HIV-1 Pseudovirus Research Articles

Establishment of a humanized mouse model of HIV-1 infection and quantification of integrated HIV proviral DNA in vivo

In recent years, virological, pathological, and immunological studies need to be carried out for the emerging anti-human immunodeficiency virus (HIV) therapies such as gene therapy, broadly neutralizing antibodies, and the derived chimeric antigen receptor (CAR)-T immunotherapy, which necessitates suitable, simple, and inexpensive small-animal models and methods for accurate quantification of the viral genome in the HIV-1 infected. In our research, the HIV-∆ENV-Jurkat-EGFP-mCherry cell line was engineered through the infection with a dual-labelled HIV pseudovirus. A nested quantitative PCR (nested-qPCR) method with the cellular genome as the integrated standard was established for the quantification of HIV proviral copies. We administered intravenous injections of healthy human peripheral blood mononuclear cell (PBMC) into NOD/Prkdcscid/IL2rgnull (NPG) mice. To verify engraftment kinetics, we analyzed the percentages of hCD45+, hCD3+, hCD4+, and hCD8+ cells in the peripheral blood of hu-PBMC-NPG mice. To evaluate HIV-1 infection in hu-PBMC-NPG mice, we inoculated these mice with HIV NL4-3-NanoLuc by intraperitoneal (IP) injection. We then monitored the luciferase expression by the small animal imaging system and measured the viral load in the spleen by qPCR. The infiltration of human PBMCs in mice was detected 3-5 weeks after intravenous injection, and the percentage of hCD45 in humanized mouse PBMCs were more than 25% five weeks after IP inoculation. The expression of the virus-associated luciferase protein was detected by luciferase imaging 27 days post infection. Moreover, the viral total DNA, RNA, and proviral DNA copies reached 18 000 copies/106 cells, 15 000 copies/μg RNA, and 15 000 copies/106 cells, respectively, in the mouse spleen. Taken together, we reported a convenient method for building a simple humanized mouse model of HuPBMC-NPG/severe combined immunodeficiency (SCID) by intravenous injection with hu-PBMCs without advanced surgical skills and irradiation. Furthermore, we established a convenient method for the efficient determination of proviral DNA to assess HIV replication in vivo, viral reservoir sizes, and efficacy of novel anti-HIV therapies including CAR-T immunotherapy and gene therapy.

Human CD4-binding site antibody elicited by polyvalent DNA prime-protein boost vaccine neutralizes cross-clade tier-2-HIV strains

The vaccine elicitation of HIV tier-2-neutralization antibodies has been a challenge. Here, we report the isolation and characterization of a CD4-binding site (CD4bs) specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent DNA prime-protein boost HIV vaccine. HmAb64 is derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39. It has a third heavy chain complementarity-determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 20 (10%), including tier-2 strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 in complex with a CNE40 SOSIP trimer revealed details of its recognition; HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4bs. This study demonstrates that a gp120-based vaccine can elicit antibodies capable of tier 2-HIV neutralization.

Advancing Natural Product Discovery: A Structure-Oriented Fractions Screening Platform for Compound Annotation and Isolation.

Natural product discovery is hindered by the lack of tools that integrate untargeted nuclear magnetic resonance and mass spectrometry data on a library scale. This article describes the first application of the innovative NMR/MS-based machine learning tool, the "Structure-Oriented Fractions Screening Platform (SFSP)", enabling functional-group-guided fractionation and accelerating the discovery and characterization of undescribed natural products. The concept was applied to the extract of a marine fungus known to be a prolific producer of diverse natural products. With the assistance of SFSP, we isolated 24 flavipidin derivatives and five phenalenone analogues from Aspergillus sp. GE2-6, revealing 27 undescribed compounds. Compounds 7-22 were proposed as isomeric derivatives featuring a 5/6-ring fusion, formed by the dimerization of flavipidin E (5). Compounds 23 and 24 were envisaged as isomeric derivatives with a 6/5/6-ring fusion, generated through the degradation of two flavipidin E molecules. Furthermore, flavipidin A (1) and asperphenalenone E (28) exhibited potent anti-influenza (PR8) activities, with IC50 values of 21.9 ± 0.2 and 12.9 ± 0.1 μM, respectively. Meanwhile, asperphenalenone (26) and asperphenalenone P (27) treatments exhibited significant inhibition of HIV pseudovirus infection in 293FT cells, boasting IC50 values of 6.1 ± 0.9 and 4.6 ± 1.1 μM, respectively. Overall, SFSP streamlines natural product isolation through NMR and MS data integration, as showcased by the discovery of numerous undescribed flavipidins and phenalenones based on NMR olefinic signals and low-field hydroxy signals.

