We report the discovery and crystal structure of a human mycoplasma protein, Protein M, which binds with high affinity to antibodies, predominantly through attachment to the variable region of the κ and λ light chains. Protein M broadly blocks antibody-antigen union and its mechanism of inhibition is of considerable interest because, as a diversity system, the binding mode of each antibody is different. Protein M thus appears to function by a mechanism that is independent of the sequences of members of the extensive antibody repertoire. By anchoring to conserved regions of the antibody light chains, Protein M is in a position to extend its large C-terminal domain over the antibody combining site and block entrance to macromolecular antigens.
Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell-wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of two CwlT from pathogens Staphylococcus aureus mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific for tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release, or closing during catalysis.
bifunctional cell-wall lysin; bacterial lysozyme; muramidase; NlpC/P60 endopeptidase; Tn916 family conjugative transposons
Telehealth appears to be an ideal mechanism for assisting rural patients and doctors and medical students/registrars in accessing specialist services. Telehealth is the use of enhanced broadband technology to provide telemedicine and education over distance. It provides accessible support to rural primary care providers and medical educators. A telehealth consultation is where a patient at a general practice, with the assistance of the general practitioner or practice nurse, undertakes a consultation by videoconference with a specialist located elsewhere. Multiple benefits of telehealth consulting have been reported, particularly those relevant to rural patients and health care providers. However there is a paucity of research on the benefits of telehealth to medical education and learning.
This protocol explains in depth the process that will be undertaken by a collaborative group of universities and training providers in this unique project.
Training sessions in telehealth consulting will be provided for participating practices and students. The trial will then use telehealth consulting as a real-patient learning experience for students, general practitioner trainees, general practitioner preceptors, and trainees.
Results will be available when the trial has been completed in 2015.
The protocol has been written to reflect the overarching premise that, by building virtual communities of practice with users of telehealth in medical education, a more sustainable and rigorous model can be developed. The Telehealth Skills Training and Implementation Project will implement and evaluate a theoretically driven model of Internet-facilitated medical education for vertically integrated, community-based learning environments
telehealth; medical education; enhanced broadband
PF10014 is a novel family of 2-oxyglutarate-Fe2+-dependent dioxygenases that are involved in biosynthesis of antibiotics and regulation of biofilm formation, likely by catalyzing hydroxylation of free amino acids or other related ligands. The crystal structure of a PF10014 member from Methylibium petroleiphilum at 1.9 Å resolution shows strong structural similarity to cupin dioxygenases in overall fold and active site, despite very remote homology. However, one of the β-strands of the cupin catalytic core is replaced by a loop that displays conformational isomerism that likely regulates the active site.
PF10014/BsmA; cupin dioxygenase; free amino acids; 2-oxyglutarate; ferrous iron
The HIV-1 envelope glycoprotein (Env) trimer contains the receptor binding sites and membrane fusion machinery that introduce the viral genome into the host cell. As the only target for broadly neutralizing antibodies (bnAbs), Env is a focus for rational vaccine design. We present a cryo-electron microscopy reconstruction and structural model of a cleaved, soluble SOSIP gp140 trimer in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 Å resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1 and HR2 domains, and shielding glycans. The structure also provides insights into trimer assembly, gp120-gp41 interactions, and the CD4bs epitope cluster for bnAbs, which covers a more extensive area and defines a more complex site of vulnerability than previously described.
HIV-1 entry into CD4+ target cells is mediated by cleaved envelope glycoprotein (Env) trimers that have been challenging to characterize structurally. Here, we describe the crystal structure at 4.7 Å of an antigenically near-native, cleaved, stabilized, soluble Env trimer (termed BG505 SOSIP.664 gp140) in complex with a potent broadly neutralizing antibody, PGT122. The structure shows a pre-fusion state of gp41, the interaction between the component gp120 and gp41 subunits, and how a close association between the gp120 V1/V2/V3 loops stabilizes the trimer apex around the three-fold axis. The complete epitope of PGT122 on the trimer involves gp120 V1, V3 and several surrounding glycans. This trimer structure advances our understanding of how Env functions and is presented to the immune system, and provides a blueprint for structure-based vaccine design.
The 2013 outbreak of avian-origin H7N9 influenza in eastern China has raised concerns about its ability to transmit in the human population. The hemagglutinin glycoprotein of most human H7N9 viruses carries Leu226, a residue linked to adaptation of H2N2 and H3N2 pandemic viruses to human receptors. However, glycan array analysis of the H7 hemagglutinin reveals negligible binding to human-like α2-6-linked receptors and strong preference for a subset of avian-like α2-3-linked glycans recognized by all avian H7 viruses. Crystal structures of H7N9 hemagglutinin and six hemagglutinin-glycan complexes have elucidated the structural basis for preferential recognition of avian-like receptors. These findings suggest that the current human H7N9 viruses are poorly adapted for efficient human-to-human transmission.
