PET/CT targeted tissue sampling reveals virus specific dIgA can alter the distribution and localization of HIV after rectal exposure.

Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection.

Combined PET and whole-tissue imaging of lymphatic-targeting vaccines in non-human primates.

Antigen accumulation in lymph nodes (LNs) is critical for vaccine efficacy, but understanding of vaccine biodistribution in humans or large animals remains limited. Using the rhesus macaque model, we employed a combination of positron emission tomography (PET) and fluorescence imaging to characterize the whole-animal to tissue-level biodistribution of a subunit vaccine comprised of an HIV envelope trimer protein nanoparticle (trimer-NP) and lipid-conjugated CpG adjuvant (amph-CpG). Following immunization in the thigh, PET imaging revealed vaccine uptake primarily in inguinal and iliac LNs, reaching distances up to 17 cm away from the injection site. Within LNs, trimer-NPs exhibited striking accumulation on the periphery of follicular dendritic cell (FDC) networks in B cell follicles. Comparative imaging of soluble Env trimers (not presented on nanoparticles) in naïve or previously-immunized animals revealed diffuse deposition of trimer antigens in LNs following primary immunization, but concentration on FDCs in pre-immunized animals with high levels of trimer-specific IgG. These data demonstrate the capacity of nanoparticle or "albumin hitchhiking" technologies to concentrate vaccines in genitourinary tract-draining LNs, which may be valuable for promoting mucosal immunity.

In Vivo Evaluation of Site-Specifically PEGylated Chemically Self-Assembled Protein Nanostructures.

Chemically self-assembled nanorings (CSANs) are made of dihydrofolate reductase (DHFR) fusion proteins and have been successfully used in vitro for cellular cargo delivery and cell surface engineering applications. However, CSANs have yet to be evaluated for their in vivo stability, circulation, and tissue distribution. In an effort to evaluate CSANs in vivo, we engineered a site-specifically PEGylated epidermal growth factor receptor (EGFR) targeting DHFR molecules, characterized their self-assembly into CSANs with bivalent methotrexates (bis-MTX), visualized their in vivo tissue localization by microPET/CT imaging, and determined their ex vivo organ biodistribution by tissue-based gamma counting. A dimeric DHFR (DHFR(2)) molecule fused with a C-terminal EGFR targeting peptide (LARLLT) was engineered to incorporate a site-specific ketone functionality using unnatural amino acid mutagenesis. Aminooxy-PEG, of differing chain lengths, was successfully conjugated to the protein using oxime chemistry. These proteins were self-assembled into CSANs with bis-MTX DHFR dimerizers and characterized by size exclusion chromatography and dynamic light scattering. In vitro binding studies were performed with fluorescent CSANs assembled using bis-MTX-FITC, while in vivo microPET/CT imaging was performed with radiolabeled CSANs assembled using bis-MTX-DOTA[(64)Cu]. PEGylation reduced the uptake of anti-EGFR CSANs by mouse macrophages (RAW 264.7) up to 40% without altering the CSAN's binding affinity toward U-87 MG glioblastoma cells in vitro. A significant time dependent tumor accumulation of (64)Cu labeled anti-EGFR-CSANs was observed by microPET/CT imaging and biodistribution studies in mice bearing U-87 MG xenografts. PEGylated CSANs demonstrated a reduced uptake by the liver, kidneys, and spleen resulting in high contrast tumor imaging within an hour of intravenous injection (9.6% ID/g), and continued to increase up to 24 h (11.7% ID/g) while the background signal diminished. CSANs displayed an in vivo profile between those of rapidly clearing small molecules and slow clearing antibodies. Thus, CSANs offer a modular, programmable, and stable protein based platform that can be used for in vivo drug delivery and imaging applications.

Simultaneous dual protein labeling using a triorthogonal reagent.

