Determination of Binding Affinity of Antibodies to HIV-1 Recombinant Envelope Glycoproteins, Pseudoviruses, Infectious Molecular Clones, and Cell-Expressed Trimeric gp160 Using Microscale Thermophoresis.

Developing a preventative vaccine for HIV-1 has been a global priority. The elicitation of broadly neutralizing antibodies (bNAbs) against a broad range of HIV-1 envelopes (Envs) from various strains appears to be a critical requirement for an efficacious HIV-1 vaccine. To understand their ability to neutralize HIV-1, it is important to characterize the binding characteristics of bNAbs. Our work is the first to utilize microscale thermophoresis (MST), a rapid, economical, and flexible in-solution temperature gradient method to quantitatively determine the binding affinities of bNAbs and non-neutralizing monoclonal antibodies (mAbs) to HIV-1 recombinant envelope monomer and trimer proteins of different subtypes, pseudoviruses (PVs), infectious molecular clones (IMCs), and cells expressing the trimer. Our results demonstrate that the binding affinities were subtype-dependent. The bNAbs exhibited a higher affinity to IMCs compared to PVs and recombinant proteins. The bNAbs and mAbs bound with high affinity to native-like gp160 trimers expressed on the surface of CEM cells compared to soluble recombinant proteins. Interesting differences were seen with V2-specific mAbs. Although they recognize linear epitopes, one of the antibodies also bound to the Envs on PVs, IMCs, and a recombinant trimer protein, suggesting that the epitope was not occluded. The identification of epitopes on the envelope surface that can bind to high affinity mAbs could be useful for designing HIV-1 vaccines and for down-selecting vaccine candidates that can induce high affinity antibodies to the HIV-1 envelope in their native conformation.

Investigating host-virus interaction mechanism and phylogenetic analysis of viral proteins involved in the pathogenesis.

Since the emergence of yellow fever in the Americas and the devastating 1918 influenza pandemic, biologists and clinicians have been drawn to human infecting viruses to understand their mechanisms of infection better and develop effective therapeutics against them. However, the complex molecular and cellular processes that these viruses use to infect and multiply in human cells have been a source of great concern for the scientific community since the discovery of the first human infecting virus. Viral disease outbreaks, such as the recent COVID-19 pandemic caused by a novel coronavirus, have claimed millions of lives and caused significant economic damage worldwide. In this study, we investigated the mechanisms of host-virus interaction and the molecular machinery involved in the pathogenesis of some common human viruses. We also performed a phylogenetic analysis of viral proteins involved in host-virus interaction to understand the changes in the sequence organization of these proteins during evolution for various strains of viruses to gain insights into the viral origin's evolutionary perspectives.

New Insights into the Structure of Kappa/Beta-Carrageenan: A Novel Potential Inhibitor of HIV-1.

New insights into the structure of the hybrid κ/ß-carrageenan (κ/ß-CRG) of the red alga Tichocarpus crinitus have been obtained. Carrageenan oligosaccharides were prepared through the chemical and enzymatic depolymerization of κ/ß-CRG with κ-carrageenase and its the enzyme-resistant fraction. The composition and distribution of the repetition units of κ/ß- CRG were investigated by using the negative ion tandem MALDI-TOFMS and ESIMS method, which made it possible to prove and characterize the hybrid structure of this polysaccharide. An analysis revealed the blockwise distribution of the long ß-blocks along the polysaccharide chain, with the inclusion of κ/ß, µ/ν-blocks and some ι-blocks. Furthermore, the desulfated κ/ß-CRG was shown to contain of -G-D- repeating units up to 3.5 kDa. Previous studies have demonstrated that CRGs suppress the replication of several viruses. Here, we established that κ/ß-CRG and its oligosaccharides significantly inhibit the transduction efficiency of replication-defective lentiviral particles pseudotyped with the envelope proteins of three different viruses. We found that the polysaccharide and its oligosaccharides strongly reduced the transduction efficiency of lentiviral particles pseudotyped with GP160-the envelope protein of the human immunodeficiency virus HIV-1-when added to T-lymphocyte Jurkat cells. The CRG oligosaccharides displayed significantly higher antiviral activity.

HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals.