Rational Design and Characterization of Trispecific Antibodies Targeting the HIV-1 Receptor and Envelope Glycoprotein.

Multitudinous broadly neutralizing antibodies (bNAbs) against HIV-1 have been developed as novel antiviral prophylactic and therapeutic agents. Combinations of bNAbs are generally even more effective than when they are applied individually, showing excellent neutralization coverage and limiting the emergence of escape mutants. In this study, we investigated the design and characterization of three trispecific antibodies that allow a single molecule to interact with independent HIV-1 envelope determinants-(1) the host receptor CD4, (2) the host co-receptor CCR5 and (3) distinct domains in the envelope glycoprotein of HIV-1-using an ELISA, an HIV-1 pseudovirus neutralization assay and in vivo antiviral experiments in humanized mice. We found that trispecific bNAbs and monovalent ones all had satisfactory binding activities against the corresponding antigens in the ELISA, exhibited higher potency and breadth than any previously described single bnAb in the HIV-1 pseudovirus neutralization assay and showed an excellent antiviral effect in vivo. The trispecific antibodies simultaneously recognize the host receptor CD4, host co-receptor CCR5 and HIV-1 envelope glycoprotein, which could mean they have promise as prophylactic and therapeutic agents against HIV-1.

Serinc5 Restricts HIV Membrane Fusion by Altering Lipid Order and Heterogeneity in the Viral Membrane.

The host restriction factor, Serinc5, incorporates into budding HIV particles and inhibits their infection by an incompletely understood mechanism. We have previously reported that Serinc5 but not its paralogue, Serinc2, blocks HIV cell entry by membrane fusion, specifically by inhibiting fusion pore formation and dilation. A body of work suggests that Serinc5 may alter the conformation and clustering of the HIV fusion protein, Env. To contribute an additional perspective to the developing model of Serinc5 restriction, we assessed Serinc2 and Serinc5's effects on HIV pseudoviral membranes. By measuring pseudoviral membrane thickness via cryo-electron microscopy and order via the fluorescent dye, FLIPPER-TR, Serinc5 was found to increase membrane heterogeneity, skewing the distribution toward a larger fraction of the viral membrane in an ordered phase. We also directly observed for the first time the coexistence of membrane domains within individual viral membrane envelopes. Using a total internal reflection fluorescence-based single particle fusion assay, we found that treatment of HIV pseudoviral particles with phosphatidylethanolamine (PE) rescued HIV pseudovirus fusion from restriction by Serinc5, which was accompanied by decreased membrane heterogeneity and order. This effect was specific for PE and did not depend on acyl chain length or saturation. Together, these data suggest that Serinc5 alters multiple interrelated properties of the viral membrane─lipid chain order, rigidity, line tension, and lateral pressure─which decrease the accessibility of fusion intermediates and disfavor completion of fusion. These biophysical insights into Serinc5 restriction of HIV infectivity could contribute to the development of novel antivirals that exploit the same weaknesses.

Curcuma Longa Induces the Transcription Factor FOXP3 to Downregulate Human Chemokine CCR5 Expression and Inhibit HIV-1 Infection.

