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The unprecedented public health and economic impact of the COVID-19 pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been met with an equally unprecedented scientific response. Much of this response has focused, appropriately, on the mechanisms of SARS-CoV-2 entry into host cells, and in particular the binding of the spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2), and subsequent membrane fusion. This Review provides the structural and cellular foundations for understanding the multistep SARS-CoV-2 entry process, including S protein synthesis, S protein structure, conformational transitions necessary for association of the S protein with ACE2, engagement of the receptor-binding domain of the S protein with ACE2, proteolytic activation of the S protein, endocytosis and membrane fusion. We define the roles of furin-like proteases, transmembrane protease, serine 2 (TMPRSS2) and cathepsin L in these processes, and delineate the features of ACE2 orthologues in reservoir animal species and S protein adaptations that facilitate efficient human transmission. We also examine the utility of vaccines, antibodies and other potential therapeutics targeting SARS-CoV-2 entry mechanisms. Finally, we present key outstanding questions associated with this critical process., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

We report a mass without neurologic findings and moderate pain in a 33-year-old male's wrist at the 3 rd zone of the ulnar nerve due to schwannoma that was excised successfully, and the patient was free of symptoms. We further discuss the prevalence and management of this lesion.

Background: The novel coronavirus SARS-CoV2 that causes COVID-19 has resulted in the death of more than 2.5 million people, but no cure exists. Although passive immunization with COVID-19 convalescent plasma (CCP) provides a safe and viable therapeutic option, the selection of optimal units for therapy in a timely fashion remains a barrier., Study Design and Methods: Since virus neutralization is a necessary characteristic of plasma that can benefit recipients, the neutralizing titers of plasma samples were measured using a retroviral-pseudotype assay. Binding antibody titers to the spike (S) protein were also determined by a clinically available serological assay (Ortho-Vitros total IG), and an in-house ELISA. The results of these assays were compared to a measurement of antibodies directed to the receptor binding domain (RBD) of the SARS-CoV2 S protein (Promega Lumit Dx)., Results: All measures of antibodies were highly variable, but correlated, to different degrees, with each other. However, the anti-RBD antibodies correlated with viral neutralizing titers to a greater extent than the other antibody assays., Discussion: Our observations support the use of an anti-RBD assay such as the Lumit Dx assay, as an optimal predictor of the neutralization capability of CCP., Competing Interests: Lumit Dx assay reagents/kits were provided by Promega Corporation (Madison, WI). No members of the Promega Organization were involved in the study design, conduct of the experiments described, the interpretation of the data/results, or the conclusions drawn by the authors. The providing of these resources does not alter our adherence to PLOS ONE policies on sharing data and materials.

Three variable 2 (V2) loops of HIV-1 envelope glycoprotein (Env) trimer converge at the Env apex to form the epitope of an important classes of HIV-1 broadly neutralizing antibodies (bNAbs). These V2-glycan/apex antibodies are exceptionally potent but less broad (∼60 to 75%) than many other bNAbs. Their CDRH3 regions are typically long, acidic, and tyrosine sulfated. Tyrosine sulfation complicates efforts to improve these antibodies through techniques such as phage or yeast display. To improve the breadth of CAP256-VRC26.25 (VRC26.25), a very potent apex antibody, we adapted and extended a B cell display approach. Specifically, we used CRISPR/Cas12a to introduce VRC26.25 heavy- and light-chain genes into their respective loci in a B cell line, ensuring that each cell expresses a single VRC26.25 variant. We then diversified these loci through activation-induced cytidine deaminase-mediated hypermutation and homology-directed repair using randomized CDRH3 sequences as templates. Iterative sorting with soluble Env trimers and further randomization selected VRC26.25 variants with successively improving affinities. Three mutations in the CDRH3 region largely accounted for this improved affinity, and VRC26.25 modified with these mutations exhibited greater breadth and potency than the original antibody. Our data describe a broader and more-potent form of VRC26.25 as well as an approach useful for improving the breadth and potency of antibodies with functionally important posttranslational modifications., Competing Interests: The authors declare no competing interest.

Vectors developed from adeno-associated virus (AAV) are powerful tools for in vivo transgene delivery in both humans and animal models, and several AAV-delivered gene therapies are currently approved for clinical use. However, AAV-mediated gene therapy still faces several challenges, including limited vector packaging capacity and the need for a safe, effective method for controlling transgene expression during and after delivery. Riboswitches, RNA elements which control gene expression in response to ligand binding, are attractive candidates for regulating expression of AAV-delivered transgene therapeutics because of their small genomic footprints and non-immunogenicity compared to protein-based expression control systems. In addition, the ligand-sensing aptamer domains of many riboswitches can be exchanged in a modular fashion to allow regulation by a variety of small molecules, proteins, and oligonucleotides. Riboswitches have been used to regulate AAV-delivered transgene therapeutics in animal models, and recently developed screening and selection methods allow rapid isolation of riboswitches with novel ligands and improved performance in mammalian cells. This review discusses the advantages of riboswitches in the context of AAV-delivered gene therapy, the subsets of riboswitch mechanisms which have been shown to function in human cells and animal models, recent progress in riboswitch isolation and optimization, and several examples of AAV-delivered therapeutic systems which might be improved by riboswitch regulation.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines. IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines., (Copyright © 2021 Guo et al.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: M.F., H.M. and B.D.Q had filed a patent for the application of ACE2-Fc variants as a SARS-CoV-2 treatment. M.R.G., C.C.B., M.D.A., and M.F. are all cofounders of, and have an equity interest in Emmune Inc., a biotech company that specializes in the development of antibody-like antiviral therapies.

Background: The novel coronavirus, SARS-CoV2 that causes COVID-19 has resulted in the death of more than 2.31 million people within the last year and yet no cure exists. Whereas passive immunization with COVID-19 convalescent plasma (CCP) provides a safe and viable option, selection of optimal units for therapy and lack of clear therapeutic benefit from transfusion remain as barriers to the use of CCP., Study Design and Methods: To identify plasma that is expected to benefit recipients, we measured anti-SARS-CoV2 antibody levels using clinically available serological assays and correlated with the neutralizing activity of CCP from donors. Neutralizing titer of plasma samples was measured by assaying infectivity of SARS-CoV-2 spike protein pseudotyped retrovirus particles in the presence of dilutions of plasma samples. We also used this assay to identify evidence of passive transfusion of neutralizing activity in CCP recipients., Results: Viral neutralization and anti-spike protein antibodies in 109 samples from 87 plasma donors were highly varied but modestly correlated with each other. Recipients who died of COVID-19 were found to have been transfused with units with lower anti-spike antibody levels and neutralizing activity. Passive transfer of neutralization activity was documented in 62% of antibody naive plasma recipients., Conclusions: Since viral neutralization is the goal of CCP transfusion, our observations not only support the use of anti-spike SARS-CoV2 serology tests to identify beneficial CCP units, but also support the therapeutic value of convalescent plasma with high titers of anti-spike antibodies.