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound "closed" conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature "opening" of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the "closed" conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

CD4+ T Cells Are Dispensable for Induction of Broad Heterologous HIV Neutralizing Antibodies in Rhesus Macaques.

Induction of broadly neutralizing antibodies (bNAbs) is a major goal for HIV vaccine development. HIV envelope glycoprotein (Env)-specific bNAbs isolated from HIV-infected individuals exhibit substantial somatic hypermutation and correlate with T follicular helper (Tfh) responses. Using the VC10014 DNA-protein co-immunization vaccine platform consisting of gp160 plasmids and gp140 trimeric proteins derived from an HIV-1 infected subject that developed bNAbs, we determined the characteristics of the Env-specific humoral response in vaccinated rhesus macaques in the context of CD4+ T cell depletion. Unexpectedly, both CD4+ depleted and non-depleted animals developed comparable Tier 1 and 2 heterologous HIV-1 neutralizing plasma antibody titers. There was no deficit in protection from SHIV challenge, no diminution of titers of HIV Env-specific cross-clade binding antibodies, antibody dependent cellular phagocytosis, or antibody-dependent complement deposition in the CD4+ depleted animals. These collective results suggest that in the presence of diminished CD4+ T cell help, HIV neutralizing antibodies were still generated, which may have implications for developing effective HIV vaccine strategies.

Combinations of Single Chain Variable Fragments From HIV Broadly Neutralizing Antibodies Demonstrate High Potency and Breadth.

Broadly neutralizing antibodies (bNAbs) are currently being assessed in clinical trials for their ability to prevent HIV infection. Single chain variable fragments (scFv) of bNAbs have advantages over full antibodies as their smaller size permits improved diffusion into mucosal tissues and facilitates vector-driven gene expression. We have previously shown that scFv of bNAbs individually retain significant breadth and potency. Here we tested combinations of five scFv derived from bNAbs CAP256-VRC26.25 (V2-apex), PGT121 (N332-supersite), 3BNC117 (CD4bs), 8ANC195 (gp120-gp41 interface) and 10E8v4 (MPER). Either two or three scFv were combined in equimolar amounts and tested in the TZM-bl neutralization assay against a multiclade panel of 17 viruses. Experimental IC50 and IC80 data were compared to predicted neutralization titers based on single scFv titers using the Loewe additive and the Bliss-Hill model. Like full-sized antibodies, combinations of scFv showed significantly improved potency and breadth compared to single scFv. Combinations of two or three scFv generally followed an independent action model for breadth and potency with no significant synergy or antagonism observed overall although some exceptions were noted. The Loewe model underestimated potency for some dual and triple combinations while the Bliss-Hill model was better at predicting IC80 titers of triple combinations. Given this, we used the Bliss-Hill model to predict the coverage of scFv against a 45-virus panel at concentrations that correlated with protection in the AMP trials. Using IC80 titers and concentrations of 1µg/mL, there was 93% coverage for one dual scFv combination (3BNC117+10E8v4), and 96% coverage for two of the triple combinations (CAP256.25+3BNC117+10E8v4 and PGT121+3BNC117+10E8v4). Combinations of scFv, therefore, show significantly improved breadth and potency over individual scFv and given their size advantage, have potential for use in passive immunization.

Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint.

Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the C terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the ∼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis.

Prevalence of gp160 polymorphisms known to be related to decreased susceptibility to temsavir in different subtypes of HIV-1 in the Los Alamos National Laboratory HIV Sequence Database.

