Characterization of the MN gp120 HIV-1 Vaccine: Antigen Binding to Alum

Purpose. The characterization of recombinant MN gp120/alum vaccine requires the study of the gp120-alum interaction for the successful formulation of an alum-based HIV-1 vaccine. Methods. Several observations suggest that the gpl20-alum interaction is weak, wherein buffer counterions such as phosphate, sulfate, bicarbonate may cause the desorption of gp120 from alum. Comparison of gp120 with other proteins using particle mobility measurements shows that the weak binding of gp120 to alum is not an anomaly. Serum and plasma also cause desorption of gp120 from alum with a half-life of only a few minutes, wherein this half-life may be faster than the in-vivo recruitment of antigen presenting cells to the site of immunization. Results. Immunization of guinea pigs, rabbits and baboons with gp120 formulated in alum or saline demonstrated that alum provides adjuvant activity for gp120, particularly after early immunizations, but the adjuvant effect is attenuated after several boosts. Conclusions. These observations indicate that both the antigen and the adjuvant require optimization together.     

Small-molecule HIV-1 entry inhibitors targeting gp120 and gp41: a patent review (2010-2015)

Introduction: It is essential to discover and develop small-molecule HIV-1 entry inhibitors with suitable pharmaceutical properties.

Areas covered: We review the development of small-molecule HIV-1 entry inhibitors as evidenced in patents, patent applications, and related research articles published between 2010 and 2015.

Expert opinion: HIV-1 Env gp120 and gp41 are important targets for development of HIV-1 entry inhibitors. The Phe43 pocket in gp120 and the highly conserved hydrophobic pocket on gp41 NHR-trimer are important targets for identification of HIV-1 attachment and fusion inhibitors, respectively. Compounds that bind to Phe43 pocket can block viral gp120 binding to CD4 on T cells, thus inhibiting HIV-1 attachment. However, most compounds targeting Phe43 pocket identified so far are HIV-1 entry agonists with the ability to enhance infectivity of HIV-1 in CD4-negative cells. Therefore, it is essential to identify HIV-1 entry antagonist-based HIV-1 attachment/entry inhibitors. Compounds binding to the gp41 hydrophobic pocket may inhibit CHR binding to the gp41 NHR trimer, thus blocking six-helix bundle formation and gp41-mediated virus-cell fusion. However, most lead compounds targeting this pocket have low potency, possibly because the pocket is too big or too deep. Therefore, it is necessary to identify other pockets in gp41 for developing HIV-1 fusion/entry inhibitors.     

Short peptides with induced beta-turn inhibit the interaction between HIV-1 gp120 and CD4

To identify novel peptides that inhibit the interaction between human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 and CD4, we constructed a targeted phage-displayed peptide library in which phenylalanine and proline were fixed at the fourth and sixth positions, respectively, because Phe43 and the adjacent beta-turn of CD4 are critical for interaction with gp120. Two synthetic peptides were selected after three rounds of biopanning against gp120, and one of them, G1 peptide (ARQPSFDLQCGF), exhibited specific inhibition of the interaction between gp120 and CD4 with an IC(50) of about 50 microM. Structural analysis using NMR demonstrated that G1 peptide forms a compact cyclic structure similar to the CD4 region interacting with gp120. Two derivatives of G1 peptide, a linear hexameric peptide (G1-6) and a cyclic nonameric peptide (G1-c), were synthesized based on the structure of the G1 peptide. Interestingly, they showed higher inhibitory activities than did G1 peptide with IC(50)'s of 6 and 1 microM, respectively. Thus, this study might provide a new insight into the development of anti-HIV-1 inhibitors.     

Galactosylceramide and transmembrane signalling in enterocytes: Calcium response induced by HIV-1 surface-envelope glycoprotein gp120

