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SummaryHIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1Å sub-tomogram averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers plus a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

SummaryHIV-1 clade C envelope immunogens that elicit both neutralizing and non-neutralizing V1V2-scaffold specific antibodies (protective correlates from RV144 human trial) are urgently needed due to the prevalence of this clade in the most impacted regions worldwide. To achieve this, we introduced structure-guided changes followed by consensus-C sequence-guided optimizations at the V2-region to generate UFO-v2-RQH173 trimer. This improved the abundance of native-like trimers and carried an intrinsic dynamic V2-loop. Following immunization of rabbits, the wild-type protein failed to elicit any autologous neutralizing antibodies but UFO-v2-RQH173 elicited both autologous neutralizing and broad V1V2-scaffold antibodies. The variant with 173Y modification in V2-region, most prevalent among HIV-1 sequences, showed decreased ability in displaying native-like V1V2 epitope with time in-vitro and elicited antibodies with lower neutralizing and higher V1V2-scaffold activities. Our results identify a clade C C.1086-UFO-v2-RQH173 trimer capable of eliciting improved neutralizing and V1V2-scaffold antibodies, and reveal the importance of V2-region in tuning this.

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

HIV-1 clade C envelope immunogens that elicit both neutralizing and non-neutralizing V1V2-scaffold-specific antibodies (protective correlates from RV144 human trial) are urgently needed due to the prevalence of this clade in the most impacted regions worldwide. To achieve this, we introduce structure-guided changes followed by consensus-C-sequence-guided optimizations at the V2 region to generate UFO-v2-RQH

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers ten-fold higher than the prefusion-stabilized spike despite a five-fold lower dose. Antibodies elicited by the RBD-nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic., Highlights • Two-component nanoparticle platform enabled rapid generation of SARS-CoV-2 vaccines • The RBD-nanoparticle vaccines elicit potent neutralizing antibody responses • Nanoparticle vaccine-elicited antibodies target multiple non-overlapping epitopes • The lead nanoparticle vaccine candidate is being manufactured for clinical trials, Walls et al. describe a potential nanoparticle vaccine for COVID-19, made of a self-assembling protein nanoparticle displaying the SARS-CoV-2 receptor-binding domain in a highly immunogenic array reminiscent of the natural virus. Their nanoparticle vaccine candidate elicits a diverse, potent, and protective antibody response, including neutralizing antibody titers ten-fold higher than the prefusion-stabilized spike ectodomain trimer.

Much of our understanding of protein structure and mechanistic function has been derived from static high-resolution structures. As structural biology has continued to evolve it has become clear that high-resolution structures alone are unable to fully capture the mechanistic basis for protein structure and function in solution. Recently Hydrogen/Deuterium-exchange Mass Spectrometry (HDX-MS) has developed into a powerful and versatile tool for structural biologists that provides novel insights into protein structure and function. HDX-MS enables direct monitoring of a protein's structural fluctuations and conformational changes under native conditions in solution even as it is carrying out its functions. In this review, we focus on the use of HDX-MS to monitor these dynamic changes in proteins. We examine how HDX-MS has been applied to study protein structure and function in systems ranging from large, complex assemblies to intrinsically disordered proteins, and we discuss its use in probing conformational changes during protein folding and catalytic function. STATEMENT FOR A BROAD AUDIENCE: The biophysical and structural characterization of proteins provides novel insight into their functionalities. Protein motions, ranging from small scale local fluctuations to larger concerted structural rearrangements, often determine protein function. Hydrogen/Deuterium-exchange Mass Spectrometry (HDX-MS) has proven a powerful biophysical tool capable of probing changes in protein structure and dynamic protein motions that are often invisible to most other techniques.

The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy minimum, the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.