HIV mutations occur frequently despite the substantial success of combination antiretroviral therapy, which significantly impairs HIV progression. Failure to develop specific vaccines, the occurrence of drug-resistant strains, and the high incidence of adverse effects due to combination antiviral therapy regimens call for novel and safer antivirals. Natural products are an important source of new anti-infective agents. For instance, curcumin inhibits HIV and inflammation in cell culture assays. Curcumin, the principal constituent of the dried rhizomes of Curcuma longa L. (turmeric), is known as a strong anti-oxidant and anti-inflammatory agent with different pharmacological effects. This work aims to assess curcumin's inhibitory effects on HIV in vitro and to explore the underpinning mechanism, focusing on CCR5 and the transcription factor forkhead box protein P3 (FOXP3). First, curcumin and the RT inhibitor zidovudine (AZT) were evaluated for their inhibitory properties. HIV-1 pseudovirus infectivity was determined by green fluorescence and luciferase activity measurements in HEK293T cells. AZT was used as a positive control that inhibited HIV-1 pseudoviruses dose-dependently, with IC50 values in the nanomolar range. Then, a molecular docking analysis was carried out to assess the binding affinities of curcumin for CCR5 and HIV-1 RNase H/RT. The anti-HIV activity assay showed that curcumin inhibited HIV-1 infection, and the molecular docking analysis revealed equilibrium dissociation constants of [Formula: see text]9.8[Formula: see text]kcal/mol and [Formula: see text]9.3[Formula: see text]kcal/mol between curcumin and CCR5 and HIV-1 RNase H/RT, respectively. To examine curcumin's anti-HIV effect and its mechanism in vitro, cell cytotoxicity, transcriptome sequencing, and CCR5 and FOXP3 amounts were assessed at different concentrations of curcumin. In addition, human CCR5 promoter deletion constructs and the FOXP3 expression plasmid pRP-FOXP3 (with an EGFP tag) were generated. Whether FOXP3 DNA binding to the CCR5 promoter was blunted by curcumin was examined using transfection assays employing truncated CCR5 gene promoter constructs, a luciferase reporter assay, and a chromatin immunoprecipitation (ChIP) assay. Furthermore, micromolar concentrations of curcumin inactivated the nuclear transcription factor FOXP3, which resulted in decreased expression of CCR5 in Jurkat cells. Moreover, curcumin inhibited PI3K-AKT activation and its downstream target FOXP3. These findings provide mechanistic evidence encouraging further assessment of curcumin as a dietary agent used to reduce the virulence of CCR5-tropic HIV-1. Curcumin-mediated FOXP3 degradation was also reflected in its functions, namely, CCR5 promoter transactivation and HIV-1 virion production. Furthermore, curcumin inhibition of CCR5 and HIV-1 might constitute a potential therapeutic strategy for reducing HIV progression.

Endodomain truncation of the HIV-1 envelope protein improves the packaging efficiency of pseudoviruses

HIV-1 remains one of the most devastating infectious pathogens without available vaccines. A valid neutralization assay using multiple representative virus strains is prerequisite for antibody response analysis in HIV-1 vaccine development, where HIV pseudoviruses (PsVs) commonly serve as surrogate agents for the authentic HIV, offering a safer manipulation in Biosafety Level 2+. However, PsV production is of low efficiency and is unstable in this field. Here, we optimize PsV production conditions via the use of alternative host cells, packaging ratios and gene truncation. We show that a 153-aa truncation of the endodomain substantially enhances the packaging efficiency of HIV PsVs, providing 4 to 25 times higher infection titers than the full-length Env. Further, we obtained a robust HIV-1 PsV panel covering 12 representative global strains for neutralization assay testing. This work sheds light on how to optimize HIV PsV packaging, and provides functional insight into the cytoplasmic domain of HIV-1.

Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation.

