Analysis of SARS-CoV-2 Emergent Variants Following AZD7442 (Tixagevimab/Cilgavimab) for Early Outpatient Treatment of COVID-19 (TACKLE Trial).

INTRODUCTION: AZD7442 (tixagevimab/cilgavimab) comprises neutralising monoclonal antibodies (mAbs) that bind to distinct non-overlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Viral evolution during mAb therapy can select for variants with reduced neutralisation susceptibility. We examined treatment-emergent SARS-CoV-2 variants during TACKLE (NCT04723394), a phase 3 study of AZD7442 for early outpatient treatment of coronavirus disease 2019 (COVID-19). METHODS: Non-hospitalised adults with mild-to-moderate COVID-19 were randomised and dosed ≤ 7 days from symptom onset with AZD7442 (n = 452) or placebo (n = 451). Next-generation sequencing of the spike gene was performed on SARS-CoV-2 reverse-transcription polymerase chain reaction-positive nasopharyngeal swabs at baseline and study days 3, 6, and 15 post dosing. SARS-CoV-2 lineages were assigned using spike nucleotide sequences. Amino acid substitutions were analysed at allele fractions (AF; % of sequence reads represented by substitution) ≥ 25% and 3% to 25%. In vitro susceptibility to tixagevimab, cilgavimab, and AZD7442 was evaluated for all identified treatment-emergent variants using a pseudotyped microneutralisation assay. RESULTS: Longitudinal spike sequences were available for 461 participants (AZD7442, n = 235; placebo, n = 226) and showed that treatment-emergent variants at any time were rare, with 5 (2.1%) AZD7442 participants presenting ≥ 1 substitution in tixagevimab/cilgavimab binding sites at AF ≥ 25%. At AF 3% to 25%, treatment-emergent variants were observed in 15 (6.4%) AZD7442 and 12 (5.3%) placebo participants. All treatment-emergent variants showed in vitro susceptibility to AZD7442. CONCLUSION: These data indicate that AZD7442 creates a high genetic barrier for resistance and is a feasible option for COVID-19 treatment.

Molecular Characterization of AZD7442 (Tixagevimab-Cilgavimab) Neutralization of SARS-CoV-2 Omicron Subvariants.

Therapeutic anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (MAbs) provide immunosuppressed and vulnerable populations with prophylactic and treatment interventions against coronavirus disease 2019 (COVID-19). AZD7442 (tixagevimab-cilgavimab) is a combination of extended-half-life neutralizing MAbs that bind to distinct epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The Omicron variant of concern carries mutations at >35 positions in the spike protein and has undergone further genetic diversification since its emergence in November 2021. Here, we characterize the in vitro neutralization activity of AZD7442 toward major viral subvariants circulating worldwide during the first 9 months of the Omicron wave. BA.2 and its derived subvariants showed the highest susceptibility to AZD7442, while BA.1 and BA.1.1 showed a lower susceptibility. BA.4/BA.5 had a susceptibility level intermediate between BA.1 and BA.2. Mutagenesis of parental Omicron subvariant spike proteins was performed to establish a molecular model to describe the underlying determinants of neutralization by AZD7442 and its component MAbs. The concurrent mutation of residues at positions 446 and 493, located in the tixagevimab and cilgavimab binding sites, was sufficient to enhance in vitro susceptibility of BA.1 to AZD7442 and its component MAbs to levels similar to the Wuhan-Hu-1+D614G virus. AZD7442 maintained neutralization activity against all Omicron subvariants tested up to and including BA.5. The evolving nature of the SARS-CoV-2 pandemic warrants continuing real-time molecular surveillance and assessment of in vitro activity of MAbs used in prophylaxis against and the treatment of COVID-19. IMPORTANCE MAbs are key therapeutic options for COVID-19 prophylaxis and treatment in immunosuppressed and vulnerable populations. Due to the emergence of SARS-CoV-2 variants, including Omicron, it is vital to ensure that neutralization is maintained for MAb-based interventions. We studied the in vitro neutralization of AZD7442 (tixagevimab-cilgavimab), a cocktail of two long-acting MAbs targeting the SARS-CoV-2 spike protein, toward Omicron subvariants circulating from November 2021 to July 2022. AZD7442 neutralized major Omicron subvariants up to and including BA.5. The mechanism of action responsible for the lower in vitro susceptibility of BA.1 to AZD7442 was investigated using in vitro mutagenesis and molecular modeling. A combination of mutations at two spike protein positions, namely, 446 and 493, was sufficient to enhance BA.1 susceptibility to AZD7442 to levels similar to the Wuhan-Hu-1+D614G ancestral virus. The evolving nature of the SARS-CoV-2 pandemic warrants continuing real-time global molecular surveillance and mechanistic studies of therapeutic MAbs for COVID-19.