BACKGROUND: Fostemsavir, a prodrug of the gp120-directed attachment inhibitor temsavir, is indicated for use in heavily treatment-experienced individuals with MDR HIV-1. Reduced susceptibility to temsavir in the clinic maps to discrete changes at amino acid positions in gp160: S375, M426, M434 and M475. OBJECTIVES: To query the Los Alamos National Laboratory (LANL) HIV Sequence Database for the prevalence of polymorphisms at gp160 positions of interest. METHODS: Full-length gp160 sequences (N = 7560) were queried for amino acid polymorphisms relative to the subtype B consensus at positions of interest; frequencies were reported for all sequences and among subtypes/circulating recombinant forms (CRFs) with ≥10 isolates in the database. RESULTS: Among 239 subtypes in the database, the 5 most prevalent were B (n = 2651, 35.1%), C (n = 1626, 21.5%), CRF01_AE (n = 674, 8.9%), A1 (n = 273, 3.6%) and CRF02_AG (n = 199, 2.6%). Among all 7560 sequences, the most prevalent amino acids at positions of interest (S375, 73.5%; M426, 82.1%; M434, 88.2%; M475, 89.9%) were the same as the subtype B consensus. Specific polymorphisms with the potential to decrease temsavir susceptibility (S375H/I/M/N/T/Y, M426L/P, M434I/K and M475I) were found in <10% of isolates of subtypes D, G, A6, BC, F1, CRF07_BC, CRF08_BC, 02A, CRF06_cpx, F2, 02G and 02B. S375H and M475I were predominant among CRF01_AE (S375H, 99.3%; M475I, 76.3%; consistent with previously reported low temsavir susceptibility of this CRF) and 01B (S375H, 71.7%; M475I, 49.5%). CONCLUSIONS: Analysis of the LANL HIV Sequence Database found a low prevalence of gp160 amino acid polymorphisms with the potential to reduce temsavir susceptibility overall and among most of the common subtypes.

Design of immunogens to elicit broadly neutralizing antibodies against HIV targeting the CD4 binding site.

A vaccine which is effective against the HIV virus is considered to be the best solution to the ongoing global HIV/AIDS epidemic. In the past thirty years, numerous attempts to develop an effective vaccine have been made with little or no success, due, in large part, to the high mutability of the virus. More recent studies showed that a vaccine able to elicit broadly neutralizing antibodies (bnAbs), that is, antibodies that can neutralize a high fraction of global virus variants, has promise to protect against HIV. Such a vaccine has been proposed to involve at least three separate stages: First, activate the appropriate precursor B cells; second, shepherd affinity maturation along pathways toward bnAbs; and, third, polish the Ab response to bind with high affinity to diverse HIV envelopes (Env). This final stage may require immunization with a mixture of Envs. In this paper, we set up a framework based on theory and modeling to design optimal panels of antigens to use in such a mixture. The designed antigens are characterized experimentally and are shown to be stable and to be recognized by known HIV antibodies.

Dendronized Gold Nanoparticles as Carriers for gp160 (HIV-1) Peptides: Biophysical Insight into Complex Formation.

The unavailability of effective and safe human immunodeficiency virus (HIV) vaccines incites several approaches for development of the efficient antigen/adjuvant vaccination composite. In this study, three different dendronized gold nanoparticles (AuNPs 13-15) were investigated for a complexation ability with gp160 synthetic peptides derived from an HIV envelope. It has been shown that HIV peptides interacted with nanoparticles as evident from the changes in their secondary structures, restricted the mobility of the attached fluorescence dye, and enhanced peptide helicity confirmed by the fluorescence polarization and circular dichroism results. Transmission electron microscopy visualized complexes as cloud-like structures with attached nanoparticles. AuNP 13-15 nanoparticles bind negatively charged peptides depending on the number of functional groups; the fastest saturation and peptide retardation were observed for the most dendronized nanoparticle as indicated from dynamic light scattering, laser Doppler velocimetry, and agarose gel electrophoresis experiments. Dendronized gold nanoparticles can be considered one of the potential HIV peptide-based vaccination platforms.

Mutational networks of escape from transmitted HIV-1 infection.

Human immunodeficiency virus (HIV) is subject to immune selective pressure soon after it establishes infection at the founder stage. As an individual progresses from the founder to chronic stage of infection, immune pressure forces a history of mutations that are embedded in envelope sequences. Determining this pathway of coevolving mutations can assist in understanding what is different with the founder virus and the essential pathways it takes to maintain infection. We have combined operations research and bioinformatics methods to extract key networks of mutations that differentiate founder and chronic stages for 156 subtype B and 107 subtype C envelope (gp160) sequences. The chronic networks for both subtypes revealed strikingly different hub-and-spoke topologies compared to the less structured transmission networks. This suggests that the hub nodes are impacted by the immune response and the resulting loss of fitness is compensated by mutations at the spoke positions. The major hubs in the chronic C network occur at positions 12, 137 (within the N136 glycan), and 822, and at position 306 for subtype B. While both founder networks had a more heterogeneous connected network structure, interestingly founder B subnetworks around positions 640 and 837 preferentially contained CD4 and coreceptor binding domains. Finally, we observed a differential effect of glycosylation between founder and chronic subtype B where the latter had mutational pathways significantly driven by N-glycosylation. Our study provides insights into the mutational pathways HIV takes to evade the immune response, and presents features more likely to establish founder infection, valuable for effective vaccine design.

