Quantifying and Modulating Protein Encapsulation in Guanosine-Based Supramolecular Particles.

The encapsulation of proteins is an effective way to preserve their structure and enhance their function. One exciting possibility is adjusting the protective agent to match the specific protein's characteristics to influence its properties. In a recent study, we developed a flow cytometry-based method to quantify the encapsulation of small-molecule dyes in colloidal particles made from guanosine derivatives (supramolecular hacky sacks (SHS) particles). We aimed to determine whether this method could quantify protein encapsulation and track changes and if the particles could be tuned to bind to specific proteins. Our results showed that fluorescein isothiocyanate (FITC)-labeled proteins had apparent association constants in the micromolar range with hydrophobicity as the dominant factor enhancing the affinities. Confocal laser scanning microscopy (CLSM) imaging supported these results and provided additional information about the protein distribution within the particles. We also tested the feasibility of tuning the avidin affinity (AVI) for SHS particles with a biotin ligand. We found that increasing the amount of biotin initially enhanced AVI binding, but then reached saturation, which we hypothesize results from noncovalent cross-linking caused by strong biotin/AVI interactions. CLSM images showed that the linker also impacted the AVI distribution within the particles. Our strategy provides an advantage over other methods for quantifying protein encapsulation by being suitable for high-throughput analysis with high reproducibility. We anticipate that future efforts to use lower-affinity ligands would result in better strategies for modulating protein affinity for drug delivery applications.

Chronic wound healing by controlled release of chitosan hydrogels loaded with silver nanoparticles and calendula extract.

As the skin is the main protective organ of the body, it is exposed to wounds or injuries which carry out a healing process during a period of approximately 15 days depending on the severity of the injury. In the present research, the development of chitosan-based hydrogels loaded with silver nanoparticles and calendula extract (Ch-AgNPs-Ce) was proposed. This can be used to fulfill the hemostatic, anti-infective, antibacterial, healing and anti-inflammatory functions through controlled release of the nanoparticles and calendula extract in substitution of commonly used drugs. The physical properties of the silver nanoparticles were analyzed by UV-visible spectroscopy, scanning and transmission electron microscopy, showing a size between 50 and 100 nm. The antibacterial properties were evaluated by the agar well diffusion method. Antimicrobial testing of the hydrogels showed that the inclusion of silver nanoparticles provides concentration-dependent antibacterial behavior against E. coli and S. aureus. The healing properties of the system were tested in two diabetic patients to whom said hydrogels were placed, obtaining a positive curative result after a few weeks. Therefore, it can be concluded that Ch-AgNPs-Ce hydrogels can achieve healing in chronic or exposed wounds after a period of time which can be used in alternative treatments in patients with poor healing capacity.

Screening and Quantification of the Encapsulation of Dyes in Supramolecular Particles.

The encapsulation of therapeutic agents, such as drugs and vaccines, into colloidal particles offers an attractive strategy to enhance their efficacy. Previously, we reported the development of guanosine-based supramolecular colloidal particles suitable for encapsulating a broad array of guests ranging from small molecule drugs, like doxorubicin, to proteins, like GFP. Many biomedical applications of such particles require a precise determination of the amount of encapsulated therapeutic agents. Despite many studies describing the development of particle-based delivery systems, a general method for the precise and quick quantification of the encapsulated payload is still lacking. Here, we report a method based on flow cytometry measurements for complexes made from guanosine-based particles and a variety of commercially available fluorescent dyes. This method allows us to determine the apparent affinities of such dyes for two variants of these particles, which in turn provides insightful structure-affinity relationships. In contrast to the current methods, such as those that rely on fluorescence microscopy based on measurements of absorption/fluorescence of dissolved particles or on the supernatant of the solution, the reported method is suitable for high-throughput screening and more reproducible results. The protocol described here should be applicable to a wide variety of colloidal particles being developed around the world. Our group is currently expanding the scope to quantify the encapsulation of other molecules of biomedical interest, such as proteins and nucleic acids.

Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction.