CRISPR effector proteins introduce double-stranded breaks into the mammalian genome, facilitating gene editing by non-homologous end-joining or homology-directed repair. Unlike the more commonly studied Cas9, the CRISPR effector protein Cas12a/Cpf1 recognizes a T-rich protospacer adjacent motif (PAM) and can process its own CRISPR RNA (crRNA) array, simplifying the use of multiple guide RNAs. We observed that the Cas12a ortholog of Lachnospiraceae bacterium MA2020 (Lb2Cas12a) edited mammalian genes with efficiencies comparable to those of AsCas12a and LbCas12a. Compared to these well-characterized Cas12a orthologs, Lb2Cas12a is smaller and recognizes a narrow set of PAM TTTV. We introduced two mutations into Lb2Cas12a, Q571K and C1003Y, that increased its cleavage efficiency for a range of target sequences beyond those of the commonly used Cas12a orthologs AsCas12a and LbCas12a. In addition to the canonical TTTV PAM, this variant, Lb2-KY, also efficiently cleaved target regions with CTTN PAMs. Finally, we demonstrated that Lb2-KY ribonucleoprotein (RNP) complexes edited two hemoglobin target regions useful for correcting common forms of sickle-cell anemia more efficiently than commercial AsCas12a RNP complexes. Thus, Lb2-KY has distinctive properties useful for modifying a range of clinically relevant targets in the human genome., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 is also a key negative regulator of the renin-angiotensin system that modulates vascular functions. We report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of ~60 pM for the spike protein of SARS‑CoV‑2 (compared with 77 nM for monomeric ACE2 and 12-22 nM for dimeric ACE2 constructs), and its peptidase activity and the ability to block activation of angiotensin II receptor type 1 in the renin-angiotensin system are preserved. Moreover, the engineered ACE2 potently inhibits SARS‑CoV‑2 infection in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus which binds its cellular receptor angiotensin-converting enzyme 2 (ACE2) and enters hosts cells through the action of its spike (S) glycoprotein displayed on the surface of the virion. Compared to the reference strain of SARS-CoV-2, the majority of currently circulating isolates possess an S protein variant characterized by an aspartic acid-to-glycine substitution at amino acid position 614 (D614G). Residue 614 lies outside the receptor binding domain (RBD) and the mutation does not alter the affinity of monomeric S protein for ACE2. However, S(G614), compared to S(D614), mediates more efficient ACE2-mediated transduction of cells by S-pseudotyped vectors and more efficient infection of cells and animals by live SARS-CoV-2. This review summarizes and synthesizes the epidemiological and functional observations of the D614G spike mutation, with focus on the biochemical and cell-biological impact of this mutation and its consequences for S protein function. We further discuss the significance of these recent findings in the context of the current global pandemic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated or when it is introduced into the SARS-CoV-1 S protein. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19., Competing Interests: The authors have declared that no competing interests exist.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 has triggered an ongoing global pandemic whereby infection may result in a lethal severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, millions of confirmed cases and hundreds of thousands of deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. The purported development of a vaccine could require at least 1-4 years, while the typical timeline from hit finding to drug registration of an antiviral is >10 years. Thus, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we developed and initiated a high-throughput cell-based screen that incorporates the essential viral papain-like protease (PLpro) and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or US Food and Drug Administration (FDA)-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Here, we report the identification of four clinically relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral PLpro.

Interferon-induced transmembrane protein 3 (IFITM3) has previously been identified as an endosomal protein that blocks viral infection 1-3 . Here we studied clinical cohorts of patients with B cell leukaemia and lymphoma, and identified IFITM3 as a strong predictor of poor outcome. In normal resting B cells, IFITM3 was minimally expressed and mainly localized in endosomes. However, engagement of the B cell receptor (BCR) induced both expression of IFITM3 and phosphorylation of this protein at Tyr20, which resulted in the accumulation of IFITM3 at the cell surface. In B cell leukaemia, oncogenic kinases phosphorylate IFITM3 at Tyr20, which causes constitutive localization of this protein at the plasma membrane. In a mouse model, Ifitm3 -/- naive B cells developed in normal numbers; however, the formation of germinal centres and the production of antigen-specific antibodies were compromised. Oncogenes that induce the development of leukaemia and lymphoma did not transform Ifitm3 -/- B cells. Conversely, the phosphomimetic IFITM3(Y20E) mutant induced oncogenic PI3K signalling and initiated the transformation of premalignant B cells. Mechanistic experiments revealed that IFITM3 functions as a PIP3 scaffold and central amplifier of PI3K signalling. The amplification of PI3K signals depends on IFITM3 using two lysine residues (Lys83 and Lys104) in its conserved intracellular loop as a scaffold for the accumulation of PIP3. In Ifitm3 -/- B cells, lipid rafts were depleted of PIP3, which resulted in the defective expression of over 60 lipid-raft-associated surface receptors, and impaired BCR signalling and cellular adhesion. We conclude that the phosphorylation of IFITM3 that occurs after B cells encounter antigen induces a dynamic switch from antiviral effector functions in endosomes to a PI3K amplification loop at the cell surface. IFITM3-dependent amplification of PI3K signalling, which in part acts downstream of the BCR, is critical for the rapid expansion of B cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signalling complexes and amplify PI3K signalling for malignant transformation.

SARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (S G614 ) with the original (S D614 ). We report here pseudoviruses carrying S G614 enter ACE2-expressing cells more efficiently than those with S D614 . This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion.

The SARS-coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing the angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino acid fragment of the 1273-amino acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD expresses inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) expresses markedly more efficiently, and generates a more potent neutralizing responses as a DNA vaccine antigen, than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers such as an H. pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines., Competing Interests: Competing interests: A patent for gRBD has been filed by The Scripps Research Institute in which B.D.Q, W.L., H.M., and M.F. are listed as inventors.

Adeno-associated virus (AAV) is one of the most commonly used vectors for gene therapy, and the applications for AAV-delivered therapies are numerous. However, the current state of technology is limited by the low efficiency with which most AAV vectors transduce skeletal muscle tissue. We demonstrate that vector efficiency can be enhanced by modifying the AAV capsid with a peptide that binds a receptor highly expressed in muscle tissue. When an insulin-mimetic peptide, S519, previously characterized for its high affinity to insulin receptor (IR), was inserted into the capsid, the AAV9 transduction efficiency of IR-expressing cell lines as well as differentiated primary human muscle cells was dramatically enhanced. This vector also exhibited efficient transduction of mouse muscle in vivo , resulting in up to 18-fold enhancement over AAV9. Owing to its superior transduction efficiency in skeletal muscle, we named this vector "enhanced AAV9" (eAAV9). We also found that the modification enhanced the transduction efficiency of several other AAV serotypes. Together, these data show that AAV transduction of skeletal muscle can be improved by targeting IR. They also show the broad utility of this modular strategy and suggest that it could also be applied to next-generation vectors that have yet to be engineered., Competing Interests: C.B.J., M.F., and H.C. are listed as inventors on a provisional patent for this technology. C.C.B., M.D.A., and M.F. have a financial interest in Emmune, which has licensed the technology from The Scripps Research Institute., (© 2020 The Author(s).)

