P24 Levels Research Articles

Alcohol and Cannabinoids Differentially Affect HIV Infection and Function of Human Monocyte-Derived Dendritic Cells (MDDC).

During human immunodeficiency virus (HIV) infection, alcohol has been known to induce inflammation while cannabinoids have been shown to have an anti-inflammatory role. For instance cannabinoids have been shown to reduce susceptibility to HIV-1 infection and attenuate HIV replication in macrophages. Recently, we demonstrated that alcohol induces cannabinoid receptors and regulates cytokine production by monocyte-derived dendritic cells (MDDC). However, the ability of alcohol and cannabinoids to alter MDDC function during HIV infection has not been clearly elucidated yet. In order to study the potential impact of alcohol and cannabinoids on differentiated MDDC infected with HIV, monocytes were cultured for 7 days with GM-CSF and IL-4, differentiated MDDC were infected with HIV-1Ba-L and treated with EtOH (0.1 and 0.2%), THC (5 and 10 μM), or JWH-015 (5 and 10 μM) for 4–7 days. HIV infection of MDDC was confirmed by p24 and Long Terminal Repeats (LTR) estimation. MDDC endocytosis assay and cytokine array profiles were measured to investigate the effects of HIV and substances of abuse on MDDC function. Our results show the HIV + EtOH treated MDDC had the highest levels of p24 production and expression when compared with the HIV positive controls and the cannabinoid treated cells. Although both cannabinoids, THC and JWH-015 had lower levels of p24 production and expression, the HIV + JWH-015 treated MDDC had the lowest levels of p24 when compared to the HIV + THC treated cells. In addition, MDDC endocytic function and cytokine production were also differentially altered after alcohol and cannabinoid treatments. Our results show a differential effect of alcohol and cannabinoids, which may provide insights into the divergent inflammatory role of alcohol and cannabinoids to modulate MDDC function in the context of HIV infection.

Pharmacokinetic and Pharmacodynamic Evaluation following Vaginal Application of IQB3002, a Dual-Chamber Microbicide Gel Containing the Nonnucleoside Reverse Transcriptase Inhibitor IQP-0528 in Rhesus Macaques.

We evaluated the in vivo pharmacokinetics and used a complementary ex vivo coculture assay to determine the pharmacodynamics of IQB3002 gel containing 1% IQP-0528, a nonnucleoside reverse transcriptase inhibitor (NNRTI), in rhesus macaques (RM). The gel (1.5 ml) was applied vaginally to 6 simian-human immunodeficiency (SHIV)-positive female RM. Blood, vaginal fluids, and rectal fluids were collected at 0, 1, 2, and 4 h. RM were euthanized at 4 h, and vaginal, cervical, rectal, and regional lymph node tissues were harvested. Anti-human immunodeficiency virus (HIV) activity was evaluated ex vivo by coculturing fresh or frozen vaginal tissues with activated human peripheral blood mononuclear cells (PBMCs) and measuring the p24 levels for 10 days after an HIV-1Ba-L challenge. The median levels of IQP-0528, determined using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) methods, were between 10(4) and 10(5) ng/g in vaginal and cervical tissue, between 10(3) and 10(4) ng/g in rectal tissues, and between 10(5) and 10(7) ng/ml in vaginal fluids over the 4-h period. The vaginal tissues protected the cocultured PBMCs from HIV-1 infection ex vivo, with a viral inhibition range of 81 to 100% in fresh and frozen tissues that were proximal, medial, and distal relative to the cervix. No viral inhibition was detected in untreated baseline tissues. Collectively, the median drug levels observed were 5 to 7 logs higher than the in vitro 50% effective concentration (EC50) range (0.21 ng/ml to 1.29 ng/ml), suggesting that 1.5 ml of the gel delivers IQP-0528 throughout the RM vaginal compartment at levels that are highly inhibitory to HIV-1. Importantly, antiviral activity was observed in both fresh and frozen vaginal tissues, broadening the scope of the ex vivo coculture model for future NNRTI efficacy studies.