A human monoclonal heterosubtypic antibody, MAb 3.1, with its heavy chain encoded by VH3-30, was isolated using phage display with immobilized hemagglutinin (HA) from influenza virus A/Japan/305/1957(H2N2) as the target. Antibody 3.1 potently neutralizes influenza viruses from the H1a clade (i.e., H1, H2, H5, H6) but has little neutralizing activity against the H1b clade. Its crystal structure in complex with HA from a pandemic H1N1 influenza virus, A/South Carolina/1/1918(H1N1), revealed that like other heterosubtypic anti-influenza virus antibodies, MAb 3.1 contacts a hydrophobic groove in the HA stem, primarily using its heavy chain. However, in contrast to the closely related monoclonal antibody (Mab) FI6 that relies heavily on HCDR3 for binding, MAb 3.1 utilizes residues from HCDR1, HCDR3, and framework region 3 (FR3). Interestingly, HCDR1 of MAb 3.1 adopts an α-helical conformation and engages in hydrophobic interactions with the HA very similar to those of the de novo in silico-designed and affinity-matured synthetic protein HB36.3. These findings improve our understanding of the molecular requirements for binding to the conserved epitope in the stem of the HA protein and, therefore, aid the development of more universal influenza vaccines targeting these epitopes.
IMPORTANCE Influenza viruses rapidly evade preexisting immunity by constantly altering the immunodominant neutralizing antibody epitopes (antigenic drift) or by acquiring new envelope serotypes (antigenic shift). As a consequence, the majority of antibodies elicited by immunization or infection protect only against the immunizing or closely related strains. Here, we describe a novel monoclonal antibody that recognizes the conserved heterosubtypic epitope in the stem of influenza A virus hemagglutinin. This antibody, referred to as MAb 3.1, recognizes its epitope in a manner that resembles recognition of a similar epitope by the de novo in silico-designed and affinity-matured synthetic protein HB36.3. Thus, besides providing novel insights into the molecular interactions between heterosubtypic antibodies and influenza virus hemagglutinin, MAb 3.1 demonstrates that de novo in silico-designed and affinity-matured synthetic proteins can foretell naturally selected antibody binding. This knowledge will aid development of a pan-influenza virus vaccine.
Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four human IgG isotypes. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.
Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 Å reveals a compact architecture composed of a central Ig-fold β-sandwich flanked by two additional protein layers. The CD81 receptor-binding site was identified by EM and by site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and EM E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide invaluable information for HCV drug and vaccine design.
Small molecules that react selectively with a specific non-enzyme drug-target protein in a complex biological environment without displacement of a leaving group (tracelessly) are rare and highly desirable. Herein, we describe the development of a family of fluorogenic stilbene-based vinyl amides and sulfonamides that covalently modify transthyretin (TTR) tracelessly. These small molecules bind selectively to TTR in complex biological environments and then undergo a rapid and chemoselective 1,4-Michael addition with the pKa-perturbed Lys-15 ε-amino group of TTR. Replacing the vinyl amide in 2 with the more reactive vinyl sulfonamide in 4 hastens conjugation kinetics. X-ray co-crystallography verifies formation of the secondary amine bond mediating conjugation in the case of 2 and 4 and confirms the expected orientation of the stilbene within the TTR binding sites. Vinyl amide 2 and sulfonamide 4 potently inhibit TTR dissociation and amyloid fibril formation in vitro. The TTR binding selectivity, modification yield, and reaction chemoselectivity of vinyl sulfonamide 4 is good enough in human plasma to serve as a starting point for medicinal chemistry efforts. Moreover, vinyl sulfonamide 4 is fluorogenic, i.e. it exhibits minimal background fluorescence in complex biological environments, remains dark upon binding to TTR, and only becomes fluorescent upon reaction with TTR. The fluorogenicity of 4 was utilized to accurately quantify the native TTR concentration in E. coli lysate using a fluorescence plate reader.
We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents.
