Recapitulation of HIV-1 Env-antibody coevolution in macaques leading to neutralization breadth.

Neutralizing antibodies elicited by HIV-1 coevolve with viral envelope proteins (Env) in distinctive patterns, in some cases acquiring substantial breadth. We report that primary HIV-1 envelope proteins-when expressed by simian-human immunodeficiency viruses in rhesus macaques-elicited patterns of Env-antibody coevolution very similar to those in humans, including conserved immunogenetic, structural, and chemical solutions to epitope recognition and precise Env-amino acid substitutions, insertions, and deletions leading to virus persistence. The structure of one rhesus antibody, capable of neutralizing 49% of a 208-strain panel, revealed a V2 apex mode of recognition like that of human broadly neutralizing antibodies (bNAbs) PGT145 and PCT64-35S. Another rhesus antibody bound the CD4 binding site by CD4 mimicry, mirroring human bNAbs 8ANC131, CH235, and VRC01. Virus-antibody coevolution in macaques can thus recapitulate developmental features of human bNAbs, thereby guiding HIV-1 immunogen design.

Crystal structure of the metazoan Nup62•Nup58•Nup54 nucleoporin complex.

Nuclear pore complexes (NPCs) conduct nucleocytoplasmic transport and gain transport selectivity through nucleoporin FG domains. Here, we report a structural analysis of the FG Nup62•58•54 complex, which is a crucial component of the transport system. It comprises a ≈13 nanometer-long trimerization interface with an unusual 2W3F coil, a canonical heterotrimeric coiled coil, and a kink that enforces a compact six-helix bundle. Nup54 also contains a ferredoxin-like domain. We further identified a heterotrimeric Nup93-binding module for NPC anchorage. The quaternary structure alternations in the Nup62 complex, which were previously proposed to trigger a general gating of the NPC, are incompatible with the trimer structure. We suggest that the highly elongated Nup62 complex projects barrier-forming FG repeats far into the central NPC channel, supporting a barrier that guards the entire cross section.

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation.

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks ­ each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.

Identification and characterization of immunodominant B-cell epitope of the C-terminus of protective antigen of Bacillus anthracis.

Bacillus anthracis is the etiological agent of anthrax. Protective antigen (PA) has been established as the key protective immunogen and is the major component of anthrax vaccine. Prior studies have indicated that C-terminus host cell receptor binding region contains dominant protective epitopes of PA. In the present study, we focused our attention on determining B-cell epitopes from this region, which could be employed as a vaccine. Using B-cell epitope prediction systems, three regions were identified; ID-I: 604-622, ID-II: 626-676 and ID-III: 707-723 aa residues. These epitopes elicited potent B-cell response in BALB/c mice. ID-II in particular was found to be highly immunogenic in terms of IgG antibody titre, with a predominantly IgG1/IgG2a subclass distribution indicating Th2 bias and high affinity/avidity index. Effective cellular immunity was additionally generated which also signified its Th2 bias. Further, ID-II induced high level of lethal toxin neutralizing antibodies and robust protective immunity (66%) against in vivo lethal toxin challenge. Thus, ID-II can be classified as an immunodominant B-cell epitope and may prove significant in the development of an effective immunoprophylactic strategy against anthrax.