Anti-HIV-1 antibodies based confirmatory results in Wuhan, China, 2012-2018.

The number, intensity and order of emergence of HIV-1 specific antibodies in serum or plasma were associated with the stage of HIV-1 infection. In this study, we retrospectively analyzed the HIV-1 confirmatory results tested by western blot (WB) or recombination immunoblot assay (RIBA) in Wuhan, 2012-2018, to access the profiles of HIV-1 specific antibodies. A total of 14432 HIV-suspected serum or plasma samples collected from local hospitals and other HIV screening laboratories were further screened by two 4th generation enzyme-linked immunosorbent assay (ELISA) kits in our laboratory, of which 11068 specimens (76.69%) had at least one positive ELISA result and thereby were finally confirmed with WB or RIBA. RIBA had identified 652 (81.09%) positive and 13 (1.62%) indeterminate cases from July 1, 2014 to January 7, 2015, while WB had identified 8358 (81.43%) positive and 643 (6.26%) indeterminate cases in the other times during 2012-2018. The indeterminate rate of WB was significant higher than that of RIBA (p<0.001). Although the number of HIV-1 infected subjects increased significantly from 2012 (n = 911) to 2018 (n = 1578), the positive rate of HIV-1 antibodies decreased markedly from 70.08% in 2012 to 58.79% in 2018 (p<0.001). The most commonly observed antibody profile was gp160+gp120+p66+(p55+)p51+gp41+p31+p24+p17+ (4131, 49.43%) for WB-MP and gp160+gp120+gp41+p31+p24+p17+ (382, 58.59%) for RIBA-WANTAI, and the absence of reactivity to three possible serologic markers for recent HIV-1 infection, p31, p66, and p51, increased significantly from 2012 to 2018, with the overall rate of 17.03%, 9.40%, and 15.15%, respectively. The suspected acute HIV-1 infection was also observed to be increased in recent years, with an overall rate of 1.00%. Our results indicated the detection rate had decreased for HIV-1 infection, but increased for suspected recent and acute HIV-1 infection during 2012-2018, reflecting the efforts of intervention among high risk population.

Probing the Structure of the HIV-1 Envelope Trimer Using Aspartate Scanning Mutagenesis.

HIV-1 envelope (Env) glycoprotein gp160 exists as a trimer of heterodimers on the viral surface. In most structures of the soluble ectodomain of trimeric HIV-1 envelope glycoprotein, the regions from 512 to 517 of the fusion peptide and from 547 to 568 of the N-heptad repeat are disordered. We used aspartate scanning mutagenesis of subtype B strain JRFL Env as an alternate method to probe residue burial in the context of cleaved, cell surface-expressed Env, as buried residues should be intolerant to substitution with Asp. The data are inconsistent with a fully disordered 547 to 568 stretch, as residues 548, 549, 550, 555, 556, 559, 562, and 566 to 569 are all sensitive to Asp substitution. In the fusion peptide region, residues 513 and 515 were also sensitive to Asp substitution, suggesting that the fusion peptide may not be fully exposed in native Env. gp41 is metastable in the context of native trimer. Introduction of Asp at residues that are exposed in the prefusion state but buried in the postfusion state is expected to destabilize the postfusion state and any intermediate states where the residue is buried. We therefore performed soluble CD4 (sCD4)-induced gp120 shedding experiments to identify Asp mutants at residues 551, 554 to 559, 561 to 567, and 569 that could prevent gp120 shedding. We also observed similar mutational effects on shedding for equivalent mutants in the context of clade C Env from isolate 4-2J.41. These substitutions can potentially be used to stabilize native-like trimer derivatives that are used as HIV-1 vaccine immunogens.IMPORTANCE In most crystal structures of the soluble ectodomain of the HIV-1 Env trimer, some residues in the fusion and N-heptad repeat regions are disordered. Whether this is true in the context of native, functional Env on the virion surface is not known. This knowledge may be useful for stabilizing Env in its prefusion conformation and will also help to improve understanding of the viral entry process. Burial of the charged residue Asp in a protein structure is highly destabilizing. We therefore used Asp scanning mutagenesis to probe the burial of apparently disordered residues in native Env and to examine the effect of mutations in these regions on Env stability and conformation as probed by antibody binding to cell surface-expressed Env, CD4-induced shedding of HIV-1 gp120, and viral infectivity studies. Mutations that prevent shedding can potentially be used to stabilize native-like Env constructs for use as vaccine immunogens.

