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Breaching HIV-1 reservoirs with nanotechnology

Introduction
HIV-1 reservoirs are cells, typically CD4+ T cells, where the virus persists despite low blood plasma concentrations of HIV-1 DNA and RNA. These reservoirs are found in anatomical sanctuary sites where anti-retroviral drugs (ARV) penetrate poorly, which, ultimately, lead to ARV resistance. Nanotechnology, one of the new strategies, is currently being explored to completely eradicate the virus and seems to hold the most promise.

HIV-1 reservoirs and sanctuary sites
Bilibana (2010) described the molecular mechanisms involved in the establishment of HIV latency. He also discussed a strategy that involves rousing the virus from its latent state in order to eradicate it completely.

That HIV infects CD4+ T cells is commonly known (Wong et al. 2010). During sexual female-to-male transmission, for example, the virus primarily invades the Langerhans cells (Rappersberger et al. 1988) which inadvertently transport the virus to the lymph nodes. Here the virus comes into contact with CD4+ T cells which it subsequently infects (Kawamura et al. 2005). Langerhans cells are dendritic cells and belong to the antigen presenting class of lymphocytes (Rappersberger et al. 1988 , Zaitseva et al. 1997).

From there the infected CD4+ T cells and other infected lymphocytes disperse throughout the body. Latency is established during early, primary infection (Pace et al. 2011). In their review, these authors build a strong case as to why CD4+ T cells are the main cells that make up the reservoir even though HIV infects other leukocytes. Chomont and colleagues (2009) found that the HIV reservoir is composed of central memory, transitional memory, effector memory and naïve cells among other types of CD4+ T cells.

Sanctuary sites are established in the central nervous system (Churchill & Nath 2013), the genital tract (Halfon et al. 2010 , Launay et al. 2011), gut- and rectal-associated lymphoid tissue (Chun et al. 2008 , Imamichi et al. 2011 , Zalar et al. 2010) among other areas (Cory et al. 2013). Sanctuaries are established at these sites because of a poor delivery of antiretroviral drugs (ARVs). As a result the virus continues to replicate at these sites while blood plasma remains clear of virus (Churchill & Nath 2013 , Katlama et al. 2013). Consequently, different viral strains can evolve at the various sanctuary sites with various ARV-resistance properties.

The role of HAART intensification in response to HIV-1 sanctuary sites
In their review Katlama and co-authors (2013) outline three strategies that can be employed to eradicate HIV-1 from sanctuary sites. The first is HAART (highly active antiretroviral therapy) intensification. Patients on HAART receive three to four ARVs: two nucleoside analogues and a protease inhibitor (PI). A second PI is sometimes added to boost the function of the primary PI (Wong et al. 2010). To intensify HAART an integrase inhibitor is added to the regimen (Luo et al. 2013).

Raltegravir prevents the viral enzyme, integrase, from inserting the processed proviral DNA into the host cell's genome (Reigadas et al. 2010) by binding to processed proviral DNA (Ammar et al. 2012). This does not prevent HIV-1 integrase from clipping two nucleotides from the 3’ ends of proviral DNA strands. 2-long terminal repeats (2-LTR) DNA circles develop from these processed proviral DNA via the cell's DNA end-joining system (Reigadas et al. 2010). The interaction between raltegravir and the DNA end-joining system is still to be investigated (Ammar et al 2012). An increase in 2-LTRs is observed in patients receiving HAART intensification, but integrase inhibitors do not stop persistent low-level viremia (Luo et al. 2013 , Patterson et al. 2013 , Reigadas et al. 2010). Patterson et al. (2013) found that raltegravir concentrates in the gastrointestinal tract, with the highest concentration (2240 mg*h/ mL) in the splenic flexure after multiple doses. The concentration in the splenic flexture was 650-fold higher than in blood plasma.

Compartment targeting
Since raltegravir concentrates in the gastrointestinal tract, it can be used to target the HIV-1 reservoirs in gut- and rectal-associated lymphoid tissues (Cory et al. 2013). This rationale is another strategy that can be used to respond to HIV-1 reservoirs. Although this strategy has not been employed in the response to HIV and AIDS, it has been used to treat childhood acute lymphoblastic leukaemia and cytomegalovirus retinitis (Cory et al. 2013).

