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Neutralizing antibodies (NAbs) to multiple epitopes on the HIV-1-envelope glycoprotein (Env) have been isolated from infected persons. The potency of NAbs is measured more often than the size of the persistent fraction of infectivity at maximum neutralization, which may also influence preventive efficacy of active or passive immunization and the therapeutic outcome of the latter. Many NAbs neutralize HIV-1 CZA97.012, a clone of a Clade-C isolate, to ~100%. But here NAb PGT151, directed to a fusion-peptide epitope, left a persistent fraction of 15%. NAb PGT145, ligating the Env-trimer apex, left no detectable persistent fraction. The divergence in persistent fractions was further analyzed by depletion of pseudoviral populations of the most PGT151- and PGT145-reactive virions. Thereby, neutralization by the non-depleting NAb increased, whereas neutralization by the depleting NAb decreased. Furthermore, depletion by PGT151 increased sensitivity to autologous neutralization by sera from rabbits immunized with soluble native-like CZA97.012 trimer: substantial persistent fractions were reduced. NAbs in these sera target epitopes comprising residue D411 at the V4-β19 transition in a defect of the glycan shield on CZA97.012 Env. NAb binding to affinity-fractionated soluble native-like CZA97.012 trimer differed commensurately with neutralization in analyses by ELISA and surface plasmon resonance. Glycan differences between PGT151- and PGT145-purified trimer fractions were then demonstrated by mass spectrometry, providing one explanation for the differential antigenicity. These differences were interpreted in relation to a new structure at 3.4-Å resolution of the soluble CZA97.012 trimer determined by cryo-electron microscopy. The trimer adopted a closed conformation, refuting apex opening as the cause of reduced PGT145 binding to the PGT151-purified form. The evidence suggests that differences in binding and neutralization after trimer purification or pseudovirus depletion with PGT145 or PGT151 are caused by variation in glycosylation, and that some glycan variants affect antigenicity through direct effects on antibody contacts, whereas others act allosterically.

Not all persons recovering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection develop SARS-CoV-2–specific antibodies. We show that nonseroconversion is associated with younger age and higher reverse transcription PCR cycle threshold values and identify SARS-CoV-2 viral loads in the nasopharynx as a major correlate of the systemic antibody response.

Mapping polyclonal serum responses is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual antibodies, which incompletely defines the polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. We used mutational antigenic profiling to determine how all mutations in Env affected viral neutralization and electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize serum Fabs bound to Env trimers. The dominant neutralizing specificities were generally only a subset of the more diverse binding specificities. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Furthermore, we refined residue-level epitope specificity directly from sera, revealing subtle differences across sera. Together, mutational antigenic profiling and EMPEM yield a holistic view of the binding and neutralizing specificity of polyclonal sera.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Fusion between the viral and target cell membranes is an obligatory step for the infectivity of all enveloped virus, and blocking this process is a clinically validated therapeutic strategy.Viral fusion is driven by specialized proteins which, although specific to each virus, act through a common mechanism, the formation of a complex between two heptad repeat (HR) regions. The HR regions are initially separated in an intermediate termed "prehairpin", which bridges the viral and cell membranes, and then fold onto each other to form a 6-helical bundle (6HB), driving the two membranes to fuse. HR-derived peptides can inhibit viral infectivity by binding to the prehairpin intermediate and preventing its transition to the 6HB.The antiviral activity of HR-derived peptides differs considerably among enveloped viruses. For weak inhibitors, potency can be increased by peptide engineering strategies, but sequence-specific optimization is time-consuming. In seeking ways to increase potency without changing the native sequence, we previously reported that attachment to the HR peptide of a cholesterol group ("cholesterol-tagging") dramatically increases its antiviral potency, and simultaneously increases its half-life in vivo. We show here that antiviral potency may be increased by combining cholesterol-tagging with dimerization of the HR-derived sequence, using as examples human parainfluenza virus, Nipah virus, and HIV-1. Together, cholesterol-tagging and dimerization may represent strategies to boost HR peptide potency to levels that in some cases may be compatible with in vivo use, possibly contributing to emergency responses to outbreaks of existing or novel viruses.