Isolation of state-dependent monoclonal antibodies against the 12-transmembrane domain glucose transporter 4 using virus-like particles.

The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes.

High-Throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers.

Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies.IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.

Characterization of NLRP12 during the in vivo host immune response to Klebsiella pneumoniae and Mycobacterium tuberculosis.

The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1ß production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of Nlrp12(-/-) mice following airway exposure to LPS, Klebsiella pneumoniae and Mycobacterium tuberculosis. In response to E. coli LPS, Nlrp12(-/-) mice showed a slight decrease in IL-1ß and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in Nlrp12(-/-) mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the Nlrp12(-/-) mice. Likewise, Nlrp12(-/-) mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis.

Granuloma formation and host defense in chronic Mycobacterium tuberculosis infection requires PYCARD/ASC but not NLRP3 or caspase-1.

The NLR gene family mediates host immunity to various acute pathogenic stimuli, but its role in chronic infection is not known. This paper addressed the role of NLRP3 (NALP3), its adaptor protein PYCARD (ASC), and caspase-1 during infection with Mycobacterium tuberculosis (Mtb). Mtb infection of macrophages in culture induced IL-1beta secretion, and this requires the inflammasome components PYCARD, caspase-1, and NLRP3. However, in vivo Mtb aerosol infection of Nlrp3(-/-), Casp-1(-/-), and WT mice showed no differences in pulmonary IL-1beta production, bacterial burden, or long-term survival. In contrast, a significant role was observed for Pycard in host protection during chronic Mtb infection, as shown by an abrupt decrease in survival of Pycard(-/-) mice. Decreased survival of Pycard(-/-) animals was associated with defective granuloma formation. These data demonstrate that PYCARD exerts a novel inflammasome-independent role during chronic Mtb infection by containing the bacteria in granulomas.