The ongoing coronavirus disease 2019 (COVID-19) pandemic has caused >20 million infections and >750,000 deaths. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, has been found closely related to the bat coronavirus strain RaTG13 (Bat-CoV RaTG13) and a recently identified pangolin coronavirus (Pangolin-CoV-2020). Here, we first investigated the ability of SARS-CoV-2 and three related coronaviruses to utilize animal orthologs of angiotensin-converting enzyme 2 (ACE2) for cell entry. We found that ACE2 orthologs of a wide range of domestic and wild mammals, including camels, cattle, horses, goats, sheep, cats, rabbits, and pangolins, were able to support cell entry of SARS-CoV-2, suggesting that these species might be able to harbor and spread this virus. In addition, the pangolin and bat coronaviruses, Pangolin-CoV-2020 and Bat-CoV RaTG13, were also found able to utilize human ACE2 and a number of animal-ACE2 orthologs for cell entry, indicating risks of spillover of these viruses into humans in the future. We then developed potently anticoronavirus ACE2-Ig proteins that are broadly effective against the four distinct coronaviruses. In particular, through truncating ACE2 at its residue 740 but not 615, introducing a D30E mutation, and adopting an antibody-like tetrameric-ACE2 configuration, we generated an ACE2-Ig variant that neutralizes SARS-CoV-2 at picomolar range. These data demonstrate that the improved ACE2-Ig variants developed in this study could potentially be developed to protect from SARS-CoV-2 and some other SARS-like viruses that might spillover into humans in the future. IMPORTANCE The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the currently uncontrolled coronavirus disease 2019 (COVID-19) pandemic. It is important to study the host range of SARS-CoV-2, because some domestic species might harbor the virus and transmit it back to humans. In addition, insight into the ability of SARS-CoV-2 and SARS-like viruses to utilize animal orthologs of the SARS-CoV-2 receptor ACE2 might provide structural insight into improving ACE2-based viral entry inhibitors. In this study, we found that ACE2 orthologs of a wide range of domestic and wild animals can support cell entry of SARS-CoV-2 and three related coronaviruses, providing insights into identifying animal hosts of these viruses. We also developed recombinant ACE2-Ig proteins that are able to potently block these viral infections, providing a promising approach to developing antiviral proteins broadly effective against these distinct coronaviruses., (Copyright © 2020 American Society for Microbiology.)

SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4 + and CD8 + T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19., Competing Interests: Declaration of Interests In accordance with the University of Pennsylvania policies and procedures and our ethical obligations as researchers, we report that D.W. and K.K. are named on patents that describe the use of nucleoside-modified mRNA as a platform to deliver therapeutic proteins. D.W. and N.P. are also named on a patent describing the use of nucleoside-modified mRNA in lipid nanoparticles as a vaccine platform. We have disclosed those interests fully to the University of Pennsylvania, and we have an approved plan for managing any potential conflicts arising from licensing of our patents in place. K.K. is an employee of BioNTech. P.J.C.L. and Y.K.T. are employees of Acuitas Therapeutics, a company involved in the development of mRNA-LNP therapeutics. Y.K.T. is named on patents that describe lipid nanoparticles for delivery of nucleic acid therapeutics including mRNA and the use of modified mRNA in lipid nanoparticles as a vaccine platform., (Published by Elsevier Inc.)

Oligonucleotide aptamers are found in prokaryotes and eukaryotes, and they can be selected from large synthetic libraries to bind protein or small-molecule ligands with high affinities and specificities. Aptamers can function as biosensors, as protein recognition elements, and as components of riboswitches allowing ligand-dependent control of gene expression. One of the best studied laboratory-selected aptamers binds the antibiotic tetracycline, but it binds with a much lower affinity to the closely related but more bioavailable antibiotic doxycycline. Here we report enrichment of doxycycline binding aptamers from a selectively randomized library of tetracycline aptamer variants over four selection rounds. Selected aptamers distinguish between doxycycline, which they bind with dissociation constants of approximately 7 nM, and tetracycline, which they bind undetectably. They thus function as orthogonal complements to the original tetracycline aptamer. Unexpectedly, doxycycline aptamers adopt a conformation distinct from that of the tetracycline aptamer and depend on constant regions originally installed as primer binding sites. We show that the fluorescence emission intensity of doxycycline increases upon aptamer binding, permitting their use as biosensors. This new class of aptamers can be used in multiple contexts where doxycycline detection, or doxycycline-mediated regulation, is necessary.

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that forms a dimer and serves as the cellular receptor for SARS-CoV-2. It is also a key negative regulator of the renin-angiotensin system (RAS), conserved in mammals, which modulates vascular functions. We report here the properties of a trimeric ACE2 variant, created by a structure-based approach, with binding affinity of ~60 pM for the spike (S) protein of SARS-CoV-2, while preserving the wildtype peptidase activity as well as the ability to block activation of angiotensin II receptor type 1 in the RAS. Moreover, the engineered ACE2 potently inhibits infection of SARS-CoV-2 in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.

Despite the wide-spread use of liposomal drug delivery systems, application of these systems for oral purposes is limited due to their large-scale formulation and storage issues. Proliposomes are one of the formulation approaches for achieving solid powders that readily form liposomes upon hydration. In this work, we investigated a dry powder formulation of a model low-soluble drug with phospholipids loaded in porous functionalized calcium carbonate microparticles. We characterized the liposome formation under conditions that mimic the different gastrointestinal stages and studied the factors that influence the dissolution rate of the model drug. The liposomes that formed upon direct contact with the simulated gastric environment had a capacity to directly encapsulate 25% of the drug in situ. The emerged liposomes allowed complete dissolution of the drug within 15 min. We identified a negative correlation between the phospholipid content and the rate of water uptake. This correlation corroborated the results obtained for the rate of dissolution and liposome encapsulation efficiency. This approach allows for the development of solid proliposomal dosage formulations, which can be scaled up with regular processes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus ) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related SARS-CoV-2, has been isolated from one horseshoe-bat species. Here we characterize the ability of S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, and RaTG13 to bind a range of ACE2 orthologs. We observed that the SARS-CoV-2 RBD bound human, pangolin, and horseshoe bat ( R. macrotis) ACE2 more efficiently than the SARS-CoV-1 or RaTG13 RBD. Only the RaTG13 RBD bound rodent ACE2 orthologs efficiently. Five mutations drawn from ACE2 orthologs of nine Rhinolophus species enhanced human ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 by an immunoadhesin form of human ACE2 (ACE2-Fc). Two of these mutations impaired neutralization of SARS-CoV-1. An ACE2-Fc variant bearing all five mutations neutralized SARS-CoV-2 five-fold more efficiently than human ACE2-Fc. These data narrow the potential SARS-CoV-2 reservoir, suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of ACE2-Fc.