Although invasive cytomegalovirus (CMV) disease is uncommon in the era of antiretroviral therapy (ART), asymptomatic CMV coinfection is nearly ubiquitous in HIV infected individuals. While microbial translocation and gut epithelial barrier dysfunction may promote persistent immune activation in treated HIV infection, potentially contributing to morbidity and mortality, it has been unclear whether CMV replication in individuals with no symptoms of CMV disease might play a role in this process. We hypothesized that persistent CMV replication in the intestinal epithelium of HIV/CMV-coinfected individuals impairs gut epithelial barrier function. Using a combination of state-of-the-art in situ hybridization technology (RNAscope) and immunohistochemistry, we detected CMV DNA and proteins and evidence of intestinal damage in rectosigmoid samples from CMV-positive individuals with both untreated and ART-suppressed HIV infection. Two different model systems, primary human intestinal cells differentiated in vitro to form polarized monolayers and a humanized mouse model of human gut, together demonstrated that intestinal epithelial cells are fully permissive to CMV replication. Independent of HIV, CMV disrupted tight junctions of polarized intestinal cells, significantly reducing transepithelial electrical resistance, a measure of monolayer integrity, and enhancing transepithelial permeability. The effect of CMV infection on the intestinal epithelium is mediated, at least in part, by the CMV-induced proinflammatory cytokine IL-6. Furthermore, letermovir, a novel anti-CMV drug, dampened the effects of CMV on the epithelium. Together, our data strongly suggest that CMV can disrupt epithelial junctions, leading to bacterial translocation and chronic inflammation in the gut and that CMV could serve as a target for therapeutic intervention to prevent or treat gut epithelial barrier dysfunction during HIV infection.

Tracking the formation of supramolecular G-quadruplexes via self-assembly enhanced emission.

We report the synthesis and self-assembly of two lipophilic 2'-deoxyguanosine (G) derivatives whose fluorescence intensity is modulated by self-assembly into supramolecular G-quadruplexes (SGQs). Whereas both derivatives self-assemble isostructurally, one shows up to 100% emission enhancement while the other shows an initial enhancement, followed by 10% quenching. Thus, the rotational restrictions resulting from self-assembly are enough to induce significant changes in emission, but it is critical to consider the specific interactions between fluorophores since they will determine the ultimate emission signature. These findings could open the door to the development of luminescent supramolecular sensors and probes.

Probing the Limits of Supramolecular G-Quadruplexes Using Atomistic Molecular Dynamics Simulations.

Guanosine and related derivatives self-assemble in the presence of cations like potassium into supramolecular G-quadruplexes (SGQs), where four guanine moieties form planar tetrads (T) that coaxially stack into columnar aggregates with broad size distributions. However, SGQs made from 8-aryl-2'-deoxyguanosine derivatives (8ArGs), form mostly octamers, or two-tetrad (2T)-SGQs, while some form dodecamers (3T-SGQs), or hexadecamers (4T-SGQs), and none reported to date form higher assemblies. A theoretical model that addresses the configurational space available for the multiple pathways available for 8ArGs to self-assemble into SGQs is used to frame a series of molecular dynamics simulations (MDS) with selected SGQs. Some key insights from this work include: (a) The predicted entropic costs are not significantly higher for SGQs with more subunits due to their hierarchical assembly pathways; (b) The multiple isomeric SGQs vary in the interfacial contacts between consecutive tetrads, due to their two distinct sides (head, h; tail, t), with the MDS supporting the predicted order of stability of hh > ht > tt for octamers. (c) Such order also applies to dodecamers and hexadecamers, but with context-dependent exceptions due to strong allosteric effects. (d) The main factor disfavoring the tt interface is the repulsive dipolar interactions between the O4' from ribose moieties on adjacent tetrads. (e) SGQs with 5 or more tetrads are disfavored because the attractive interactions are not large or strong enough to overcome the many repulsive forces resulting from the addition of further tetrads. We expect these findings provide some guidelines to enable the further development of SGQs into functional materials.

Organic Nanoflowers from a Wide Variety of Molecules Templated by a Hierarchical Supramolecular Scaffold.