Summary Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were measured in single-cultured human epithelial intestinal HT-29-D4 cells by digital microscopy using the Ca²⁺-sensitive fluorescent dye Fura-2. Exposure of these cells to HIV-1 surface-envelope glycoprotein gp120, or to a soluble form of its precursor (gp160), resulted in a significant, dose-dependent rise in [Ca²⁺]_i. gp120 or gp160 specifically abrogated the [Ca²⁺]_i response to the neuropeptide agonist neurotensin, which is a stimulator of chloride secretion via inositol trisphosphate-mediated Ca²⁺ mobilization. By contrast, upon exposure to neurotensin gp120 failed to show any increase in [Ca²⁺]_i within the same cells, suggesting that both neurotensin and gp120 stimulate a common pathway of [Ca²⁺]_i mobilization. gp120-/gp160-induced [Ca²⁺]_i responses were abolished by preincubation with neutralizing antibodies directed against the third variable domain of gp120. These antibodies inhibited the binding of gp120/gp160 to galactosylceramide (GalCer), the alternative HIV-1 receptor in HT-29-D4 cells. Furthermore, HT-29-D4 cells displayed an important increase in [Ca²⁺]_i to anti-GalCer mAb alone, which rendered the cells insensitive to gp120. By contrast, HT-29-D4 cells became insensitive to anti-GalCer mAb after exposure to gp120. These data indicate that HIV-1 may directly alter enterocytic functions through interaction with the GalCer receptor.     

Morphine and gp120 Toxic Interactions in Striatal Neurons are Dependent on HIV-1 Strain

A rigorously controlled, cell culture paradigm was used to assess the role of HIV-1 gp120 ± morphine in mediating opioid-HIV interactive toxicity in striatal neurons. Computerized time-lapse microscopy tracked the fate of individual neurons co-cultured with mixed-glia from mouse striata during opioid and gp120 exposure. Subpopulations of neurons and astroglia displayed ??-opioid receptor, CXCR4, and CCR5 immunoreactivity. While gp120 alone was or tended to be neurotoxic irrespective of whether X4-tropic gp120_IIIB, R5-tropic gp120_ADA, or dual-tropic gp120_MN was administered, interactive toxicity with morphine differed depending on HIV-1 strain. For example, morphine only transiently exacerbated gp120_IIIB-induced neuronal death; however, in combination with gp120_MN, morphine caused sustained increases in the rate of neuronal death compared to gp120_MN alone that were prevented by naloxone. Alternatively, gp120_ADA significantly increased the rate of neuron death, but gp120_ADA toxicity was unaffected by morphine. The transient neurotoxic interactions between morphine and gp120_IIIB were abrogated in the absence of glia suggesting that glia contribute significantly to the interactive pathology with chronic opiate abuse and neuroAIDS. To assess how mixed-glia might contribute to the neurotoxicity, the effects of morphine and/or gp120 on the production of reactive oxygen species (ROS) and on glutamate buffering were examined. All gp120 variants, and to a lesser extent morphine, increased ROS and/or decreased glutamate buffering, but together failed to show any interaction with morphine. Our findings indicate that HIV-1 strain-specific differences in gp120 are critical determinants in shaping both the timing and pattern of neurotoxic interactions with opioid drugs.     

gp120 of HIV-1 induces apoptosis in rat cortical cell cultures: prevention by memantine.

After incubation of rat cortical cell cultures with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 for 12 h, cells showed fragmentation of DNA at internucleosomal linkers, the characteristic feature of apoptosis. In a quantitative approach, it was determined that the percentage of DNA fragmentation increased from 7%, in the absence of gp120, to 62% following incubation with 24 ng/ml of gp120. Simultaneously, the percentage of viable cells decreased from 94% to 33%. Memantine (1-amino-3,5-dimethyladamantane), a drug currently used in the therapy of spasticity and Parkinson's disease as well as the NMDA antagonist MK-801 both prevented the effects of gp120 at micromolar concentrations. In human cultured astrocytes, gp120 was ineffective with respect to DNA fragmentation and cell toxicity. From these data, we conclude that the gp120-induced apoptosis may contribute to the neurological complications frequently associated with the immunodeficiency syndrome. The cytoprotective effect of memantine in cortical cell cultures may qualify the drug for the treatment of AIDS-related dementia.     

HIV-1 Gag-gp120嵌合基因在重组杆状病毒系统中的嵌合表达

将中国株HIV-1B亚型gag基因序列克隆到杆状病毒表达载体pFASTBAC1中,将gp120基因以相同的读框克隆到gag基因的3／端下游,构建了重组表达质粒pFge,利用大肠杆菌埃希菌杆状病毒表达系统筛选重组杆状病毒,在昆虫细胞sf9中表达了HIV-1的Gag-gp120嵌合蛋白。     

人CCR5受体与CD4抗原和HIV-1包膜糖蛋白gp120复合物的分子模型(英文)