Impact of Integrase Sequences from HIV-1 Subtypes A6/A1 on the In Vitro Potency of Cabotegravir or Rilpivirine.

The FLAIR study demonstrated noninferiority of monthly long-acting cabotegravir + rilpivirine versus daily oral dolutegravir/abacavir/lamivudine for maintaining virologic suppression. Three participants who received long-acting therapy had confirmed virologic failure (CVF) at Week 48, and all had HIV-1 that was originally classified as subtype A1 and contained the baseline integrase polymorphism L74I; updated classification algorithms reclassified all 3 as HIV-1 subtype A6. Retrospectively, the impact of L74I on in vitro sensitivity and durability of response to cabotegravir in HIV-1 subtype B and A6 backgrounds was studied. Site-directed L74I and mutations observed in participants with CVF were generated in HIV-1 subtype B and a consensus integrase derived from 3 subtype A6 CVF baseline sequences. Rilpivirine susceptibility was assessed in HIV-1 subtype B and A1 containing reverse transcriptase mutations observed in participants with CVF. HIV-1 subtype B L74I and L74I/G140R mutants and HIV-1 subtype A6 I74L and I74/G140R mutants remained susceptible to cabotegravir; L74I/Q148R double mutants exhibited reduced susceptibility in HIV-1 subtypes B and A6 (half maximal effective capacity fold change, 4.4 and 4.1, respectively). Reduced rilpivirine susceptibility was observed across HIV-1 subtypes B and A1 with resistance-associated mutations K101E or E138K (half maximal effective capacity fold change, 2.21 to 3.09). In cabotegravir breakthrough experiments, time to breakthrough was similar between L74 and I74 viruses across HIV-1 subtypes B and A6; Q148R was selected at low cabotegravir concentrations. Therefore, the L74I integrase polymorphism did not differentially impact in vitro sensitivity to cabotegravir across HIV-1 subtype B and A6 integrase genes (ClinicalTrials.gov identifier: NCT02938520).

Long-acting and extended-release implant and nanoformulations with a synergistic antiretroviral two-drug combination controls HIV-1 infection in a humanized mouse model.

The HIV pandemic has affected over 38 million people worldwide with close to 26 million currently accessing antiretroviral therapy (ART). A major challenge in the long-term treatment of HIV-1 infection is nonadherence to ART. Long-acting antiretroviral (LA-ARV) formulations, that reduce dosing frequency to less than once a day, are an urgent need that could tackle the adherence issue. Here, we have developed two LA-ART interventions, one an injectable nanoformulation, and the other, a removable implant, for the delivery of a synergistic two-drug ARV combination comprising a pre-clinical nonnucleoside reverse transcriptase inhibitor (NNRTI), Compound I, and the nucleoside reverse transcriptase inhibitor (NRTI), 4'-ethynyl-2-fluoro-2'-deoxyadenosine. The nanoformulation is poly(lactide-co-glycolide)-based and the implant is a copolymer of ω-pentadecalactone and p-dioxanone, poly(PDL-co-DO), a novel class of biocompatible, biodegradable materials. Both the interventions, packaged independently with each ARV, released sustained levels of the drugs, maintaining plasma therapeutic indices for over a month, and suppressed viremia in HIV-1-infected humanized mice for up to 42 days with maintenance of CD4+ T cells. These data suggest promise in the use of these new drugs as LA-ART formulations in subdermal implant and injectable mode.