A Universal CAR-NK Cell Targeting Various Epitopes of HIV-1 gp160.

Engineering T cells and natural killer (NK) cells with anti-HIV chimeric antigen receptors (CAR) has emerged as a promising strategy to eradicate HIV-infected cells. However, current anti-HIV CARs are limited by targeting a single epitope of the HIV envelope glycoprotein gp160, which cannot counter the enormous diversity and mutability of viruses. Here, we report the development of a universal CAR-NK cell, which recognizes 2,4-dinitrophenyl (DNP) and can subsequently be redirected to target various epitopes of gp160 using DNP-conjugated antibodies as adaptor molecules. We show that this CAR-NK cell can recognize and kill mimic HIV-infected cell lines expressing subtypes B and C gp160. We additionally find that anti-gp160 antibodies targeting membrane-distal epitopes (including V1/V2, V3, and CD4bs) are more likely to activate universal CAR-NK cells against gp160+ target cells, compared with those targeting membrane-proximal epitopes located in the gp41 MPER. Finally, we confirm that HIV-infected primary human CD4+ T cells can be effectively killed using the same approach. Given that numerous anti-gp160 antibodies with different antigen specificities are readily available, this modular universal CAR-NK cell platform can potentially overcome HIV diversity, thus providing a promising strategy to eradicate HIV-infected cells.

Rapid Induction of Multifunctional Antibodies in Rabbits and Macaques by Clade C HIV-1 CAP257 Envelopes Circulating During Epitope-Specific Neutralization Breadth Development.

We report here on HIV-1 immunization results in rabbits and macaques co-immunized with clade C gp160 DNA and gp140 trimeric envelope vaccines, a strategy similar to a recent clinical trial that showed improved speed and magnitude of humoral responses. Clade C envelopes were isolated from CAP257, an individual who developed a unique temporal pattern of neutralization breadth development, comprising three separate "Waves" targeting distinct Env epitopes and different HIV clades. We used phylogeny and neutralization criteria to down-select envelope vaccine candidates, and confirmed antigenicity of our antigens by interaction with well-characterized broadly neutralizing monoclonal antibodies. Using these envelopes, we performed rabbit studies that screened for immunogenicity of CAP257 Envs from timepoints preceding peak neutralization breadth in each Wave. Selected CAP257 envelopes from Waves 1 and 2, during the first 2 years of infection that were highly immunogenic in rabbits were then tested in macaques. We found that in rabbits and macaques, co-immunization of DNA, and protein envelope-based vaccines induced maximum binding and neutralizing antibody titers with three immunizations. No further benefit was obtained with additional immunizations. The vaccine strategies recapitulated the Wave-specific epitope targeting observed in the CAP257 participant, and elicited Tier 1A, 1B, and Tier 2 heterologous neutralization. CAP257 envelope immunogens also induced the development of ADCC and TFH responses in macaques, and these responses positively correlated with heterologous neutralization. Together, the results from two animal models in this study have implications for identifying effective vaccine immunogens. We used a multi-step strategy to (1) select an Env donor with well-characterized neutralization breadth development; (2) study Env phylogeny for potential immunogens circulating near peak breadth timepoints during the first 2 years of infection; (3) test down-selected Envs for antigenicity; (4) screen down-selected Envs in an effective vaccine regimen in rabbits; and (5) advance the most immunogenic Envs to NHP studies. The results were an induction of high titers of HIV-1 envelope-specific antibodies with increasing avidity and cross-clade neutralizing antibodies with effector functions that together may improve the potential for protection in a pre-clinical SHIV model.