SARS coronavirus 2 (SARS-CoV-2) isolates encoding a D614G mutation in the viral spike (S) protein predominate over time in locales where it is found, implying that this change enhances viral transmission. We therefore compared the functional properties of the S proteins with aspartic acid (S D614 ) and glycine (S G614 ) at residue 614. We observed that retroviruses pseudotyped with S G614 infected ACE2-expressing cells markedly more efficiently than those with S D614 . This greater infectivity was correlated with less S1 shedding and greater incorporation of the S protein into the pseudovirion. Similar results were obtained using the virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, S G614 did not bind ACE2 more efficiently than S D614 , and the pseudoviruses containing these S proteins were neutralized with comparable efficiencies by convalescent plasma. These results show S G614 is more stable than S D614 , consistent with epidemiological data suggesting that viruses with S G614 transmit more efficiently.

Widespread use of gene therapy technologies is limited in part by the lack of small genetic switches with wide dynamic ranges that control transgene expression without the requirement of additional protein components 1-5 . In this study, we engineered a class of type III hammerhead ribozymes to develop RNA switches that are highly efficient at cis-cleaving mammalian mRNAs and showed that they can be tightly regulated by a steric-blocking antisense oligonucleotide. Our variant ribozymes enabled in vivo regulation of adeno-associated virus (AAV)-delivered transgenes, allowing dose-dependent and up to 223-fold regulation of protein expression over at least 43 weeks. To test the potential of these reversible on-switches in gene therapy for anemia of chronic kidney disease 6 , we demonstrated regulated expression of physiological levels of erythropoietin with a well-tolerated dose of the inducer oligonucleotide. These small, modular and efficient RNA switches may improve the safety and efficacy of gene therapies and broaden their use.

Broadly neutralizing antibodies (bNAbs) can prevent and control an HIV-1 infection, but their breadth is invariably too limited for use as monotherapy. To address this problem, bi- and trispecific antibody-like constructs have been developed. These engineered antibodies typically have greater breadth than the native bNAbs from which they were derived, but they are not more potent because they do not, in most cases, simultaneously engage more than a single epitope of the HIV-1 envelope glycoprotein (Env). Here, we describe a new class of bispecific antibodies targeting the V2-glycan (apex) and V3-glycan regions of the HIV-1 envelope glycoprotein (Env). Specifically, bispecific antibodies with a single-chain (scFv) form of the CAP256.VRC26.25 V2-glycan (apex) antibody on one antibody arm and a full V3-glycan Fab on the other arm neutralizes more HIV-1 isolates than the bNAbs from which they were derived. Moreover, these bispecific antibodies are markedly more potent than their parental bNAbs, likely because they simultaneously engage both the apex and V3-glycan epitopes of Env. Our data show that simultaneous engagement of two critical epitopes of a single Env trimer can markedly increase the potency of a bispecific antibody. IMPORTANCE Broadly neutralizing antibodies (bNAbs) can prevent a new HIV-1 infection and can at least temporarily suppress an established infection. However, antibody-resistant viruses rapidly emerge in infected persons treated with any single bNAb. Several bispecific antibodies have been developed to increase the breadth of these antibodies, but typically only one arm of these bispecific constructs binds the HIV-1 envelope glycoprotein trimer (Env). Here, we develop and characterize bispecific constructs based on well-characterized V2-glycan and V3-glycan bNAbs and show that at least one member of this class is more potent than its parental antibodies, indicating that they can simultaneously bind both of these epitopes of a single Env trimer. These data show that bispecific antibody-like proteins can achieve greater neutralization potency than the bNAbs from which they were derived., (Copyright © 2020 Davis-Gardner et al.)

Malignant transformation of glomus tumor is exceedingly rare and most common reported in lower extremity and abdominal viscera. We could find only 6 previous case report of malignant glomus tumor in the hand. Although large and deeply located glomus tumors are considered to be malignant, evidence has shown that most of these cases were clinically benign. These lesions are better considered as glomus tumor of uncertain malignant potential. Due to rarity of malignant glomus tumor, decision for operative treatment must be based on few case reports. In this article we review the literature for malignant glomus tumor of hand and their management, also we report a 49 year-old man with glomus tumor of uncertain malignant potential in thumb who was treated by wide amputation.

A number of simian and simian human immunodeficiency viruses (SIV and SHIV, respectively) have been used to assess the efficacy of HIV-1 vaccine strategies. Among these, SIVmac239 is considered among the most stringent because, unlike SHIV models, its full genome has coevolved in its macaque host and its tier 3 envelope glycoprotein (Env) is exceptionally hard to neutralize. Here, we investigated the ability of eCD4-Ig, an antibody-like entry inhibitor that emulates the HIV-1 and SIV receptor and coreceptor, to prevent SIVmac239 infection. We show that rh-eCD4-Ig I39N expressed by recombinant adeno-associated virus (AAV) vectors afforded four rhesus macaques complete protection from high-dose SIVmac239 challenges that infected all eight control macaques. However, rh-eCD4-Ig I39N -expressing macaques eventually succumbed to serial escalating challenge doses that were 2, 8, 16, and 32 times the challenge doses that infected the control animals. Despite receiving greater challenge doses, these macaques had significantly lower peak and postpeak viral loads than the control group. Virus isolated from three of four macaques showed evidence of strong immune pressure from rh-eCD4-Ig I39N , with mutations located in the CD4-binding site, which, in one case, exploited a point-mutation difference between rh-eCD4-Ig I39N and rhesus CD4. Other escape pathways associated with clear fitness costs to the virus. Our data report effective protection of rhesus macaques from SIVmac239., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Preexisting immunity against dengue virus or West Nile virus was previously reported to mediate antibody-dependent enhancement (ADE) of Zika virus (ZIKV) infection in a mouse model. We show here that ZIKV-immune plasma samples from both symptomatic and asymptomatic individuals mediated ZIKV ADE of infection in vitro and in mice. In a lethal infection model with a viral inoculum 10 times higher, both ADE and protection were observed, depending on the amount of infused immune plasma. In a vertical-transmission model, ZIKV-immune plasma infused to timed pregnant mice increased fetal demise and decreased the body weight of surviving fetuses. Depletion of IgG from an immune plasma abolished ADE of infection, and the presence of purified IgG alone mediated ADE of infection. Higher viral loads and proinflammatory cytokines were detected in mice treated with ZIKV-immune plasma samples compared to those receiving control plasma. Together, these data show that passive immunization with homotypic ZIKV antibodies, depending on the concentration, could either worsen or limit a subsequent ZIKV infection. IMPORTANCE Antibody-dependent enhancement (ADE) of virus infection is common to many viruses and is problematic when plasma antibody levels decline to subneutralizing concentrations. ADE of infection is especially important among flaviviruses, many of which are the cause of global health problems. Recently, human plasma samples immune to heterologous flaviviruses were shown to promote Zika virus (ZIKV) infection. Here we showed in immunocompromised mouse models that homologous immune plasma samples protect mice from subsequent infection at high antibody concentrations but that they mediate ADE of infection and increase ZIKV pathogenesis in adult mice and fetal demise during pregnancy at low concentrations., (Copyright © 2019 Shim et al.)