Langerhans cells are specialized antigen-presenting cells localized within the epidermis and mucosal epithelium. Upon contact with Langerhans cells, pathogens are captured by the C-type lectin langerin and internalized into a structurally unique vesicle known as a Birbeck granule. Although the immunological role of Langerhans cells and Birbeck granules have been extensively studied, the mechanism by which the characteristic zippered membrane structure of Birbeck granules is formed remains elusive. In this study, we observed isolated Birbeck granules using cryo-electron tomography and reconstructed the 3D structure of the repeating unit of the honeycomb lattice of langerin at 6.4 Å resolution. We found that the interaction between the two langerin trimers was mediated by docking the flexible loop at residues 258-263 into the secondary carbohydrate-binding cleft. Mutations within the loop inhibited Birbeck granule formation and the internalization of HIV pseudovirus. These findings suggest a molecular mechanism for membrane zippering during Birbeck granule biogenesis and provide insight into the role of langerin in the defense against viral infection.

Transcriptomic Evidence of the Immune Response Activation in Individuals With Limb Girdle Muscular Dystrophy Dominant 2 (LGMDD2) Contributes to Resistance to HIV-1 Infection.

LGMDD2 is a rare form of muscular dystrophy characterized by one of the three heterozygous deletions described within the TNPO3 gene that result in the addition of a 15-amino acid tail in the C-terminus.TNPO3 is involved in the nuclear import of splicing factors and acts as a host cofactor for HIV-1 infection by mechanisms not yet deciphered. Further characterization of the crosstalk between HIV-1 infection and LGMDD2 disease may contribute to a better understanding of both the cellular alterations occurring in LGMDD2 patients and the role of TNPO3 in the HIV-1 cycle. To this regard, transcriptome profiling of PBMCs from LGMDD2 patients carrying the deletion c.2771delA in the TNPO3 gene was compared to healthy controls. A total of 545 differentially expressed genes were detected between LGMDD2 patients and healthy controls, with a high representation of G protein-coupled receptor binding chemokines and metallopeptidases among the most upregulated genes in LGMDD2 patients. Plasma levels of IFN-β and IFN-γ were 4.7- and 2.7-fold higher in LGMDD2 patients, respectively. An increase of 2.3-fold in the expression of the interferon-stimulated gene MxA was observed in activated PBMCs from LGMDD2 patients after ex vivo HIV-1 pseudovirus infection. Thus, the analysis suggests a pro-inflammatory state in LGMDD2 patients also described for other muscular dystrophies, that is characterized by the alteration of IL-17 signaling pathway and the consequent increase of metallopeptidases activity and TNF response. In summary, the increase in interferons and inflammatory mediators suggests an antiviral environment and resistance to HIV-1 infection but that could also impair muscular function in LGMDD2 patients, worsening disease evolution. Biomarkers of disease progression and therapeutic strategies based on these genes and mechanisms should be further investigated for this type of muscular dystrophy.

Engineering pan–HIV-1 neutralization potency through multispecific antibody avidity

Deep mining of B cell repertoires of HIV-1-infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC50 value of 0.0009 µg/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 µg/mL cutoff-a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability invivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity.

Synthesis and In Vitro Study of Antiviral Activity of Glycyrrhizin Nicotinate Derivatives against HIV-1 Pseudoviruses and SARS-CoV-2 Viruses.

When developing drugs against SARS-CoV-2, it is important to consider the characteristics of patients with different co-morbidities. People infected with HIV-1 are a particularly vulnerable group, as they may be at a higher risk than the general population of contracting COVID-19 with clinical complications. For such patients, drugs with a broad spectrum of antiviral activity are of paramount importance. Glycyrrhizinic acid (Glyc) and its derivatives are promising biologically active compounds for the development of such broad-spectrum antiviral agents. In this work, derivatives of Glyc obtained by acylation with nicotinic acid were investigated. The resulting preparation, Glycyvir, is a multi-component mixture containing mainly mono-, di-, tri- and tetranicotinates. The composition of Glycyvir was characterized by HPLC-MS/MS and its toxicity assessed in cell culture. Antiviral activity against three strains of SARS-CoV-2 was tested in vitro on Vero E6 cells by MTT assay. Glycyvir was shown to inhibit SARS-CoV-2 replication in vitro (IC502–8 μM) with an antiviral activity comparable to the control drug Remdesivir. In addition, Glycyvir exhibited marked inhibitory activity against HIV pseudoviruses of subtypes B, A6 and the recombinant form CRF63_02A (IC50 range 3.9–27.5 µM). The time-dependence of Glycyvir inhibitory activity on HIV pseudovirus infection of TZM-bl cells suggested that the compound interfered with virus entry into the target cell. Glycyvir is a promising candidate as an agent with low toxicity and a broad spectrum of antiviral action.