Sulfonate modified Lactoferrin nanoparticles as drug carriers with dual activity against HIV-1.

Intriguing properties and structural dynamics of Lactoferrin have been exploited in numerous applications, including its use as self-assembling, pH sensitive nanoparticles to deliver intended cargo at the disease site. In this study, we explore the possibility of surface modification of Lactoferrin nanoparticles to hone its specificity to target HIV-1 infected cells. Existence of free cysteine groups on Lactoferrin nanoparticles available for reaction with external molecules facilitates conjugation on the surface with Sodium 2-mercaptoethanesulfonate (MES). Conjugation with MES is used to edge a negative charge that can mimic CCR5 and Heparan sulfate (initial point of contact of HIV-1 env to host cell surface) electrostatic charge (Sulfate group). A simple sono-chemical irradiation method was employed for self-assembly of Nanoparticles and for surface modification. The nanoparticles serve dual purpose to abrogate extracellular entry and to target viral enzymes, when loaded with ART drugs. The morphology and size distribution of the formed particles were explored using Transmission Electron Microscope (TEM), Scanning Electron Microscope (SEM) and Dynamic Light Scattering. Raman SERS was employed to understand the difference in the protein upon surface modification. The anti-HIV property of the particles was confirmed in-vitro. The modified device demonstrated acceptable nanoparticle properties with controlled release and higher effective concentration in the area of infection.

Quantitative HIV-1-specific antibodies as predictors of peripheral blood cell-associated HIV-1 DNA concentrations.

OBJECTIVE: This study evaluated HIV-1 antibody levels as predictors of cell-associated HIV-1 DNA levels in perinatally infected (PHIV) children with long-term viral suppression on antiretroviral therapy (ART). DESIGN: HIV-1 antibody and HIV-1 DNA levels were measured in blood specimens from 61 children and adolescents from the Pediatric HIV/AIDS Cohort Study: Adolescent Master Protocol. Twenty perinatally HIV-1-exposed, uninfected children studied through 2 years served as controls. METHODS: HIV-1 IgG antibodies to six HIV-1 proteins were measured by quantitative ELISA; HIV-1 DNA levels were measured by droplet digital PCR. RESULTS: Among 13 children with viral suppression at less than 1 year, antibodies to gp160 and gp41 were low but stable longitudinally; antibodies to p17, p24, and RT decreased, and antibodies to p31 were low or undetectable. Among 48 children with viral suppression between 1 and 5 years, antibody levels to all six HIV-1 proteins were higher than in children with earlier viral suppression and remained high over time. A receiver operator curve approach identified gp41 and gp160 as useful predictors of HIV-1 DNA less than 10 or less than 100 copies per million PBMC (cpm); C-statistics including all antibodies ranged from 0.75 to 0.77. An ensemble learning approach also identified gp41 and gp160 as important predictors of HIV-1 DNA less than 10 or less than 100 cpm; area under the curve estimates utilizing all HIV-1 antibodies ranged from 0.70 to 0.81. CONCLUSION: Quantitative HIV-1 gp41 and gp160 antibody levels may serve as rapid, inexpensive screening tools for low PBMC HIV-1 DNA levels in children with viral suppression on ART, facilitating inclusion into remission protocols.

Cryo-EM Structure of Full-length HIV-1 Env Bound With the Fab of Antibody PG16.

The HIV-1 envelope protein (Env) is the target of neutralizing antibodies and the template for vaccine immunogen design. The dynamic conformational equilibrium of trimeric Env influences its antigenicity and potential immunogenicity. Antibodies that bind at the trimer apex stabilize a "closed" conformation characteristic of the most difficult to neutralize isolates. A goal of vaccine development is therefore to mimic the closed conformation in a designed immunogen. A disulfide-stabilized, trimeric Env ectodomain-the "SOSIP" construct-has many of the relevant properties; it is also particularly suitable for structure determination. Some single-molecule studies have, however, suggested that the SOSIP trimer is not a good representation of Env on the surface of a virion or an infected cell. We isolated Env (fully cleaved to gp120 and gp41) from the surface of expressing cells using tagged, apex-binding Fab PG16 and determined the structure of the PG16-Env complex by cryo-EM to an overall resolution of 4.6 Å. Placing the only purification tag on the Fab ensured that the isolated Env was continuously stabilized in its closed, native conformation. The Env structure in this complex corresponds closely to the SOSIP structures determined by both x-ray crystallography and cryo-EM. Although the membrane-interacting elements are not resolved in our reconstruction, we can make inferences about the connection between ectodomain and membrane-proximal external region (MPER) by reference to the published cryo-tomography structure of an Env "spike" and the NMR structure of the MPER-transmembrane segment. We discuss these results in view of the conflicting interpretations in the literature.

