Current HIV Research - Volume 9, Issue 8, 2011

In the current “Hot Topic” series published in the Current HIV Research, the readers will find a number of intriguing reviews that are related to the current state of HIV latency. These reviews are written by some of the leading investigators who have contributed significantly to the HIV latency field. At the outset, we also would like to acknowledge that many other critical topics related to latency were left out mainly due to space and time limitations. The five reviews start with a manuscript from Mbonye and Karn who describe some of the current complexities related to HIV reservoirs in patients including the estimated long half-life of 45 months. These authors do an excellent job of describing the latest mechanisms that regulate both viral promoter and epigenetic associated phenotypes. They also accurately describe a set of evidence from the literature regarding modes of activation in both cell line as well as primary cells. Finally, some of the other possible latency mechanisms including RNA export are included and later discussed in more detail from a review by Ajamian and Mouland. The excellent figures and diagrams in this particular review also do a great job of describing the state of the art in a snap shot. The second review by Planelles, Wolschendorf and Kutsch describes assays that are required for high throughput screening and how some of these assays could effectively be used to screen for activators of latent virus. The authors discuss issues related to integration profiles and contrast patient samples to cell lines in vitro. Previous work from Planelles' lab described use of labeled virus and its utility in creating primary latent T-cells in vitro. The review also provides a table that gives an overview of various modes of activation using antibodies, chemicals and cytokines. These authors also add new technological advances in the high throughput screening assays both in 96 well as well as 384 well formats. Methods of screening that are the most sensitive for high throughput assays are described and detailed (i.e., fluorescence flow cytometry). These authors further consider latent or persistent HIV reservoirs to likely reside in multiple cell types in vivo therefore complicating the mode of action when trying to activate the latent viruses. The next review is by Tyagi and Romerio that defines multiple modes of latency that could occur at transcription initiation, elongation and some of the post transcriptional regulations. The authors also have a table comparing various in vitro models of latency and viral strains that have been used to create these infections. They cite some of the early work from Gallo's group that detected HIV RNA after various early HAART treatments. They pay close attention to both X4 and R5 viruses throughout the review and the distinction between choosing replication competent and single round virus infection are well described. From some of the literature reviews as well as their own work they conclude the expansion of immunological memory cells may be a slow and step-wise process rather than an “all or none” process. Finally, the authors acknowledge that the literature has focused heavily on a narrow window of latency, mainly on transcription of HIV and other restriction points, including downstream events (post transcriptional) which could become significant targets for controlling latency. The next review is by Ajamian and Mouland which discusses some of the post transcriptional events that may control latency. The authors site a number of important papers including one that identified five cellular miRNA that are up regulated in resting T-cells during latency and control HIV production. The miRNA arena which has a natural segway to RNA helicases are described and could potentially control infection. For instance, the RIG-I pathway which controls the innate immunity of the cell is highly regulated by DExD/H RNA helicases. These RNA helicases also directly bind to HIV proteins including Gag and Rev open reading frames. They control multiple mechanisms including RNA surveillance, cytoplasmic export, translation and stability of genomic RNA. Perhaps one of the best cited examples of RNA helicases and Rev contributing to latency comes from studies done in Astrocytes. All in all, there is considerable evidence for the involvement of RNA helicases that may control latency in cells tested to date. One caveat however, is that very few of these studies utilize primary cells or field isolates of HIV. The final review by Van Duyne et al. describes some of the latest findings in humanized mouse models that could potentially contribute to latency. The review discusses various animal models as well as some of the cell line data that have been utilized regarding latency and further describes newer models using different mouse strains. The discussions on in vivo animal models of latency touch on various tissues that could potentially be source of latent populations. Some of the highlighted areas include lymphoid tissues, CNS, Gut, and reproductive tract. Finally, the future direction section provides a more detailed description of newer animal models and the various cell types including T-cells and macrophages that could be the source of viral reservoirs. Although these reviews describe the current state of the art, many possibly significant mechanisms of latency have still not been fully exploited. For instance, issues related to DNA methylation of HIV DNA [1], modifications of Tat protein including methylation [2-3], presence of virus specific miRNA which could control both transcriptional silencing and mRNA regulation [4-8], a clear distinction between wild type and possible mutant pools from in vivo models, and other more pressing distinctions between definition of latency of T-cells in blood stream (∼2-3% of all T-cells) versus T-cells in tissues such as lymph nodes (∼65% of all T-cells), and the incomplete definition of mechanisms that regulate macrophage