Public antibodies to malaria antigens generated by two LAIR1 insertion modalities.

In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.

High-avidity IgA protects the intestine by enchaining growing bacteria.

Vaccine-induced high-avidity IgA can protect against bacterial enteropathogens by directly neutralizing virulence factors or by poorly defined mechanisms that physically impede bacterial interactions with the gut tissues ('immune exclusion'). IgA-mediated cross-linking clumps bacteria in the gut lumen and is critical for protection against infection by non-typhoidal Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium). However, classical agglutination, which was thought to drive this process, is efficient only at high pathogen densities (≥108 non-motile bacteria per gram). In typical infections, much lower densities (100-107 colony-forming units per gram) of rapidly dividing bacteria are present in the gut lumen. Here we show that a different physical process drives formation of clumps in vivo: IgA-mediated cross-linking enchains daughter cells, preventing their separation after division, and clumping is therefore dependent on growth. Enchained growth is effective at all realistic pathogen densities, and accelerates pathogen clearance from the gut lumen. Furthermore, IgA enchains plasmid-donor and -recipient clones into separate clumps, impeding conjugative plasmid transfer in vivo. Enchained growth is therefore a mechanism by which IgA can disarm and clear potentially invasive species from the intestinal lumen without requiring high pathogen densities, inflammation or bacterial killing. Furthermore, our results reveal an untapped potential for oral vaccines in combating the spread of antimicrobial resistance.

A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens.

Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies. Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different P. falciparum isolates and opsonize these cells by binding to members of the RIFIN family. These antibodies acquired broad reactivity through a novel mechanism of insertion of a large DNA fragment between the V and DJ segments. The insert, which is both necessary and sufficient for binding to RIFINs, encodes the entire 98 amino acid collagen-binding domain of LAIR1, an immunoglobulin superfamily inhibitory receptor encoded on chromosome 19. In each of the two donors studied, the antibodies are produced by a single expanded B-cell clone and carry distinct somatic mutations in the LAIR1 domain that abolish binding to collagen and increase binding to infected erythrocytes. These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine.

Identification of an N-terminal inhibitory extension as the primary mechanosensory regulator of twitchin kinase.

Titin-like kinases are an important class of cytoskeletal kinases that intervene in the response of muscle to mechanical stimulation, being central to myofibril homeostasis and development. These kinases exist in autoinhibited states and, allegedly, become activated during muscle activity by the elastic unfolding of a C-terminal regulatory segment (CRD). However, this mechano-activation model remains controversial. Here we explore the structural, catalytic, and tensile properties of the multidomain kinase region of Caenorhabditis elegans twitchin (Fn(31)-Nlinker-kinase-CRD-Ig(26)) using X-ray crystallography, small angle X-ray scattering, molecular dynamics simulations, and catalytic assays. This work uncovers the existence of an inhibitory segment that flanks the kinase N-terminally (N-linker) and that acts synergistically with the canonical CRD tail to silence catalysis. The N-linker region has high mechanical lability and acts as the primary stretch-sensor in twitchin kinase, while the CRD is poorly responsive to pulling forces. This poor response suggests that the CRD is not a generic mechanosensor in this kinase family. Instead, the CRD is shown here to be permissive to catalysis and might protect the kinase active site against mechanical damage. Thus, we put forward a regulatory model where kinase inhibition results from the combined action of both N- and C-terminal tails, but only the N-terminal extension undergoes mechanical removal, thereby affording partial activation. Further, we compare invertebrate and vertebrate titin-like kinases and identify variations in the regulatory segments that suggest a mechanical speciation of these kinase classes.

Exceptionally potent human monoclonal antibodies are effective for prophylaxis and treatment of tetanus in mice.