目的:研究人CCR5与CD4抗原和HIV-1包膜糖蛋白gp120的相互作用。方法:人CCR5受体的结构保守区由SYBYL软件中的Biopolymer模块建立,非保守区由LOOP SEARCH方法建立。将得到的结构模型与CD4抗原和gp120结合形成复合物。结果:人CCR5受体既可以与CD4抗原相互作用,也可以和gp120相互作用,其N-末端残基通过静电和氢键方式与CD4相互作用,并深埋在一个疏水中心里,被碱性基团包围。而且有7个氨基酸残基通过范德华作用、疏水性作用和氢键与6个gp120残基相互作用。结论:该模型将有助于设计作用更强的抗艾滋病药物。     

Molecular modeling on human CCR5 receptors and complex with CD4 antigens and HIV-1 envelope glycoprotein gp120

AIM: To investigate the interaction between human CCR5 receptors (CCR5) and HIV-1 envelope glycoprotein gp120 (HIV-1 gp120) and HIV-1 receptor CD4 antigens (CD4). METHODS: The structurally conserved regions (SCR) of human CCR5 was built by the SYBYL/Biopolymer module using the corresponding transmembrane (TM) domain of bacteriorhodopsin (bR) as the template. The coordinates for amino-terminal residue sequence, and carboxyl-terminal residue sequence, extracellular and cytoplasmic loops were generated using LOOP SEARCH algorithm. Subsequently the structural model was merged into the complex with HIV-1 gp120 and CD4. RESULTS: Human CCR5 interacted with both an HIV-1 gp120 and CD4. The N-terminal residues (especially Met1 and Gln4) of human CCR5 contacted with CD4 residues, mainly with one span (56-59) of CD4 in electrostatic interaction and hydrogen-bonds. The binding sites of human CCR5 were buried in a hydrophobic center surrounded by a highly basic periphery. On the other hand, direct interatomic contacts were made between 7 CCR5 residues and 6 gp120 amino-acid residues, which included van der Waals contacts, hydrophobic interaction, and hydrogen bonds. CONCLUSION: The interaction model should be helpful for rational design of novel anti-HIV drugs.     

Glycosylation of HIV-1 gp120 V3 loop: towards the rational design of a synthetic carbohydrate vaccine

A wide variety of proteins can bind high-mannose oligosaccharides and are broadly neutralizing against HIV-1. However, success in eliciting broadly neutralizing antibodies against HIV-1 has been limited to date. The rational design of an HIV-1 vaccine is based on the information gained through the structural analysis of antibodies complexed with their epitopes. Of particular interest to this review are the binding of mannosides to human monoclonal antibody 2G12 recognizing Man(9)GlcNAc(2) from HIV-1 gp120. It is widely recognized that T-cell-independent antigens carbohydrates are poorly immunogenic, and fail to induce memory. To increase the immunogenicity, carbohydrate antigens have to be coupled to a highly immunogenic carrier. The design of peptide carbohydrate mimotopes (mimetics of carbohydrate antigens) is one approach that is currently explored to elicit neutralizing antibodies. This work is concerned with existing structural data on Man(9)GlcNAc(2) as the most promising epitope (or glycotope). Structural analysis of various torsion angles of Man(9)GlcNAc(2) is explored. The focus is made primarily on the third variable region (V3 loop) of gp120 due to its crucial relevance for coreceptor usage, as a principal neutralizing determinant (PND), and for its conserved glycosylation sites N295, N302 and N332. Valuable structural information from glycosylation effects is taken into account for the development of a V3 loop rational structure-based vaccine strategy using N295 and N302 as potential conformational epitope.     

HIV-1 gp120Bal down-Regulates Phosphorylated NMDA Receptor Subunit 1 in Cortical Neurons via Activation of Glutamate and Chemokine Receptors

HIV-1 envelope glycoprotein gp120 (gp120) is a major virulence protein implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND). Although gp120 has been suggested to cause synaptic and neuronal injuries by disrupting NMDA receptor (NMDAR) function, the underlying mechanism is unclear. Here, we show that gp120Bal down-regulates the phosphorylation of the NMDAR subunit1 NR1 (at Ser896 and Ser897), which is essential for NMDAR function. This effect of gp120Bal is blocked by specific antagonists of both NMDA and AMPA receptors, indicating a critical role of synaptic activation. Furthermore, AMD3100 and maraviroc, antagonists of CCR5 and CXCR4 chemokine receptors, respectively, inhibit the effect of gp120Bal on NR1, suggesting that CXCR4 and CCR5 activation are involved. These findings may provide mechanistic insights into the synaptopathogenesis caused by HIV-1 infection.     