Fluorogen Activating Protein; quaternary structure; cooperative binding; directed evolution; yeast surface display
Broadly neutralizing monoclonal antibodies (bnMAbs) that target the high-mannose patch centered around the glycan at position 332 on HIV Env are promising vaccine leads and therapeutic candidates as they effectively protect against mucosal SHIV challenge and strongly suppress SHIV viraemia in established infection in macaque models. However, these antibodies demonstrate varying degrees of dependency on the N332 glycan site and the origins of their neutralization breadth are not always obvious. By measuring neutralization on an extended range of glycan site viral variants, we found that some bnMAbs can utilize alternate N-linked glycans in the absence of the N332 glycan site and therefore neutralize a substantial number of viruses lacking the site. Furthermore, many of the antibodies can neutralize viruses in which the N332 glycan site is shifted to the 334 position. Finally, we found that a combination of three antibody families that target the high-mannose patch can lead to 99% neutralization coverage of a large panel of viruses containing the N332/334 glycan site and up to 66% coverage for viruses that lack the N332/334 glycan site. The results indicate that a diverse response against the high-mannose patch may provide near equivalent coverage as a combination of bnMAbs targeting multiple epitopes. Additionally, the ability of some bnMAbs to utilize other N-linked glycan sites can help counter neutralization escape mediated by shifting of glycosylation sites. Overall, this work highlights the importance of promiscuous glycan binding properties in bnMAbs to the high-mannose patch for optimal anti-viral activity either in protective or therapeutic modalities.
We describe a coumarin derivative that is non-fluorescent in aqueous buffers and that very selectively binds to transthyretin (TTR) in complex biological environments potently inhibiting TTR amyloidogenesis while also exhibiting sensitive off-on fluorescent sensing of the properly folded quaternary structure of TTR.
Animal models are invaluable tools which allow us to investigate the microbiome-host dialogue. However, experimental design introduces biases in the data that we collect, also potentially leading to biased conclusions. With obesity at pandemic levels animal models of this disease have been developed; we investigated the role of experimental design on one such rodent model. We used 454 pyrosequencing to profile the faecal bacteria of obese (n = 6) and lean (homozygous n = 6; heterozygous n = 6) Zucker rats over a 10 week period, maintained in mixed-genotype cages, to further understand the relationships between the composition of the intestinal bacteria and age, obesity progression, genetic background and cage environment. Phylogenetic and taxon-based univariate and multivariate analyses (non-metric multidimensional scaling, principal component analysis) showed that age was the most significant source of variation in the composition of the faecal microbiota. Second to this, cage environment was found to clearly impact the composition of the faecal microbiota, with samples from animals from within the same cage showing high community structure concordance, but large differences seen between cages. Importantly, the genetically induced obese phenotype was not found to impact the faecal bacterial profiles. These findings demonstrate that the age and local environmental cage variables were driving the composition of the faecal bacteria and were more deterministically important than the host genotype. These findings have major implications for understanding the significance of functional metagenomic data in experimental studies and beg the question; what is being measured in animal experiments in which different strains are housed separately, nature or nurture?
Kanadaptin is a nuclear protein of unknown function that is widely expressed in mammalian tissues. The crystal structure of the forkhead-associated (FHA) domain of human kanadaptin was determined to 1.6 Å resolution. The structure reveals an asymmetric dimer in which one monomer is complexed with a phosphopeptide mimic derived from a peptide segment from the N-terminus of a symmetry-related molecule as well as a sulfate bound to the structurally conserved phosphothreonine recognition cleft. This structure provides insights into the molecular recognition features utilized by this family of proteins and represents the first evidence that kanadaptin is likely involved in a phosphorylation-mediated signaling pathway. These results will be of use for designing experiments to further probe the function of kanadaptin.
Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers 10S and 10R methylthio-DDACTHF bound to human GAR Tfase, including the highest resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (10R Ki = 210 nM, 10S Ki = 180 nM) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC50 = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high and these lipophilic C10-substituted analogs show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC50 = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diasterisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogs also indicates that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase, but also in selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for future design of potent and selective anti-folate drugs that target GAR Tfase.
De novo purine biosynthesis; glycinamide ribonucleotide transformylase; (6R)-N10-formyltetrahydrofolic; C10 stereochemistry anti-folate therapeutics; x-ray crystallography; in silico modeling
Two ferret-adapted H5N1 viruses capable of respiratory droplet transmission have been reported with mutations in the hemagglutinin receptor-binding site and stalk domains. Glycan microarray analysis reveals that both viruses exhibit a strong shift toward binding to “human-type” α2-6 sialosides but with notable differences in fine specificity. Crystal structure analysis further shows that the stalk mutation causes no obvious perturbation of the receptor-binding pocket, consistent with its impact on hemagglutinin stability without affecting receptor specificity.