Synergistic Inhibition of R5 HIV-1 by the Fusion Protein (FLSC) IgG1 Fc and Maraviroc in Primary Cells: Implications for Prevention and Treatment.

Antiretroviral (ARV) drugs targeting retroviral enzymes have been extensively employed to treat HIV-1 infection. Drawbacks of this approach include cost, toxicity, and the eventual emergence of resistant strains that threaten prophylactic and/or therapeutic efficacy. Accordingly, efforts to develop next-generation ARV approaches are warranted, particularly if they can offer a higher threshold of resistance. We have previously shown that FLSC, a fusion protein containing gp120(BAL) and the D1 and D2 domains of human CD4, specifically binds CCR5, an important cellular co-receptor, and inhibits the entry of R5 HIV isolates. (FLSC) IgG1, a fusion of FLSC and the hinge-C(H)2-C(H)3 region of human IgG1, has an increased antiviral activity, likely due to the resultant bivalency. In this study, we show CCR5 reduction upon (FLSC) IgG1 treatment both by standard flow cytometry and visualized using a novel nanoparticle method. A β-lactamase virus-cell fusion assay was used to quantify (FLSC) IgG1 inhibition of HIV-1 entry into both cell lines and primary cells. Synergistic anti-viral activities of (FLSC) IgG1 and MVC in primary cells were evaluated by measuring supernatant p24 levels via ELISA and calculated using the MacSynergy™ II program. We previously reported that treatment with the CCR5 small molecule antagonist Maraviroc (MVC) increased the apparent exposure of the (FLSC) IgG1 binding sites on CCR5, leading us to wonder if the two compounds used in combination might synergize in their anti-viral activity. Here we show that this is indeed the case. We demonstrate that fusion protein (FLSC) IgG1, strongly synergizes with the CCR5 antagonist Maraviroc to successfully inhibit both MVC-sensitive and MVC-resistant R5 HIV-1. Observed synergy between (FLSC) IgG1 and MVC was high in both, cell lines and primary PBMCs. This has relevance for future in vivo studies. In addition, synergy occurred both with MVC-sensitive viruses and MVC-resistant viruses, partially restoring the inhibitory effect of MVC. These findings suggest that a combinatorial treatment based on these two compounds has potential merit and that future in vivo studies are warranted.

ID: 41: Treatment with interferon alpha subtype 14 potently suppresses HIV infection in humanized mice

There are twelve human interferon alpha subtypes that differentially trigger intrinsic, innate, and adaptive immune responses to viral infections. IFN α 2 is currently in HIV clinical trials, but the other subtypes have not been tested and may be better HIV therapeutics. We used HIV-1-infected humanized mice to compare the antiviral properties of IFN α 2 with IFN α 14, the subtype we found to induce the most potent anti-HIV effect in vitro. After ten days of post-exposure prophylaxis, IFN α 14 suppressed virus much better than IFN α 2 with no detectable plasma HIV p24 and greatly reduced or undetectable plasma RNA and provirus levels. Furthermore, CXCL10, an inflammatory chemokine indicative of pathological immune activation, was elevated in IFN α 2-treated but not IFN α 14-treated animals. IFN α 14 efficacy was associated with up-regulated BST2 (tetherin) and MX2 transcripts and TRAIL expression on NK cells but no significant activation of adaptive immune responses. Treatment of chronically HIV-1-infected humanized mice with IFN α 14 resulted in significant decrease in plasma HIV p24 and plasma RNA levels. IFN α 14 strongly outperformed IFN α 2 as an HIV anti-viral in humanized mice.

Baculovirus Infection of Human Monocyte-Derived Dendritic Cells Restricts HIV-1 Replication.

