. 16. Shan L, Dend K, Shroff NS, Durand CM, Alireza S, et al. Stimulation of HIV-1-Specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation. Immunity 2012;36:1-11. 17. Vyas TK, Shah L, Amiji MM. Nanoparticulate drug carriers for delivery of HIV/AIDS therapy to viral reservoir sites. Expert Opin Drug Deliv 2006;3:613-28. 18. Amiji MM, Vyas TK, Shah LK. Role of nanotechnology in HIV/AIDS treatment: potential to overcome the viral reservoir challenge. Discov Med 2006;6:157-62. 19. Nowacek A, Gendelman HE. NanoART, neuroAIDS and CNS drug delivery. Nanomedicine 2009;4:557-74. 20. Kovochich M, Marsden MD, Zack JA. Activation of latent HIV using drug-loaded nanoparticles. PLoS One 2011;6:e18270. 21. Choi SU, Bui T, Ho RJ. pH-dependent interactions of indinavir and lipids in nanoparticles and their ability to enatrap a solute. J Pharm Sci 2008;97: 931-43. 22. Kinman L, Brodie SJ, Tsai CC, Bui T, Larsen K, et al. Lipid-drug association enhanced HIV-1 protease inhibitor indinavir localization in lymphoid tissues and viral load reduction: a proof of concept study i HIV2287-infected macaques. J Acquir Immune Defic Syndr 2003;34:387-97. 23. Ma X, Wang D, Wu Y, Ho RJY, Jia L, et al. AIDS treatment and novel anti-HIV compounds improved by nanotechnology. AAPS J 2010;12: 272-8. 24. Chiappetta DA, Hocht C, Taira C, Sosnik A. Efavirenz-loaded polymeric micelles for pediatric anti-HIV pharmacotherapy with significantly higher oral bioavailabilty. Nanomedicine 2010;5:11-23. 25. Baert L, van't Klooster G, Dries W, Franois M, Wouters A, et al. Development of a long-acting injectable formulation with nanoparticles of rilpivirine (TMC278) for HIV treatment. Eur J Pharm Biopharm 2009;72:502-8. 26. Abdool Karim Q, Abdool Karim SS, Frohlich JA, Grobler AC, Baxter C, et al. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 2010;329:1168-74.

take time to substantially decrease the infected cells from the reservoirs and fully reconstitute HIV-specic immune responses. We envision that repeated DermaVir immune intensication in combination with Activators could eliminate signicant amount of infected cells from the reservoirs. Consequently, patients could decrease ARV exposure, and their immune system could maintain the HIV RNA level under the detection limit. During remission, demonstrated by undetectable HIV RNA after interruption of cART, maintenance of high-level T-cell responses might require the repeated administration of DermaVir immunotherapy (e.g., 4 times yearly). Potential advantages of immunotherapy in comparison with cART include its excellent safety prole, potentially higher specicity, the longevity of an immune response, and likely cost savings as well as at least theoretically the chance to achieve a cure (remission). However, despite decades of research, no therapeutic vaccine has reached the market. Challenges include (i) the disease mechanisms and interaction with the immune system; (ii) immune escape from T-cell recognition based on the high genetic diversity of the virus and the HLA diversity of the host; and (iii) the shortage of funding in comparison with prophylactic vaccine development. Any treatment that can eradicate the virus from infected patients or cure the disease (remission) would have a huge commercial opportunity. Nanotechnology offers opportunities to develop new treatment approaches that could contribute to the cure of HIV/ AIDS. To affect public health the new approaches must signicantly improve the health of HIV-infected patients at lower cost than the that of the current cART. One concern regarding to the systemic nanomedicine administrations is toxicity, because a new immunotoxicity was observed during the treatment of cancer with liposomal nanomedicine formulations. 43 Would the improvement in half-life or the increase of bioavailability of the presently used ARV justify a new potential toxicity? Another concern is an increase in price due to the need of sophistication in manufacturing and quality control that is usually accompanied by more difcult scale-up and higher production costs. We envision that disease modifying topical nanomedicines developed for immunotherapy and microbicides might have the safety, efcacy, and cost features to contribute to the cure of HIV/AIDS and signicantly improve public health.

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