HIV-1 Gp120 Protein Research Articles

An MVA Vector Expressing HIV-1 Envelope under the Control of a Potent Vaccinia Virus Promoter as a Promising Strategy in HIV/AIDS Vaccine Design.

Highly attenuated poxviral vectors, such as modified vaccinia virus ankara (MVA), are promising vaccine candidates against several infectious diseases. One of the approaches developed to enhance the immunogenicity of poxvirus vectors is increasing the promoter strength and accelerating during infection production levels of heterologous antigens. Here, we have generated and characterized the biology and immunogenicity of an optimized MVA-based vaccine candidate against HIV/AIDS expressing HIV-1 clade B gp120 protein under the control of a novel synthetic late/early optimized (LEO) promoter (LEO160 promoter; with a spacer length of 160 nucleotides), termed MVA-LEO160-gp120. In infected cells, MVA-LEO160-gp120 significantly increased the expression levels of HIV-1 gp120 mRNA and protein, compared to the clinical vaccine MVA-B vector expressing HIV-1 gp120 under the control of the commonly used synthetic early/late promoter. When mice were immunized with a heterologous DNA-prime/MVA-boost protocol, the immunization group DNA-gp120/MVA-LEO160-gp120 induced an enhancement in the magnitude of gp120-specific CD4+ and CD8+ T-cell responses, compared to DNA-gp120/MVA-B; with most of the responses being mediated by the CD8+ T-cell compartment, with a T effector memory phenotype. DNA-gp120/MVA-LEO160-gp120 also elicited a trend to a higher magnitude of gp120-specific CD4+ T follicular helper cells, and modest enhanced levels of antibodies against HIV-1 gp120. These findings revealed that this new optimized vaccinia virus promoter could be considered a promising strategy in HIV/AIDS vaccine design, confirming the importance of early expression of heterologous antigen and its impact on the antigen-specific immunogenicity elicited by poxvirus-based vectors.

Identification of functional mimetics of anti-hiv antibody n6 by methods of virtual screening and molecular modeling

Three chemical compounds presenting functional mimetics of neutralizing anti-HIV-1 antibody N6 were identified by an integrated approach including virtual screening, high-throughput docking and molecular dynamics. Using molecular docking, the identified compounds are predicted to be able to block three key regions of the HIV-1 gp120 protein by formation of a wide network of intermolecular contacts with the glycoprotein residues critical for the virus binding to primary receptor CD4. It is shown that the complexes of these compounds with gp120 exhibit low values of dissociation constant and Gibbs free energy, which validates a high efficacy of intermolecular interactions stabilizing these supramolecular structures. Based on the data obtained, the identified compounds are assumed to form promising basic structures for the development of novel, potent and broad anti-HIV-1 drugs mimicking structural and functional properties of the cross-reactive neutralizing antibody N6.

In silico Identification of High-Affinity Ligands of the Hiv-1 Gp120 Protein, Potential Peptidomimetics of Neutralizing Antibody N6

Six potential peptidomimetics of the cross-reactive neutralizing anti-HIV-1 antibody N6 that are able to mimic the pharmacophoric features of this immunoglobulin by specific and effective interactions with the CD4-binding site of the viral gp120 protein were identified by virtual screening and molecular modeling. The key role in the interaction of these compounds with gp120 is shown to play multiple van der Waals contacts with conserved residues of the gp120 Phe-43 cavity critical for the HIV binding to cellular receptor CD4, as well as hydrogen bond with Asp-368gp120 that increase the chemical affinity without activating unwanted allosteric effect. According to the data of molecular dynamics, the complexes of the identified ligands with gp120 are energetically stable and show the lower values of binding free energy compared with the HIV-1 inhibitors NBD-11021 and DMJ-II-121 used in the calculations as a positive control. Based on the data obtained, it was concluded that the identified compounds may be considered as promising candidates for detailed experimental studies to their further use in the design of novel antiviral drugs presenting HIV-1 inhibitors that block the early stages of the development of HIV infection.