Acquired immune deficiency syndrome (AIDS) is mainly caused by infection with human immunodeficiency virus-1 (HIV-1) and still poses a global threat for which we lack a protective or therapeutic vaccine. Dendritic cells (DCs) play a major role in the onset of HIV infection, providing one of the primary sites of HIV replication, and also act as viral reservoirs in vivo. Previous studies have shown that baculovirus (BV) induces strong host immune responses against infections and malignancies. In this study, we infected human monocyte-derived DCs with recombinant BV (AcCAG-gag) and showed that AcCAG-gag-infected human DCs underwent maturation and produced interferon alpha and other proinflammatory cytokines accompanied by increases in the mRNA and protein expression levels of APOBEC3 (A3A, A3F, and A3G), proteins associated with the inhibition of HIV-1 replication. Surprisingly, HIV-1 inhibition is also observed in human DCs infected with a wild-type BV, as determined by the production of inflammatory cytokines, the expression of A3, and a reduction in the p24 level. Our findings outline the mechanism underlying the inhibition of HIV-1 in BV-infected human DCs and pave the way for the use of BV as an effective tool for immunotherapy against HIV-1.

62. Aptamer-siRNA Conjugate Directed Transcriptional Gene Silencing in HIV-1 Infected T Cells

The primary standard of care against human immunodeficiency virus (HIV) -1 is highly active antiretroviral therapy which is plagued by toxicity and patient compliance. As such, alternative therapies are sought to provide sustained viral inhibition with reduced treatment intervention. One new potential approach, termed transcriptional gene silencing (TGS), involves the use of small non-coding RNAs (sncRNAs) to inhibit gene expression via the induction of concomitant silent-state epigenetic marks on histones and DNA. We previously demonstrated the ability to silence HIV-1 via TGS by targeting sites within the 5’ LTR promoter [1, 2]. However, the lack of efficient, systemic delivery of these RNAs to infected cells hinders successful clinical progression. To address this hurdle, we describe here the development of chimeric molecules based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV siRNA (dsiRNA) to deliver these sncRNAs to virally infected cells. The efficacy of such a system had been previously demonstrated both in vitro and in vivo to target selectively gp120-expressing cells and to deliver siRNAs for the inhibition of HIV-1 through post-transcriptional gene silencing (PTGS) [3]. To validate the ability for nuclear delivery of sncRNAs, we demonstrated, by fluorescent confocal microscopy, 15% nuclear delivery for our novel chimera-delivered dsiRNAs in a gp120 expressing CHO cells. Using nuclear-cytoplasmic fractionation of HIV-infected PBMCs, dsiRNAs targeting the susceptible regions within the 5’ LTR showed preferential nuclear localization when compared to a scrambled siRNAs. Additionally, two 800 nM doses at day 5 and 7 post-infection (p.i.) induced 10-fold suppression of viral p24 levels as measured at day 12 p.i. in CEM cells. The LTR-362 27-mer aptamer chimera proved more potent than A-1 tat/rev 27mer chimera, a positive control that inhibits HIV via PTGS. To validate the inhibition occurred via TGS, 5’-Azacytidine and trichostatin A were added at day 5 p.i, and resulted in transcriptional derepression by the A-1 LTR-263 27-mer, but not the A-1 Tat/Rev 27mer. Nuclear run-on analysis provided further confirmation of TGS inhibition. This is the first study to demonstrate RNA-induced TGS of an existing viral infection in both human PBMC and CEM cell lines following application of aptamer-siRNA chimeras.

Molecular mechanisms linking high dose medroxyprogesterone with HIV-1 risk.

