Haemagglutinin substitutions N125D, D127E, D222G and R223Q improve replicative fitness and vaccine effectiveness of an A/H1N1pdm09 live attenuated influenza vaccine virus by enhancing α-2,6 receptor binding.

During 2013-14 and 2015-16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type (wt) reference strains. Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses.

Preclinical development of a high affinity α-synuclein antibody, MEDI1341, that can enter the brain, sequester extracellular α-synuclein and attenuate α-synuclein spreading in vivo.

There are no approved drug therapies that can prevent or slow the progression of Parkinson's disease (PD). Accumulation and aggregation of α-synuclein protein is observed throughout the nervous system in PD. α-Synuclein is a core component of Lewy bodies and neurites that neuropathologically define PD, suggesting that α-synuclein may be a key causative agent in PD. Recent experimental data suggest that PD progression may arise due to spreading of pathological forms of extracellular α-synuclein throughout the brain via a cellular release, uptake and seeding mechanism. We have developed a high affinity α-synuclein antibody, MEDI1341, that can enter the brain, sequester extracellular α-synuclein and attenuate α-synuclein spreading in vivo. MEDI1341 binds both monomeric and aggregated forms of α-synuclein. In vitro, MEDI1341 blocks cell-to-cell transmission of pathologically relevant α-synuclein preformed fibrils (pffs). After intravenous injection into rats and cynomolgus monkeys, MEDI1341 rapidly enters the central nervous system and lowers free extracellular α-synuclein levels in the interstitial fluid (ISF) and cerebrospinal fluid (CSF) compartments. Using a novel lentiviral-based in vivo mouse model of α-synuclein spreading in the brain, we show that treatment with MEDI1341 significantly reduces α-synuclein accumulation and propagation along axons. In this same model, we demonstrate that an effector-null version of the antibody was equally as effective as one with effector function. MEDI1341 is now in Phase 1 human clinical trial testing as a novel treatment for α-synucleinopathies including PD with the aim to slow or halt disease progression.

Group B Streptococcus pilus sortase regulation: a single mutation in the lid region induces pilin protein polymerization in vitro.

Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date, and their mechanisms of transpeptidation and regulation need to be further investigated. The available 3-dimensional structures of these enzymes reveal a typical sortase fold, except for the presence of a unique feature represented by an N-terminal highly flexible loop known as the "lid." This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an autoinhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work, we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme.

Understanding the molecular determinants driving the immunological specificity of the protective pilus 2a backbone protein of group B streptococcus.

The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response.

Structure-based approach to rationally design a chimeric protein for an effective vaccine against Group B Streptococcus infections.

Structural vaccinology is an emerging strategy for the rational design of vaccine candidates. We successfully applied structural vaccinology to design a fully synthetic protein with multivalent protection activity. In Group B Streptococcus, cell-surface pili have aroused great interest because of their direct roles in virulence and importance as protective antigens. The backbone subunit of type 2a pilus (BP-2a) is present in six immunogenically different but structurally similar variants. We determined the 3D structure of one of the variants, and experimentally demonstrated that protective antibodies specifically recognize one of the four domains that comprise the protein. We therefore constructed a synthetic protein constituted by the protective domain of each one of the six variants and showed that the chimeric protein protects mice against the challenge with all of the type 2a pilus-carrying strains. This work demonstrates the power of structural vaccinology and will facilitate the development of an optimized, broadly protective pilus-based vaccine against Group B Streptococcus by combining the uniquely generated chimeric protein with protective pilin subunits from two other previously identified pilus types. In addition, this work describes a template procedure that can be followed to develop vaccines against other bacterial pathogens.

New insights into the role of the glutamic acid of the E-box motif in group B Streptococcus pilus 2a assembly.

Group B Streptococcus pili are covalently linked structures assembled via a sortase-catalyzed transpeptidation mechanism involving specific residues and motifs. A sequence element containing a conserved glutamic acid, called the E-box, has been described to be involved in pilus formation. Although it is known that the glutamic acid is involved in stabilizing the internal isopeptide bonds, its role in pilus assembly still needs to be investigated. Using site-specific mutagenesis and complementation studies of knockout strains, we found that the E-box glutamic residue of the backbone and the major ancillary proteins is essential for pilus protein polymerization. NMR analysis revealed that the mutation of this residue seriously affected the folding of the protein. By contrast, the mutation of the lysine involved in the same isopeptide bond did not engender a structural destabilization, and the native fold was preserved. Moreover, molecular dynamics simulations on the E-box-containing domain of the backbone protein showed that the E-box glutamic acid is necessary to maintain the appropriate dryness of the domain core and that its mutation favors an unfolded state. The data provide the first direct evidence that the E-box has an additional and key role in maintaining the correct protein fold independently of isopeptide bond formation.