Enhancement of Immune Responses by Guanosine-Based Particles in DNA Plasmid Formulations against Infectious Diseases.

Immunogenicity of DNA vaccines can be efficiently improved by adding adjuvants into their formulations. In this regard, the application of nano- and microparticles as vaccines adjuvants, or delivery systems, provides a powerful tool in designing modern vaccines. In the present study, we examined the role of “Supramolecular Hacky Sacks” (SHS) particles, made via the hierarchical self-assembly of a guanosine derivative, as a novel immunomodulator for DNA plasmid preparations. These plasmids code for the proteins HIV-1 Gag (pGag), the wild-type vaccinia virus Western Reserve A27 (pA27L), or a codon-optimized version of the latter (pOD1A27Lopt), which is also linked to the sequence of the outer domain-1 (OD1) from HIV-1 gp120 protein. We evaluated the enhancement of the immune responses generated by our DNA plasmid formulations in a murine model through ELISpot and ELISA assays. The SHS particles increased the frequencies of IFN-γ-producing cells in mice independently immunized with pGag and pA27L plasmids. Moreover, the addition of SHS to pGag and pA27L DNA plasmid formulations enhanced the production of IFN-γ (Th1-type) over IL-4 (Th2-type) cellular immune responses. Furthermore, pGag and pA27L plasmids formulated with SHS, triggered the production of antigen-specific IgG in mice, especially the IgG2a isotype. However, no improvement of either of those adaptive immune responses was observed in mice receiving pOD1A27Lopt+SHS. Here, we demonstrated that SHS particles have the ability to improve both arms of adaptive immunity of plasmid coding “wild-type” antigens without additional strategies to boost their immunogenicity. To the best of our knowledge, this is the first report of SHS guanosine-based particles as DNA plasmid adjuvants.

HIV-1 Protein gp120 Induces Mouse Lung Fibroblast-to-Myofibroblast Transdifferentiation via CXCR4 Activation

BackgroundIndividuals with HIV have ∼2-fold increased risk of developing pulmonary fibrosis. The mechanism(s) by which this occurs has yet to be determined. HIV-1 protein gp120 activates CXCR4 in the lymphocyte, promoting a variety of intracellular signaling pathways including those common to TGFβ1 associated with lung fibroblast-to-myofibroblast transdifferentiation. We hypothesized that gp120 promotes pulmonary fibrotic changes via activation of CXCR4 in the lung fibroblast. MethodsMouse primary lung fibroblasts (PLFs) were cultured ± gp120, then analyzed for α-SMA expression and stress fiber formation. In parallel, PLFs were cultured ± gp120 ± AMD3100 (a CXCR4 antagonist), and α-SMA, pan and phospho-Akt, and total and phospho-MAPK (or ERK1/2) protein expression was quantified. Finally, lungs and PLFs from wild-type and HIV-1 transgenic mice were analyzed for hydroxyproline and α-SMA content. Resultsgp120 treatment increased α-SMA expression and myofibroblast differentiation in PLFs. gp120 treatment activated phosphorylation of ERK1/2, but not PI3K-Akt. Pretreatment with AMD3100 inhibited gp120-induced ERK1/2 phosphorylation and gp120-induced α-SMA expression. In parallel, there was a significant increase in hydroxyproline content in lungs from older HIV-1 transgenic mice and a >3-fold increase in α-SMA expression in PLFs isolated from HIV-1 transgenic mice. Conclusionsgp120 induces α-SMA expression and fibroblast-to-myofibroblast transdifferentiation by activating the CXCR4-ERK1/2 signaling pathway in mouse PLFs. Lungs of older HIV-1 transgenic mice contain higher hydroxyproline content and their PLFs have a striking increase in α-SMA expression. These results suggest a mechanism by which individuals with HIV are at increased risk of developing pulmonary fibrotic changes as they age.