Unlike humans or mice, some species have limited genome encoded combinatorial diversity potential, yet mount a robust antibody response. Cows are unusual in having exceptionally long CDR H3 loops and few V-regions, but the mechanism for creating diversity is not understood. Deep sequencing revealed that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded mini-domains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a β-strand “stalk” that supports a structurally diverse, disulfide-bonded, “knob” domain. Sequence analysis suggests that diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias towards mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of CDR H3s of unprecedented length that fold into a diversity of mini-domains generated through combinations of somatically generated disulfides.
Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab′ fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection.
Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld-carboxypeptidase A (LdcA) catabolizes the bond between the last two l- and d-amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA (NaLdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The NaLdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling.
Despite available antivirals and vaccines, influenza infections continue to be a major cause of mortality worldwide. Vaccination generally induces an effective, but strain-specific antibody response. As the virus continually evolves, new vaccines have to be administered almost annually when a novel strain becomes dominant. Furthermore, the sporadic emerging resistance to neuraminidase inhibitors among circulating strains suggests an urgent need for new therapeutic agents. Recently, several cross-reactive antibodies have been described, which neutralize an unprecedented spectrum of influenza viruses. These broadly neutralizing antibodies generally target conserved functional regions on the major influenza surface glycoprotein hemagglutinin (HA). The characterization of their neutralization breadth and epitopes on HA could stimulate the development of new antibody-based antivirals and broader influenza vaccines.
The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically.
We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T332N pseudovirus.
The antigenicity and structural integrity of cleaved BG505 SOSIP.664 trimers render these proteins good mimics of functional Env spikes on virions. In contrast, uncleaved gp140s antigenically resemble individual gp120-gp41ECTO protomers and gp120 monomers, but not native trimers. Although NAb binding to functional trimers may thus be both necessary and sufficient for neutralization, the kinetics and stoichiometry of the interaction influence the neutralizing efficacy of individual NAbs.
Mitochondrial DNA (mtDNA) is highly polymorphic at the population level, and specific mtDNA variants affect mitochondrial function. With emerging evidence that mitochondrial mechanisms are central to common human diseases, it is plausible that mtDNA variants contribute to the “missing heritability” of several complex traits. Given the central role of mtDNA genes in oxidative phosphorylation, the same genetic variants would be expected to alter the risk of developing several different disorders, but this has not been shown to date. Here we studied 38,638 individuals with 11 major diseases, and 17,483 healthy controls. Imputing missing variants from 7,729 complete mitochondrial genomes, we captured 40.41% of European mtDNA variation. We show that mtDNA variants modifying the risk of developing one disease also modify the risk of developing other diseases, thus providing independent replication of a disease association in different case and control cohorts. High-risk alleles were more common than protective alleles, indicating that mtDNA is not at equilibrium in the human population, and that recent mutations interact with nuclear loci to modify the risk of developing multiple common diseases.
There is a growing body of evidence indicating that mitochondrial dysfunction, a result of genetic variation in the mitochondrial genome, is a critical component in the aetiology of a number of complex traits. Here, we take advantage of recent technical and methodological advances to examine the role of common mitochondrial DNA variants in several complex diseases. By examining over 50,000 individuals, from 11 different diseases we show that mitochondrial DNA variants can both increase or decrease an individual's risk of disease, replicating and expanding upon several previously reported studies. Moreover, by analysing several large disease groups in tandem, we are able to show a commonality of association, with the same mitochondrial DNA variants associated with several distinct disease phenotypes. These shared genetic associations implicate a shared underlying functional effect, likely changing cellular energy, which manifests as distinct phenotypes. Our study confirms the important role that mitochondrial DNA variation plays on complex traits and additionally supports the utility of a GWAS-based approach for analysing mitochondrial genetics.
Vaccine development to induce broadly neutralizing antibodies (bNAbs) against HIV-1 is a
global health priority. Potent VRC01-class bNAbs against the CD4 binding site of HIV gp120 have been
isolated from HIV-1-infected individuals; however, such bNAbs have not been induced by vaccination.
Wild-type gp120 proteins lack detectable affinity for predicted germline precursors of VRC01-class
bNAbs, making them poor immunogens to prime a VRC01-class response. We employed computation-guided,
in vitro screening to engineer a germline-targeting gp120 outer domain immunogen that binds to
multiple VRC01-class bNAbs and their germline precursors. When multimerized on nanoparticles, this
immunogen (eOD-GT6) activates both germline and mature VRC01-class B cells. Thus, eOD-GT6
nanoparticles have promise as a vaccine prime candidate. In principle, similar germline-targeting
strategies can be applied to other epitopes and pathogens.