Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. 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-1JR-FL strain in complex with the antibody PGT15119. 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.

Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2.

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.

Mayo Clinic VT calculator: A practical tool for accurate wide complex tachycardia differentiation.

The discrimination of ventricular tachycardia (VT) versus supraventricular wide complex tachycardia (SWCT) via 12-lead electrocardiogram (ECG) is crucial for achieving appropriate, high-quality, and cost-effective care in patients presenting with wide QRS complex tachycardia (WCT). Decades of rigorous research have brought forth an expanding arsenal of applicable manual algorithm methods for differentiating WCTs. However, these algorithms are limited by their heavy reliance on the ECG interpreter for their proper execution. Herein, we introduce the Mayo Clinic ventricular tachycardia calculator (MC-VTcalc) as a novel generalizable, accurate, and easy-to-use means to estimate VT probability independent of ECG interpreter competency. The MC-VTcalc, through the use of web-based and mobile device platforms, only requires the entry of computerized measurements (i.e., QRS duration, QRS axis, and T-wave axis) that are routinely displayed on standard 12-lead ECG recordings.

AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 µg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 µg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 µg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

eCD4-Ig Limits HIV-1 Escape More Effectively than CD4-Ig or a Broadly Neutralizing Antibody.

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.

A Coreceptor-Mimetic Peptide Enhances the Potency of V3-Glycan Antibodies.

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 (IC50) 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.

Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies.

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.

Anti-drug Antibody Responses Impair Prophylaxis Mediated by AAV-Delivered HIV-1 Broadly Neutralizing Antibodies.

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.

Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging.

Herpesviral DNA packaging into nascent capsids requires multiple conserved viral proteins that coordinate genome encapsidation. Here, we investigated the role of the ORF68 protein of Kaposi's sarcoma-associated herpesvirus (KSHV), a protein required for viral DNA encapsidation whose function remains largely unresolved across the herpesviridae. We found that KSHV ORF68 is expressed with early kinetics and localizes predominantly to viral replication compartments, although it is dispensable for viral DNA replication and gene expression. However, in agreement with its proposed role in viral DNA packaging, KSHV-infected cells lacking ORF68 failed to cleave viral DNA concatemers, accumulated exclusively immature B capsids, and released no infectious progeny virions. ORF68 has no predicted domains aside from a series of putative zinc finger motifs. However, in vitro biochemical analyses of purified ORF68 protein revealed that it robustly binds DNA and is associated with nuclease activity. These activities provide new insights into the role of KSHV ORF68 in viral genome encapsidation.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma and several B-cell cancers, causing significant morbidity and mortality in immunocompromised individuals. A critical step in the production of infectious viral progeny is the packaging of the newly replicated viral DNA genome into the capsid, which involves coordination between at least seven herpesviral proteins. While the majority of these packaging factors have been well studied in related herpesviruses, the role of the KSHV ORF68 protein and its homologs remains unresolved. Here, using a KSHV mutant lacking ORF68, we confirm its requirement for viral DNA processing and packaging in infected cells. Furthermore, we show that the purified ORF68 protein directly binds DNA and is associated with a metal-dependent cleavage activity on double-stranded DNA in vitro These activities suggest a novel role for ORF68 in herpesviral genome processing and encapsidation.

eCD4-Ig Variants That More Potently Neutralize HIV-1.

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.

eCD4-Ig promotes ADCC activity of sera from HIV-1-infected patients.

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.

Conditional Regulation of Gene Expression by Ligand-Induced Occlusion of a MicroRNA Target Sequence.

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.

Simian Immunodeficiency Virus SIVmac239, but Not SIVmac316, Binds and Utilizes Human CD4 More Efficiently than Rhesus CD4.

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.

CD4-Induced Antibodies Promote Association of the HIV-1 Envelope Glycoprotein with CD4-Binding Site Antibodies.