In silico search, chemical characterization and immunogenic evaluation of amino-terminated G4-PAMAM-HIV peptide complexes using three-dimensional models of the HIV-1 gp120 protein.

Peptide epitopes have been widely used to develop synthetic vaccines and immunotherapies. However, peptide epitopes may exhibit poor absorption or immunogenicity due to their low molecular weights. Conversely, fourth-generation polyamidoamine (G4-PAMAM) dendrimers are nonimmunogenic and relatively nontoxic synthetic nanoparticles that have been used as adjuvants and nanocarriers of small peptides and to improve nasal absorption. Based on this information, we hypothesized that the combination of intranasal immunization and G4-PAMAM dendrimers would be useful for enhancing the antibody responses of HIV-1 gp120 peptide epitopes. Therefore, we first used structural data, peptide epitope predictors and docking and MD simulations on MHC-II to identify two peptide epitopes on the CD4 binding site of HIV-1 gp120. The formation of G4-PAMAM-peptide complexes was evaluated in silico (molecular docking studies using different G4-PAMAM conformations retrieved from MD simulations as well as the MMGBSA approach) and validated experimentally (electrophoresis, 1H NMR and cryo-TEM). Next, the G4-PAMAM dendrimer-peptide complexes were administered intranasally to groups of female BALB/cJ mice. The results showed that both peptides were immunogenic at the systemic and mucosal levels (nasal and vaginal), and G4-PAMAM dendrimer-peptide complexes improved IgG and IgA responses in serum and nasal washes. Thus, G4-PAMAM dendrimers have potential for use as adjuvants and nanocarriers of peptides.

Human Immunodeficiency Virus Type 1 gp120 and Tat Induce Mitochondrial Fragmentation and Incomplete Mitophagy in Human Neurons.

HIV enters the central nervous system (CNS) during the early stages of infection and can cause neurological dysfunction, including neurodegeneration and neurocognitive impairment. The specific autophagy responsible for removal of damaged mitochondria (mitophagy) and mitochondrial dynamics constitute neuronal mitochondrial quality control mechanisms and are impaired in neurodegenerative disorders and numerous other diseases. The release of HIV proteins gp120 and Tat from infected cells is thought to play an important role in HIV-associated neurocognitive disorders (HAND), but the mechanism(s) leading to impairment are poorly understood. Here, we report that exposure of human primary neurons (HPNs) to HIV gp120 and Tat accelerates the balance of mitochondrial dynamics toward fission (fragmented mitochondria) and induces perinuclear aggregation of mitochondria and mitochondrial translocation of dynamin-related protein 1 (DRP1), leading to neuronal mitochondrial fragmentation. HIV gp120 and Tat increased the expression of microtubule-associated protein 1 light chain 3 beta (LC3B) protein and induced selective recruitment of Parkin/SQSTM1 to the damaged mitochondria. Using either a dual fluorescence reporter system expressing monomeric red fluorescent protein and enhanced green fluorescent protein targeted to mitochondria (mito-mRFP-EGFP) or a tandem light chain 3 (LC3) vector (mCherry-EGFP-LC3), both HIV proteins were found to inhibit mitophagic flux in human primary neurons. HIV gp120 and Tat induced mitochondrial damage and altered mitochondrial dynamics by decreasing mitochondrial membrane potential (ΔΨm). These findings indicate that HIV gp120 and Tat initiate the activation and recruitment of mitophagy markers to damaged mitochondria in neurons but impair the delivery of mitochondria to the lysosomal compartment. Altered mitochondrial dynamics associated with HIV infection and incomplete neuronal mitophagy may play a significant role in the development of HAND and accelerated aging associated with HIV infection.IMPORTANCE Despite viral suppression by antiretrovirals, HIV proteins continue to be detected in infected cells and neurologic complications remain common in infected people. Although HIV is unable to infect neurons, viral proteins, including gp120 and Tat, can enter neurons and can cause neuronal degeneration and neurocognitive impairment. Neuronal health is dependent on the functional integrity of mitochondria, and damaged mitochondria are subjected to mitochondrial control mechanisms. Multiple lines of evidence suggest that specific elimination of damaged mitochondria through mitophagy and mitochondrial dynamics play an important role in CNS diseases. Here, we show that in human primary neurons, gp120 and Tat favor the balance of mitochondrial dynamics toward enhanced fragmentation through the activation of mitochondrial translocation of DRP1 to the damaged mitochondria. However, mitophagy fails to go to completion, leading to neuronal damage. These findings support a role for altered mitophagy in HIV-associated neurological disorders and provide novel targets for potential intervention.