latency/persistence in blood stream versus tissues all require further research. Finally, in future more intense research is needed to better describe additional HIV cellular reservoirs and sanctuary sites that exist in vivo other than resting CD4+ T cells and monocytes/macrophages. This is significant in light of the fact that resting CD4+ T cells, monocytes, or other “blood” cell type may not be the source of resurgent HIV, highlighting the presence of additional reservoirs for HIV [9-10]. One such reservoir which remains elusive could be the central nervous system (CNS). HIV invades the brain within weeks of infection and persists in the CNS at a steady state, despite cART in cellular reservoirs such as perivascular macrophages, microglia, and astrocytes [11-13]. Because cART typically has lower penetration effectiveness into the CNS [14], the CNS microenvironment may be a breeding ground for drug resistant HIV and/or low level of HIV replication that can drive persistent inflammation in the CNS. Indeed, despite cART, 52% of HIV infected individuals are diagnosed with a spectrum of neurologic disease that range from mild to moderate neurocognitive disorder to more severe encephalitis/dementia [15]. Along these lines knowledge about HIV latency and cell types in CNS is in its infancy. For instance, CD16+ monocytes and other lymphocytes, including CD4+ T cells infiltrate the brain and disseminate HIV into the CNS [16]. Within the brain, microglia, which are resident macrophages, are the primary target for productive HIV infection. However, considerable evidence from in vitro and in vivo studies also indicate that astrocytes harbor HIV DNA [17-20]. In fact, 1-3% of astrocytes from patients without a diagnosis of HIV-associated encephalitis harbor HIV provirus [17]. The size of this pool is impressive considering that the size of the resting CD4+ T cell pool harboring HIV provirus is quite small constituting approximately 1 cell/million resting CD4+ T cells [21]. Therefore astrocytes may constitute a considerable reservoir for HIV in vivo. Further research is needed on this possible source of latent cells which will be important for any therapeutic or “shock and kill” approach to succeed. REFERENCES [1] Kauder SE, Bosque A, Lindqvist A, Planelles V, Verdin E. Epigenetic regulation of HIV-1 latency by cytosine methylation. PLoS Pathog 2009; 5(6): e1000495. [2] Sakane N, Kwon HS, Pagans S, et al. Activation of HIV transcription by the viral Tat protein requires a demethylation step mediated by lysinespecific demethylase 1 (LSD1/KDM1). PLoS Pathog 2011; 7(8): e1002184. [3] Van Duyne R, Easley R, Wu W, et al. Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR. Retrovirology 2008; 5: 40. [4] Klase Z, Kale P, Winograd R, et al. HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR. BMC Mol Biol 2007; 8: 63. [5] Klase Z, Winograd R, Davis J, et al. HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression. Retrovirology 2009; 6: 18. [6] Ouellet DL, Plante I, Landry P, et al. Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element. Nucleic Acids Res 2008; 36(7): 2353-65. [7] Schopman NC, Willemsen M, Liu YP, et al. Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs. Nucleic Acids Res 2011. [8] Weinberg MS, Villeneuve LM, Ehsani A, et al. The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. RNA 2006; 12(2): 256-62. [9] Brennan TP, Woods JO, Sedaghat AR, et al. Analysis of human immunodeficiency virus type 1 viremia and provirus in resting CD4+ T cells reveals a novel source of residual viremia in patients on antiretroviral therapy. J Virol 2009; 83(17): 8470-81. [10] Chun TW, Davey RT, Jr., Ostrowski M, et al. Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy. Nat Med 2000; 6(7): 757-61. [11] Roberts ES, Burudi EM, Flynn C, et al. Acute SIV infection of the brain leads to upregulation of IL6 and interferon-regulated genes: expression patterns throughout disease progression and impact on neuroAIDS. J Neuroimmunol 2004; 157(1-2): 81-92. [12] Chiodi F, Sonnerborg A, Albert J, et al. Human immunodeficiency virus infection of the brain. I. Virus isolation and detection of HIV specific antibodies in the cerebrospinal fluid of patients with varying clinical conditions. J Neurol Sci 1988; 85(3): 245-57. [13] Clements JE, Babas T, Mankowski JL, et al. The central nervous system as a reservoir for simian immunodeficiency virus (SIV): steady-state levels of SIV DNA in brain from acute through asymptomatic infection. J Infect Dis 2002; 186(7): 905-13. [14] Letendre S, Marquie-Beck J, Capparelli E, et al. Validation of the CNS Penetration-Effectiveness rank for quantifying antiretroviral penetration into the central nervous system. Arch Neurol 2008; 65(1): 65-70. [15] Heaton RK, Clifford DB, Franklin DR, Jr., et al. HIV-associated neurocognitive disorders persist in the era of potent antiretroviral therapy: CHARTER Study. Neurology 75(23): 2087-96. [16] Fischer-Smith T, Bell C, Croul S, Lewis M, Rappaport J. Monocyte/macrophage trafficking in acquired immunodeficiency syndrome encephalitis: lessons from human and nonhuman primate studies. J Neurovirol 2008; 14(4): 318-26. [17] Churchill MJ, Wesselingh SL, Cowley D, et al. Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia. Ann Neurol 2009; 66(2): 253-8. [18] Dewhurst S, Sakai K, Bresser J, et al. Persistent productive infection of human glial cells by human immunodeficiency virus (HIV) and by infectious molecular clones of HIV. J Virol 1987; 61(12): 3774-82. [19] Dewhurst S, Bresser J, Stevenson M, et al. Susceptibility of human glial cells to infection with human immunodeficiency virus (HIV). FEBS Lett 1987; 213(1): 138-43. [20] Trillo-Pazos G, Diamanturos A, Rislove L, et al. Detection of HIV-1 DNA in microglia/macrophages, astrocytes and neurons isolated from brain tissue with HIV-1 encephalitis by laser capture microdissection. Brain Pathol 2003; 13(2): 144-54. [21] Siliciano JD, Kajdas J, Finzi D, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med 2003; 9(6): 727-8.