UNLABELLED: The HIV-1 envelope glycoprotein (Env) is a trimer of gp120/gp41 heterodimers that mediates viral entry. Env binds cellular CD4, an association which stabilizes a conformation favorable to its subsequent association with a coreceptor, typically CCR5 or CXCR4. The CD4- and coreceptor-binding sites serve as epitopes for two classes of HIV-1-neutralizing antibodies: CD4-binding site (CD4bs) and CD4-induced (CD4i) antibodies, respectively. Here we observed that, at a fixed total concentration, mixtures of the CD4i antibodies (E51 or 412d) and the CD4bs antibody VRC01 neutralized the HIV-1 isolates 89.6, ADA, SG3, and SA32 more efficiently than either antibody alone. We found that E51, and to a lesser extent 412d and 17b, promoted association of four CD4bs antibodies to the Env trimer but not to monomeric gp120. We further demonstrated that the binding of the sulfotyrosine-binding pocket by CCR5mim2-Ig was sufficient for promoting CD4bs antibody binding to Env. Interestingly, the relationship is not reciprocal: CD4bs antibodies were not as efficient as CD4-Ig at promoting E51 or 412d binding to Env trimer. Consistent with these observations, CD4-Ig, but none of the CD4bs antibodies tested, substantially increased HIV-1 infection of a CD4-negative, CCR5-positive cell line. We conclude that the ability of CD4i antibodies to promote VRC01 association with Env trimers accounts for the increase potency of VRC01 and CD4i antibody mixtures. Our data further suggest that potent CD4bs antibodies avoid inducing Env conformations that bind CD4i antibodies or CCR5. IMPORTANCE: Potent HIV-1-neutralizing antibodies can prevent viral transmission and suppress an ongoing infection. Here we show that CD4-induced (CD4i) antibodies, which recognize the conserved coreceptor-binding site of the HIV-1 envelope glycoprotein (Env), can increase the association of Env with potent broadly neutralizing antibodies that recognize the CD4-binding site (CD4bs antibodies). We further show that, unlike soluble forms of CD4, CD4bs antibodies poorly induce envelope glycoprotein conformations that efficiently bind CCR5. This study provides insight into the properties of potent CD4bs antibodies and suggests that, under some conditions, CD4i antibodies can improve their potency. These observations may be helpful to the development of vaccines designed to elicit specific antibody classes.

Envelope Glycoprotein Internalization Protects Human and Simian Immunodeficiency Virus-Infected Cells from Antibody-Dependent Cell-Mediated Cytotoxicity.

UNLABELLED: The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface. IMPORTANCE: This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals.

A double-mimetic peptide efficiently neutralizes HIV-1 by bridging the CD4- and coreceptor-binding sites of gp120.

UNLABELLED: The HIV-1 envelope glycoprotein binds cooperatively to its cellular receptor CD4 and a coreceptor, principally CXCR4 or CCR5. We have previously improved a natural amino-acid form of a scorpion toxin-derived CD4-mimetic peptide and in parallel generated sulfopeptide mimetics of the CCR5 amino terminus. Here we show that some fusions of these CCR5- and CD4-mimetic peptides, expressed as immunoadhesins, neutralize HIV-1 more efficiently than CD4-Fc or equimolar mixtures of immunoadhesin forms of each peptide. Specifically, double-mimetic peptides with linkers of 11 amino acids or greater, and with the CCR5-mimetic component preceding the CD4-mimetic component, were more efficient than constructs with shorter linkers or in a reverse orientation. The potency of these constructs derives from (i) their ability to simultaneously and cooperatively bind the CD4- and CCR5-binding sites of a single gp120 monomer of the HIV-1 envelope glycoprotein trimer and (ii) the ability of the CCR5-mimetic component to prevent the CD4-mimetic peptide from promoting infection when cellular CD4 is limiting. Thus, there is a significant advantage to simultaneously targeting both conserved regions of the HIV-1 envelope glycoprotein. IMPORTANCE: This report describes a novel class of peptides that potently inhibit HIV-1 entry. These peptides simultaneously target the receptor- and coreceptor-binding sites of the HIV-1 envelope glycoprotein gp120. Peptides of this class overcome key limitations of inhibitors that target only one gp120 binding region and illustrate the utility of binding the sulfotyrosine-binding pockets of gp120.