The engineered antibody-like entry inhibitor eCD4-Ig neutralizes every human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus isolate it has been tested against. The exceptional breadth of eCD4-Ig derives from its ability to closely and simultaneously emulate the HIV-1 receptor CD4 and coreceptors, either CCR5 or CXCR4. Here we investigated whether viral escape from eCD4-Ig is more difficult than that from CD4-Ig or the CD4-binding site antibody NIH45-46. We observed that a viral swarm selected with high concentrations of eCD4-Ig was increasingly resistant to but did not fully escape from eCD4-Ig. In contrast, viruses selected under the same conditions with CD4-Ig or NIH45-46 fully escaped from those inhibitors. eCD4-Ig-resistant viruses acquired unique changes in the V2 apex, V3, V4, and CD4-binding regions of the HIV-1 envelope glycoprotein (Env). Most of the alterations did not directly affect neutralization by eCD4-Ig or neutralizing antibodies. However, alteration of Q428 to an arginine or lysine resulted in markedly greater resistance to eCD4-Ig and CD4-Ig, with correspondingly dramatic losses in infectivity and greater sensitivity to a V3 antibody and to serum from an infected individual. Compensatory mutations in the V3 loop (N301D) and in the V2 apex (K171E) partially restored viral fitness without affecting serum or eCD4-Ig sensitivity. Collectively, these data suggest that multiple mutations will be necessary to fully escape eCD4-Ig without loss of viral fitness. IMPORTANCE HIV-1 broadly neutralizing antibodies (bNAbs) and engineered antibody-like inhibitors have been compared for their breadths, potencies, and in vivo half-lives. However, a key limitation in the use of antibodies to treat an established HIV-1 infection is the rapid emergence of fully resistant viruses. Entry inhibitors of similar breadths and potencies can differ in the ease with which viral escape variants arise. Here we show that HIV-1 escape from the potent and exceptionally broad entry inhibitor eCD4-Ig is more difficult than that from CD4-Ig or the bNAb NIH45-46. Indeed, full escape was not observed under conditions under which escape from CD4-Ig or NIH45-46 was readily detected. Moreover, viruses that were partially resistant to eCD4-Ig were markedly less infective and more sensitive to antibodies in the serum of an infected person. These data suggest that eCD4-Ig will be more difficult to escape and that even partial escape will likely extract a high fitness cost., (Copyright © 2019 American Society for Microbiology.)

Recombinant adeno-associated virus (rAAV)-mediated gene delivery can efficiently target muscle tissues to serve as "biofactories" for secreted proteins in prophylactic and therapeutic scenarios. Nevertheless, efficient rAAV-mediated gene delivery is often limited by host immune responses against the transgene product. The development of strategies to prevent anti-transgene immunity is therefore crucial. The employment of endogenous microRNA (miRNA)-mediated regulation to detarget transgene expression from antigen presenting cells (APCs) has shown promise for reducing immunogenicity. However, the mechanisms underlying miRNA-mediated modulation of anti-transgene immunity by APC detargeting are not fully understood. Using the highly immunogenic ovalbumin (OVA) protein as a proxy for foreign antigens, we show that rAAV vectors containing miR142 binding sites efficiently repress co-stimulatory signals in dendritic cells, significantly blunt the cytotoxic T cell response, allow for sustained transgene expression in skeletal myoblasts, and attenuate clearance of transduced muscle cells in mice. Furthermore, the blunting of humoral immunity against circulating OVA correlates with detargeting of OVA expression from APCs. This demonstrates that incorporating APC-specific miRNA binding sites into rAAV vectors provides an effective strategy for reducing transgene-specific immune response. This approach holds promise for clinical applications where the safe and efficient delivery of a prophylactic or therapeutic protein is desired.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic 1-8 . Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3) 8-10 . It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers) 5,11-18 . Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1 JR-FL strain in complex with the antibody PGT151 19 . Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.

Adeno-associated virus (AAV) delivery of potent and broadly neutralizing antibodies (bNAbs is a promising approach for the prevention of HIV-1 infection. The immunoglobulin G (IgG)1 subtype is usually selected for this application, because it efficiently mediates antibody effector functions and has a somewhat longer half-life. However, the use of IgG1-Fc has been associated with the generation of anti-drug antibodies (ADAs) that correlate with loss of antibody expression. In contrast, we have shown that expression of the antibody-like molecule eCD4-Ig bearing a rhesus IgG2-Fc domain showed reduced immunogenicity and completely protected rhesus macaques from simian-HIV (SHIV)-AD8 challenges. To directly compare the performance of the IgG1-Fc and the IgG2-Fc domains in a prophylactic setting, we compared AAV1 expression of rhesus IgG1 and IgG2 forms of four anti-HIV bNAbs: 3BNC117, NIH45-46, 10-1074, and PGT121. Interestingly, IgG2-isotyped bNAbs elicited significantly lower ADA than their IgG1 counterparts. We also observed significant protection from two SHIV-AD8 challenges in macaques expressing IgG2-isotyped bNAbs, but not from those expressing IgG1. Our data suggest that monoclonal antibodies isotyped with IgG2-Fc domains are less immunogenic than their IgG1 counterparts, and they highlight ADAs as a key barrier to the use of AAV1-expressed bNAbs., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Broadly neutralizing antibodies (bNAbs) target five major epitopes on the HIV-1 envelope glycoprotein (Env). The most potent bNAbs have median half-maximal inhibitory concentration (IC 50 ) values in the nanomolar range, and the broadest bNAbs neutralize up to 98% of HIV-1 strains. The engineered HIV-1 entry inhibitor eCD4-Ig has greater breadth than bNAbs and similar potency. eCD4-Ig is markedly more potent than CD4-Ig due to its C-terminal coreceptor-mimetic peptide. Here we investigated whether the coreceptor-mimetic peptide mim6 improved the potency of bNAbs with different epitopes. We observed that when mim6 was appended to the C terminus of the heavy chains of bNAbs, this sulfopeptide improved the potency of all classes of bNAbs against HIV-1 isolates that are sensitive to neutralization by the sulfopeptide alone. However, mim6 did not significantly enhance neutralization of other isolates when appended to most classes of bNAbs, with one exception. Specifically, mim6 improved the potency of bNAbs of the V3-glycan class, including PGT121, PGT122, PGT128, and 10-1074, by an average of 2-fold for all HIV-1 isolates assayed. Despite this difference, 10-1074 does not induce exposure of the coreceptor-binding site, and addition of mim6 to 10-1074 did not promote shedding of the gp120 subunit of Env. Mixtures of 10-1074 and an Fc domain fused to mim6 neutralized less efficiently than a 10-1074/mim6 fusion, indicating that mim6 enhances the avidity of this fusion. Our data show that mim6 can consistently improve the potency of V3-glycan antibodies and suggest that these antibodies bind in an orientation that facilitates mim6 association with Env. IMPORTANCE HIV-1 requires both the cellular receptor CD4 and a tyrosine-sulfated coreceptor to infect its target cells. CD4-Ig is a fusion of the HIV-1-binding domains of CD4 with an antibody Fc domain. Previous studies have demonstrated that the potency of CD4-Ig is markedly increased by appending a coreceptor-mimetic sulfopeptide to its C terminus. We investigated whether this coreceptor-mimetic peptide improves the potency of broadly neutralizing antibodies (bNAbs) targeting five major epitopes on the HIV-1 envelope glycoprotein (Env). We observed that inclusion of the sulfopeptide dramatically improved the potency of all bNAb classes against isolates with more-open Env structures, typically those that utilize the coreceptor CXCR4. In contrast, the sulfopeptide improved only V3-glycan antibodies when neutralizing primary isolates, on average by 2-fold. These studies improve the potency of one class of bNAbs, show that coreceptor-mimetic sulfopeptides enhance neutralization through distinct mechanisms, and provide insight for the design of novel multispecific entry inhibitors., (Copyright © 2019 American Society for Microbiology.)