BackgroundEpidemiological studies suggest that medroxyprogesterone acetate (MPA) may increase the risk of HIV-1. The current studies were designed to identify potential underlying biological mechanisms.MethodsHuman vaginal epithelial (VK2/E6E7), peripheral blood mononuclear (PBMC), and polarized endometrial (HEC-1-A) cells were treated with a range of concentrations of MPA (0.015-150 μg/ml) and the impact on gene expression, protein secretion, and HIV infection was evaluated.ResultsTreatment of VK2/E6E7 cells with high doses (>15μg/ml] of MPA significantly upregulated proinflammatory cytokines, which resulted in a significant increase in HIV p24 levels secreted by latently infected U1 cells following exposure to culture supernatants harvested from MPA compared to mock-treated cells. MPA also increased syndecan expression by VK2/E6E7 cells and cells treated with 15 μg/ml of MPA bound and transferred more HIV-1 to T cells compared to mock-treated cells. Moreover, MPA treatment of epithelial cells and PBMC significantly decreased cell proliferation resulting in disruption of the epithelial barrier and decreased cytokine responses to phytohaemagglutinin, respectively.ConclusionWe identified several molecular mechanisms that could contribute to an association between DMPA and HIV including proinflammatory cytokine and chemokine responses that could activate the HIV promoter and recruit immune targets, increased expression of syndecans to facilitate the transfer of virus from epithelial to immune cells and decreased cell proliferation. The latter could impede the ability to maintain an effective epithelial barrier and adversely impact immune cell function. However, these responses were observed primarily following exposure to high (15-150 μg/ml) MPA concentrations. Clinical correlation is needed to determine whether the prolonged MPA exposure associated with contraception activates these mechanisms in vivo.

Sustained-release nanoART formulation for the treatment of neuroAIDS.

A novel approach was developed for the coencapsulation of an anti-HIV drug (tenofovir) and a latency-breaking agent (vorinostat), using magnetically guided layer-by-layer (LbL) assembled nanocarriers for the treatment of neuroAIDS. Ultrasmall iron oxide (Fe3O4) nanoparticles (10±3 nm) were synthesized and characterized. The LbL technique was used to achieve a sustained release profile, and application of 2 bilayers ([tenofovir+dextran sulphate]2+vorinostat) to magnetic nanoparticles resulted in a 2.8 times increase in drug (tenofovir) loading and also resulted in an increase in the drug release period by 30-fold, with 100% drug release in sustained manner over a period of 5 days with the simultaneous stimulation of latent HIV expression. Nanoformulation showed a good blood–brain barrier transmigration ability (37.95%±1.5%) with good in vitro antiviral efficacy (~33% reduction of p24 level) over a period of 5 days after HIV infection in primary human astrocytes, with good cell viability (>90%). Hence, LbL arrangements of drugs on magnetic nanoparticles provides sustained release and, therefore, may improve the patient’s adherence to therapy and lead to better compliance.

Transduction of ferret airway epithelia using a pre-treatment and lentiviral gene vector.

BackgroundThe safety and efficiency of gene therapies for cystic fibrosis (CF) need to be assessed in pre-clinical models. Using the normal ferret, this study sought to determine whether ferret airway epithelia could be transduced with a lysophosphatidylcholine (LPC) pre-treatment followed by a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector, in preparation for future studies in CF ferrets.MethodsSix normal ferrets (7 -8 weeks old) were treated with a 150 μL LPC pre-treatment, followed one hour later by a 500 μL LV vector dose containing the LacZ transgene. LacZ gene expression in the conducting airways and lung was assessed by X-gal staining after 7 days. The presence of transduction in the lung, as well as off-target transduction in the liver, spleen and gonads, were assessed by qPCR. The levels of LV vector p24 protein bio-distribution in blood sera were assessed by ELISA at 0, 1, 3, 5 and 7 days.ResultsThe dosing protocol was well tolerated. LacZ gene expression was observed en face in the trachea of all animals. Histology showed that ciliated and basal cells were transduced in the trachea, with rare LacZ transduced single cells noted in lung. p24 levels was not detectable in the sera of 5 of the 6 animals. The LacZ gene was not detected in the lung tissue and no off-target transduction was detected by qPCR.ConclusionsThis study shows that ferret airway epithelia are transducible using our unique two-step pre-treatment and LV vector dosing protocol. We have identified a number of unusual anatomical factors that are likely to influence the level of transduction that can be achieved in ferret airways. The ability to transduce ferret airway epithelium is a promising step towards therapeutic LV-CFTR testing in a CF ferret model.

Cenicriviroc blocks HIV entry but does not lead to redistribution of HIV into extracellular space like maraviroc.