Enhancement of NMDA Receptor-Mediated Excitatory Postsynaptic Currents by gp120-Treated Macrophages: Implications for HIV-1-Associated Neuropathology

A plethora of prior studies has linked HIV-1-infected and immune activated brain mononuclear phagocytes (MP; blood borne macrophages and microglia) to neuronal dysfunction. These are modulated by N-methyl-D-aspartate receptor (NMDAR) antagonists and supporting their relevance for HIV-1-associated nervous system disease. The role of NMDAR subsets in HIV-1-induced neuronal injury, nonetheless, is poorly understood. To this end, we investigated conditioned media from HIV-1gp120-treated human monocyte-derived-macrophages (MDM) for its abilities to affect NMDAR-mediated excitatory postsynaptic currents (EPSC_NMDAR) in rat hippocampal slices. Bath application of gp120-treated MDM-conditioned media (MCM) produced an increase of EPSC_NMDAR. In contrast, control (untreated) MCM had limited effects on EPSC_NMDAR. Testing NR2A NMDAR (NR2AR)-mediated EPSC (EPSC_NR2AR) and NR2B NMDAR (NR2BR)-mediated EPSC (EPSC_NR2BR) for MCM showed significant increased EPSC_NR2BR when compared to EPSC_NR2AR enhancement. When synaptic NR2AR-mediated EPSC was blocked by bath application of MK801 combined with low frequency stimulations, MCM retained its ability to enhance EPSC_NMDAR evoked by stronger stimulations. This suggested that increase in EPSC_NMDAR was mediated, in part, through extra-synaptic NR2BR. Further analyses revealed that the soluble factors with low (<3 kD) to medium (3–10 kD) molecular weight mediated the observed increases in EPSC_NMDAR. The link between activation of NR2BRs and HIV-1gp120 MCM for neuronal injury was demonstrated by NR2BR but not NR2AR blockers. Taken together, these results indicate that macrophage secretory products induce neuronal injury through extra-synaptic NR2BRs.     

Systematic protein-protein docking and molecular dynamics studies of HIV-1 gp120 and CD4: insights for new drug development

Background and the purpose of the study

The interactions between HIV-1 gp120 and mutated CD4 proteins were investigated in order to identify a lead structure for therapy based on competitive blocking of the HIV binding receptor for human T-cells. Crystal structures of HIV gp120-CD4 complexes reveal a close interaction of the virus receptor with CD4 Phe43, which is embedded in a pocket of the virus protein.

Methods

This study applies computer simulations to determine the best binding of amino acid 43 CD4 mutants to HIV gp120. Besides natural CD4, three mutants carrying alternate aromatic residues His, Trp and Tyr at position 43 were investigated. Several docking programs were applied on isolated proteins based on selected crystal structures of gp120-CD4 complexes, as well as a 5 ns molecular dynamics study on the protein complexes. The initial structures were minimized in Gromacs to avoid crystal packing effects, and then subjected to docking experiments using AutoDock4, FireDock, ClusPro and ZDock. In molecular dynamics, the Gibbs free binding energy was calculated for the gp120-CD4 complexes. The docking outputs were analyzed on energy within the respective docking software.

Results and conclusion

Visualization and hydrogen bonding analysis were performed using the Swiss-PdbViewer. Strong binding to HIV gp120 can be achieved with an extended aromatic group (Trp). However, the sterical demand of the interaction affects the binding kinetics. In conclusion, a ligand for an efficient blocking of HIV gp120 should involve an extended but conformational flexible aromatic group, i.e. a biphenyl. A docking study on biphenylalanine-43 confirms this expectation.     

Caspase-1 inhibitors abolish deleterious enhancement of COX-2 expression induced by HIV-1 gp120 in human neuroblastoma cells

The human CHP100 neuroblastoma cell line has been shown to provide an useful in vitro model to elucidate the mechanisms underlying HIV-1 gp120 neurotoxicity. Here we report western blotting evidence demonstrating that exposure to a cytotoxic concentration of the viral coat protein up-regulates expression of the inducible isoform of cyclooxygenase (COX-2) in neuroblastoma cells and this seems to be due to the previously observed increase in secreted IL-1beta. In fact, here we show that acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK) and t-butoxycarbonyl-L-aspartic acid benzyl ester-chloromethylketone (Boc-Asp-(OBzl)-CMK), two inhibitors of Interleukin-1 Converting Enzyme (ICE; also referred to as caspase-1), abolish COX-2 expression enhanced by gp120 and consequent cell death. In addition, NS-398, a selective inhibitor of COX-2 activity, affords neuroprotection strengthening the role of COX-2 in the mechanisms of death. In conclusion, the present data support the notion that IL-1beta is the signal through which gp120 elevates COX-2 expression and the latter is strongly implicated in the mechanisms underlying cytotoxicity.     