Clinical evidence for a lack of cross-resistance between temsavir and ibalizumab or maraviroc.

BACKGROUND: Temsavir (TMR), the active agent of the gp120-directed attachment inhibitor fostemsavir (FTR), the CD4-directed attachment inhibitor ibalizumab (IBA), and the CCR5 antagonist maraviroc (MVC) are antiretroviral agents that target steps in HIV-1 viral entry. Although mechanisms of inhibition of the three agents are different, it is important to understand whether there is potential for cross-resistance between these agents, as all involve interactions with gp120. METHODS: Envelopes derived from plasma samples from participants in the BRIGHTE study who experienced protocol-derived virologic failure (PDVF) and were co-dosed with FTR and either IBA or MVC were analyzed for susceptibility to the agents. Also, CCR5-tropic MVC-resistant envelopes from the MOTIVATE trials were regenerated and studies were performed to understand whether susceptibility to multiple agents were linked. RESULTS: The cloned envelopes exhibited reduced susceptibility to TMR and resistance to the co-dosed agent. At PDVF, emergent or preexisting amino acid substitutions were present at TMR positions of interest. When amino acid substitutions at these positions were reverted to the consensus sequence, full susceptibility to TMR was restored without effecting resistance to the co-dosed agent. In addition, five envelopes from MOTIVATE were regenerated and exhibited R5-tropic-MVC-resistance. Only one exhibited reduced susceptibility to TMR and it contained an M426L polymorphism. When reverted to 426M, full sensitivity for TMR was restored, but it remained MVC resistant. CONCLUSION: The data confirm that decreased susceptibility to TMR and resistance to IBA or MVC are not linked and that there is no cross-resistance between either of these two agents and FTR.

Modulating HIV-1 envelope glycoprotein conformation to decrease the HIV-1 reservoir.

Small CD4-mimetic compounds (CD4mc) sensitize HIV-1-infected cells to antibody-dependent cellular cytotoxicity (ADCC) by facilitating antibody recognition of epitopes that are otherwise occluded on the unliganded viral envelope (Env). Combining CD4mc with two families of CD4-induced (CD4i) antibodies, which are frequently found in plasma of HIV-1-infected individuals, stabilizes Env in a conformation that is vulnerable to ADCC. We employed new-generation SRG-15 humanized mice, supporting natural killer (NK) cell and Fc-effector functions to demonstrate that brief treatment with CD4mc and CD4i-Abs significantly decreases HIV-1 replication, the virus reservoir and viral rebound after ART interruption. These effects required Fc-effector functions and NK cells, highlighting the importance of ADCC. Viral rebound was also suppressed in HIV-1+-donor cell-derived humanized mice supplemented with autologous HIV-1+-donor-derived plasma and CD4mc. These results indicate that CD4mc could have therapeutic utility in infected individuals for decreasing the size of the HIV-1 reservoir and/or achieving a functional cure.

Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice.

Non-invasive bioluminescent imaging (NIBLI) of HIV-1 infection dynamics allows for real-time monitoring of viral spread and the localization of infected cell populations in living animals. In this report, we describe full-length replication-competent GFP and Nanoluciferase (Nluc) expressing HIV-1 reporter viruses from two clinical transmitted / founder (T/F) strains: TRJO.c and Q23.BG505. By infecting humanized mice with these HIV-1 T/F reporter viruses, we were able to directly monitor longitudinal viral spread at whole-animal resolution via NIBLI at a sensitivity of as few as 30-50 infected cells. Bioluminescent signal strongly correlated with HIV-1 infection and responded proportionally to virus suppression in vivo in animals treated daily with a combination antiretroviral therapy (cART) regimen. Longitudinal NIBLI following cART withdrawal visualized tissue-sites that harbored virus during infection recrudescence. Notably, we observed rebounding infection in the same lymphoid tissues where infection was first observed prior to ART treatment. Our work demonstrates the utility of our system for studying in vivo viral infection dynamics and identifying infected tissue regions for subsequent analyses.