HIV-induced matrix metalloproteinase-9 activation through mitogen-activated protein kinase signalling promotes HSV-1 cell-to-cell spread in oral epithelial cells.

We have shown that cell-free HIV-1 and viral proteins tat and gp120 activate mitogen-activated protein kinases (MAPKs) in tonsil epithelial cells, disrupting their tight and adherens junctions. This causes liberation of the HSV-1 receptor nectin-1 from assembled adherens junctions, leading to promotion of HSV-1 infection and spread. In the present study, we show that HIV-associated activation of MAPK leads to upregulation of transcription factor NF-κB and matrix metalloproteinase-9 (MMP-9). This induces the disruption of tight and adherens junctions, increasing HSV-1 cell-to-cell spread. Inhibition of HIV-associated MAPK activation by U0126 abolishes NF-κB and MMP-9 upregulation and reduces HSV-1 spread. Inactivation of MMP-9 also reduced HIV-promoted HSV-1 spread. These results indicate that HIV-1-activated MAPK/NF-κB and MMP-9 play a critical role in the disruption of oral epithelial junctions and HSV-1 cell-to-cell spread. Inhibition of MMP-9 expression in the oral epithelium of HIV-infected individuals may prevent the development of diseases caused by HSV-1, such as ulcers, necrotic lesions and gingivostomatitis.

Scaling Synapses in the Presence of HIV.

A defining feature of HIV-associated neurocognitive disorder (HAND) is the loss of excitatory synaptic connections. Synaptic changes that occur during exposure to HIV appear to result, in part, from a homeostatic scaling response. Here we discuss the mechanisms of these changes from the perspective that they might be part of a coping mechanism that reduces synapses to prevent excitotoxicity. In transgenic animals expressing the HIV proteins Tat or gp120, the loss of synaptic markers precedes changes in neuronal number. In vitro studies have shown that HIV-induced synapse loss and cell death are mediated by distinct mechanisms. Both in vitro and animal studies suggest that HIV-induced synaptic scaling engages new mechanisms that suppress network connectivity and that these processes might be amenable to therapeutic intervention. Indeed, pharmacological reversal of synapse loss induced by HIV Tat restores cognitive function. In summary, studies indicate that there are temporal, mechanistic and pharmacological features of HIV-induced synapse loss that are consistent with homeostatic plasticity. The increasingly well delineated signaling mechanisms that regulate synaptic scaling may reveal pharmacological targets suitable for normalizing synaptic function in chronic neuroinflammatory states such as HAND.

Serum glycan-binding IgG antibodies in HIV-1 infection and during the development of broadly neutralizing responses.

The HIV-1 envelope is covered with glycans that provide structural integrity and protect conserved regions from host antibody responses. However, these glycans are often the target of broadly neutralizing antibodies (bNAbs) that emerge in some HIV-infected individuals. We aimed to determine whether antiglycan IgG antibodies are a general response to HIV-1 infection or specific to individuals who develop bNAbs. IgG binding to glycans was assessed using arrays that contained 245 unique components including N-linked carbohydrates, glycolipids, and Tn-peptides. Sera from 20 HIV-negative and 27 HIV-positive women (including 12 individuals who developed bNAbs) were profiled longitudinally. HIV-1 gp120 proteins were used to compete for binding to the array. Antiglycan IgG antibodies fluctuated over a 3-year period, irrespective of HIV infection. However, HIV-positive individuals had elevated binding to 40 components on the array that included Man8, Man9, Tn-peptides, heat shock protein, and glycolipids. Competition experiments confirmed that a proportion of these glycan-binding IgG antibodies were HIV-1-specific, some of which were higher in individuals who developed bNAbs. HIV-1 infection is associated with elevated levels of IgG antibodies to specific glycans. Furthermore, some antiglycan IgG antibodies were more abundant in individuals with bNAbs, suggesting a unique phenotype that may be informative for HIV vaccine design.