Intensive antiretroviral therapy successfully suppresses viral replication but is unable to eradicate the virus. HIV persists in a small number of resting memory T cells where HIV has been transcriptionally silenced. This review will focus on recent insights into the HIV transcriptional control mechanisms that provide the biochemical basis for understanding latency. There are no specific repressors of HIV transcription encoded by the virus, instead latency arises when the regulatory feedback mechanism driven by HIV Tat expression is disrupted. Small changes in transcriptional initiation, induced by epigenetic silencing, lead to profound restrictions in Tat levels and force the entry of proviruses into latency. In resting memory T cells, which carry the bulk of the latent viral pool, additional restrictions, especially the limiting cellular levels of the essential Tat cofactor P-TEFb and the transcription initiation factors NF-κB and NFAT ensure that the provirus remains silenced unless the host cell is activated. The detailed understanding of HIV transcription is providing a framework for devising new therapeutic strategies designed to purge the latent viral pool. Importantly, the recognition that there are multiple restrictions imposed on latent proviruses suggest that proviral reactivation will not be achieved when only a single reactivation step is targeted and that any optimal activation strategy will require both removal of epigenetic blocks and the activation of P-TEFb.

A curative therapy for HIV-1 infection will have to include measures to eliminate the reservoir of latently HIV- 1 infected cells that allow the virus to persist despite otherwise successful therapy. To date, all efforts to deplete the latent reservoir by triggering viral reactivation have used preexisting drugs that are believed to potentially target molecular mechanisms controlling HIV-1 infection. These therapeutic attempts were not clinically successful. Only in the last few years have cellular models of latent HIV-1 infection suitable for high throughput screening been developed and concerted drug discovery efforts were initiated to discover new HIV-1 reactivating drugs. We here provide a historic overview about the development of cell models with latent HIV-1 infection that lend themselves to drug discovery. We provide an overview from the first reported latently infected cell lines to current in vitro models of latent HIV-1 infection in primary T cells, and compare their potential to be used in future large-scale drug screening efforts.

The limitations of current anti-retroviral therapies (ART) and the lack of a valid anti-HIV-1 vaccine candidate underscore the need for new therapeutic concepts aiming at the eradication of HIV-1, which represents at the same time an ideal goal and a major challenge for AIDS research. At present, this aim is unattainable due to the existence of cellular and anatomical reservoirs of persistent infection. Memory CD4+ T cells comprise the largest pool of cells harboring silent, stably integrated HIV-1, which remains undetected by the immune system and refractory to conventional anti-retroviral drugs. The eradication of latent HIV-1 reservoirs will require new, potent and specific therapeutic strategies, which in turn must rely upon a deeper understanding of HIV-1 latency. To facilitate the advancement of our knowledge in this new area of research, several in vitro models of HIV-1 latency in CD4+ T cells have been established. Here, we dissect and critically compare the rationale behind each experimental approach. Furthermore, we outline new avenues of research that will benefit from these models, including the push toward the development of new classes of viral eradication drugs.

HAART treatment has greatly improved life expectancy of HIV-1-infected individuals. Unfortunately, latency still remains the major barrier towards HIV-1 eradication. Efforts to identify viral and host cell proteins involved in latency remain important research areas to win this war against HIV-1. Here, we review the contributions of several factors in the establishment of latently infected cells. In addition, we also raise the possibility that RNA helicases, while playing important roles at almost every step of the HIV-1 replication cycle, could be implicated in the processes governing the establishment of these latent reservoirs.