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System.

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 µL of solution in 1-2 µL droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Structural and pharmacological evaluation of a novel non-nucleoside reverse transcriptase inhibitor as a promising long acting nanoformulation for treating HIV.

Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48 h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.

Intranasal delivery of a Fas-blocking peptide attenuates Fas-mediated apoptosis in brain ischemia.

Ischemic stroke-induced neuronal cell death results in the permanent disabling of brain function. Apoptotic mechanisms are thought to play a prominent role in neuronal injury and ample evidence implicates Fas signaling in mediating cell death. In this study, we describe the neuroprotective effects of a Fas-blocking peptide (FBP) that by obstructing Fas signaling in cerebral ischemia inhibits apoptosis. Using an intranasal administration route in a rat model of focal cerebral ischemia, we demonstrate that nose-to-brain delivery of FBP after middle cerebral artery occlusion (MCAO) surgery results in the delivery and retention of FBP in Fas-expressing ischemic areas of the brain. A single intranasal administration of 2 mg/kg FBP resulted in significantly reduced neuronal cell death by inhibiting Fas-mediated apoptosis leading to decreased infarct volumes, reduced neurologic deficit scores and recovery from cerebral ischemia. Intranasally delivered FBP might be a promising strategy for the treatment of cerebral ischemic stroke.

Small Interfering RNA-Mediated Control of Virus Replication in the CNS Is Therapeutic and Enables Natural Immunity to West Nile Virus.

No vaccines or therapeutics are licensed for West Nile virus (WNV), a mosquito-transmitted neuroencephalitic flavivirus. The small interfering RNA siFvEJW targets a conserved sequence within the WNV E protein and limits virus infection. Using a rabies virus-derived neuron-targeting peptide (RVG9R) and an intranasal route for delivering siFvEJW to the CNS, we demonstrate that treatment of WNV-infected mice at late stages of neuroinvasive disease results in recovery. Selectively targeting virus in the CNS lowers viral burdens in the brain, reduces neuropathology, and results in a 90% survival rate at 5-6 days post-infection (when viral titers peak in the CNS), while placebo-treated mice succumb by days 9-10. Importantly, CNS virus clearance is achieved by humoral and cell-mediated immune responses to WNV infection in peripheral tissues, which also engender sterilizing immunity against subsequent WNV infection. These results indicate that intranasal RVG9R-siRNA treatment offers efficient late-stage therapy and facilitates natural long-term immunity against neuroinvasive flaviviruses.

From in silico hit to long-acting late-stage preclinical candidate to combat HIV-1 infection.

The HIV-1 pandemic affecting over 37 million people worldwide continues, with nearly one-half of the infected population on highly active antiretroviral therapy (HAART). Major therapeutic challenges remain because of the emergence of drug-resistant HIV-1 strains, limitations because of safety and toxicity with current HIV-1 drugs, and patient compliance for lifelong, daily treatment regimens. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral polymerase have been a key component of the current HIV-1 combination drug regimens; however, these issues hamper them. Thus, the development of novel more effective NNRTIs as anti-HIV-1 agents with fewer long-term liabilities, efficacy on new drug-resistant HIV-1 strains, and less frequent dosing is crucial. Using a computational and structure-based design strategy to guide lead optimization, a 5 µM virtual screening hit was transformed to a series of very potent nanomolar to picomolar catechol diethers. One representative, compound I, was shown to have nanomolar activity in HIV-1-infected T cells, potency on clinically relevant HIV-1 drug-resistant strains, lack of cytotoxicity and off-target effects, and excellent in vivo pharmacokinetic behavior. In this report, we show the feasibility of compound I as a late-stage preclinical candidate by establishing synergistic antiviral activity with existing HIV-1 drugs and clinical candidates and efficacy in HIV-1-infected humanized [human peripheral blood lymphocyte (Hu-PBL)] mice by completely suppressing viral loads and preventing human CD4+ T-cell loss. Moreover, a long-acting nanoformulation of compound I [compound I nanoparticle (compound I-NP)] in poly(lactide-coglycolide) (PLGA) was developed that shows sustained maintenance of plasma drug concentrations and drug efficacy for almost 3 weeks after a single dose.