Nanoflowers (NFs) are flowered-shaped particles with overall sizes or features in the nanoscale. Beyond their pleasing aesthetics, NFs have found a number of applications ranging from catalysis, to sensing, to drug delivery. Compared to inorganic based NFs, their organic and hybrid counterparts are relatively underdeveloped mostly because of the lack of a reliable and versatile method for their construction. We report here a method for constructing NFs from a wide variety of biologically relevant molecules (guests), ranging from small molecules, like doxorubicin, to biomacromolecules, like various proteins and plasmid DNA. The method relies on the encapsulation of the guests within a hierarchically structured particle made from supramolecular G-quadruplexes. The size and overall flexibility of the guests dictate the broad morphological features of the resulting NFs, specifically, small and rigid guests favor the formation of NFs with spiky petals, while large and/or flexible guests promote NFs with wide petals. The results from experiments using confocal fluorescence microscopy, and scanning electron microscopy provides the basis for the proposed mechanism for the NF formation.

Structural Studies of Supramolecular G-Quadruplexes Formed from 8-Aryl-2'-deoxyguanosine Derivatives.

Self-assembly is a powerful tool for the construction of complex nanostructures. Despite advances in the field, the development of precise self-assembled structures remains a challenge. We have shown that, in the presence of suitably sized cations like K(+), 8-aryl-2'-deoxyguanosine (8ArG) derivatives self-assemble into sets of coaxially stacked planar tetramers, which we term supramolecular G-quadruplexes (SGQs). Previously, we reported that, when the 8-aryl group is a phenyl ring with a meta-carbonyl group, the resulting supramolecule is a hexadecamer, which is remarkably robust as illustrated by its isostructural assembly in both organic and aqueous environments. We report here a detailed three-dimensional structure of the SGQs formed by lipophilic, and hydrophilic, 8ArG derivatives with either 8-(meta-acetylphenyl), 8-(para-acetylphenyl), or 8-(meta-ethoxycarbonylphenyl) groups. The chirality and close contacts between the subunits impose different levels of steric and electrostatic constraints on opposite sides of the tetrads, which determine their preferred relative orientation. The balance between attractive noncovalent interactions juxtaposed with repulsive steric and electrostatic interactions explains the high cooperativity, fidelity, and stability of these SGQs. These structural studies, together with titration experiments and molecular dynamics simulations, provide insight into the mechanism of formation of these SGQs.

Oral administration of the nucleoside EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) provides rapid suppression of HIV viremia in humanized mice and favorable pharmacokinetic properties in mice and the rhesus macaque.

Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) has a 3'-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIVJR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.25 nM and a selectivity index of 184,000; similar antiviral potency was found against 12 different HIV clinical isolates from multiple clades (A, B, C, D, and CRF01_AE). EFdA was readily absorbed after oral dosing (5 mg/kg of body weight) in both mice and the rhesus macaque, with micromolar levels of the maximum concentration of drug in serum (Cmax) attained at 30 min and 90 min, respectively. Trough levels were at or above 90% inhibitory concentration (IC90) levels in the macaque at 24 h, suggesting once-daily dosing. EFdA showed reasonable penetration of the blood-brain barrier in the rhesus macaque, with cerebrospinal fluid levels at approximately 25% of plasma levels 8 h after single oral dosing. Rhesus PBMCs isolated 24 h following a single oral dose of 5 mg/kg EFdA were refractory to SIV infection due to sufficiently high intracellular EFdA-triphosphate levels. The intracellular half-life of EFdA-triphosphate in PBMCs was determined to be >72 h following a single exposure to EFdA. Daily oral administration of EFdA at low dosage levels (1 to 10 mg/kg/day) was highly effective in protecting humanized mice from HIV infection, and 10 mg/kg/day oral EFdA completely suppressed HIV RNA to undetectable levels within 2 weeks of treatment.

Tuning thermoresponsive supramolecular G-quadruplexes.