Structure analysis and site-directed mutagenesis of defined key residues and motives for pilus-related sortase C1 in group B Streptococcus.

In group B Streptococcus (GBS), 3 structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular-weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies, and in vivo mutagenesis, we performed a broad characterization of GBS sortase C1 of pilus island 2a. The high-resolution X-ray structure of the enzyme revealed that the active site, into the ß-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the "lid." We show that the catalytic triad and the predicted N- and C-terminal transmembrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies, we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild-type protein.

Human plasmacytoid dendritic cells are unresponsive to bacterial stimulation and require a novel type of cooperation with myeloid dendritic cells for maturation.

Dendritic cell (DC) populations play unique and essential roles in the detection of pathogens, but information on how different DC types work together is limited. In this study, 2 major DC populations of human blood, myeloid (mDCs) and plasmacytoid (pDCs), were cultured alone or together in the presence of pathogens or their products. We show that pDCs do not respond to whole bacteria when cultured alone, but mature in the presence of mDCs. Using purified stimuli, we dissect this cross-talk and demonstrate that mDCs and pDCs activate each other in response to specific induction of only one of the cell types. When stimuli for one or both populations are limited, they synergize to reach optimal activation. The cross-talk is limited to enhanced antigen presentation by the nonresponsive population with no detectable changes in the quantity and range of cytokines produced. We propose that each population can be a follower or leader in immune responses against pathogen infections, depending on their ability to respond to infectious agents. In addition, our results indicate that pDCs play a secondary role to induce immunity against human bacterial infections, which has implications for more efficient targeting of DC populations with improved vaccines and therapeutics.

A direct role for SNX9 in the biogenesis of filopodia.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia.

Group B Streptococcus vaccine: state of the art.

Group B Streptococcus (GBS) is cause of neonatal invasive diseases as well as of severe infections in the elderly and immune-compromised patients. Despite significant advances in the prevention and treatment of neonatal disease, sepsis and meningitis caused by GBS still represent a significant public health care concern globally and additional prevention and therapeutic strategies against infection are highly desirable. The introduction of national recommended guidelines in several countries to screen pregnant women for GBS carriage and the use of antibiotics during delivery significantly reduced disease occurring within the first hours of life (early-onset disease), but it has had no effect on the late-onset diseases occurring after the first week and is not feasible in most countries. Availability of an effective vaccine against GBS would provide an effective means of controlling GBS disease. This review provides an overview of the burden of invasive disease caused by GBS in infants and adults, and highlights the strategies for the development of an effective vaccine against GBS infections.

Structure and assembly of group B streptococcus pilus 2b backbone protein.

Group B Streptococcus (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.

Sortase A substrate specificity in GBS pilus 2a cell wall anchoring.

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(ΔN40)) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(ΔN40) does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.

Opposite effects of simvastatin on the bactericidal and inflammatory response of macrophages to opsonized S. aureus.

Besides lowering circulating cholesterol, statins act as immunomodulators. Although the effects of statins on lymphocyte activation and differentiation have been clearly defined, there is no consensus as to effects of these drugs on phagocytes. We have addressed the outcome of simvastatin treatment on the activation and effector function of human macrophages in the pathophysiologically relevant context of challenge with an opportunistic pathogen. We provide evidence that: simvastatin blocks the biological effects rapidly triggered by IgG-opsonized bacteria (phagocytosis and oxidative burst) while enhancing the delayed effects elicited by FcgammaR stimulation (production of proinflammatory mediators); these opposite effects of simvastatin result from enhancement of the JNK pathway and concomitant impairment of other signaling modules activated by FcgammaR engagement; and these activities are dependent on the capacity of simvastatin to block protein prenylation. The results provide novel mechanistic insight into the activities of statins on phagocytes and are of relevance to the assessment of potential side-effects in patients undergoing long-term hypocholesterolemic therapy.