IntroductionCenicriviroc (CVC), a once-daily, dual CCR5/CCR2 co-receptor antagonist, has completed Phase 2b development. CVC demonstrated favourable safety and similar efficacy compared with efavirenz (EFV) in Study 202 (NCT01338883); an ex vivo sub-analysis evaluated treatment effects on HIV entry, measured by intracellular HIV DNA declines, in subjects with virologic success at Week 24. In addition, in vitro assays determined and compared the extent of any cell-free virion redistribution that CVC or maraviroc (MVC) may cause.MethodsEx vivo: intracellular DNA (frozen PBMCs) from 30 subjects with virologic success at Week 24 (10, 13 and 7 subjects on CVC 100 mg, CVC 200 mg and EFV, respectively). Early (strong-stop) and late (full-length) reverse transcript levels were measured by qPCR. In vitro: PM-1 cells were infected with CCR5-tropic HIV-1 BaL in the presence or absence of inhibitory concentrations of CVC (20 nM), MVC (50 nM) or controls. P24 and viral load levels were measured by ELISA and qRT-PCR after 4 hours.ResultsEx vivo analysis showed full-length HIV DNA declines were similar across all groups (CVC 100 mg, CVC 200 mg and EFV) at Week 24. Strong-stop HIV DNA declines (a marker of HIV entry) at Week 24 were pronounced for both CVC arms (CVC 100 mg, 51% decline; CVC 200 mg, 37% decline) compared to no decline for the EFV arm. In vitro experiments revealed that CVC-treated cells had lower levels of supernatant P24 at 4 hours versus baseline (0 hrs: 506 ng/mL; 4 hrs: 192 ng/mL), but P24 levels remained constant for MVC-treated cells after 4 hours (0 hrs: 506 ng/mL; 4 hrs: 520 ng/mL). Viral load levels for CVC-treated cells remained stable after 4 hours (0 hrs: 1.19×1010 copies/mL; 4 hrs: 1.26×1010 copies/mL). MVC-treated cells exhibited a slight increase in viral load after 4 hours (0 hrs: 1.19×1010 copies/mL; 4 hrs: 1.67×1010 copies/mL).ConclusionsEx vivo analysis confirmed that CVC treatment blocks HIV entry (strong-stop HIV DNA declines), while in vitro analysis showed that CVC-treated cells do not repel virus back into the extracellular space, as seen with MVC. Experiments are underway to determine whether or not interactions between CVC and HIV at the binding site may explain these unanticipated findings.

Fetal exposure to HIV-1 alters chemokine receptor expression by CD4+T cells and increases susceptibility to HIV-1.

Absolute numbers of lymphocytes are decreased in uninfected infants born to HIV-1-infected women (HIV-1-exposed). Although the exact mechanism is unknown, fetal exposure to maternal HIV-1-infection could prime the immune system and affect T cell trafficking. We compared the expression of chemokine receptors on cord blood CD4+ T cells from HIV-1-exposed children and healthy controls. At baseline CD4+ T cells had a largely naïve phenotype. However, stimulation with cytokines resulted in an upregulation of inflammatory response-related chemokine receptors on CD4+ T cells, with HIV-1-exposed infants having a significantly higher frequency of CD4+ T cells expressing, in particularly Th2 associated chemokine receptors (CCR3 p < 0.01, CCR8 p = 0.03). Numbers of naive CCR7+ CD4+ T cells were reduced (p = 0.01) in HIV-1-exposed infants. We further assessed whether the inflammatory phenotype was associated with susceptibility to HIV-1 and detected higher levels of p24 upon in in vitro infection of stimulated CD4+ T cells of HIV-1-exposed infants. In summary, fetal exposure to HIV-1 primes the immune system in the infant leading to an enhanced immune activation and altered T cell homing, with potential ramifications regarding T cell responses and the acquisition of HIV-1 as an infant.

Morphine enhances HIV-1SF162-mediated neuron death and delays recovery of injured neurites.

