Broad protection against clade 1 sarbecoviruses after a single immunization with cocktail spike-protein-nanoparticle vaccine.

The 2002 SARS outbreak, the 2019 emergence of COVID-19, and the continuing evolution of immune-evading SARS-CoV-2 variants together highlight the need for a broadly protective vaccine against ACE2-utilizing sarbecoviruses. While updated variant-matched formulations are a step in the right direction, protection needs to extend beyond SARS-CoV-2 and its variants to include SARS-like viruses. Here, we introduce bivalent and trivalent vaccine formulations using our spike protein nanoparticle platform that completely protect female hamsters against BA.5 and XBB.1 challenges with no detectable virus in the lungs. The trivalent cocktails elicit highly neutralizing responses against all tested Omicron variants and the bat sarbecoviruses SHC014 and WIV1. Finally, our 614D/SHC014/XBB trivalent spike formulation completely protects human ACE2-transgenic female hamsters against challenges with WIV1 and SHC014 with no detectable virus in the lungs. Collectively, these results illustrate that our trivalent protein-nanoparticle cocktail can provide broad protection against SARS-CoV-2-like and SARS-CoV-1-like sarbecoviruses.

The mating pilus of E. coli pED208 acts as a conduit for ssDNA during horizontal gene transfer.

IMPORTANCE: Bacteria are constantly exchanging DNA, which constitutes horizontal gene transfer. While some of these occurs by a non-specific process called natural transformation, some occurs by a specific mating between a donor and a recipient cell. In specific conjugation, the mating pilus is extended from the donor cell to make contact with the recipient cell, but whether DNA is actually transferred through this pilus or by another mechanism involving the type IV secretion system complex without the pilus has been an open question. Using Escherichia coli, we show that DNA can be transferred through this pilus between a donor and a recipient cell that has not established a tight mating junction, providing a new picture for the role of this pilus.

Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Tad and toxin-coregulated pilus structures reveal unexpected diversity in bacterial type IV pili.

Type IV pili (T4P) are ubiquitous in both bacteria and archaea. They are polymers of the major pilin protein, which has an extended and protruding N-terminal helix, α1, and a globular C-terminal domain. Cryo-EM structures have revealed key differences between the bacterial and archaeal T4P in their C-terminal domain structure and in the packing and continuity of α1. This segment forms a continuous α-helix in archaeal T4P but is partially melted in all published bacterial T4P structures due to a conserved helix breaking proline at position 22. The tad (tight adhesion) T4P are found in both bacteria and archaea and are thought to have been acquired by bacteria through horizontal transfer from archaea. Tad pilins are unique among the T4 pilins, being only 40 to 60 residues in length and entirely lacking a C-terminal domain. They also lack the Pro22 found in all high-resolution bacterial T4P structures. We show using cryo-EM that the bacterial tad pilus from Caulobacter crescentus is composed of continuous helical subunits that, like the archaeal pilins, lack the melted portion seen in other bacterial T4P and share the packing arrangement of the archaeal T4P. We further show that a bacterial T4P, the Vibrio cholerae toxin coregulated pilus, which lacks Pro22 but is not in the tad family, has a continuous N-terminal α-helix, yet its α1 s are arranged similar to those in other bacterial T4P. Our results highlight the role of Pro22 in helix melting and support an evolutionary relationship between tad and archaeal T4P.

Correlative montage parallel array cryo-tomography for in situ structural cell biology.

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here we present robust tools for montage parallel array cryo-tomography (MPACT) tailored for vitrified specimens. The combination of correlative cryo-fluorescence microscopy, focused-ion-beam milling, substrate micropatterning, and MPACT supports studies that contextually define the three-dimensional architecture of cells. To further extend the flexibility of MPACT, tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

Integrated Fluorescence Microscopy (iFLM) for Cryo-FIB-milling and In-situ Cryo-ET.