Accurate analysis of intraparticle distribution of substances within porous drug carriers is important to optimize loading and subsequent processing. Mercury intrusion porosimetry, a common technique used for characterization of porous materials, assumes cylindrical pore geometry, which may lead to misinterpretation. Therefore, imaging techniques such as focused ion beam scanning electron microscopy (FIB-SEM) help to better interpret these results. The purpose of this study was to investigate the differences between mercury intrusion and scanning electron microscopy and to identify the limitations of each method. Porous microparticles, functionalized calcium carbonate, were loaded with bovine serum albumin and dipalmitoylphosphatidylcholine (DPPC) by solvent evaporation and results of the pore size distribution obtained by both methods were compared. The internal structure of the novel pharmaceutical excipient, functionalized calcium carbonate, was revealed for the first time. Our results demonstrated that image analysis provides a closer representation of the material distribution since it was possible to discriminate between blocked and filled pores. The physical nature of the loaded substances is critical for the deposition within the pores of functionalized calcium carbonate. We conclude, that a combination of mercury intrusion porosimetry and focused ion beam scanning electron microscopy allows for a reliable analysis of sub-micron porous structures of particulate drug carriers.

Human Immunodeficiency Virus-1 (HIV-1) entry is dependent on the envelope glycoprotein (Env) that is present on the virion and facilitates fusion between the envelope and the cellular membrane. The protein consists of two subunits, gp120 and gp41, with the former required for binding the CD4 receptor and either the CXCR4 or CCR5 coreceptor, and the latter for mediating fusion. The requirement of fusion for infection has made Env an attractive target for HIV therapy development and led to the FDA approval of enfuvirtide, a fusion inhibitor. Continued development of entry inhibitors is warranted because enfuvirtide resistant HIV-1 strains have emerged. In this study, a novel HIV-1 fusion assay was validated using neutralizing antibodies and then used to investigate the mechanism of action of eCD4-Igmim2, an HIV-1 inhibitor proposed to cooperatively bind the CD4 binding site and the sulfotyrosine-binding pocket of gp120. Greater reduction in fusion levels was observed with eCD4-Igmim2 in the fusion assay than all of the gp120 antibodies evaluated. Lab adapted isolates, HIV-1HXB2 and HIV-1YU2, were sensitive to eCD4-Igmim2 in the fusion assay, while primary isolates, HIV-1BG505 and HIV-1ZM651 were resistant. These results correlated with greater IC50 values for primary isolates compared to the lab adapted isolates observed in a virus neutralization assay. Analysis of gp120 models identified differences in the V1 and V2 domains that are associated with eCD4-Igmim2 sensitivity. This study highlights the use of a fusion assay to identify key areas for improving the potency of eCD4-Igmim2., Competing Interests: The authors have declared that no competing interests exist.

Many broadly neutralizing antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1) were shown effective in animal models, and are currently evaluated in clinical trials. However, use of these antibodies in humans is hampered by the rapid emergence of resistant viruses. Here we show that soft-randomization can be used to accelerate the parallel identification of viral escape pathways. As a proof of principle, we soft-randomized the epitope regions of VRC01-class bNAbs in replication-competent HIV-1 and selected for resistant variants. After only a few passages, a surprisingly diverse population of antibody-resistant viruses emerged, bearing both novel and previously described escape mutations. We observed that the escape variants resistant to some VRC01-class bNAbs are resistant to most other bNAbs in the same class, and that a subset of variants was completely resistant to every well characterized VRC01-class bNAB, including VRC01, NIH45-46, 3BNC117, VRC07, N6, VRC-CH31, and VRC-PG04. Thus, our data demonstrate that soft randomization is a suitable approach for accelerated detection of viral escape, and highlight the challenges inherent in administering or attempting to elicit VRC01-class antibodies., Competing Interests: The authors have declared that no competing interests exist.

The human immunodeficiency virus type 1 (HIV-1) entry inhibitor eCD4-Ig is a fusion of CD4-Ig and a coreceptor-mimetic peptide. eCD4-Ig is markedly more potent than CD4-Ig, with neutralization efficiencies approaching those of HIV-1 broadly neutralizing antibodies (bNAbs). However, unlike bNAbs, eCD4-Ig neutralized all HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates that it has been tested against, suggesting that it may be useful in clinical settings, where antibody escape is a concern. Here, we characterize three new eCD4-Ig variants, each with a different architecture and each utilizing D1.22, a stabilized form of CD4 domain 1. These variants were 10- to 20-fold more potent than our original eCD4-Ig variant, with a construct bearing four D1.22 domains (eD1.22-HL-Ig) exhibiting the greatest potency. However, this variant mediated less efficient antibody-dependent cell-mediated cytotoxicity (ADCC) activity than eCD4-Ig itself or several other eCD4-Ig variants, including the smallest variant (eD1.22-Ig). A variant with the same architecture as the original eCD4-Ig (eD1.22-D2-Ig) showed modestly higher thermal stability and best prevented the promotion of infection of CCR5-positive, CD4-negative cells. All three variants, and eCD4-Ig itself, mediated more efficient shedding of the HIV-1 envelope glycoprotein gp120 than did CD4-Ig. Finally, we show that only three D1.22 mutations contributed to the potency of eD1.22-D2-Ig and that introduction of these changes into eCD4-Ig resulted in a variant 9-fold more potent than eCD4-Ig and 2-fold more potent than eD1.22-D2-Ig. These studies will assist in developing eCD4-Ig variants with properties optimized for prophylaxis, therapy, and cure applications. IMPORTANCE HIV-1 bNAbs have properties different from those of antiretroviral compounds. Specifically, antibodies can enlist immune effector cells to eliminate infected cells, whereas antiretroviral compounds simply interfere with various steps in the viral life cycle. Unfortunately, HIV-1 is adept at evading antibody recognition, limiting the utility of antibodies as a treatment for HIV-1 infection or as part of an effort to eradicate latently infected cells. eCD4-Ig is an antibody-like entry inhibitor that closely mimics HIV-1's obligate receptors. eCD4-Ig appears to be qualitatively different from antibodies, since it neutralizes all HIV-1, HIV-2, and SIV isolates. Here, we characterize three new structurally distinct eCD4-Ig variants and show that each excels in a key property useful to prevent, treat, or cure an HIV-1 infection. For example, one variant neutralized HIV-1 most efficiently, while others best enlisted natural killer cells to eliminate infected cells. These observations will help generate eCD4-Ig variants optimized for different clinical applications., (Copyright © 2018 American Society for Microbiology.)