Domain-Scan: Combinatorial Sero-Diagnosis of Infectious Diseases Using Machine Learning.

The presence of pathogen-specific antibodies in an individual's blood-sample is used as an indication of previous exposure and infection to that specific pathogen (e.g., virus or bacterium). Measurement of the diagnostic antibodies is routinely achieved using solid phase immuno-assays such as ELISA tests and western blots. Here, we describe a sero-diagnostic approach based on phage-display of epitope arrays we term "Domain-Scan". We harness Next-generation sequencing (NGS) to measure the serum binding to dozens of epitopes derived from HIV-1 and HCV simultaneously. The distinction of healthy individuals from those infected with either HIV-1 or HCV, is modeled as a machine-learning classification problem, in which each determinant ("domain") is considered as a feature, and its NGS read-out provides values that correspond to the level of determinant-specific antibodies in the sample. We show that following training of a machine-learning model on labeled examples, we can very accurately classify unlabeled samples and pinpoint the domains that contribute most to the classification. Our experimental/computational Domain-Scan approach is general and can be adapted to other pathogens as long as sufficient training samples are provided.

Neutralization Breadth and Potency of Single-Chain Variable Fragments Derived from Broadly Neutralizing Antibodies Targeting Multiple Epitopes on the HIV-1 Envelope.

Passive administration of HIV-directed broadly neutralizing antibodies (bNAbs) can prevent infection in animal models, and human efficacy trials are under way. Single-chain variable fragments (scFv), comprised of only the variable regions of antibody heavy and light chains, are smaller molecules that may offer advantages over full-length IgG. We designed and expressed scFv of HIV bNAbs prioritized for clinical testing that target the V2-apex (CAP256-VRC26.25), V3-glycan supersite (PGT121), CD4 binding site (3BNC117), and MPER (10E8v4). The use of either a 15- or 18-amino-acid glycine-serine linker between the heavy- and light-chain fragments provided adequate levels of scFv expression. When tested against a 45-multisubtype virus panel, all four scFv retained good neutralizing activity, although there was variable loss of function compared to the parental IgG antibodies. For CAP256-VRC26.25, there was a significant 138-fold loss of potency that was in part related to differential interaction with charged amino acids at positions 169 and 170 in the V2 epitope. Potency was reduced for the 3BNC117 (13-fold) and PGT121 (4-fold) scFv among viruses lacking the N276 and N332 glycans, respectively, and in viruses with a longer V1 loop for PGT121. This suggested that scFv interacted with their epitopes in subtly different ways, with variation at key residues affecting scFv neutralization more than the matched IgGs. Remarkably, the scFv of 10E8v4 maintained breadth of 100% with only a minor reduction in potency. Overall, scFv of clinically relevant bNAbs had significant neutralizing activity, indicating that they are suitable for passive immunization to prevent HIV-1 infection.IMPORTANCE Monoclonal antibodies have been isolated against conserved epitopes on the HIV trimer and are being investigated for passive immunization. Some of the challenges associated with full-sized antibody proteins may be overcome by using single-chain variable fragments (scFv). These smaller forms of antibodies can be produced more efficiently, may show fewer off-target effects with increased tissue penetration, and are more adaptable to vectored-mediated expression than IgG. Here, we demonstrate that scFv of four HIV-directed bNAbs (CAP256-VRC26.25, PGT121, 3BNC117, and 10E8v4) had significant neutralizing activity against diverse global strains of HIV. Loss of potency and/or breadth was shown to be due to increased dependence of the scFv on key residues within the epitope. These smaller antibody molecules with functional activity in the therapeutic range may be suitable for further development as passive immunity for HIV prevention.