Broadly neutralizing antibodies (bNAbs) against HIV-1-Env V1V2 arise in multiple donors. However, atomic-level interactions had only been determined with antibodies from a single donor, making commonalities in recognition uncertain. Here we report the co-crystal structure of V1V2 with antibody CH03 from a second donor and model Env interactions of antibody CAP256-VRC26 from a third. These V1V2-directed bNAbs utilized strand-strand interactions between a protruding antibody loop and a V1V2 strand, but differed in their N-glycan recognition. Ontogeny analysis indicated protruding loops to develop early, with glycan interactions maturing over time. Altogether, the multidonor information suggested V1V2-directed bNAbs to form an ‘extended class’, for which we engineered ontogeny-specific antigens: Env trimers with chimeric V1V2s that interacted with inferred ancestor and intermediate antibodies. The ontogeny-based design of vaccine antigens described here may provide a general means for eliciting antibodies of a desired class.

This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

Elastin-like polypeptides (ELPs) are stimulus responsive peptide polymers that exhibit inverse temperature phase transition behavior, causing an ELP to aggregate above its inverse transition temperature (Tt). Although this property has been exploited in a variety of biotechnological applications, de novo design of ELPs that display a specific Tt is not trivial because the Tt of an ELP is a complex function of several variables, including its sequence, chain length, polypeptide concentration, and the type and concentration of cosolutes in solution. This paper provides a quantitative model that predicts the Tt of a family of ELPs (Val-Pro-Gly-Xaa-Gly, where Xaa = Ala and/or Val) from their composition, chain length, and concentration in phosphate buffered saline. This model will enable de novo prediction of the amino acid sequence and chain length of ELPs that will display a predetermined Tt in physiological buffer within a specified concentration regime, thereby greatly facilitating the design of new ELPs for applications in medicine and biotechnology.

New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100 nm size, near monodisperse nanoparticles upon conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a four-fold higher maximum tolerated dose than free drug, and induce nearly complete tumor regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents, and targeting moieties into multifunctional nanomedicines.

We present a technology to screen natively-paired human antibody repertoires from millions of B cells. Libraries of natively-paired variable region heavy and light (VH:VL) amplicons are expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)–directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of “public” antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that “public” NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.

Packaging clinically relevant hydrophobic drugs into a self-assembled nanoparticle can improve their aqueous solubility, plasma half-life, tumor specific uptake and therapeutic potential. To this end, here we conjugated paclitaxel (PTX) to recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into ~60-nm diameter near-monodisperse nanoparticles that increased the systemic exposure of PTX by 7-fold compared to free drug and 2-fold compared to the FDA approved taxane nanoformulation (Abraxane®). The tumor uptake of the CP-PTX nanoparticle was 5-fold greater than free drug and 2-fold greater than Abraxane. In a murine cancer model of human triple negative breast cancer and prostate cancer, CP-PTX induced near complete tumor regression after a single dose in both tumor models, whereas at the same dose, no mice treated with Abraxane survived for more than 80 days (breast) and 60 days (prostate) respectively. These results show that a molecularly engineered nanoparticle with precisely engineered design features outperforms Abraxane, the current gold standard for paclitaxel delivery.

High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCRβ/δ+TCRα/γ) at the single-cell level for typical samples containing >10(4) lymphocytes is problematic, because immune receptors comprise two polypeptide chains that are encoded by separate mRNAs. Here we present a technology that allows rapid and low-cost determination of a paired immune receptor repertoire from millions of cells with high precision (>97%). Flow focusing is used to encapsulate single cells in emulsions containing magnetic beads for mRNA capture. The mRNA transcripts are then reverse-transcribed, physically linked to their partners by overlap extension PCR, and interrogated by high-throughput paired-end Illumina sequencing. This protocol describes the construction and operation of the flow-focusing device in detail, as well as the bioinformatics pipeline used to interpret the data. The entire procedure can be performed by a single researcher in under 12 h of effort per sample.