The existence of long-lasting cellular reservoirs of HIV-1 is one of the major hurdles in developing effective anti-retroviral therapies. These latently infected cells and tissues efficiently evade immune responses and remain dormant until activated, upon which they can generate a productive HIV-1 infection. This classic scenario of viral latency becomes even more difficult to study and model due to the extreme complexity of translating in vivo virus-cell interactions into a controlled in vitro system. The recent developments and constant improvements upon hematopoietic engraftment of human cells and tissues onto recipient immunocompromised murine scaffolds have made it possible to model complex human innate and adaptive immune responses in a small animal model. Specifically, HIV-1 infection has been successfully modeled in these humanized mice to mimic transmission, pathogenesis, host immune responses, and treatment. Here, we review the complexities surrounding modeling HIV-1 latency in vitro and in vivo and highlight the most recent humanized mouse models that support retroviral infection.

Introduction: Previous studies have shown that HIV infection is related to changes in the immune status of the mucosal surfaces. Such changes may also occur in the genital tract, since patients infected by HIV have the virus in their cervical secretions. Methods: Fragments of the uterine cervix of 29 autopsied women were collected at a university hospital from 1985 to 2008, and were divided in groups with and without AIDS. Image J software was used to measure the cervical epithelium and to count the epithelial cellular layers. Langerhans cells (LCs) and IgG positive cells were respectively immunostained with anti-S100 and anti-IgG. Results: Women with AIDS, when compared with women without AIDS, had thinner cervical epithelium (103.32 vs 116.71 μm), lower number of cellular layers (10.41 vs 13.66 μm), lower mean cell diameter (10.09 vs 11.51 μm), less number of total LCs (11.19 vs 23.08 LCs/mm² ), and higher percentage of IgG positive cells (22.64% vs 16.06%). All these results were significant. Conclusion: AIDS causes alterations in the structure of the cervical epithelium and in its extracellular matrix, leading to alterations in the local and systemic immunity, and triggering signs and opportunistic infections in the uterine cervix in the course of the disease.

The lower gastrointestinal tract is a major mucosal site of HIV entry and initial infection. Thus, the induction of strong cellular immune responses at this mucosal site will be an important feature of an effective HIV vaccine. We have used a novel prime-boost vaccination approach to induce immune responses at mucosal sites. Orally delivered recombinant Clostridium perfringens expressing HIV-1 gag (Cp-Gag) was evaluated for induction of HIV-1 Gag specific T cell responses in a prime-boost model with intranasal inoculation of HIV-1 virus like particles (VLP). HIV-1 specific cellular immune responses in both the effector (Lamina propria) and inductive sites (Peyer's patches) of the gastrointestinal (GI) tract were significantly higher in mice immunized using Cp-Gag and VLPs in a prime-boost approach compared to mice immunized with either Cp-Gag or VLPs alone. Such cellular immune response was found to be mediated by both CD8+ and CD4+ T cells. Such a strong mucosal immune response could be very useful in developing a mucosal vaccine against HIV-1.

HIV is known to have the ability to adapt rapidly its genome under drug pressure, resulting in clinical treatment failure. We present the case of an HIV-infected patient who developed mutations of resistance to nevirapine although he always had an undetectable viral load and without context of inobservance. The concepts of undetectability and virological success in HIV infection must be balanced by the possible appearance of resistance under a treatment considered as effective.