We used human monoclonal antibodies (humAbs) to study the mechanism of neuron intoxication by tetanus neurotoxin and to evaluate these antibodies as a safe preventive and therapeutic substitute for hyperimmune sera to treat tetanus in mice. By screening memory B cells from immune donors, we selected 2 tetanus neurotoxin-specific mAbs with exceptionally high neutralizing activities and extensively characterized them both structurally and functionally. We found that these antibodies interfered with the binding and translocation of the neurotoxin into neurons by interacting with 2 epitopes, whose identification pinpoints crucial events in the cellular pathogenesis of tetanus. Our observations explain the neutralization ability of these antibodies, which we found to be exceptionally potent in preventing experimental tetanus when injected into mice long before the toxin. Moreover, their Fab derivatives neutralized tetanus neurotoxin in post-exposure experiments, suggesting their potential for therapeutic use via intrathecal injection. As such, we believe these humAbs, as well as their Fab derivatives, meet the requirements to be considered for prophylactic and therapeutic use in human tetanus and are ready for clinical trials.

Structural Basis for Broad HIV-1 Neutralization by the MPER-Specific Human Broadly Neutralizing Antibody LN01.

Potent and broadly neutralizing antibodies (bnAbs) are the hallmark of HIV-1 protection by vaccination. The membrane-proximal external region (MPER) of the HIV-1 gp41 fusion protein is targeted by the most broadly reactive HIV-1 neutralizing antibodies. Here, we examine the structural and molecular mechansims of neutralization by anti-MPER bnAb, LN01, which was isolated from lymph-node-derived germinal center B cells of an elite controller and exhibits broad neutralization breadth. LN01 engages both MPER and the transmembrane (TM) region, which together form a continuous helix in complex with LN01. The tilted TM orientation allows LN01 to interact simultaneously with the peptidic component of the MPER epitope and membrane via two specific lipid binding sites of the antibody paratope. Although LN01 carries a high load of somatic mutations, most key residues interacting with the MPER epitope and lipids are germline encoded, lending support for the LN01 epitope as a candidate for lineage-based vaccine development.

A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein.

Immunization with attenuated Plasmodium falciparum sporozoites (PfSPZs) has been shown to be protective against malaria, but the features of the antibody response induced by this treatment remain unclear. To investigate this response in detail, we isolated IgM and IgG monoclonal antibodies from Tanzanian volunteers who were immunized with repeated injection of Sanaria PfSPZ Vaccine and who were found to be protected from controlled human malaria infection with infectious homologous PfSPZs. All isolated IgG monoclonal antibodies bound to P. falciparum circumsporozoite protein (PfCSP) and recognized distinct epitopes in its N terminus, NANP-repeat region, and C terminus. Strikingly, the most effective antibodies, as determined in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the PfCSP N-terminal junction that is not in the RTS,S vaccine. These dual-specific antibodies were isolated from different donors and were encoded by VH3-30 or VH3-33 alleles that encode tryptophan or arginine at position 52. Using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. Our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies.

Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor.

Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.

The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition.

Protein scaffolds that support molecular recognition have multiple applications in biotechnology. Thus, protein frames with robust structural cores but adaptable surface loops are in continued demand. Recently, notable progress has been made in the characterization of Ig domains of intracellular origin--in particular, modular components of the titin myofilament. These Ig belong to the I(intermediate)-type, are remarkably stable, highly soluble and undemanding to produce in the cytoplasm of Escherichia coli. Using the Z1 domain from titin as representative, we show that the I-Ig fold tolerates the drastic diversification of its CD loop, constituting an effective peptide display system. We examine the stability of CD-loop-grafted Z1-peptide chimeras using differential scanning fluorimetry, Fourier transform infrared spectroscopy and nuclear magnetic resonance and demonstrate that the introduction of bioreactive affinity binders in this position does not compromise the structural integrity of the domain. Further, the binding efficiency of the exogenous peptide sequences in Z1 is analyzed using pull-down assays and isothermal titration calorimetry. We show that an internally grafted, affinity FLAG tag is functional within the context of the fold, interacting with the anti-FLAG M2 antibody in solution and in affinity gel. Together, these data reveal the potential of the intracellular Ig scaffold for targeted functionalization.

Distal-to-proximal NO conversion in hemoproteins: the role of the proximal pocket.