Accelerated Clearance and Degradation of Cell-Free HIV by Neutralizing Antibodies Occurs via FcγRIIb on Liver Sinusoidal Endothelial Cells by Endocytosis.

Neutralizing Abs suppress HIV infection by accelerating viral clearance from blood circulation in addition to neutralization. The elimination mechanism is largely unknown. We determined that human liver sinusoidal endothelial cells (LSEC) express FcγRIIb as the lone Fcγ receptor, and using humanized FcγRIIb mouse, we found that Ab-opsonized HIV pseudoviruses were cleared considerably faster from circulation than HIV by LSEC FcγRIIb. Compared with humanized FcγRIIb-expressing mice, HIV clearance was significantly slower in FcγRIIb knockout mice. Interestingly, a pentamix of neutralizing Abs cleared HIV faster compared with hyperimmune anti-HIV Ig (HIVIG), although the HIV Ab/Ag ratio was higher in immune complexes made of HIVIG and HIV than pentamix and HIV. The effector mechanism of LSEC FcγRIIb was identified to be endocytosis. Once endocytosed, both Ab-opsonized HIV pseudoviruses and HIV localized to lysosomes. This suggests that clearance of HIV, endocytosis, and lysosomal trafficking within LSEC occur sequentially and that the clearance rate may influence downstream events. Most importantly, we have identified LSEC FcγRIIb-mediated endocytosis to be the Fc effector mechanism to eliminate cell-free HIV by Abs, which could inform development of HIV vaccine and Ab therapy.

In vitro Study on Synergistic Interactions Between Free and Encapsulated Q-Griffithsin and Antiretrovirals Against HIV-1 Infection.

IntroductionHuman immunodeficiency virus (HIV) remains a persistent global challenge, impacting 38 million people worldwide. Antiretrovirals (ARVs) including tenofovir (TFV), raltegravir (RAL), and dapivirine (DAP) have been developed to prevent and treat HIV-1 via different mechanisms of action. In parallel, a promising biological candidate, griffithsin (GRFT), has demonstrated outstanding preclinical safety and potency against HIV-1. While ARV co-administration has been shown to enhance virus inhibition, synergistic interactions between ARVs and the oxidation-resistant variant of GRFT (Q-GRFT) have not yet been explored. Here, we co-administered Q-GRFT with TFV, RAL, and DAP, in free and encapsulated forms, to identify unique protein-drug synergies.MethodsNanoparticles (NPs) were synthesized using a single or double-emulsion technique and release from each formulation was assessed in simulated vaginal fluid. Next, each ARV, in free and encapsulated forms, was co-administered with Q-GRFT or Q-GRFT NPs to evaluate the impact of co-administration in HIV-1 pseudovirus assays, and the combination indices were calculated to identify synergistic interactions. Using the most synergistic formulations, we investigated the effect of agent incorporation in NP-fiber composites on release properties. Finally, NP safety was assessed in vitro using MTT assay.ResultsAll active agents were encapsulated in NPs with desirable encapsulation efficiency (15–100%), providing ~20% release over 2 weeks. The co-administration of free Q-GRFT with each free ARV resulted in strong synergistic interactions, relative to each agent alone. Similarly, Q-GRFT NP and ARV NP co-administration resulted in synergy across all formulations, with the most potent interactions between encapsulated Q-GRFT and DAP. Furthermore, the incorporation of Q-GRFT and DAP in NP-fiber composites resulted in burst release of DAP and Q-GRFT with a second phase of Q-GRFT release. Finally, all NP formulations exhibited safety in vitro.ConclusionsThis work suggests that Q-GRFT and ARV co-administration in free or encapsulated forms may improve efficacy in achieving prophylaxis.