Perceptions of video-based appointments from the patient's home: a patient survey.

BACKGROUND: We examined patient interest in a telehealth model in which the patient supplies the hardware and Internet connectivity to meet with a healthcare provider from his or her home via video call (video appointment). We hoped to understand prospectively the desirability, feasibility, and viability from the patient perspective. MATERIALS AND METHODS: A phone survey was conducted of a random sample of patients who had been seen in the outpatient setting at a single institution. The sample was stratified by proximity to the local institution with oversampling for patients living outside a 120-mile radius. RESULTS: Out of 500 total patients, 301 patients responded, and 263 met the inclusion criteria. Of those 263 respondents, 38% indicated "very likely" to accept an invitation to see their provider via video, 28.1% "somewhat likely," and 33.8% "not at all likely." Of respondents, 75% have broadband, although only 36% reported having a Web camera. The primary factors affecting willingness to participate in a video appointment include comfort in setting up a video call, age, and distance participants would have traveled for an in-clinic appointment. CONCLUSIONS: Patient survey data indicate that most patients are likely to be accepting of telehealth care to the home using video call and that most have the required technology. Nevertheless, there are still significant hurdles to effectively implement this adaptation of telehealth care as part of mainstream practice.

Direct expression and validation of phage-selected peptide variants in mammalian cells.

Phage display is a key technology for the identification and maturation of high affinity peptides, antibodies, and other proteins. However, limitations of bacterial expression restrict the range and sensitivity of assays that can be used to evaluate phage-selected variants. To address this problem, selected genes are typically transferred to mammalian expression vectors, a major rate-limiting step in the iterative improvement of peptides and proteins. Here we describe a system that combines phage display and efficient mammalian expression in a single vector, pDQ1. This system permits immediate expression of phage-selected genes as IgG1-Fc fusions in mammalian cells, facilitating the rapid, sensitive characterization of a large number of library outputs for their biochemical and functional properties. We demonstrate the utility of this system by improving the ability of a CD4-mimetic peptide to bind the HIV-1 envelope glycoprotein and neutralize HIV-1 entry. We further improved the potency of the resulting peptide, CD4mim6, by limiting its ability to induce the CD4-bound conformation of the envelope glycoprotein. Thus, CD4mim6 and its variants can be used to investigate the properties of the HIV-1 envelope glycoprotein, and pDQ1 can accelerate the discovery of new peptides and proteins through phage display.

Adeno-associated viral delivery of Env-specific antibodies prevents SIV rebound after discontinuing antiretroviral therapy.

An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here we show that adeno-associated virus (AAV)-delivery of two rhesus macaque antibodies to the SIV envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIVmac239M after discontinuing suppressive ART. Following AAV administration, sustained antibody expression with minimal anti-drug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within two weeks in all of the control animals but remained below the threshold of detection in plasma (<15 copies/mL) for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals with delayed rebound exhibited restricted barcode diversity and antibody escape. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.

Enhanced recognition and neutralization of HIV-1 by antibody-derived CCR5-mimetic peptide variants.

A tyrosine-sulfated CCR5-mimetic peptide, CCR5mim1, inhibits HIV-1 infection more efficiently than sulfopeptides based on the CCR5 amino terminus. Here we characterized sulfopeptide chimeras of CCR5mim1 and the heavy-chain CDR3 of the antibody PG16. Two chimeras bound a range of envelope glycoproteins and neutralized HIV-1 more efficiently than CCR5mim1. An immunoadhesin form of one of these, CCR5mim2-Ig, synergized with CD4-Ig to neutralize HIV-1. These sulfopeptides are among the broadest and most potent CCR5-mimetic peptides described to date.