HIV-1 enters the CNS soon after initial systemic infection; within the CNS parenchyma infected and/or activated perivascular macrophages, microglia and astrocytes release viral and cellular toxins that drive secondary toxicity in neurons and other cell types. Our previous work has largely modeled HIV-neuropathology using the individual viral proteins Tat or gp120, with murine striatal neurons as targets. To model disease processes more closely, the current study uses supernatant from HIV-1-infected cells. Supernatant from HIV-1SF162-infected differentiated-U937 cells (HIV+ sup) was collected and p24 level was measured by ELISA to assess the infection. Injection drug abuse is a significant risk factor for HIV-infection, and opiate drug abusers show increased HIV-neuropathology, even with anti-retroviral treatments. We therefore assessed HIV+ sup effects on neuronal survival and neurite growth/pruning with or without concurrent exposure to morphine, an opiate that preferentially acts through µ-opioid receptors. Effects of HIV+ sup ± morphine were assessed on neuronal populations, and also by time-lapse imaging of individual cells. HIV+ sup caused dose-dependent toxicity over a range of p24 levels (10–500 pg/ml). Significant interactions occurred with morphine at lower p24 levels (10 and 25 pg/ml), and GSK3β was implicated as a point of convergence. In the presence of glia, selective neurotoxic measures were significantly enhanced and interactions with morphine were also augmented, perhaps related to a decreased level of BDNF. Importantly, the arrest of neurite growth that occurred with exposure to HIV+ sup was reversible unless neurons were continuously exposed to morphine. Thus, while reducing HIV-infection levels may be protective, ongoing exposure to opiates may limit recovery. Opiate interactions observed in this HIV-infective environment were similar, though not entirely concordant, with Tat/gp120 interactions reported previously, suggesting unique interactions with virions or other viral or cellular proteins released by infected and/or activated cells.

Endosomal trafficking of nanoformulated antiretroviral therapy facilitates drug particle carriage and HIV clearance.

Limitations of antiretroviral therapy (ART) include poor patient adherence, drug toxicities, viral resistance, and failure to penetrate viral reservoirs. Recent developments in nanoformulated ART (nanoART) could overcome such limitations. To this end, we now report a novel effect of nanoART that facilitates drug depots within intracellular compartments at or adjacent to the sites of the viral replication cycle. Poloxamer 407-coated nanocrystals containing the protease inhibitor atazanavir (ATV) were prepared by high-pressure homogenization. These drug particles readily accumulated in human monocyte-derived macrophages (MDM). NanoATV concentrations were ∼1,000 times higher in cells than those that could be achieved by the native drug. ATV particles in late and recycling endosome compartments were seen following pulldown by immunoaffinity chromatography with Rab-specific antibodies conjugated to magnetic beads. Confocal microscopy provided cross validation by immunofluorescent staining of the compartments. Mathematical modeling validated drug-endosomal interactions. Measures of reverse transcriptase activity and HIV-1 p24 levels in culture media and cells showed that such endosomal drug concentrations enhanced antiviral responses up to 1,000-fold. We conclude that late and recycling endosomes can serve as depots for nanoATV. The colocalization of nanoATV at endosomal sites of viral assembly and its slow release sped antiretroviral activities. Long-acting nanoART can serve as a drug carrier in both cells and subcellular compartments and, as such, can facilitate viral clearance. The need for long-acting ART is significant and highlighted by limitations in drug access, toxicity, adherence, and reservoir penetrance. We propose that targeting nanoformulated drugs to infected tissues, cells, and subcellular sites of viral replication may improve clinical outcomes. Endosomes are sites for human immunodeficiency virus assembly, and increasing ART concentrations in such sites enhances viral clearance. The current work uncovers a new mechanism by which nanoART can enhance viral clearance over native drug formulations.