Thermoresponsive systems are attractive due to their suitability for fundamental studies as well as their practical uses in a wide variety of applications. While much progress has been achieved using polymers, alternative strategies such as the use of well-defined nonpolymeric supramolecules are still underdeveloped. Here we report three 8-aryl-2'-deoxyguanosine derivatives (8ArGs) that self-assemble in aqueous media into precise thermoresponsive supramolecular G-quadruplexes (SGQs). We report the synthesis of such derivatives, studies of their isothermal self-assembly, and the thermally induced assembly to form higher-order meso-globular assemblies we term supramolecular hacky sacks (SHS). The lower critical solution temperature (LCST) that indicates the formation of the SHS was modulated by changing (a) intrinsic parameters (i.e., structure of the 8ArGs); (b) extrinsic parameters such as the salt used to promote the formation of the SGQ; and (c) supramolecular parameters such as the coassembly different 8ArGs to form heteromeric SGQs. Changes in the intrinsic parameters lead to LCST variations in the range of 28-59 °C. Modulating extrinsic parameters such as replacing KI with KSCN abolishes the thermoresponsive phenomenon whereas changing the cation from K(+) to Na(+) or adjusting the pH (in the range of 6-8) has negligible effects on the LCST. Modulating supramolecular parameters results in transition temperatures that are intermediate between those obtained by the respective homomeric SGQs, although the specific proportions of the subunits are critical in determining the reversibility of the process. Given the extensive applications of thermoresponsive polymers, the nonpolymeric supramolecular counterparts presented here may represent an attractive alternative for fundamental studies and biorelevant applications.

Tuning supramolecular G-quadruplexes with mono- and divalent cations.

Supramolecular G-quadruplexes (SGQs) are formed via the cation promoted self-assembly of guanine derivatives into stacks of planar hydrogen-bonded tetramers. Here, we present results on the formation of SGQs made from the 8-(m-acetylphenyl)-2'-deoxyguanosine (mAGi) derivative in the presence of various mono- and divalent cations. NMR and HR ESI-MS data indicate that varying the cation can efficiently tune the molecularity, the fidelity and stability (thermal and kinetic) of the resulting SGQs. The results show that, parallel to the previously reported potassium-templated hexadecamer (mAGi16·3K+), Na+, Rb+ and [Formula: see text] also promote the formation of similar supramolecules with high fidelity and molecularity. In contrast, the divalent cations Pb2+, Sr2+ and Ba2+ template the formation of octamers (mAGi8), with the latter two inducing higher thermal stabilities. Molecular dynamics simulations for the hexadecamers containing monovalent cations enabled critical insights that help explain the experimental observations.

Patchy supramolecules as versatile tools to probe hydrophobicity in nanoglobular systems.

We describe precise supramolecules that enable the evaluation of the effective hydrophobicity of amphiphilic or "patchy" nanoglobular systems. These supramolecules exhibit the lower critical solution temperature phenomenon, which provides a quantitative measure of their effective hydrophobicity. Specifically, two isomeric 8-aryl-2'-deoxyguanosine derivatives with a transposed pair of methylene groups self-assemble into hexadecameric nanoglobular supramolecular G-quadruplexes (SGQs) that show large differences in their transition temperatures as determined by turbidity and differential scanning calorimetry studies. Molecular modeling studies suggested that differential clustering of the hydrophobic patches on the surface is responsible for the striking differences between the two isomeric supramolecules.

Movement and behavioral states of common carp (Cyprinus carpio) in response to a behavioral deterrent in a navigational lock.

BACKGROUND: Freshwater ecosystems are some of the most affected by biological invasions due, in part, to the introduction of invasive carp worldwide. Where carp have become established, management programs often seek to limit further range expansion into new areas by reducing their movement through interconnected rivers and waterways. Lock and dams are important locations for non-physical deterrents, such as carbon dioxide (CO2), to reduce unwanted fish passage without disrupting human use. The purpose of this study was to evaluate the behavioral responses of common carp (Cyprinus carpio) to non-physical deterrents within a navigation structure on the Fox River, Wisconsin. Acoustic telemetry combined with hidden Markov models (HMMs) was used to analyze variation in carp responses to treatments. Outcomes may inform CO2 effectiveness at preventing invasive carp movement through movement pinch-points. METHODS: Carbon dioxide (CO2) was recently registered as a pesticide in the United States for use as a deterrent to invasive carp movement. As a part of a multi-component study to test a large-scale CO2 delivery system within a navigation lock, we characterized the influence of elevated CO2 and forced water circulation in the lock chamber on carp movements and behavior. Through time-to-event analyses, we described the responses of acoustic-tagged carp to experimental treatments including (1) CO2 injection in water with forced water circulation, (2) forced water circulation without CO2 and (3) no forced water circulation or CO2. We then used hidden Markov models (HMMs) to define fine-scale carp movement and evaluate the relationships between carp behavioral states and CO2 concentration, forced water circulation, and temperature. RESULTS: Forced water circulation with and without CO2 injection were effective at expelling carp from the lock chamber relative to null treatments where no stimulus was applied. A portion of carp exposed to forced water circulation with CO2 transitioned from an exploratory to an encamped behavioral state with shorter step-lengths and a unimodal distribution in turning angles, resulting in some carp remaining in the lock chamber. Whereas carp exposed to forced water circulation only remained primarily in an exploratory behavioral state, resulting in all carp exiting the lock chamber. CONCLUSION: Our findings illustrate the potential of forced water circulation, alone, as a non-physical deterrent and the efficacy of CO2 injection with forced water circulation in expelling carp from a navigation lock. Results demonstrate how acoustic telemetry and HMMs in an experimental context can describe fish behavior and inform management strategies.