Construction of heterofunctional proteins is a rapidly emerging area of biotherapeutics. Combining a protein with other moieties, such as a targeting element, a toxic protein or small molecule, and a fluorophore or polyethylene glycol (PEG) group, can improve the specificity, functionality, potency, and pharmacokinetic profile of a protein. Protein farnesyl transferase (PFTase) is able to site-specifically and quantitatively prenylate proteins containing a C-terminal CaaX-box amino acid sequence with various modified isoprenoids. Here, we describe the design, synthesis, and application of a triorthogonal reagent, 1, that can be used to site-specifically incorporate an alkyne and aldehyde group simultaneously into a protein. To illustrate the capabilities of this approach, a protein was enzymatically modified with compound 1 followed by oxime ligation and click reaction to simultaneously incorporate an azido-tetramethylrhodamine (TAMRA) fluorophore and an aminooxy-PEG moiety. This was performed with both a model protein [green fluorescent protein (GFP)] as well as a therapeutically useful protein [ciliary neurotrophic factor (CNTF)]. Next, a protein was enzymatically modified with compound 1 followed by coupling to an azido-bis-methotrexate dimerizer and aminooxy-TAMRA. Incubation of that construct with a dihydrofolate reductase (DHFR)-DHFR-anti-CD3 fusion protein resulted in the self-assembly of nanoring structures that were endocytosed into T-leukemia cells and visualized therein. These results highlight how complex multifunctional protein assemblies can be prepared using this facile triorthogonal approach.

Targeted delivery of antisense oligonucleotides by chemically self-assembled nanostructures.

Synthetic nucleic acids have shown great potential in the treatment of various diseases. Nevertheless, the selective delivery to a target tissue has proved challenging. The coupling of nucleic acids to targeting peptides, proteins, and antibodies has been explored as an approach for their selective tissue delivery. Nevertheless, the preparation of covalently coupled peptides and proteins that can also undergo intracellular release as well as deliver more than one copy of the nucleic acid has proved challenging. Recently, we have developed a novel method for the rapid noncovalent conjugation of nucleic acids to targeting single chain antibodies (scFv) using chemically self-assembled nanostructures (CSANs). CSANs have been prepared by the self-assembly of two dihydrofolate reductase molecules (DHFR(2)) and a targeting scFv in the presence of bis-methotrexate (bis-MTX). The valency of the nanorings can be tuned from one to eight subunits, depending on the length and composition of the linker between the dihydrofolate reductase molecules. To explore their potential for the therapeutic delivery of nucleic acids as well as the ability to expand the capabilities of CSANs by incorporating smaller cyclic targeting peptides, we prepared DHFR(2) proteins fused through a flexible peptide linker to cyclic-RGD, which targets αvß3 integrins, and a bis-MTX chemical dimerizer linked to an antisense oligonucleotide (bis-MTX-ASO) that has been shown to silence expression of eukaryotic translation initiation factor 4E (eIF4E). Monomeric and multimeric cRGD-CSANs were then prepared with bis-MTX-ASO and shown to undergo endocytosis in the breast cancer cell line, MDA-MB-231, which overexpresses αvß3. The bis-MTX-ASO was shown to undergo endosomal escape resulting in the knock down of eIF4E with at least the same efficiency as ASO delivered by oligofectamine. The modularity, flexibility, and common method of conjugation may prove to be a useful general approach for the targeted delivery of ASOs, as well as other nucleic acids to cells.

Chemically self-assembled antibody nanostructures as potential drug carriers.

Chemically self-assembled antibody nanorings (CSANs) displaying multiple copies of single-chain variable fragments can be prepared from dihydrofolate reductase (DHFR) fusion proteins and bis-methotrexate (bisMTX). We have designed and synthesized a bisMTX chemical dimerizer (bisMTX-NH(2)) that contains a third linker arm that can be conjugated to fluorophores, radiolabels, and drugs. Monovalent, divalent, and higher-order AntiCD3 CSANs were assembled with a fluorescein isothiocyanate (FITC)-labeled bis-methotrexate ligand (bisMTX-FITC) and found to undergo rapid internalization and trafficking by HPB-MLT, a CD3+ T-leukemia cell line, to the early and late endosome and lysosome. Because the fluorescence of bisMTX-FITC when incorporated into CSANs was found to be significantly greater than that of the free ligand, the stability of the endocytosed AntiCD3 CSANs could be monitored. The internalized CSANs were found to be stable for several hours, while treatment with the nontoxic DHFR inhibitor trimethoprim resulted in a rapid loss (>80%) of cellular fluorescence within minutes, consistent with efficient intracellular disassembly of the nanorings. Over longer time periods (24 h), cellular fluorescence decreased by 75-90%, regardless of whether cells had been treated with DMSO or trimethoprim. Although bisMTX is a potent inhibitor of DHFR, it was found to be nontoxic (GI(50) > 20 µM) to HPB-MLT cells. In contrast, AntiCD3 CSANs prepared with bisMTX were found to be at least 13-fold more cytotoxic (GI(50) = 0.5-1.5 µM) than bisMTX at 72 h. Consistent with our findings from CSAN stability studies, no increase in cytotoxicity was observed upon treatment with trimethoprim. Taken together, our results suggest that cell receptor targeting CSANs prepared with trifunctional bisMTX could be used as potential tissue selective drug carriers.