Hemoproteins play central roles in the formation and utilization of nitric oxide (NO) in cellular signaling, as well as in protection against nitrosative stress. Key to heme-nitrosyl function and reactivity is the Fe coordination number (5 or 6). For (five-coordinate) 5c-NO complexes, the potential for NO to bind on either heme face exists, as in the microbial cytochrome c' from Alcaligenes xylosoxidans (AxCYTcp), which forms a stable proximal 5c-NO complex via a distal six-coordinate NO intermediate and a putative dinitrosyl species. Strong parallels between the NO-binding kinetics of AxCYTcp, the eukaryotic NO sensor soluble guanylate cyclase, and the ferrocytochrome c/cardiolipin complex have led to the suggestion that a distal-to-proximal NO switch could contribute to the selective ligand responses in gas-sensing hemoproteins. The proximal NO-binding site in AxCYTcp is close to a conserved basic (Arg124) residue that is postulated to modulate NO reactivity. We have replaced Arg124 by five different amino acids and have determined high-resolution (1.07-1.40 Å) crystallographic structures with and without NO. These, together with kinetic and resonance Raman data, provide new insights into the mechanism of distal-to-proximal heme-NO conversion, including the determinants of Fe-His bond scission. The Arg124Ala variant allowed us to determine the structure of an analog of the previously unobserved key 5c-NO distal intermediate species. The very high resolution structures combined with the extensive spectroscopic and kinetic data have allowed us to provide a fresh insight into heme reactivity towards NO, a reaction that is of wide importance in biology.

Characterization of cycP gene expression in Achromobacter xylosoxidans NCIMB 11015 and high-level heterologous synthesis of cytochrome c' in Escherichia coli.

The cycP gene encoding a periplasmic cytochrome c' from the denitrifying beta-proteobacterium Achromobacter xylosoxidans was characterized. The genes flanking cycP encode components of a mobile genetic element characteristic of the beta-proteobacteria, suggesting that cycP has inserted within a transposon or insertion element. The gene therefore does not form part of a denitrification operon or gene cluster. The level of expression of the cycP gene and the level of synthesis of its corresponding gene product were found to increase by maximally 3-fold anaerobically. Expression of cycP appears to occur mainly by non-specific read-through transcription from portions of the insertion element. Conditions were developed for high-level overproduction of cytochrome c' in Escherichia coli, which resulted in signal peptide cleavage concomitant with secretion of the protein into the periplasm. Using a single-step purification, 20-30 mg of pure protein were isolated from a 1-litre culture. Based on UV-visible spectrophotometry the dimeric protein was shown to have a full complement of haem and to be indistinguishable from the native protein purified from A. xylosoxidans. This system provides an excellent platform to facilitate biochemical and structural dissection of the mechanism underlying the novel specificity of NO binding to the proximal face of the haem.

Modulation of NO binding to cytochrome c' by distal and proximal haem pocket residues.

We have cloned and expressed the cycP gene encoding cytochrome c' from Alcaligenes xylosoxidans and generated mutations in Arg-124 and Phe-59, residues close to the haem, to probe their involvement in modulating the unusual spin-state equilibrium of the haem Fe and the unique proximal mode of binding of NO to form a stable five-coordinate adduct. Arg-124 is located in the proximal pocket of the haem and forms a hydrogen bond to the stable five-coordinated bound NO. Phe-59 provides steric hindrance at the distal face where NO binds initially to form a six-coordinate adduct. Optical spectroscopy showed altered electronic properties of the oxidised haem centre resulting from the mutations of both residues. The high affinity of the ferrous proteins for NO remained unchanged and all of the mutational variants formed a stable five-coordinate NO species (lambda(Soret) 395 nm) in the presence of stoichiometric concentrations of NO. However, the kinetics of the reactivity towards NO were altered, with mutation of the distal Phe-59 residue resulting in the transient six-coordinate distally bound NO adduct (lambda(Soret) 415 nm) not being detected. Surprisingly, substitution of the proximal residue Arg-124 with Phe, Ala, Gln or Glu also resulted in the six-coordinate adduct not being detected, showing that this proximal residue also modulates reactivity towards NO on the opposite haem face. In contrast, the R124L substitution retained the property of the native protein in the initial formation of a six-coordinate NO adduct, a finding of functional importance since a Lys or an Arg residue is invariant in these proteins.