Allosteric SHP2 inhibition increases apoptotic dependency on BCL2 and synergizes with venetoclax in FLT3- and KIT-mutant AML.

Mutations in the receptor tyrosine kinases (RTKs) FLT3 and KIT are frequent and associated with poor outcomes in acute myeloid leukemia (AML). Although selective FLT3 inhibitors (FLT3i) are clinically effective, remissions are short-lived due to secondary resistance characterized by acquired mutations constitutively activating the RAS/MAPK pathway. Hereby, we report the pre-clinical efficacy of co-targeting SHP2, a critical node in MAPK signaling, and BCL2 in RTK-driven AML. The allosteric SHP2 inhibitor RMC-4550 suppresses proliferation of AML cell lines with FLT3 and KIT mutations, including cell lines with acquired resistance to FLT3i. We demonstrate that pharmacologic SHP2 inhibition unveils an Achilles' heel of RTK-driven AML, increasing apoptotic dependency on BCL2 via MAPK-dependent mechanisms, including upregulation of BMF and downregulation of MCL1. Consequently, RMC-4550 and venetoclax are synergistically lethal in AML cell lines and in clinically relevant xenograft models. Our results provide mechanistic rationale and pre-clinical evidence for co-targeting SHP2 and BCL2 in RTK-driven AML.

Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles.

We have developed a pipeline to express, purify, and characterize HIV envelope protein (Env) gp145 from Chinese hamster ovary cells, to accelerate the production of a promising vaccine candidate. First in shake flasks, then in bioreactors, we optimized the growth conditions. By adjusting the pH to 6.8, we increased expression levels to 101 mg/L in a 50 L bioreactor, nearly twice the previously reported titer value. A battery of analytical methods was developed in accordance with current good manufacturing practices to ensure a quality biopharmaceutical. Imaged capillary isoelectric focusing verified proper glycosylation of gp145; dynamic light scattering confirmed the trimeric arrangement; and bio-layer interferometry and circular dichroism analysis demonstrated native-like properties (i.e., antibody binding and secondary structure). MALDI-TOF mass spectrometry was used as a multi-attribute platform for accurate mass determination, glycans analysis, and protein identification. Our robust analysis demonstrates that our gp145 product is very similar to a reference standard and emphasizes the importance of accurate characterization of a highly heterogeneous immunogen for the development of an effective vaccine. Finally, we present a novel guanosine microparticle with gp145 encapsulated and displayed on its surface. The unique properties of our gp145 microparticle make it amenable to use in future preclinical and clinical trials.

An aquatic host-guest complex between a supramolecular G-quadruplex and the anticancer drug doxorubicin.

We describe the synthesis of a fluorescent deoxyguanosine derivative that co-assembles (in water) with an unlabeled analogue into a heteromeric supramolecular G-quadruplex, which forms a host-guest complex with doxorubicin as evidenced by FRET experiments.

Aquatic self-assembly of sixteen subunits into a 39-kDa dendrimer.