DC SIGNR silencing reduces HIV-1 infection in Dendritic cells

Results High expression of DC SIGN/R was observed on DCs infected with HIV-1 (p= 0.01). DC sIGNR expression in DCs was maximally downregulated by siRNA at 24 hrs (p=0.002) and was associated with significant reduction in expression of CD40 (p= 0.003), CD80 (p=0.008), CD86 (p=0.007) and P38 MAPK (p=0.005). Transfection of DC SIGNR on DCs at 24 hours followed by infection with clade C HIV-1 demonstrated lower levels of p24 compared to that in untransfected DCs (p=0.0008).

Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity following DNA-Vaccination of BALB/c Mice

The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation.

HIV Protective KIR3DL1/S1-HLA-B Genotypes Influence NK Cell-Mediated Inhibition of HIV Replication in Autologous CD4 Targets

Carriage of the genetic combination encoding a high expression inhibitory Killer Immunoglobulin-like Receptor (KIR)3DL1 with its ligand, HLA-B*57 (*h/*y+B*57) is associated with slower time to AIDS and better HIV viral load control than being a Bw6 homozygote (Bw6hmz). Natural Killer (NK) cells from *h/*y+B*57 carriers receive potent educational signals through HLA-B*57 KIR3DL1 ligation leading to high functional potential. NK cells from Bw6hmz are not educated through KIR3DL1 because Bw6 antigens do not interact with this inhibitory receptor. To better understand the impact of KIR/HLA combinations on NK cell mediated anti-viral activity we measured NK cell mediated inhibition of HIV replication in autologous infected CD4 (iCD4) cells by assessing the frequency of p24 positive CD4 targets and supernatant levels of HIV p24 longitudinally in the presence versus absence of NK cells. Forty-seven HIV uninfected subjects were studied, including carriers of *h/*y+B*57, a low expression KIR3DL1 genotype with HLA-B*57 termed *l/*x+B*57, a genotype designated 3DS1+*80I and Bw6hmz. NK cells from *h/*y+B*57 carriers, like those from 3DS1+*80I subjects, inhibited HIV replication in autologous iCD4 cells better than those from Bw6hmz and *l/*x+B*57 carriers. Cell contact between NK and iCD4 cells activated NK cells to inhibit viral replication in a non-contact dependent fashion through secretion of CC-chemokines. iCD4 stimulated NK cells from *h/*y+B*57 and 3DS1+*80I carriers produced higher levels of CC-chemokines than those from Bw6hmz or *l/*x+B*57 carriers. Higher levels of CC-chemokines were produced by KIR3DL1+ than KIR3DL1− NK cells. We conclude that NK-mediated inhibition of viral replication in autologous iCD4 cells is partially due to a block at the level of HIV entry into new targets by secreted CC-chemokines.

Naturally occurring compounds elicit HIV-1 replication in chronically infected promonocytic cells.

Since antiretroviral therapy suppresses but does not eradicate HIV-1 infection, methods to purge viral reservoirs are required. Many strategies involve the reactivation of chronically HIV infected cells to induce the expression of integrated viral genome. In this study, five bioactive compounds, the plant derivatives 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), nordihydroguaiaretic acid (NDGA), and curcumin (Cur) and the synthetic stigmasterol analogs (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3β-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2), were evaluated for their ability to elicit HIV replication in promonocytic (U1) and lymphocytic (H9+) HIV-1 chronically infected cells. The results revealed that natural compounds CDM, NDGA, and Cur were able to increase HIV-1 p24 antigen, determined by ELISA, only in latently infected promonocytic cells. CDM would reactivate HIV from latency by modulating the release of IL-6 and TNF-α, since the amount of both cytokines measured through ELISA significantly increased in U1 treated cells. Besides, NDGA increased ROS production, which might be related to the increase on p24 level observed in NDGA treated U1. These findings suggest that CDM, NDGA, and Cur might be candidates for further studies on latency-reversing therapeutics to eliminate latently HIV-1 reservoirs.