HIV-1 Gp120 clade B/C induces a GRP78 driven cytoprotective mechanism in astrocytoma.

HIV-1 clades are known to be one of the key factors implicated in modulating HIV-associated neurocognitive disorders. HIV-1 B and C clades account for the majority of HIV-1 infections, clade B being the most neuropathogenic. The mechanisms behind HIV-mediated neuropathogenesis remain the subject of active research. We hypothesized that HIV-1 gp120 clade B and C proteins may exert differential proliferation, cell survival and NeuroAIDS effects in human astrocytoma cells via the Unfolded Protein Response, an endoplasmic reticulum- based cytoprotective mechanism. The differential effect of gp120 clade B and C was evaluated using for the first time a Tandem Mass Tag isobaric labeling quantitative proteomic approach. Flow cytometry analyses were performed for cell cycle and cell death identification. Among the proteins differentiated by HIV-1 gp120 proteins figure cytoskeleton, oxidative stress, UPR markers and numerous glycolytic metabolism enzymes. Our results demonstrate that HIV-1 gp120 B induced migration, proliferative and protective responses granted by the expression of GRP78, while HIV-1 gp120 C induced the expression of key inflammatory and pro-apoptotic markers. These novel findings put forward the first evidence that GRP78 is a key player in HIV-1 clade B and C neuropathogenic discrepancies and can be used as a novel target for immunotherapies.

HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates.

The nonreplicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens (NYVAC-C) showed limited immunogenicity in phase I clinical trials. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in nonhuman primates immunized with two replicating NYVAC vectors that have been modified by the insertion of the K1L and C7L vaccinia virus host range genes and express the clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of the type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking that of the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4) and booster doses with NYVAC-C-KC vectors plus the clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, and high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4+ and CD8+ T cells, the induction of some cytokines, and the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions.IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1 protection. Here we developed novel replicating poxvirus NYVAC-based HIV/AIDS vaccine candidates expressing clade C HIV-1 antigens, with one of them lacking the vaccinia virus B19 protein, an inhibitor of the type I interferon response. Immunization of nonhuman primates with these novel NYVAC-C-KC vectors and the protein component gp120 elicited high levels of T cell and humoral immune responses, with the vector containing a deletion in B19R inducing a trend toward a higher magnitude of CD4+ and CD8+ T cell responses and neutralization of some HIV-1 strains. These poxvirus vectors could be considered HIV/AIDS vaccine candidates based on their activation of potential immune correlates of protection.

Dendritic Cell Response to HIV-1 Is Controlled by Differentiation Programs in the Cells and Strain-Specific Properties of the Virus