We have developed the 8-(m-acetylphenyl)-2'-deoxyguanosine (mAG) scaffold for the self-assembly of supramolecules in water and for the synthesis of self-assembled dendrimers (SADs) in organic media. Previously, reported mAG assemblies showed promising characteristics for the construction of SADs. Yet, none of these SADs had large enough dendrons to reach a fractal geometry characteristic of high-generation dendrimers. Here we present the synthesis as well as the molecular and supramolecular characterization of a fourth-generation hydrophilic self-assembled hexadecameric dendrimer [mAGD(4)(OH)(16)](16)·3KI (3(16)) with a size and shape akin to those of globular proteins. The diameter of 3(16) (5.0 nm) was measured by pulsed field gradient NMR and dynamic light scattering experiments, which enabled the construction of a computer-generated molecular model. This SAD represents an attractive platform for biomedical applications due to its water solubility, discreteness, well-defined structure, thermal stability (T(m) = 68 °C), and functional core.

Surface Proteomics Reveals CD72 as a Target for In Vitro-Evolved Nanobody-Based CAR-T Cells in KMT2A/MLL1-Rearranged B-ALL.

Alternative strategies are needed for patients with B-cell malignancy relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-cell acute lymphoblastic leukemia (B-ALL), which we further found to be expressed in other B-cell malignancies. Using a recently described, fully in vitro system, we selected synthetic CD72-specific nanobodies, incorporated them into chimeric antigen receptors (CAR), and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of the role of CD72 in inhibiting B-cell receptor signaling, we found that SHIP1 inhibition increased CD72 surface density. We establish that CD72-nanobody CAR-T cells are a promising therapy for MLLr B-ALL. SIGNIFICANCE: Patients with MLLr B-ALL have poor prognoses despite recent immunotherapy advances. Here, surface proteomics identifies CD72 as being enriched on MLLr B-ALL but also widely expressed across B-cell cancers. We show that a recently described, fully in vitro nanobody platform generates binders highly active in CAR-T cells and demonstrate its broad applicability for immunotherapy development.This article is highlighted in the In This Issue feature, p. 1861.

Self-assembled cation transporters made from lipophilic 8-phenyl-2'-deoxyguanosine derivatives.

We have previously reported that 8-phenyl-2'-deoxyguanosine derivatives (8PhGs) are able to extract metal cations from an aqueous phase into an organic phase. Herein we report on the ability of 8PhGs to transport metal cations across a bulk lipophilic liquid membrane. The experiments were performed using lithium, sodium, potassium, and strontium picrate salts with the parent lipophilic Gi, two isomeric 8PhG derivatives, cis-dicyclohexano-18-crown-6 (CD18C6) and [2•2•2] cryptand as reference compounds. The relative amounts of the picrate salts were measured by UV spectroscopy in both, the source phase and the receiving phase over a period of 24 h. The results show that the transport efficiency of the self-assembled ionophores formed by 8PhGs is either similar or superior to that of CD18C6, and in all but one case higher than the parent compound Gi. The varying efficiencies between the derivatives can be attributed to the stability (kinetic and thermodynamic) and the different molecularities of the supramolecules formed by these 8PhGs. The ease of the synthesis of 8PhGs, their anion independent assembly and the fact that the transport efficiency can be modulated as a function of the structure of the 8PhGs bode well for the use of such compounds in the development of novel antimicrobial agents and cation sensing devices.

Immunotherapeutic Blockade of CD47 Inhibitory Signaling Enhances Innate and Adaptive Immune Responses to Viral Infection.

Paradoxically, early host responses to infection include the upregulation of the antiphagocytic molecule, CD47. This suggests that CD47 blockade could enhance antigen presentation and subsequent immune responses. Indeed, mice treated with anti-CD47 monoclonal antibody following lymphocytic choriomeningitis virus infections show increased activation of both macrophages and dendritic cells (DCs), enhancement of the kinetics and potency of CD8+ T cell responses, and significantly improved virus control. Treatment efficacy is critically dependent on both APCs and CD8+ T cells. In preliminary results from one of two cohorts of humanized mice infected with HIV-1 for 6 weeks, CD47 blockade reduces plasma p24 levels and restores CD4+ T cell counts. The results indicate that CD47 blockade not only enhances the function of innate immune cells but also links to adaptive immune responses through improved APC function. As such, immunotherapy by CD47 blockade may have broad applicability to treat a wide range of infectious diseases.