Correlative cryo-FLM-FIB milling is a powerful sample preparation technique for in situ cryo-ET. However, correlative workflows that incorporate precise targeting remain challenging. Here, we demonstrate the development and use of an integrated Fluorescence Light Microscope (iFLM) module within a cryo-FIB-SEM to enable a coordinate-based two-point 3D correlative workflow. The iFLM guided targeting of regions of interest coupled with an automated milling process of the cryo-FIB-SEM instrument allows for the efficient preparation of 9-12 ∼200 nm thick lamellae within 24 hours. Using regular and montage-cryo-ET data collection schemes, we acquired data from FIB-milled lamellae of HeLa cells to examine cellular ultrastructure. Overall, this workflow facilitates on-the-fly targeting and automated FIB-milling of cryo-preserved cells, bacteria, and possibly high pressure frozen tissue, to produce lamellae for downstream cryo-ET data collection.

Handling difficult cryo-ET samples: A study with primary neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Atomic-level architecture of Caulobacter crescentus flagellar filaments provide evidence for multi-flagellin filament stabilization.

Flagella are dynamic, ion-powered machines with assembly pathways that are optimized for efficient flagella production. In bacteria, dozens of genes are coordinated at specific times in the cell lifecycle to generate each component of the flagellum. This is the case for Caulobacter crescentus, but little is known about why this species encodes six different flagellin genes. Furthermore, little is known about the benefits multi-flagellin species possess over single flagellin species, if any, or what molecular properties allow for multi-flagellin filaments to assemble. Here we present an in-depth analysis of several single flagellin filaments from C. crescentus, including an extremely well-resolved structure of a bacterial flagellar filament. We highlight key molecular interactions that differ between each bacterial strain and speculate how these interactions may alleviate or impose helical strain on the overall architecture of the filament. We detail conserved residues within the flagellin subunit that allow for the synthesis of multi-flagellin filaments. We further comment on how these molecular differences impact bacterial motility and highlight how no single flagellin filament achieves wild-type levels of motility, suggesting C. crescentus has evolved to produce a filament optimized for motility comprised of six flagellins. Finally, we highlight an ordered arrangement of glycosylation sites on the surface of the filaments and speculate how these sites may protect the ß-hairpin located on the surface exposed domain of the flagellin subunit.

The role of CenKR in the coordination of Rhodobacter sphaeroides cell elongation and division.

Cell elongation and division are essential aspects of the bacterial life cycle that must be coordinated for viability and replication. The impact of misregulation of these processes is not well understood as these systems are often not amenable to traditional genetic manipulation. Recently, we reported on the CenKR two-component system (TCS) in the Gram-negative bacterium Rhodobacter sphaeroides that is genetically tractable, widely conserved in α-proteobacteria, and directly regulates the expression of components crucial for cell elongation and division, including genes encoding subunit of the Tol-Pal complex. In this work, we show that overexpression of cenK results in cell filamentation and chaining. Using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we generated high-resolution two-dimensional (2D) images and three-dimensional (3D) volumes of the cell envelope and division septum of wild-type cells and a cenK overexpression strain finding that these morphological changes stem from defects in outer membrane (OM) and peptidoglycan (PG) constriction. By monitoring the localization of Pal, PG biosynthesis, and the bacterial cytoskeletal proteins MreB and FtsZ, we developed a model for how increased CenKR activity leads to changes in cell elongation and division. This model predicts that increased CenKR activity decreases the mobility of Pal, delaying OM constriction, and ultimately disrupting the midcell positioning of MreB and FtsZ and interfering with the spatial regulation of PG synthesis and remodeling. IMPORTANCE By coordinating cell elongation and division, bacteria maintain their shape, support critical envelope functions, and orchestrate division. Regulatory and assembly systems have been implicated in these processes in some well-studied Gram-negative bacteria. However, we lack information on these processes and their conservation across the bacterial phylogeny. In R. sphaeroides and other α-proteobacteria, CenKR is an essential two-component system (TCS) that regulates the expression of genes known or predicted to function in cell envelope biosynthesis, elongation, and/or division. Here, we leverage unique features of CenKR to understand how increasing its activity impacts cell elongation/division and use antibiotics to identify how modulating the activity of this TCS leads to changes in cell morphology. Our results provide new insight into how CenKR activity controls the structure and function of the bacterial envelope, the localization of cell elongation and division machinery, and cellular processes in organisms with importance in health, host-microbe interactions, and biotechnology.