The antisense protein of HTLV-2 positively modulates HIV-1 replication

Unlike HTLV-1, HTLV-2 does not induce leukemia and has been tentatively associated with an HTLV-1-associated myelopathy-like disorder. It has been reported that HTLV-2/HIV-1 co-infected patients progress less rapidly to AIDS than HIV-1-infected individuals. Tax2 has been suggested to mediate this protective state by inducing MIP-1α expression and blocking HIV-1 infection. As cells from HTLV-2-infected individuals mainly express Antisense Protein 2 (APH-2), our objective was to determine if this protein might also intervene in controlling HIV-1 replication in dually infected individuals. Using Jurkat cells, we first demonstrated that both APH-2 and HBZ, the HTLV-1 analogue, equally induced MIP-1α in unstimulated and stimulated Jurkat T cells. To assess if APH-2 might directly affect HIV-1 replication, a full length luciferase-expressing proviral DNA was tested in Jurkat cells. Surprisingly, upon co-transfection with an APH-2 expression vector, an increase in luciferase activity was observed, while HBZ expression rather led to reduced reporter gene expression. Western blot analyses and ELISA assay further indicated that HIV-1 p24 levels were more important in APH-2-expressing cells. To determine if APH-2 was directly modulating HIV-1 LTR activity, both NF-κB and NFAT were tested in stimulated Jurkat cells. Unexpectedly, HBZ and APH-2 inhibited NF-κB and NFAT activation, albeit at different extent. In addition, LTR activation was also inhibited by both antisense proteins although APH-2 had a more modest effect. Our results thus highlight the complex interplay between HTLV antisense transcript-encoded proteins and HIV-1 expression and further studies will be required to determine the potential impact of APH-2 in HTLV-2/HIV-1-infected individuals.

Recombinant Tax1 and Tax2 proteins inhibit HIV-1 replication in peripheral blood mononuclear cells

Patients with HIV-1 and HTLV-2 coinfections often exhibit a clinical course similar to HIV-1 infected long-term nonprogressors. This observation has been attributed in part to the ability of the HTLV Tax2 protein to activate production of antiviral chemokines and to downregulate the CCR5 co receptor on lymphocytes. Therefore we investigated the possibility that a recombinant Tax2 protein could suppress HIV-1 viral replication in vitro. R5-tropic HIV-1 (NLAD8)-infected-PBMCs were treated daily with recombinant Tax1 and Tax2 proteins (dosage range 1-100 pM). Culture supernatants were collected at intervals for 22 days post-infection and assayed for levels of HIV-1 p24 antigen. Treatment of PBMCs with Tax2 protein resulted in significant reduction in HIV-1 p24 antigen levels (p<0.05) at days 10, 14, and 18 post-infection compared to HIV-1-infected or mock-treated PBMCs. This was preceded by the detection of increased levels of CC-chemokines MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5, on days 1-7 of infection (p<0.05). Similar, but less robust inhibition was determined in Tax1 treated PBMCs. Addition of Tax2 proteins starting 48 hours previous to infection resulted in a significant inhibition of HIV-1 p24 levels (p<0.05 at days 7 to 14 compared to the untreated, R5 infected PBMCs. In contrast, when addition of Tax2 began two days after R5 infection, significant HIV-1 p24 levels were only determined at days 7 and 10 for 1 pM and at day 7 for 10 pM (p<0.05). These results support the contention that Tax1 and Tax2 play a role in generating antiviral responses against HIV-1 in vivo and in vitro.

HIV Antibody Characterization as a Method to Quantify Reservoir Size During Curative Interventions

Quantitative humoral profiling of recent samples from a human immunodeficiency virus (HIV)-infected adult who was cured following a delta32/delta32 CCR5 stem cell transplant in 2007 revealed no antibodies against p24, matrix, nucleocapsid, integrase, protease, and gp120, but low levels of antibodies against reverse transcriptase, tat, and gp41. Antibody levels to these HIV proteins persisted at high and stable levels in most noncontrollers, elite controllers, and antiretroviral-treated subjects, but a rare subset of controllers had low levels of antibodies against matrix, reverse transcriptase, integrase, and/or protease. Comprehensive HIV antibody profiles may prove useful for monitoring curative interventions.