HIV latency and its translational implications

Introduction

Acquired immunodeficiency syndrome (AIDS) is a medical condition caused by human

immunodeficiency virus (HIV), and is characterized by significantly reduced plasma CD4 T cell

count (below 150/ml) and acquisition of rare cancers or/ and infections (e.g. Non-Hodgkin

lymphoma, tuberculosis, and candidiasis)[1]. 35 million people worldwide are infected by HIV,

vast majority of them are living in developing nations, especially in Sub-Saharan Africa. Of 35

million infected individuals, 3.2 are children[2].The introduction of antiretroviral therapy in

1986 was considered as a breakthrough in HIV infection/ AIDS as it reduced plasma viral load

significantly, prolonged the life expectation, and improved the quality of life of infected

people[3]. Unfortunately, incomplete virus eradication, drug resistance, side effects and high

cost put doubts on the future prospects of antiretroviral therapy in clinics[4]. Previous studies

revealed that reactivation of latent HIV in resting T cell is responsible for constant recurrence of

infection in patients who discontinued the therapy[5], putting urgent needs for new therapies.

Intriguingly, researchers proposed one so called “ shock and kill” strategy, which describes the

eradication of latent viral pool by inducing virus reactivation (hence “shock”) in complement

with antiretroviral therapy ( hence “kill”), giving hope for the development of new therapies.

However, one of the key questions needs to be elucidated is what causes HIV enters latency.

Tat, which has long been evident to be an viral transactivator that regulates initiation,

elongation processes, is crucial HIV latency biology [6,7]. Changes in cellular microenvironment,

viral gene transcription level and chromatin configuration are known factors that force HIV

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enters latent life cycle. What is noteworthy is that Tat does not work alone, requiring

cofactors(i.e. RTEFb, P-TEFB and NF-kB), which are potential drug targets.

This report is aimed at revising the recent advance in HIV infection/AIDS treatment. Firstly, HIV

latency biology and development of antiretroviral therapy will be introduced as foundations of

what led to the breakthrough and why we need the breakthrough in HIV/AIDS treatment.

Secondly, translational implications from research and other treatment alternatives will be

discussed. Finally, limitations of the breakthrough as well as other theories will be covered in

the discussion session.

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Early treatment-Antiretroviral therapies and their limitations

A. Development history

Antiretroviral therapies are combination of chemical drugs that suppress the entry,

replication and spread of HIV in cells. Combined antiretroviral therapies consist of several

inhibitors and are referred to as highly active anti-retroviral therapy (HAART)[8, WHO]. The

history of antiretroviral therapy originated from a clinical trial about zidovudine carried out

by Fischl and colleagues in 1986[9], since then Hammer et al proposed “triple-drug anti-HIV

therapy concept” based on the significant reduction of mortality rate in HIV infected who

were given indinavir-based HAARTs[10]. Antiretroviral therapies reduce plasma viral load,

improves CD4+ T cell count (above 500 cells/ ul according to a report[11]) and the quality of

life. Nonetheless, treatment by antiretroviral therapy during the first decade is regretfully

unsuccessful[12]. The reasons for that will be discussed in the next session.

B. Limitations of antiretroviral therapies

A variety of antiretroviral drugs have been developed since its primary introduction. There

are five categories of antiretroviral drugs that are currently available: non-nucleoside

reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors

(NRTIs), protease inhibitors, entry and fusion inhibitors and integrase inhibitor[13]. Drugs of

each categories and their action mechanisms are listed below. Examples are given in

table1[14,15]. As effective and successful as antiretroviral drugs are meant to be,

observations of infection relapses, increased plasma viral load as well as reduced CD4+ T cell

count have been made in patients who discontinued their therapy. Moreover, considerable

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antiretroviral drugs that are currently being used in clinical practice have potential to cause

unwanted effects including nephrotoxicity, neurotoxicity, diarrhea, rash[16].

Table1. Examples of drugs of each categories

Table1: Names listed are the generic name of drugs. In clinical practice, different categories of

drugs are prescribed in combination to maximize the effectiveness of drugs.

Drug resistance, like in the drug treatment of any other infections caused by microorganisms,

especially in the treatment of bacterial infections, is inevitably identified in HIV infection/AIDS

treatment. The situation might be even worse regarding the fact that retrovirus, viral group to

which HIV belongs, has a very high mutation rate because of its single-stranded RNA genome,

which is not as stable as double-stranded DNA. Hence, it is strongly suggested that doctors

perform HIV genotypic resistance tests before prescribing the most appropriate and efficacious

antiretroviral drugs. Interestingly, there are debates about the availability of HIV genotypic

NNRTIs

NRTIs

Protease

inhibitor

Entry and fusion

inhibitor

Integrase

inhibitor

Examples Festinavir,

Tenofovir

Nevirapine,

Efavirenz

Simeprevir,

Boceprevir

Albuvirtide Elitegravir

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resistance tests in areas where medical settings , putting constraints on wide-spread utilization

of genotypic testing[17]. Laethem et al suggest in their research that the next generation

sequencing technique has larger throughput and is more affordable[17]. Nevertheless, HIV

genotypic testing will not relive the drug resistance that the HIV infection/AIDS treatment

community currently suffer from. New hope relies in new therapeutic strategies.