HIV-Cell Membrane Fusion Intermediates are Restricted by Serinc3 and Serinc5

Serinc3 and Serinc5 are recently described host restriction factors that can block HIV infection by incorporating into budding viral particles and decreasing their ability to infect subsequent cells. Serincs are thought to block the very earliest stages of infection, membrane fusion and cell entry, by an incompletely understood mechanism. Understanding how Serincs disrupt HIV membrane fusion will clarify the requirements for normal HIV membrane fusion and could identify new viral weaknesses and drug targets. We used giant plasma membrane vesicles (blebs) as target membranes to study “wildtype” and Serinc-disrupted HIV membrane fusion at a single-particle level by fluorescence microscopy and cryo-Electron Tomography (cryoET). Using fluorescent reporters of membrane and content mixing, we observed that Serinc3 and Serinc5 cause no defect in mixing of the outer lipid leaflets (hemifusion), but a pronounced defect in fusion pore opening. Additionally, cryo-ET of HIV pseudoviruses mixed with blebs allowed observation of multiple intermediates of HIV membrane fusion. We found that Serinc3 and Serinc5 increased the number of hemifusion and early fusion product events observed in these tomograms and many of the fusion products have a narrowed neck between former virus and bleb. These results suggest that Serinc3 and Serinc5 restrict HIV membrane fusion, not by inhibiting a particular intermediate step, but by causing broad changes to the energetics of fusion that enables more frequent observation of multiple transient intermediate states.

Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles.

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.

Probing Vulnerability of the gp41 C-Terminal Heptad Repeat as Target for Miniprotein HIV Inhibitors

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region.

Sustained-release Griffithsin nanoparticle-fiber composites against HIV-1 and HSV-2 infections

Human immunodeficiency virus (HIV-1) and herpes simplex virus 2 (HSV-2) affect hundreds of millions of people worldwide. The antiviral lectin, Griffithsin (GRFT), has been shown to be both safe and efficacious against HSV-2 and HIV-1 infections in vivo. The goal of this work was to develop a multilayered nanoparticle (NP)-electrospun fiber (EF) composite to provide sustained-release of GRFT, and to examine its safety and efficacy in a murine model of lethal HSV-2 infection. Composites were fabricated from polycaprolactone (PCL) fibers surrounding polyethylene oxide (PEO) fibers that incorporated methoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-PLGA) GRFT NPs. GRFT loading and release were determined via ELISA, showing that NP-EF composites achieved high GRFT loading, and provided sustained-release of GRFT for up to 90 d. The in vitro efficacy of GRFT NP-EFs was assessed using HIV-1 pseudovirus assays, demonstrating complete in vitro protection against HIV-1 infection. Additionally, sustained-release NP-EFs, administered 24 h prior to infection, prevented against a lethal dose of HSV-2 infection in a murine model. In parallel, histology and cytokine expression from murine reproductive tracts and vaginal lavages collected 24 and 72 h post-administration were similar to untreated mice, suggesting that NP-EF composites may be a promising and safe sustained-delivery platform to prevent HSV-2 infection. Future work will evaluate the ability to provide prolonged protection against multiple virus challenges, and different administration times with respect to infection.

Anti-HIV, antitumor and immunomodulatory activities of paclitaxel from fermentation broth using molecular imprinting technique

In this study, a single component paclitaxel was obtained from fermentation broth by molecular imprinting technique, and its antiviral, antitumor and immunomodulatory activities were studied. The results showed that paclitaxel had a good inhibitory activity on human breast cancer MCF-7 cells and showed a concentration- dependent relationship with an IC50 of about 15 μg/mL in the sulforhodamine B assay. At the same time, paclitaxel exerted a weak inhibitory activity on cervical cancer Hela cells. In addition, paclitaxel not only inhibited the invasion of HIV-1 pseudovirus into cells, but also exhibited inhibitory activity to a certain extent after viral invasion of the cells. At a paclitaxel concentration of 20 μg/mL, the inhibition of HIV-1 pseudovirus reached about 66%. The inhibition of HIV-1 protease activity was concentration-dependent. At a concentration of 20 μg/mL, the inhibitory effect of paclitaxel on HIV-1 protease was similar to that of the positive control pepstatin A, being 15.8%. The HIV-1 integrase inhibiting activity of paclitaxel was relatively weak. Paclitaxel significantly up-regulated the expression of interleukin-6.

