Design and Evaluation of Meningococcal Vaccines through Structure-Based Modification of Host and Pathogen Molecules

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps

Pirt, a TRPV1 Modulator, Is Required for Histamine-Dependent and -Independent Itch

Itch, or pruritus, is an important clinical problem whose molecular basis has yet to be understood. Recent work has begun to identify genes that contribute to detecting itch at the molecular level. Here we show that Pirt, known to play a vital part in sensing pain through modulation of the transient receptor potential vanilloid 1 (TRPV1) channel, is also necessary for proper itch sensation. Pirt−/− mice exhibit deficits in cellular and behavioral responses to various itch-inducing compounds, or pruritogens. Pirt contributes to both histaminergic and nonhistaminergic itch and, crucially, is involved in forms of itch that are both TRPV1-dependent and -independent. Our findings demonstrate that the function of Pirt extends beyond nociception via TRPV1 regulation to its role as a critical component in several itch signaling pathways

Local Control of Excitation-Contraction Coupling in Human Embryonic Stem Cell-Derived Cardiomyocytes

We investigated the mechanisms of excitation-contraction (EC) coupling in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and fetal ventricular myocytes (hFVMs) using patch-clamp electrophysiology and confocal microscopy. We tested the hypothesis that Ca2+ influx via voltage-gated L-type Ca2+ channels activates Ca2+ release from the sarcoplasmic reticulum (SR) via a local control mechanism in hESC-CMs and hFVMs. Field-stimulated, whole-cell [Ca2+]i transients in hESC-CMs required Ca2+ entry through L-type Ca2+ channels, as evidenced by the elimination of such transients by either removal of extracellular Ca2+ or treatment with diltiazem, an L-type channel inhibitor. Ca2+ release from the SR also contributes to the [Ca2+]i transient in these cells, as evidenced by studies with drugs interfering with either SR Ca2+ release (i.e. ryanodine and caffeine) or reuptake (i.e. thapsigargin and cyclopiazonic acid). As in adult ventricular myocytes, membrane depolarization evoked large L-type Ca2+ currents (ICa) and corresponding whole-cell [Ca2+]i transients in hESC-CMs and hFVMs, and the amplitude of both ICa and the [Ca2+]i transients were finely graded by the magnitude of the depolarization. hESC-CMs exhibit a decreasing EC coupling gain with depolarization to more positive test potentials, “tail” [Ca2+]i transients upon repolarization from extremely positive test potentials, and co-localized ryanodine and sarcolemmal L-type Ca2+ channels, all findings that are consistent with the local control hypothesis. Finally, we recorded Ca2+ sparks in hESC-CMs and hFVMs. Collectively, these data support a model in which tight, local control of SR Ca2+ release by the ICa during EC coupling develops early in human cardiomyocytes

Congenital Zika virus infection as a silent pathology with loss of neurogenic output in the fetal brain

Zika virus (ZIKV) is a flavivirus with teratogenic effects on fetal brain, but the spectrum of ZIKV-induced brain injury is unknown, particularly when ultrasound imaging is normal. In a pregnant pigtail macaque (Macaca nemestrina) model of ZIKV infection, we demonstrate that ZIKV-induced injury to fetal brain is substantial, even in the absence of microcephaly, and may be challenging to detect in a clinical setting. A common and subtle injury pattern was identified, including (i) periventricular T2-hyperintense foci and loss of fetal noncortical brain volume, (ii) injury to the ependymal epithelium with underlying gliosis and (iii) loss of late fetal neuronal progenitor cells in the subventricular zone (temporal cortex) and subgranular zone (dentate gyrus, hippocampus) with dysmorphic granule neuron patterning. Attenuation of fetal neurogenic output demonstrates potentially considerable teratogenic effects of congenital ZIKV infection even without microcephaly. Our findings suggest that all children exposed to ZIKV in utero should receive long-term monitoring for neurocognitive deficits, regardless of head size at birth

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or 'double' tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the 'double' tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy

Factor H Binds to the hypervariable region of many streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems

Agonist-induced Ca(2+) entry determined by inositol 1,4,5-trisphosphate recognition

It has been considered that Ca(2+) release is the causal trigger for Ca(2+) entry after receptor activation. In DT40 B cells devoid of inositol 1,4,5-trisphosphate receptors (IP(3)R), the lack of Ca(2+) entry in response to receptor activation is attributed to the absence of Ca(2+) release. We reveal in this article that IP(3)R recognition of IP(3) determines agonist-induced Ca(2+) entry (ACE), independent of its Ca(2+) release activity. In DT40 IP(3)R(–/–) cells, endogenous ACE can be rescued with type 1 IP(3)R mutants (both a ΔC-terminal truncation mutant and a D2550A pore mutant), which are defective in Ca(2+) release channel activity. Thus, in response to B cell receptor activation, ACE is restored in an IP(3)R-dependent manner without Ca(2+) store release. Conversely, ACE cannot be rescued with mutant IP(3)Rs lacking IP(3) binding (both the Δ90–110 and R265Q IP(3)-binding site mutants). We conclude that an IP(3)-dependent conformational change in the IP(3)R, not endoplasmic reticulum Ca(2+) pool release, triggers ACE

Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23∼27∼24 clusters

MicroRNAs (miRNAs) modulate complex physiological and pathological processes by repressing expression of multiple components of cellular regulatory networks. Here we demonstrate that miRNAs encoded by the miR-23∼27∼24 gene clusters are enriched in endothelial cells and highly vascularized tissues. Inhibition of miR-23 and miR-27 function by locked nucleic acid-modified anti-miRNAs represses angiogenesis in vitro and postnatal retinal vascular development in vivo. Moreover, miR-23 and miR-27 are required for pathological angiogenesis in a laser-induced choroidal neovascularization mouse model. MiR-23 and miR-27 enhance angiogenesis by promoting angiogenic signaling through targeting Sprouty2 and Sema6A proteins, which exert antiangiogenic activity. Manipulating miR-23/27 levels may have important therapeutic implications in neovascular age-related macular degeneration and other vascular disorders