Sustained and specific in vitro inhibition of HIV-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes

Selective delivery of antiretrovirals to human immunodeficiency virus (HIV) infected cells may reduce toxicities associated with long-term highly active antiretroviral therapy (HAART), may improve therapeutic compliance and delay the emergence of resistance. We developed sterically stabilized pegylated liposomes coated with targeting ligands derived from the Fab’ fragment of HIV-gp120-directed monoclonal antibody F105, and evaluated these liposomes as vehicles for targeted delivery of a novel HIV-1 protease inhibitor. We demonstrated that the immunoliposomes were selectively taken up by HIV-1-infected cells and localized intracellularly, enabling the establishment of a cytoplasmic reservoir of protease inhibitor. In antiviral experiments, the drug delivered by the immunoliposomes showed greater and longer antiviral activity than comparable concentrations of free drug or drug encapsulated in non-targeted liposomes. In conclusion, by combining a targeting moiety with drug-loaded liposomes, efficient and specific uptake by non-phagocytic HIV-infected cells was facilitated, resulting in drug delivery to infected cells. This approach to targeted delivery of antiretroviral compounds may enable the design of drug regimens for patients that allow increased therapeutic adherence and less toxic treatment of HIV infection.     

Structural and functional characterization of an epitope in the conserved C-terminal region of HIV-1 gp120

Abstract: Through an integrated study of the reactivity of a monoclonal antibody, 803-15.6, with synthetic peptides and native recombinant HIV-1 envelope glycoprotein gp120, we have obtained structure–functional information on a region of rgp120 not yet elucidated by X-ray crystallography. mAb 803-15.6 binds with high affinity and broad cross-clade specificity to the conserved C-terminal region (amino acids 502–516) of HIV-1 rgp120. Phage display selection from a random peptide library identified the core binding motif as AXXKXRH, homologous to residues 502–508. Using quantitative binding analyses, the affinity of mAb 803-15.6 for native, monomeric recombinant gp120_HXB2 (rgp120) was found to be similar to that for the synthetic gp120 peptide (502–516). Circular dichroism studies indicate that the synthetic peptide largely has a random coil conformation in solution. The results therefore suggest that the 803-15.6 epitope is fully accessible on rgp120 and that this region of rgp120 is as flexible as the synthetic peptide. Residues 502–504 are on the edge of a putative gp41 binding site that has been postulated to change conformation on CD4 binding. However, the affinity of mAb 803-15.6 for rgp120 is not affected by binding of CD4 and vice-versa. These results suggest either that the 502–504 region does not change conformation upon CD4 binding, or that recombinant gp120 does not undergo the same changes as occur in the native viral gp120–gp41 oligomer. The detailed characterization of the 803-15.6 epitope may be useful for further study of the role of the C5 region of gp120 in the viral attachment and fusion process.     

The Neural Circuitry of Executive Functions in Healthy Subjects and Parkinson's Disease

In our constantly changing environment, we are frequently faced with altered circumstances requiring generation and monitoring of appropriate strategies, when novel plans of action must be formulated and conducted. The abilities that we call upon to respond accurately to novel situations are referred to as ‘executive functions'', and are frequently engaged to deal with conditions in which routine activation of behavior would not be sufficient for optimal performance. Here, we summarize important findings that may help us understand executive functions and their underlying neuronal correlates. We focus particularly on observations from imaging technology, such as functional magnetic resonance imaging, position emission tomography, diffusion tensor imaging, and transcranial magnetic stimulation, which in the past few years have provided the bulk of information on the neurobiological underpinnings of the executive functions. Further, emphasis will be placed on recent insights from Parkinson''s disease (PD), in which the underlying dopaminergic abnormalities have provided new exciting information into basic molecular mechanisms of executive dysfunction, and which may help to disentangle the cortical/subcortical networks involved in executive processes.