Background: Since recent observations demonstrated that extended resistance to protease inhibitors, nucleosidic and non - nucleosidic retrotranscriptase inhibitors (PI, NRTI, NNRTI) is a marker of disease progression and death, it is a matter of the greatest importance that experienced human immunodeficiency virus (HIV) - infected patients with limited therapeutic options receive a suppressive therapy pending the availability of at least two new antiretroviral drugs. Aim of the present study is to evaluate if the GSS score, calculated by analyzing the resistance to historical antiretroviral drugs and drug classes, is still relevant since several new potent drugs and drug classes entered the current clinical use. Methods: Taking into account patients without suppression of HIV replication for ≥ 6 months from October 2008 and October 2009, we analyzed viroimmunological and resistance data of 38 outpatients starting their last antiretroviral regimen including at least one of the following: maraviroc, enfuvirtide, raltegravir, etravirine, darunavir/ritonavir or tipranavir/ritonavir. Mutations present in all available genotypic resistance tests were recorded for each patient and then correlated to GSS value, assessed using the last genotypic ribonucleic acid (RNA) resistance test. GSS was studied as predictor of virological treatment outcome by univariate and multivariate logistic regression. Results: At 48 weeks, undetectable viral load was obtained in 80% of patients without difference between GSS classes (HIV-RNA median <50copies/ml); 95.8% of patients with baseline HIV-RNA <50,000copies/ml obtained virological suppression (p=0.003). 48 weeks CD4+ median value was 412 cells/μl considering GSS1 and 300 cells/μl for combined GSS2 and GSS3 scores. Data also showed a > 60% recurrence of specific mutations for NRTI: M41L, M184IV, L210W, T215FY, K219EQ and 75% for D67N. K103N and Y181CIV mutations for NNRTI persisted in 35% of cases and their prevalence incresed in parallel with the number of GRTs. About 60% of tests reported L10FIRVC, M36ILV, M46IL, I54VLAMTS, V82AFTSLI, and L90M mutations in the protease region. 63P mutation was found in a total number of GRTs close to 80%. This percentages, when correlated to GSS, revealed a distinct pattern for most mutations, that showed a greater prevalence for GSS = 2. Conversely, only NNRTI 181CIV and NRTI 210W showed larger numbers in GSS1 and GSS3. Conclusions: Single drugs belonging to new antiretroviral classes did not correlate to viroimmunological success for any GSS. High frequency and recurrence over GRTs for specific mutations confirm their key role following the exposure to ARVs classes. A baseline HIV-RNA <50,000 cp/ml is a predictor of therapeutic success and a carefully selected HAART based upon the evaluation of GRTs can favorably influence the immunovirologic response.

Despite the worldwide efforts made in the field of HIV vaccine development, an efficient AIDS vaccine strategy is still vague. Virus-like particles (VLPs) are one of the introduced aspects for HIV vaccine development since the non-replicative nature of HIV VLPs, resulting from the lack of viral genomic RNA, makes them suitable for broad applications. We have previously designed and introduced non-infectious VLPs (mzNL4-3) by introduction of a deletion mutation in the reverse transcriptase and integrase coding regions of HV-1. There are evidences suggesting that an effective cellular immune response against HIV-1 is able to control and suppress viremia during primary and chronic HIV infections. In the present study we have evaluated the potency of mzNL4-3 VLPs mixed with Neisseria meningitidis serogroup B outer-membrane vesicle (OMV), which is among the microbial components with proved adjuvant properties, to induce humoral and cellular responses against HIV-1. Analysis of anti-HIV-1 responses elicited in immunized BALB/c mice following different immunization regimens indicated OMV+VLP as an immunopotent combination which significantly induced anti-HIV-1 IgG with IgG2a dominancy. Results of cytokine and ELISpot assays also showed the capability of VLP+OMV immunogen for effective induction of IFN-gamma; and IL4 secreting cells and further suggested the promotion of Th1-oriented response that was evidenced with the increased IFN-γ/IL4 secretion ratio. According to our study, HIV-1 VLPs combined with N. meningitidis B OMVs seem to be a promising approach in vaccine development against HIV-1.

Our goal in this study was to analyze position 22 of the V3 loop associated with co-receptor usage and disease progression in human immunodeficiency virus type 1 (HIV-1) subtype B infection. Bioinformatics approaches were used to compare the amino acid sequence and secondary structure of the V3 loop of the CCR5-tropic virus and CXCR4-tropic virus in HIV-1 subtype B. HIV-1 subtype B V3 amino acid sequence files in the FASTA format were collected from the HIV Sequence Database. The amino acid sequences of different tropism were multiple-aligned with CLUSTAL W program, and the frequencies of the amino acids at each position of the V3 loop sequences of two groups were calculated and sorted in descending order. The secondary structure of the consensus V3 amino acid sequences from CCR5-tropic and CXCR4-tropic viruses were predicted with the APSSP2 method. The amino acids at positions 11, 22, and 25 of V3 were different between the CCR5-tropic virus and CXCR4-tropic virus. The consensus amino acid frequencies were found to be 71.9% S, 66.7% A, and 56.0% D for the CCR5-tropic virus and 50.0% R, 57.1% T, and 26.2% Q for the CXCR4- tropic virus at positions 11, 22, and 25, respectively. There was a strong association between the identity of the residues at position 11, 22, and 25 of the V3 loop amino acid sequence and CD4+ T cell counts of different patients. The change of the residue at position 22 in the R5-tropic or X4-tropic viruses is expected to likely change the secondary structure to be similar to the X4-tropic or R5-tropic viruses. Our study indicates that position 22 of the V3 loop amino acid sequence is significantly associated with viral tropism and disease progression in HIV-1 subtype B.