Programmable self-assembly of antibody-oligonucleotide conjugates as small molecule and protein carriers.

Dihydrofolate reductase single-chain variable fragment (scFv) fusion proteins can be used for the targeted cellular delivery of oligonucleotides, conjugated small molecules, and proteins via labeling of oligonucleotides by bis-methotrexate.

A novel nucleoside analog, 1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR), exhibits efficacy against a broad range of flaviviruses in vitro.

Antiviral therapies are urgently needed to control emerging flaviviruses such as dengue, West Nile, and yellow fever. Ribavirin (RBV) has shown activity against flaviviruses in cultured cells, but efficacy in animal models has generally been poor. In a preliminary screen of novel, synthetic 1-beta-d-ribofuranosyl-azole analogs, two compounds, 1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR) and 1-beta-d-ribofuranosyl-4-ethynyl-[1,3]imidazole (IM18), significantly reduced the replication of dengue virus serotype 2 (DENV-2) in cultured Vero cells. In the current study we demonstrated that the effective concentration 50 (EC(50)) of ETAR for DENV-2 is substantially lower than both IM18 and RBV. Moreover, ETAR reduced the replication of five additional flaviviruses, including DENV serotypes 1, 3 and 4, Langat virus and Modoc virus, > or =1000-fold relative to untreated controls. Addition of exogenous guanosine to DENV-2 infected cells negated the antiviral effects of both RBV and ETAR, indicating that GTP depletion is a major mechanism of action for both drugs. ETAR represents a promising drug candidate for the treatment of flavivirus infections.

Synthesis of 1-beta-D-ribofuranosyl-3-ethynyl-[1,2,4]triazole and its in vitro and in vivo efficacy against Hantavirus.

There are no FDA approved drugs for the treatment of hemorrhagic fever with renal syndrome (HFRS), a serious human illnesses caused by hantaviruses. Clinical studies using ribavirin (RBV) to treat HFRS patients suggest that it provides an improved prognosis when given early in the course of disease. Given the unique antiviral activity of RBV and the lack of other lead scaffolds, we prepared a diverse series of 3-substituted 1,2,4-triazole-beta-ribosides and identified one with antiviral activity, 1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR). ETAR showed an EC(50) value of 10 and 4.4 microM for Hantaan virus (HTNV) and Andes virus, respectively. ETAR had weak activity against Crimean Congo hemorrhagic fever virus, but had no activity against Rift Valley fever virus. Intraperitoneally delivered ETAR offered protection to suckling mice challenged with HTNV with a approximately 25% survival at 12.5 and 25mg/kg ETAR, and a MTD of 17.1+/-0.7 days. ETAR was phosphorylated in Vero E6 cells to its 5'-triphosphate and reduced cellular GTP levels. In contrast to RBV, ETAR did not increase mutation frequency of the HTNV genome, which suggests it has a different mechanism of action than RBV. ETAR is an exciting and promising lead compound that will be elaborated in further synthetic investigations as a framework for the rational design of new antivirals for treatment of HFRS.

Structural effects on the phosphorylation of 3-substituted 1-beta-D-ribofuranosyl-1,2,4-triazoles by human adenosine kinase.

The conversion of ribavirin to the monophosphate by adenosine kinase is the rate-limiting step in activation of this broad spectrum antiviral drug. Variation of the 3-substituents in a series of bioisosteric and homologated 1-beta-D-ribofuranosyl-1,2,4-triazoles has marked effects on activity with the human adenosine kinase, and analysis of computational descriptors and binding models offers insight for the design of novel substrates.