Dendritic cells (DCs) are potent antigen-presenting cells that might play contradictory roles during HIV-1 infection, contributing not only to antiviral immunity but also to viral dissemination and immune evasion. Although DCs are characterized by enormous functional diversity, it has not been analyzed how differentially programmed DCs interact with HIV-1. We have previously described the reprogramming of DC development by endogenously produced lactic acid that accumulated in a cell culture density-dependent manner and provided a long-lasting anti-inflammatory signal to the cells. By exploiting this mechanism, we generated immunostimulatory DCs characterized by the production of TH1 polarizing and inflammatory mediators or, alternatively, suppressed DCs that produce IL-10 upon activation, and we tested the interaction of these DC types with different HIV-1 strains. Cytokine patterns were monitored in HIV-1-exposed DC cultures. Our results showed that DCs receiving suppressive developmental program strongly upregulated their capacity to produce the TH1 polarizing cytokine IL-12 and the inflammatory chemokines CCL2 and CCL7 upon interaction with HIV-1 strains IIIB and SF162. On the contrary, HIV-1 abolished cytokine production in the more inflammatory DC types. Preincubation of the cells with the HIV-1 proteins gp120 and Nef could inhibit IL-12 production irrespectively of the tested DC types, whereas MyD88- and TRIF-dependent signals stimulated IL-12 production in the suppressed DC type only. Rewiring of DC cytokines did not require DC infections or ligation of the HIV-1 receptor CD209. A third HIV-1 strain, BaL, could not modulate DC cytokines in a similar manner indicating that individual HIV-1 strains can differ in their capacity to influence DCs. Our results demonstrated that HIV-1 could not induce definite and invariable modulatory programs in DCs. Instead, interaction with the virus triggered different responses in different DC types. Thus, the outcome of DC-HIV-1 interactions might be highly variable, shaped by endogenous features of the cells and diversity of the virus.

Neuronal Cell Death and Degeneration through Increased Nitroxidative Stress and Tau Phosphorylation in HIV-1 Transgenic Rats.

The underlying mechanisms for increased neurodegeneration and neurocognitive deficits in HIV-infected people are unclear. Therefore, this study was aimed to investigate the mechanisms of increased neurodegeneration in 5-month old male HIV-1 Transgenic (Tg) rats compared to the age- and gender-matched wild-type (WT) by evaluating histological changes and biochemical parameters of the key proteins involved in the cell death signaling and apoptosis. Histological and immunohistochemical analyses revealed decreased neuronal cells with elevated astrogliosis in HIV-1 Tg rats compared to WT. Mechanistic studies revealed that increased levels of nitroxidative stress marker proteins such as NADPH-oxidase, cytochrome P450-2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), the stress-activated mitogen-activated protein kinases such as JNK and p38K, activated cell-cycle dependent CDK5, hypoxia-inducible protein-1α, nitrated proteins, hyperphosphorylated tau, and amyloid plaques in HIV-Tg rats were consistently observed in HIV-1 Tg rats. Confocal microscopy and cell viability analyses showed that treatment with an antioxidant N-acetylcysteine or a specific inhibitor of iNOS 1400W significantly prevented the increased apoptosis of neuro-2A cells by HIV-1 Tat or gp120 protein, demonstrating the causal role of HIV-1 mediated nitroxidative stress and protein nitration in promoting neuronal cell death. Immunoprecipitation and immunoblot analysis confirmed nitration of Hsp90, evaluated as an example of nitrated proteins, suggesting possible involvement of nitrated proteins in neuronal damage. Further, activated p-JNK directly binds tau and phosphorylates multiple amino acids, suggesting an important role of p-JNK in tau hyperphosphorylation and tauopathy. These changes were accompanied with elevated levels of many apoptosis-related proteins Bax and cleaved (activated) caspase-3 as well as proinflammatory cytokines including TNF-α, IL-6 and MCP-1. Collectively, these results indicate that raised nitroxidative stress accompanied by elevated inflammation, cell death signaling pathway including activated p-JNK, C-terminal C99 amyloid fragment formation and tau hyperphosphorylation are responsible for increased apoptosis of neuronal cells and neurodegeneration in 5-month old HIV-Tg rats.

