During the height of the 2009 H1N1 swine-derived influenza pandemic, a clinical trial was conducted in which seven subjects were immunized using a monovalent, MF59
®
-adjuvanted vaccine, developed ...from an egg- passaged candidate vaccine virus (CVV), A/California/07/2009 X-181. Whole blood was collected prior to immunization and at 8, 22, and 202 days post-vaccination, and subjects’ serological responses were evaluated. Here, we reconstruct and examine the longitudinal, influenza-specific circulating B cell repertoire of one subject in that study. Genotypic analysis of 390 total subject-derived antibodies (Abs) revealed a total of 29 germline genes in use among immunoglobulin heavy chain variable regions (IgHV), with the majority of those sequences isolated representing memory recall responses and two major lineages dominating the early response. In vitro phenotyping showed a diverse set of binding epitopes on the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), many of which are considered subdominant. Strong correlations were found between IgHV germline usage among non-related lineages and both binding epitope and neutralization breadth. Results here highlight the potential for Ab responses to be misdirected to egg-adaptive artifacts on CVVs while simultaneously stressing the ability to mount potent, broadly neutralizing responses to mostly novel antigens via recall of subdominant memory responses, as well as the need for evaluating alternative endpoint assays and anti-NA responses following clinical trials.
Abstract
Sickle cell disease (SCD) is caused by a mutant β-globin gene and affects more than 100,000 individuals in the USA alone. Sickled RBCs and ischemic events cause tissue damage, altered ...splenic architecture, and inflammation. When children are infected with parvovirus B19, they can develop life-threatening aplastic crisis. Previous reports have described immune defects among patients with SCD, and their potential correlations with splenic dysfunction or inflammation. In a multi-center study involving St. Jude, NIH, Novartis, and GlaxoSmithKline, we examined (i) inflammatory cytokines/chemokines in patients with SCD, treated or untreated with hydroxyurea (HU); (ii) immune responses toward parvovirus B19 infections in children with SCD; and (iii) immune responses in a mouse model for SCD against a candidate Saccharomyces cerevisiae-derived parvovirus B19 VLP vaccine. Results showed that HU treatment reduced serum cytokines/chemokines in patients with SCD (e.g. TGFα, TNFα, and sVCAM-1). Both HU-treated and untreated children, regardless of their cytokine/chemokine profile, responded to parvovirus B19. Binding antibodies were apparent in the nasal cavity (a common point-of entry for the pathogen) and sera by day 7 post-infection, and virus-specific neutralizing antibodies were induced. In transgenic mice with SCD (expressing human α, βS, and γ globin genes), we observed normal antibody activities toward the VLP vaccine. Together, data show that parvovirus B19 -specific B cells withstand the aberrant SCD environment better than their previously-described Streptococcus pneumonia-specific B cell counterparts. Data encourage rapid development of the VLP vaccine to prevent aplastic crises in children with SCD.
Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and ...gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV.
Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. ...Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments. The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).
Structural studies of quinolinate synthase suggest a model for the enzyme–substrate complex and an enzyme–intermediate complex with a 4Fe–4S cluster. Quinolinate synthase (QS) catalyzes the ...condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel β-sheet flanked by four α-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a 4Fe–4S cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii Sakuraba et al. (2005 ▶), J. Biol. Chem.280, 26645–26648. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the 4Fe–4S cluster, prior to cluster assembly.
The crystal structure of Acetobacter aceti PurE was determined to a resolution of 1.55 Å and is compared with the known structures of the class I PurEs from a mesophile, Escherichia coli, and a ...thermophile, Thermotoga maritima. Analyses of the general factors that increase protein stability are examined as potential explanations for the acid stability of A. aceti PurE. Increased inter‐subunit hydrogen bonding and an increased number of arginine‐containing salt bridges appear to account for the bulk of the increased acid stability. A chain of histidines linking two active sites is discussed in the context of the proton transfers catalyzed by the enzyme.
Electron cryomicroscopy (cryo-EM) yields images of macromolecular assemblies and their components, from which 3D structures can be determined, by using an image processing method commonly known as ..."single-particle reconstruction." During the past two decades, this technique has become an important tool for 3D structure determination, but it generally has not been possible to determine atomic models. In principle, individual molecular images contain high-resolution information contaminated by a much higher level of noise. In practice, it has been unclear whether current averaging methods are adequate to extract this information from the background. We present here a reconstruction, obtained by using recently developed image processing methods, of the rotavirus inner capsid particle ("double-layer particle" or DLP) at a resolution suitable for interpretation by an atomic model. The result establishes single-particle reconstruction as a high-resolution technique. We show by direct comparison that the cryo-EM reconstruction of viral protein 6 (VP6) of the rotavirus DLP is similar in clarity to a 3.8-Å resolution map obtained from x-ray crystallography. At this resolution, most of the amino acid side chains produce recognizable density. The icosahedral symmetry of the particle was an important factor in achieving this resolution in the cryo-EM analysis, but as the size of recordable datasets increases, single-particle reconstruction also is likely to yield structures at comparable resolution from samples of much lower symmetry. This potential has broad implications for structural cell biology.
The rotavirus inner capsid particle, known as the “double-layered particle” (DLP), is the “payload” delivered into a cell in the process of viral infection. Its inner and outer protein layers, ...composed of viral protein (VP) 2 and VP6, respectively, package the 11 segments of the double-stranded RNA (dsRNA) of the viral genome, as well as about the same number of polymerase molecules (VP1) and capping-enzyme molecules (VP3). We have determined the crystal structure of the bovine rotavirus DLP. There is one full particle (outer diameter ∼700 Å) in the asymmetric unit of the P212121 unit cell of dimensions a=740 Å, b=1198 Å, and c=1345 Å. A three-dimensional reconstruction from electron cryomicroscopy was used as a molecular replacement model for initial phase determination to about 18.5 Å resolution, and the 60-fold redundancy of icosahedral particle symmetry allowed phases to be extended stepwise to the limiting resolution of the data (3.8 Å). The structure of a VP6 trimer (determined previously by others) fits the outer layer density with very little adjustment. The T=13 triangulation number of that layer implies that there are four and one-third VP6 trimers per icosahedral asymmetric unit. The inner layer has 120 copies of VP2 and thus 2 copies per icosahedral asymmetric unit, designated VP2A and VP2B. Residues 101–880 fold into a relatively thin principal domain, comma-like in outline, shaped such that only rather modest distortions (concentrated at two “subdomain” boundaries) allow VP2A and VP2B to form a uniform layer with essentially no gaps at the subunit boundaries, except for a modest pore along the 5-fold axis. The VP2 principal domain resembles those of the corresponding shells and homologous proteins in other dsRNA viruses: λ1 in orthoreoviruses and VP3 in orbiviruses. Residues 1–80 of VP2A and VP2B fold together with four other such pairs into a “5-fold hub” that projects into the DLP interior along the 5-fold axis; residues 81–100 link the 10 polypeptide chains emerging from a 5-fold hub to the N-termini of their corresponding principal domains, clustered into a decameric assembly unit. The 5-fold hub appears to have several distinct functions. One function is to recruit a copy of VP1 (or of a VP1–VP3 complex), potentially along with a segment of plus-strand RNA, as a decamer of VP2 assembles. The second function is to serve as a shaft around which can coil a segment of dsRNA. The third function is to guide nascent mRNA, synthesized in the DLP interior by VP1 and 5′-capped by the action of VP3, out through a 5-fold exit channel. We propose a model for rotavirus particle assembly, based on known requirements for virion formation, together with the structure of the DLP and that of VP1, determined earlier.
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