Structural and Preclinical Studies of Computationally Designed Non-Nucleoside Reverse Transcriptase Inhibitors for Treating HIV infection.

The clinical benefits of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are hindered by their unsatisfactory pharmacokinetic (PK) properties along with the rapid development of drug-resistant variants. However, the clinical efficacy of these inhibitors can be improved by developing compounds with enhanced pharmacological profiles and heightened antiviral activity. We used computational and structure-guided design to develop two next-generation NNRTI drug candidates, compounds I and II, which are members of a class of catechol diethers. We evaluated the preclinical potential of these compounds in BALB/c mice because of their high solubility (510 µg/ml for compound I and 82.9 µg/ml for compound II), low cytotoxicity, and enhanced antiviral activity against wild-type (WT) HIV-1 RT and resistant variants. Additionally, crystal structures of compounds I and II with WT RT suggested an optimal binding to the NNRTI binding pocket favoring the high anti-viral potency. A single intraperitoneal dose of compounds I and II exhibited a prolonged serum residence time of 48 hours and concentration maximum (Cmax) of 4000- to 15,000-fold higher than their therapeutic/effective concentrations. These Cmax values were 4- to 15-fold lower than their cytotoxic concentrations observed in MT-2 cells. Compound II showed an enhanced area under the curve (0-last) and decreased plasma clearance over compound I and efavirenz, the standard of care NNRTI. Hence, the overall (PK) profile of compound II was excellent compared with that of compound I and efavirenz. Furthermore, both compounds were very well tolerated in BALB/c mice without any detectable acute toxicity. Taken together, these data suggest that compounds I and II possess improved anti-HIV-1 potency, remarkable in vivo safety, and prolonged in vivo circulation time, suggesting strong potential for further development as new NNRTIs for the potential treatment of HIV infection.

Trileucine residues in a ligand-CPP-based siRNA delivery platform improve endosomal escape of siRNA.

siRNA entrapment within endosomes is a significant problem encountered with siRNA delivery platforms that co-opt receptor-mediated entry pathways. Attachment of a cell-penetrating peptide (CPP), such as nona-arginine (9R) to a cell receptor-binding ligand like the Rabies virus glycoprotein, RVG, allows effective siRNA delivery to the cytoplasm by non-endocytic pathways, but a significant amount of siRNA complexes also enters the cell by ligand-induced receptor endocytosis and remain localized in endosomes. Here, we report that the incorporation of trileucine (3 Leu) residues as an endo-osmolytic moiety in the peptide improves endosomal escape and intracellular delivery of siRNA. The trileucine motif did not affect early non-endosomal mechanism of cytoplasmic siRNA delivery but enhanced target gene silencing by >20% only beyond 24 h of transfection when siRNA delivery is mostly through the endocytic route and siRNA trapped in the endosomes at later stages were subject to release into cytoplasm. The mechanism may involve endosomal membrane disruption as trileucine residues lysed RBCs selectively under endosomal pH conditions. Interestingly <3 Leu or >3 Leu residues were not as effective, suggesting that 3 Leu residues are useful for enhancing cytoplasmic delivery of siRNA routed through endosomes.

In vivo correction of anaemia in ß-thalassemic mice by γPNA-mediated gene editing with nanoparticle delivery.

The blood disorder, ß-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human ß-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% ß-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration.

Silencing CCR2 in Macrophages Alleviates Adipose Tissue Inflammation and the Associated Metabolic Syndrome in Dietary Obese Mice.