Gene therapy
The next strategy that was discussed by Katlama and her co-authors (2013) is gene therapy. Zinc-finger nucleases, transcription activator-like effector nucleases and homing endonucleases are currently being studied to either “delete the virus from infected cells or to produce cells resistant to HIV infection” (Katlama et al. 2013).

The three-tier approach
The three-tiered approach (Bilibana 2010 , Katlama et al. 2013) involves the reactivation of proviral DNA in latently infected cells, the targeting of residual virus replication and increasing the effectiveness of the host's natural mechanisms of eradicating viruses from its system.

When the HIV-1 provirus is activated its host cells transcribe proviral DNA and translate the mRNA product which results in the expression of virus-coat proteins on the cell surface. One of the most important transcription factors involved in the HIV-1 replication is the nuclear factor kappa β (NF-κβ) (Chan & Greene 2012 , Wolschendorf et al. 2012). Therefore, therapeutic mechanisms of NF-κβ activation are currently being investigated (Katlama et al. 2013).

One of the ways in which residual virus replication can be targeted is through the use of nanotechnology. Nanocarriers are particles in the nanometer size range (Cory et al. 2013 , Van’t Klooster et al. 2010 , Wong et al. 2010) and are composed of various materials. These nanocarriers should preferably be biodegradable and non-toxic, especially when they target the central nervous system (Wong et al. 2010). Nanocarrier systems that fit these criteria are those that are polymer/dendrimer-based, lipid-based or micelle-based. In context of the central nervous system these nanocarrier systems have already been reviewed (Wong et al. 2010).

The ARV is encapsulated in a nanocarrier which transports the ARV to its target sanctuary site, a site that it does not inherently target. The composition of the nanocarrier determines whether it passively diffuses across the natural barriers, is actively transported across the barrier by endocytosis or by receptor-mediated endocytosis (Wong et al. 2010).

Nano-scale ART (nanoART) was studied in mice that were reconstituted with HIV-1 human blood lymphocytes (Roy et al. 2012). Despite the shortcomings of such a study, it is evident that nanoART can be used to create drug depots that need weekly as opposed to daily replenishment (Cory et al. 2013). Research has shown that macrophages absorb the nanoART molecules and transport them to the sanctuary sites where the nanoART molecules are released over a long period of time (Batrakova et al. 2011 , Kadiu et al. 2011).  

An alternative approach to pharmacological virus eradication is that of making prodrug modifications to existing and potential ARVs (Cory et al. 2013). This approach is not cost-effective since both the ARV or potential ARV and its prodrug form have to undergo more screening tests and clinical trials (Wong et al. 2010).

One of the methods employed to utilise the body’s natural defence mechanisms to eradicate reactivated cellular reservoirs involves priming CD8+ cytotoxic cells to seek out and destroy the reactivated cellular reservoirs in the sanctuary sites (Katlama et al. 2013). A recently published study (Hansen et al. 2013) describes the use of rhesus cytomegalovirus (RhCMV) that expresses SIV surface proteins to successfully elicit a CD8+ cytotoxic response on the RhCMV host cells. The findings of the study hold great promise since the cytomegalovirus is also common in humans and can be genetically engineered to prophylactically prime the CD8+ cytotoxic cells to recognise virtually immutable HIV-1 coat proteins.

Conclusion
Despite continuous treatment with HAART and HAART intensification, low-level viremia is persistent, therefore, it is important to design and test novel approaches to eradicate the virus reservoir. Nanotechnology as part of the three-tier approach (Katlama et al. 2013) seems most promising.

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Author: Waldo Adams (BSc Hons Biochemistry)
Reviewed by: Marike Kotzé (MSc), Jean Fourie (MPhil) and Alfred Thutloa (MPhil)
Contact: afroaidsinfo@mrc.ac.za
Date: June 2013

Preferred citation
Adams, W. (2013) Breaching HIV-1 reservoirs with nanotechnology, AfroAIDSinfo. Issue 13 no. 6, Science (Open access).

Last updated: 5 June, 2013