Hints for new therapies-Understandings from HIV latency biology

A. Tat mediated transcriptional feedback loop

Tat was primary described by Sodroski and colleagues in 1985 as a viral transactivator that

regulates initiation, elongation processes of transcription[17]. Tat binds to viral DNA and recruits

transcription factors such as RTEFB and P-TEFB, for instance[18]. Hence, together they initiate

transcription of viral gene. Epigenetic modification of viral gene and viral gene transcription

interruption play crucial roles in HIV latency, although it is still controversial which one of them

is the primary cause[19,20]. Recent research shown Tat as potential causative agent of B cell

lymphoma in HIV infected individuals or AIDS patient[21], which might explain the prevalence of

B cell lymphoma in AIDS patients. That is important because studies of Tat in B cell lymphoma

will probably extend our knowledge about Tat biology in HIV latency.

B. Inducible reactivation from latency

HIV reservoir, resting CD4+ T cell, is the main obstacle for eradicating the virus by antiretroviral

therapies. Previous research carried out suggests induced virus reactivation in complement with

antiretroviral drug interventions as well as the immune system of the host could facilitate virus

eradication [22]. Indeed, methyl transferase inhibitor and acetyl transferase inhibitors which

prevent epigenetic modification of viral genes, have been proven to be effective at inducing viral

reactivation[6].Furthermore, activation of T cell receptor (TCR) stimulated by alpha-CD3/CD28

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monoclonal antibodies causes reactivation of latent HIV by activating p-TEFb, which is an essential

transcription factor for HIV, and is usually suppressed by infected host cell as a defense

system.[11]. There are debates concerning inducible HIV reactivation could potentially cause

“HIV-associated complications”[23], but this worry is less significant when considering most of

the HIV infected individuals who received antiretroviral therapies are immune-competent.

New light-Translational implications from HIV latency biology research

Researche into HIV latency biology have led to one important outcome that is of greatest

translational implications. It is Tat-pTEFb interaction [29].

Transcription factor pTEFb comprises a cyclin-dependent kinase 9, CDK9[25] ,and a partner

cyclin T1, T2, or K[26]. Burlein and colleagues conducted an elegant study whereby they

designed a homogenous assay in Alpha LISA format using His-tagged pTEFb and biotinylated

Tat, which enabled the authors to observe the interaction between Tat and its cofactor

pTEFb[24]. Tat forms a quaternary complex with pTEFb and other molecules. Together, they

initiate the phosphorylation of viral RNA polymerase II and elongation of viral RNA[24]. Upon

infection, CD4+ T cell initiates the conversion of pTEFb into RNP complex that comprises 7SK

RNA and 7SK RNP in resting T cells, resulting in dramatic decrease in intracellular pTEFb

level[25,26]. Therefore, HIV will not be able to replicate itself because of pTEFb deprivation.

Having said that, development of drugs that reverse the transformation of pTEFb into RNP

complexes or simply pTEFb transcription factor supply could induce reactivation of latent HIV.

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In addition, other therapeutic strategy such as HIV immunization is increasingly being revised as

potential new treatment. Immunization strategy includes therapeutic and preventive vaccine.

The former targets key molecules in HIV life cycle, preventing it from reactivation, replication,

and transmission to uninfected individuals[27]. Preventive vaccine, however, is dedicated to

preventing the virus from entering susceptible people[27]. Immunization strategies are more

cost-effective. This is of particular significance in under-developed area where antiretroviral

drugs are less available and affordable[38]. Examples of therapeutic and preventive vaccine

candidates (have not been proven to market) as well as their limitations and advantages are

listed in table2.

Table 2. The compare and contrast between therapeutic and preventive vaccines

Therapeutic vaccine Preventive vaccine

Examples Vacc-4x, Pep Tcell,

Thera Vax, Tat vaccine[34].

MRK rAd5, HVTN 502,

HVNT503, HVNT505[34].

Component Peptides, plasmid, viral

porteins, modified Tat.

HIV-1 Gag, Pol, Nef, and Env.

Advantages

Simple efficacy assessment,

facilitates the identification

of potential biomarkers that

can be drug targets, enhance

the effects of antiretroviral

drug[27].

Tat vaccine, which utilizes

biologically active viral Tat

protein to stimulate the body

to produce anti-Tat

antibodies, is considered as

the key to achieve anti-HIV

immunity[28,29]

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Limitations

Therapeutic vaccines are

effective only at the early

sate of HIV infection, which

contraindicates with the

current difficulties in making

early diagnosis[27].