Adipose tissue macrophage (ATM)-mediated inflammation is a key feature contributing to the adverse metabolic outcomes of dietary obesity. Recruitment of macrophages to obese adipose tissues (AT) can occur through the engagement of CCR2, the receptor for MCP-1 (monocyte chemoattractant protein-1), which is expressed on peripheral monocytes/macrophages. Here, we show that i.p. administration of a rabies virus glycoprotein-derived acetylcholine receptor-binding peptide effectively delivers complexed siRNA into peritoneal macrophages and ATMs in a mouse model of high-fat diet-induced obesity. Treatment with siRNA against CCR2 inhibited macrophage infiltration and accumulation in AT and, therefore, proinflammatory cytokines produced by macrophages. Consequently, the treatment significantly improved glucose tolerance and insulin sensitivity profiles, and also alleviated the associated symptoms of hepatic steatosis and reduced hepatic triglyceride production. These results demonstrate that disruption of macrophage chemotaxis to the AT through cell-targeted gene knockdown strategies can provide a therapeutic intervention for obesity-related metabolic diseases. The study also highlights a siRNA delivery approach for targeting specific monocyte subsets that contribute to obesity-associated inflammation without affecting the function of other tissue-resident macrophages that are essential for host homeostasis and survival.

Retroviruses use CD169-mediated trans-infection of permissive lymphocytes to establish infection.

Dendritic cells can capture and transfer retroviruses in vitro across synaptic cell-cell contacts to uninfected cells, a process called trans-infection. Whether trans-infection contributes to retroviral spread in vivo remains unknown. Here, we visualize how retroviruses disseminate in secondary lymphoid tissues of living mice. We demonstrate that murine leukemia virus (MLV) and human immunodeficiency virus (HIV) are first captured by sinus-lining macrophages. CD169/Siglec-1, an I-type lectin that recognizes gangliosides, captures the virus. MLV-laden macrophages then form long-lived synaptic contacts to trans-infect B-1 cells. Infected B-1 cells subsequently migrate into the lymph node to spread the infection through virological synapses. Robust infection in lymph nodes and spleen requires CD169, suggesting that a combination of fluid-based movement followed by CD169-dependent trans-infection can contribute to viral spread.

Cationic cell-penetrating peptides as vehicles for siRNA delivery.

RNA interference mediated gene silencing has tremendous applicability in fields ranging from basic biological research to clinical therapy. However, delivery of siRNA across the cell membrane into the cytoplasm, where the RNA silencing machinery is located, is a significant hurdle in most primary cells. Cell-penetrating peptides (CPPs), peptides that possess an intrinsic ability to translocate across cell membranes, have been explored as a means to achieve cellular delivery of siRNA. Approaches using CPPs by themselves or through incorporation into other siRNA delivery platforms have been investigated with the intent of improving cytoplasmic delivery. Here, we review the utilization of CPPs for siRNA delivery with a focus on strategies developed to enhance cellular uptake, endosomal escape and cytoplasmic localization of CPP/siRNA complexes.

Adenovirus-Vectored Broadly Neutralizing Antibodies Directed Against gp120 Prevent Human Immunodeficiency Virus Type 1 Acquisition in Humanized Mice.

Despite nearly three decades of research, a safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) has yet to be achieved. More recently, the discovery of highly potent anti-gp160 broadly neutralizing antibodies (bNAbs) has garnered renewed interest in using antibody-based prophylactic and therapeutic approaches. Here, we encoded bNAbs in first-generation adenoviral (ADV) vectors, which have the distinctive features of a large coding capacity and ease of propagation. A single intramuscular injection of ADV-vectorized bNAbs in humanized mice generated high serum levels of bNAbs that provided protection against multiple repeated challenges with a high dose of HIV-1, prevented depletion of peripheral CD4(+) T cells, and reduced plasma viral loads to below detection limits. Our results suggest that ADV vectors may be a viable option for the prophylactic and perhaps therapeutic use of bNAbs in humans.