RNA switches that modulate gene expression with small molecules have a number of scientific and clinical applications. Here, we describe a novel class of small regulatory on switches based on the ability of a ligand-bound aptamer to promote stem formation between a microRNA target sequence (miR-T) and a complementary competing strand. Two on switch architectures employing this basic concept were evaluated, differing in the location of a tetracycline aptamer and the region of a miR-21 target sequence (miR-21-T) masked by its competing strand. Further optimizations of miR-21-T and its competing strand resulted in tetracycline-regulated on switches that induced luciferase expression by 19-fold in HeLa cells. A similar switch design based on miR-122-T afforded 7-fold regulation when placed in tandem, indicating that this approach can be extended to additional miR-T. Optimized on switches introduced into adeno-associated virus (AAV) vectors afforded 10-fold regulation of two antiviral proteins in AAV-transduced cells. Our data demonstrate that small-molecule-induced occlusion of a miR-T can be used to conditionally regulate gene expression in mammalian cells and suggest that regulatory switches built on this principle can be used to dose expression of an AAV transgene., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antibody-dependent cell-mediated cytotoxity (ADCC) can eliminate HIV-1 infected cells, and may help reduce the reservoir of latent virus in infected patients. Sera of HIV-1 positive individuals include a number of antibodies that recognize epitopes usually occluded on HIV-1 envelope glycoprotein (Env) trimers. We have recently described eCD4-Ig, a potent and exceptionally broad inhibitor of HIV-1 entry that can be used to protect rhesus macaques from multiple high-dose challenges with simian-human immunodeficiency virus AD8 (SHIV-AD8). Here we show that eCD4-Ig bearing an IgG1 Fc domain (eCD4-IgG1) can mediate efficient ADCC activity against HIV-1 isolates with differing tropisms, and that it does so at least 10-fold more efficiently than CD4-Ig, even when more CD4-Ig molecules bound cell surface-expressed Env. An ADCC-inactive IgG2 form of eCD4-Ig (eCD4-IgG2) exposes V3-loop and CD4-induced epitopes on cell-expressed trimers, and renders HIV-1-infected cells susceptible to ADCC mediated by antibodies of these classes. Moreover, eCD4-IgG2, but not IgG2 forms of the broadly neutralizing antibodies VRC01 and 10-1074, enhances the ADCC activities of serum antibodies from patients by 100-fold, and significantly enhanced killing of two latently infected T-cell lines reactivated by vorinostat or TNFα. Thus eCD4-Ig is qualitatively different from CD4-Ig or neutralizing antibodies in its ability to mediate ADCC, and it may be uniquely useful in treating HIV-1 infection or reducing the reservoir of latently infected cells.

Rhesus macaques are used to model human immunodeficiency virus type 1 (HIV-1) infections, but they are not natural hosts of HIV-1 or any simian immunodeficiency virus (SIV). Rather, they became infected with SIV through cross-species transfer from sooty mangabeys in captivity. It has been shown that HIV-1 utilizes rhesus CD4 less efficiently than human CD4. However, the relative ability of SIV envelope glycoproteins to bind or utilize these CD4 orthologs has not been reported. Here we show that several SIV isolates, including SIVmac239, are more efficiently neutralized by human CD4-Ig (huCD4-Ig) than by the same molecule bearing rhesus CD4 domains 1 and 2 (rhCD4-Ig). An I39N mutation in CD4 domain 1, present in human and sooty mangabey CD4 orthologs, largely restored rhCD4-Ig neutralization of SIVmac239 and other SIV isolates. We further observed that SIVmac316, a derivative of SIVmac239, bound to and was neutralized by huCD4-Ig and rhCD4-Ig with nearly identical efficiencies. Introduction of two SIVmac316 CD4-binding site residues (G382R and H442Y) into the SIVmac239 envelope glycoprotein (Env) markedly increased its neutralization sensitivity to rhesus CD4-Ig without altering neutralization by human CD4-Ig, SIV neutralizing antibodies, or sera from SIV-infected macaques. These changes also allowed SIVmac239 Env to bind rhCD4-Ig more efficiently than huCD4-Ig. The variant with G382R and H442Y (G382R/H442Y variant) also infected cells expressing rhesus CD4 with markedly greater efficiency than did unaltered SIVmac239 Env. We propose that infections of rhesus macaques with SIVmac239 G382R/H442Y might better model some aspects of human infections. IMPORTANCE Rhesus macaque infection with simian immunodeficiency virus (SIV) has served as an important model of human HIV-1 infection. However, differences between this model and the human case have complicated the development of vaccines and therapies. Here we report the surprising observation that SIVmac239, a commonly used model virus, more efficiently utilizes human CD4 than the CD4 of rhesus macaques, whereas the closely related virus SIVmac316 uses both CD4 orthologs equally well. We used this insight to generate a form of SIVmac239 envelope glycoprotein (Env) that utilized rhesus CD4 more efficiently, while retaining its resistance to antibodies and sera from infected macaques. This Env can be used to make the rhesus model more similar in some ways to human infection, for example by facilitating infection of cells with low levels of CD4. This property may be especially important to efforts to eradicate latently infected cells., (Copyright © 2017 American Society for Microbiology.)

Cpf1 is a CRISPR effector protein that has greater specificity than Streptococcus pyogenes Cas9 (SpCas9) in genome-editing applications. Here we show that Lachnospiraceae bacterium (Lb) and Acidaminococus sp. (As) Cpf1 orthologs have RNase activities that can excise multiple CRISPR RNAs (crRNAs) from a single RNA polymerase II-driven RNA transcript expressed in mammalian cells. This property simplifies modification of multiple genomic targets and can be used to increase the efficiency of Cpf1-mediated editing.