Multivalent S2-based vaccines provide broad protection against SARS-CoV-2 variants of concern and pangolin coronaviruses.

BACKGROUND: The COVID-19 pandemic continues to cause morbidity and mortality worldwide. Most approved COVID-19 vaccines generate a neutralizing antibody response that primarily targets the highly variable receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein. SARS-CoV-2 "variants of concern" have acquired mutations in this domain allowing them to evade vaccine-induced humoral immunity. Recent approaches to improve the breadth of protection beyond SARS-CoV-2 have required the use of mixtures of RBD antigens from different sarbecoviruses. It may therefore be beneficial to develop a vaccine in which the protective immune response targets a more conserved region of the S protein. METHODS: Here we have developed a vaccine based on the conserved S2 subunit of the S protein and optimized the adjuvant and immunization regimen in Syrian hamsters and BALB/c mice. We have characterized the efficacy of the vaccine against SARS-CoV-2 variants and other coronaviruses. FINDINGS: Immunization with S2-based constructs elicited a broadly cross-reactive IgG antibody response that recognized the spike proteins of not only SARS-CoV-2 variants, but also SARS-CoV-1, and the four endemic human coronaviruses. Importantly, immunization reduced virus titers in respiratory tissues in vaccinated animals challenged with SARS-CoV-2 variants B.1.351 (beta), B.1.617.2 (delta), and BA.1 (omicron) as well as a pangolin coronavirus. INTERPRETATION: These results suggest that S2-based constructs can elicit a broadly cross-reactive antibody response resulting in limited virus replication, thus providing a framework for designing vaccines that elicit broad protection against coronaviruses. FUNDING: NIH, Japan Agency for Medical Research and Development, Garry Betty/ V Foundation Chair Fund, and NSF.

A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence.

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activity of the two immunized RM was found to be dependent on the gp120 V5 region. Interestingly, the exact same residues of V5 were also targeted by a neutralizing monoclonal antibody (nmAb) isolated from the subject Z1800M early in infection. Glycan profiling and computational modeling of the Z1800M Env gp120 immunogen provided further evidence that the V5 loop is exposed in this T/F Env and was a dominant feature that drove neutralizing antibody targeting during infection and immunization. An expanded B cell clonotype was isolated from one of the neutralization-positive RM and nmAbs corresponding to this group demonstrated V5-dependent neutralization similar to both the RM serum and the human Z1800M nmAb. The results demonstrate that neutralizing antibody responses elicited by the Z1800M T/F Env in RM converged with those in the HIV-1 infected human subject, illustrating the potential of using immunogens based on this or other T/F Envs with well-defined immunogenicity as a starting point to drive breadth.

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

Multivalent nanoparticle-based vaccines protect hamsters against SARS-CoV-2 after a single immunization.

The COVID-19 pandemic continues to wreak havoc as worldwide SARS-CoV-2 infection, hospitalization, and death rates climb unabated. Effective vaccines remain the most promising approach to counter SARS-CoV-2. Yet, while promising results are emerging from COVID-19 vaccine trials, the need for multiple doses and the challenges associated with the widespread distribution and administration of vaccines remain concerns. Here, we engineered the coat protein of the MS2 bacteriophage and generated nanoparticles displaying multiple copies of the SARS-CoV-2 spike (S) protein. The use of these nanoparticles as vaccines generated high neutralizing antibody titers and protected Syrian hamsters from a challenge with SARS-CoV-2 after a single immunization with no infectious virus detected in the lungs. This nanoparticle-based vaccine platform thus provides protection after a single immunization and may be broadly applicable for protecting against SARS-CoV-2 and future pathogens with pandemic potential.