Protocol for a randomized clinical trial exploring the effect of antimicrobial agents on the penile microbiota, immunology and HIV susceptibility of Ugandan men

BackgroundThe foreskin is the main site of HIV acquisition in a heterosexual uncircumcised man, but many men in endemic countries are reluctant to undergo penile circumcision (PC). Observational studies suggest that proinflammatory anaerobic bacteria are enriched on the uncircumcised penis, where they may enhance HIV susceptibility through increased foreskin inflammatory cytokines and the recruitment of HIV-susceptible CD4+ target cells. This trial will examine the impact of systemic and topical antimicrobials on ex vivo foreskin HIV susceptibility.Methods/designThis randomized, open-label clinical trial will randomize 125 HIV-negative Ugandan men requesting voluntary PC to one of five arms (n = 25 each). The control group will receive immediate PC, while the four intervention groups will defer PC for 1 month and be provided in the interim with either oral tinidazole, penile topical metronidazole, topical clindamycin, or topical hydrogen peroxide. The impact of these interventions on HIV entry into foreskin-derived CD4+ T cells will be quantified ex vivo at the time of PC using a clade A, R5 tropic HIV pseudovirus assay (primary endpoint); secondary endpoints include the impact of antimicrobials on immune parameters and the microbiota of the participant’s penis and of the vagina of their female partner (if applicable), assessed by multiplex enzyme-linked immunosorbent assay and 16S rRNA sequencing.DiscussionThere is a critical need to develop acceptable, simple, and effective means of HIV prevention in men unwilling to undergo PC. This trial will provide insight into the causative role of the foreskin microbiota on HIV susceptibility, and the impact of simple microbiota-focused clinical interventions. This may pave the way for future clinical trials using low-cost, nonsurgical intervention(s) to reduce HIV risk in uncircumcised heterosexual men.Trial registrationClinicalTrials.gov, NCT03412071. Retrospectively registered on 26 January 2018.

Characterization of CD4+ T cell subsets and HIV susceptibility in the inner and outer foreskin of Ugandan men.

Biological mechanisms of foreskin HIV acquisition are poorly defined. The inner foreskin is preferentially infected in explant models, so we hypothesized that this site would be enriched for HIV-susceptible CD4+ T cells and proinflammatory/chemoattractant cytokines. A total of 42 HIV-uninfected Ugandan men without genital symptoms provided foreskin tissues and swabs at the time of elective penile circumcision. The immune phenotype of foreskin-derived CD4+ T cells and entry of a CCR5-tropic HIV pseudovirus was characterized, and specific cytokine levels assayed by multiplexed chemiluminescent ELISA. Unexpectedly, outer foreskin CD4+ T cells more frequently expressed CCR5 (median 29.2% vs 22.9%, P=0.01) and CD69 (median 36.5% vs 15%, P<0.01), and on a per-cell basis, HIV entry was higher. However, overall CD4+ T cell density was approximately twofold higher in the inner foreskin, and several highly susceptible T cell subsets were increased at this site, including Th17 cells (20.0% vs 14.1%, P=0.0021). Specific pro-inflammatory cytokine levels were also higher on the inner foreskin surface (IL-17, IL-8, RANTES and IL-1β; all P<0.05). There was marked heterogeneity in CD4+ T cell populations and immune milieu between inner and outer foreskin tissues. Despite higher per-cell viral entry into CD4+ T cells from the outer foreskin, the higher target cell density and enriched pro-inflammatory cytokines of the inner foreskin suggest that this may be a preferential site for HIV acquisition.