Purpose of Review: Here we discuss recently developed HIV-1 entry inhibitors that can target multiple epitopes on the HIV-1 envelope glycoprotein (Env), with an emphasis on eCD4-Ig. Some of these inhibitors are more potent and broader than any single antibody characterized to date. We also discuss the use of recombinant adeno-associated virus (rAAV) vectors as a platform for long-term expression of these inhibitors., Recent Findings: Much of the exterior of HIV-1 Env can be targeted by broadly neutralizing antibodies (bNAbs). Recent studies combine the variable regions or Fabs from different bNAbs, often with the receptor-mimetic components, to create broad, potent, and hard-to-escape inhibitors. rAAV vectors can express these inhibitors for years in vivo, highlighting their ability to prevent or treat HIV-1 infection., Summary: By targeting multiple epitopes on Env, bispecific and antibody-like inhibitors can be broader and more potent than bNAbs. These inhibitors can provide long-term protection from, and perhaps suppression of, HIV-1 if they are administered by a delivery platform, like rAAV vectors, but only after rAAV limitations are addressed.

Background: The aim of this study was to evaluate the idiopathic congenital clubfoot deformity treated by Ponseti method to determine the different factors such as radiological investigations that may have relations with the risk of failure and recurrence in mid-term follow-up of the patients., Methods: Since 2006 to 2011, 226 feet from 149 patients with idiopathic congenital clubfoot were treated with weekly castings by Ponseti method. Anteroposterior and lateral foot radiographies were performed at the final follow-up visit and the data from clinical and radiological outcomes were analysed., Results: In our patients, 191(84.9%) feet required percutaneous tenotomy. The successful correction rate was 92% indication no need for further surgical correction. No significant correlation was found between the remained deformity rate and the severity of the deformity and compliance of using the brace ( P =0.108 and 0.207 respectively). The remained deformity rate had an inverse association with the beginning age of treatment ( P =0.049). No significant correlation was found between the percutaneous tetonomy and passive dorsiflexion range ( P =0.356)., Conclusion: According to our results treatment with the Ponseti method resulted in poor or no correlation. The diagnosis of clubfoot is a clinical judgment; therefore, the outcome of the treatment must only be clinically evaluated. Although the Ponseti method can retrieve the normal shape of the foot, it fails to treat the bone deformities and eventually leads to remained radiologic deformity. Further studiesare suggested to define a different modification that can address the abnormal angles between the foot and ankle bones to minimize the risk of recurrence.

In this study, poly(ethylene-co-vinyl alcohol) (EVOH) as a novel biocompatible polymeric scaffold was surface modified by succinylation to get EVOHS and further pegylated to improve structural properties using methoxypolyethylene glycol (5000 Da) succinate (PEGS) along with targeting with retinoic acid (RA) to get final modified active and passive targeted conjugate (PEGS-EVOHS-RA) to evaluate its ability in carrying and delivery of epirubicin to hepatocellular carcinoma cell lines in response to varying temperatures. In this regard, the PEGS-EVOHS-RA conjugate was prepared through the desired chemical reactions and its structure was confirmed using 1 H-NMR and FT-IR spectra. The micelles were prepared from PEGS-EVOHS-RA by dialysis method. The Particle size and zeta potential were measured, and entrapment efficacy along with in vitro release efficiency in different temperatures were also studied. The structural morphology of optimized nanomicelle was studied by transmission electron microscopy micrographs. The desired final micelles were evaluated for their toxicity using MTT assay on HepG2 human hepatocellular carcinoma cell lines at normal (37 °C) and elevated temperature (45 °C). The results revealed that, as the hydrophilicity of micelles increased, all characteristic properties improved. Then, these micelles can be considered as potentially effective thermo responsive delivery systems for targeted delivery of cytotoxic agents to hepatocellular carcinoma.

Objectives: Despite the progress in understanding acute pain physiology during recent decade, eighty percent of patients still suffer from post-operative discomfort. Pregabalin is an anticonvulsant agent that is approved for painful neuropathies in diabetic patients and post herpetic neuralgia. The main objective of the present study was to compare the improvement in post-operative pain management and patient lifestyle in 3 groups, as first group received placebo, second who received Pregabalin for one day and the last group those who received it for 14 days., Methods: This was a prospective single center, randomized, triple-blind, 3-arm, parallel group study. In this triple-blind study, patients were randomized to 1 of 3 groups using computer-generated random number table. 1) The first group received placebo for 14 days, the second group received Pregabalin 300mg 8h preoperatively and 150mg 12 and 24h postoperatively and for the rest of 13days received placebo and the third group received Pregabalin 300mg eight hours preoperatively and 15mg every 12h postoperatively for 14 days. Name, age, gender, height, weight, education, duration of pain, past medical history, drug history,total morphine requirement at the time of discharge and MRI findings of all the patients were recorded, also they Numerical scale system (NRS) and Oswestry low back pain disability index (ODI) questionnaire were completed for them. All the patients were operated based on standard surgery techniques, bilateral foramenotomy and interlaminar discectomy., Results: Of the 105 patients who entered the run-in period, 47 patients (44.8%) were female and 58 (55.2%) were male. The Patients radicular pain mean score based on NRS estimated before surgery was 7.22±1.95 in pregabalin14, 7.71±1.84 in pregabalin1 and 7.45±1.9 in control group. There were no statically significant differences between three groups (P-Value>0.05). The Patients back pain mean score based on NRS was 5.2±2.87 in pregabalin14, 5.11±3.23 in pregabalin1 and 6.4±3.06 in control group. This means that there were no significant differences in the overall score among those three groups (P-Value>0.05). In comparison to their preoperative pain, the average radicular pain in each group of patients improved significantly 4, 8, 12 and 24h after the operation (P-Value<0.001), but there were no significant differences in radicular pain improvements comparing three groups., Conclusion: The results of this study indicate that 1day and 2 weeks post-operative 300mg pregabalin administration may not improve acute pain, morphine consumption and quality of life of patients after surgery. It seems that the diseases cause chronic pain that requires long-term treatment with higher doses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Efforts to control mammalian gene expression with ligand-responsive riboswitches have been hindered by lack of a general method for generating efficient switches in mammalian systems. Here we describe a rational-design approach that enables rapid development of efficient cis -acting aptazyme riboswitches. We identified communication-module characteristics associated with aptazyme functionality through analysis of a 32-aptazyme test panel. We then developed a scoring system that predicts an aptazymes's activity by integrating three characteristics of communication-module bases: hydrogen bonding, base stacking, and distance to the enzymatic core. We validated the power and generality of this approach by designing aptazymes responsive to three distinct ligands, each with markedly wider dynamic ranges than any previously reported. These aptayzmes efficiently regulated adeno-associated virus (AAV)-vectored transgene expression in cultured mammalian cells and mice, highlighting one application of these broadly usable regulatory switches. Our approach enables efficient, protein-independent control of gene expression by a range of small molecules., Competing Interests: The authors declare that no competing interests exist.