Unlike therapeutic vaccines,

research into preventive

vaccine has been not fruitful

due to three reasons: 1) lack

of efficacy trial; 2) safety

concerns; 3) vaccines failed

to offer protection[30-34]

Table2. Vaccines listed are available at:

http://www.pipelinereport.org/sites/g/files/g575521/f/201407/Cure%20Immune%20Based

%20and%20Gene%20Therapies.pdf

Discussion

“Shock and kill” strategy is a new HIV/AIDS treatment characterized by induced reactivation of

latent HIV in combination with antiretroviral therapy. However, this proposal is in its infancy.

Considerable pharmaceutical and clinical trials are required to develop drugs that are capable

of inducing viral reactivation effectively and safely in patients.

Notably, there has been research suggesting virus eradication by preventing HIV reactivates

from latency. Szeto and colleagues demonstrated in their research that minocycline, an

immunomodulatory antibiotic, suppresses replication and reactivation of HIV. Hence has anti-

HIV effect[35]. Unlike conventional anti-HIV drugs which work by directly targeting HIV,

minocycline achieves its anti-HIV effect by altering cellular environment. More specifically,

minocycline causes dose-dependent decrease in viral RNA by downregulating the expression of

activation and proliferation biomarkers (e.g. CD25 and CCR5) in CD4+ cell, and by decreasing cell

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cytokine (e.g. IFN-gamma and TNF-alpha) secretion level[36]. Mousseau et al also showed in

their publication that Didehydro-Cortistatin A (cDA) prevents HIV-1 from reactivating from

latency[37]. The authors proposed that dCA probably cause epigenetic changes in HIV viral

genome (no direct evidence showed there were epigenetic modification changes), concluding

from their observation of no viral reactivation even after withdrawal of dCA[37]. Those two

teams offered an opinion that is totally opposite to the rationale this report has been

discussing. This is interesting, especially when considering the advantages and disadvantages of

those two contrast strategies. Inducing viral reactivation is more robust, but has the potential

to cause HIV-associated complications in immuno-compromised patients. Preventing virus from

reactivating is soft, but it will inevitably cause “non-AIDS-associated disease” caused by

consistent release of provirus from infected CD4+ T cell. In this regard, it is seems sensible to

seek more integrated treatment.

Amongst our limited choices, traditional Chinese medicine (TCM) is worth considering. As one

of the oldest medical systems ever been practiced by human-beings, TCM has invaluable clinical

utilization in terms of disease treatment in modern medicine system. As a matter of fact, TCM

has been proved to be effective at monitoring HIV/AIDS, cancer, and infertility[38-42]. Of

particular interest of this report, understanding of TCM in HIV/AIDS treatment will be discussed.

According to traditional Chinese medicine pattern identification, AIDS is defined as “yi bing”,

disease characterized by long latency, sudden onset, and serve symptoms[43], which is

interestingly consistent with the Western Medicine’s description of HIV/AIDS. Nevertheless,

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Western Medicine and TCM do function in different systems and adapt inconsistent therapeutic

strategies and beliefs. For example, in the treatment of AIDS, TCM focuses on the treatment of

the patient with the disease rather than the disease to which western medicine pays

attention[43]. In addition, TCM is based on the utilization of herbs and other natural exist

material, which is completely different to the artificially synthesized chemicals used by Western

Medicine. Regretfully, despite of all those advantageous facts about TCM, it has yet not been

accepted and recommended by scientists and heath-care professionals word-wide[43]. This is

because there is a considerable lack of intensive clinical trails trying to elucidate the action

mechanisms of Chinese herbs[43]. In this regard, researchers proposed Complementary and

Integrate Medicine(CIM) based on the combination between TCM and Western Medicine,

which is a fast-developing area, and has been widely utilized in China for HIV/AIDS treatment.

Conclusion

Infection relapses, increase in plasma viral load , and decrease in CD4+ T cell count are observed

in HIV infected /AIDS patients when therapies are discontinued. This is probably because of the

conventional antiretroviral therapy’s failure to eradicate the virus from the patient’s body.

Unfortunately, the further employment of conventional antiretroviral drugs in clinical practice

is constrained by its limitations that are difficult to overcome. Encouragingly, a large number of

research has been conducted and proven to be fruitful, shedding light into the understandings

of HIV latency biology. Identification of Tat-mediated transcriptional feedback system,

particularly its cofactors, P-TEFB and NF-kB, is of great significance, giving translational

implications. Methyl transferase, acetyl transferase inhibitors, and anti-TCR monoclonal

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antibodies are receiving attention as viral reactivation inducers. New therapeutic strategies

such as preventive and therapeutic vaccines and CIM are the future HIV infection/AIDS

treatment development direction.

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