Expansion of myeloid-derived suppressor cells promotes differentiation of regulatory T cells in HIV-1+ individuals

Regulatory T cells (Tregs) contribute to HIV-1 disease progression by impairing antiviral immunity; however, the precise mechanisms responsible for the development of Tregs in the setting of HIV-1 infection are incompletely understood. In this study, we provide evidence that HIV-induced expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) promote the differentiation of Foxp3 Tregs. We measured MDSC induction and cytokine expression by flow cytometry and analyzed their functions by coculturing experiments. We observed a dramatic increase in M-MDSC frequencies in the peripheral blood of HIV-1 seropositive (HIV-1) individuals, even in those on antiretroviral therapy with undetectable viremia, when compared with healthy participants. We also observed increases in M-MDSCs after incubating healthy peripheral mononuclear cells (PBMCs) with HIV-1 proteins (gp120 or Tat) or Toll-like receptor 4 ligand lipopolysaccharides in vitro, an effect that could be abrogated in the presence of the phosphorylated signal transducer and activator of transcription 3 inhibitor, STA-21. Functional analyses indicated that M-MDSCs from HIV-1 individuals express higher levels of IL-10, tumor growth factor-β, IL-4 receptor α, p47, programmed death-ligand 1, and phosphorylated signal transducer and activator of transcription 3 - all of which are known mediators of myelopoiesis and immunosuppression. Importantly, incubation of healthy CD4 T cells with MDSCs derived from HIV-1 individuals significantly increased differentiation of Foxp3 Tregs. In addition, depletion of MDSCs from PBMCs of HIV-1 individuals led to a significant reduction of Foxp3 Tregs and increase of IFNγ production by CD4 T effector cells. These results suggest that HIV-induced MDSCs promote Treg cell development and inhibit T cell function - a hallmark of many chronic infectious diseases.

Characterizing the germinal center response to electroporated DNA immunogens

Abstract Most licensed vaccines rely on the production of antibodies that mediate protection from disease. The germinal center (GC) is a dynamic cellular substructure found in lymphoid organs where follicular helper T cells (TFH) provide the necessary proliferation stimuli and cytokine signaling that promote antibody class-switching, somatic hypermutation, and affinity maturation of B cell receptors. Here, mature B cells differentiate into long-lived antibody-secreting cells and memory B cells, instituting the characteristic humoral memory response to vaccination. Although HIV-1 DNA vaccines have been shown to be highly immunogenic, leading to enhanced HIV-1 env epitope breadth and titers in pre-clinical and clinical studies, many questions remain regarding the quality of the humoral immune response and the ability of the DNA antigen/molecular adjuvant to induce long-lived anti-viral antibody that seeds mucosal secondary immune tissues. However, the magnitude and kinetics of the GC response to EP DNA has not been elucidated. Our results indicate that DNA antigens induce a GC response. Antigen-specific GC B cells (B220+CD138−IgM−IgD−PNA+CXCR5+gp120+) are successfully detected by flow cytometry using fluorophore-conjugated HIV-1 gp120 protein after two immunizations in the spleens and draining lymph nodes of vaccinated animals and by antigen-specific ELISpot assay. The presence of antigen-specific antibodies of multiple isotypes is successfully detected in the serum of vaccinated animals via ELISA. These results represent, to our knowledge, the first reports on the magnitude and kinetics of the germinal center response to EP DNA. This work is funded by a W.W. Smith Charitable Foundation grant to MK.

In Silico Identification and Molecular Validation of Putative Antimicrobial Peptides for HIV Therapy

Objective: Despite the effort to reduce the rate of HIV infection, AIDS still remains the major cause of death around the world, predominantly in Sub-Sahara Africa. Neither a cure, nor an HIV vaccine has been found to date and the disease can only be managed by using High Active Antiretroviral Therapy (HAART). The need for non-toxic regiments has brought about the necessity for additional HIV treatment to lower mortality rates. Antimicrobial Peptides (AMPs) had proven to be a promising therapeutic agent against HIV. The aim of this research was to identify AMPs, which binds gp120 at the area where gp120 interacts with CD4+, to prevent HIV invasion and HIV replication. Method: Putative AMPs were identified using an In Silico mathematical algorithm, Profile Hidden Markov Models (HMMER). The AMPS 3-D structures was carried out using I-TASSER and the modelled AMPs were docked against the HIV protein gp120 using PATCHDOCK. Subsequently, molecular method was used to show the anti-HIV ability of these putative to validate by inhibiting HIV-1 replication. Results: The In Silico results showed that 30 putative anti-HIV AMPs were identified. Furthermore, out of the 10 best ranked putative AMPs, based on their E-value, selected for In Silico docking, two AMPs proved to inhibit HIV-1 NL4- 3 with maximal effective concentration (EC50) values of 37.5 μg/ml and 93.75 μg/ml respectively. This result looks promising since 150 μg/ml AMPs could not achieved 80% toxicity of the human T cells, thus high Therapeutics Index (TI) might be obtained if 50% cytotoxic concentration (CC50) is established. Conclusion: The ability of these AMPs to inhibit HIV replication justifies the usage of HMMER in design and discovery. Additionally, these AMPs pave the way for the design of anti-HIV peptide-based drugs.

Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage.

HIV-1 infection frequently causes HIV-associated neurocognitive disorders (HAND) despite combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can themselves be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine, seems to aggravate HAND and compromise antiretroviral therapy. However, the combined effect of virus and recreational and therapeutic drugs on the brain is poorly understood. Therefore, we exposed mixed neuronal-glial cerebrocortical cells to antiretrovirals (ARVs) (zidovudine [AZT], nevirapine [NVP], saquinavir [SQV], and 118-D-24) of four different pharmacological categories and to methamphetamine and, in some experiments, the HIV-1 gp120 protein for 24 h and 7 days. Subsequently, we assessed neuronal injury by fluorescence microscopy, using specific markers for neuronal dendrites and presynaptic terminals. We also analyzed the disturbance of neuronal ATP levels and assessed the involvement of autophagy by using immunofluorescence and Western blotting. ARVs caused alterations of neurites and presynaptic terminals primarily during the 7-day incubation and depending on the specific compounds and their combinations with and without methamphetamine. Similarly, the loss of neuronal ATP was context specific for each of the drugs or combinations thereof, with and without methamphetamine or viral gp120. Loss of ATP was associated with activation of AMP-activated protein kinase (AMPK) and autophagy, which, however, failed to restore normal levels of neuronal ATP. In contrast, boosting autophagy with rapamycin prevented the long-term drop of ATP during exposure to cART in combination with methamphetamine or gp120. Our findings indicate that the overall positive effect of cART on HIV infection is accompanied by detectable neurotoxicity, which in turn may be aggravated by methamphetamine.

Computational discovery of novel HIV-1 entry inhibitors based on potent and broad neutralizing antibody VRC01

Computational prediction of novel HIV-1 entry inhibitors presenting peptidomimetics of broadly neutralizing antibody (bNAb) VRC01 was carried out based on the analysis of the X-ray complex of this antibody antigen-binding fragment with the HIV envelope gp120 core. Using these empirical data, peptidomimetic candidates of bNAb VRC01 were identified by a public web-oriented virtual screening platform (pepMMsMIMIC) and models of these candidates bound to gp120 were generated by molecular docking. At the final point, the stability of the complexes of these molecules with gp120 was estimated by molecular dynamics and binding free energy calculations. The calculations identified six molecules exhibiting a high affinity to the HIV-1 gp120 protein. These molecules were selected as the most probable peptidomimetics of bNAb VRC01. In a mechanism similar to that of bNAb VRC01, these compounds were predicted to block the functionally conserved regions of gp120 critical for the HIV-1 binding to cellular receptor CD4. The docked structures of the identified molecules with gp120 do not undergo substantial rearrangements during the molecular dynamics simulations, in agreement with the low values of free energy of their formation. Based on these findings, the selected compounds are considered as promising basic structures for the rational design of novel, potent, and broad-spectrum anti-HIV-1 therapeutics.

A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen.

In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive and memory T-cell immune responses, as well as humoral responses. This novel HIV-1 gp120-14K immunogen might be considered as an HIV vaccine candidate for broad T and B-cell immune responses.