Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding

Enveloped viruses carry one or multiple proteins with receptor-binding functionalities. Functional receptors can be glycans, proteinaceous, or both; therefore, recombinant protein approaches are instrumental in attaining new insights regarding viral envelope protein receptor-binding properties. Visualizing and measuring receptor binding typically entails antibody detection or direct labeling, whereas direct fluorescent fusions are attractive tools in molecular biology. Here, we report a suite of distinct fluorescent fusions, both N- and C-terminal, for influenza A virus hemagglutinins and SARS-CoV-2 spike RBD. The proteins contained three or six fluorescent protein barrels and were applied directly to cells to assess receptor binding properties

Differences in tetracycline resistance determinant carriage among Shigella flexneri and Shigella sonnei are not related to different plasmid Inc-type carriage

Objectives: The aim of this study was to establish the prevalence of the most common molecular mechanisms involved in tetracycline resistance as well as their relationship with plasmid incompatibility (Inc) groups in a collection of Shigella spp. causing traveller’s diarrhoea. Methods: Tetracycline susceptibility was established in 187 Shigella spp. (74 Shigella flexneri and 113 Shigella sonnei), of which 153 isolates were recovered as a confirmed cause of traveller’s diarrhoea. The prevalence of the tet(A), tet(B) and tet(G) genes was analysed by PCR. Eighteen plasmid Inc groups was determined in a subset of 59 isolates. Results: Among 154 tetracycline-resistant isolates, 122 (79.2%) harboured at least tet(A) or tet(B). The tet(B) gene was the most frequently detected, being present in 70 isolates (45.5%), whilst tet(A) was detected in 57 isolates (37.0%). The tet(G) gene was present in only 11 (7.2%) isolates. Moreover, the tet(A) gene was more frequent in S. sonnei (P = 0.0007), whilst the tet(B) gene was more frequent in S. flexneri (P < 0.0001). Plasmids belonging to Inc group B (P < 0.05) were significantly more frequent among S. flexneri, whilst those belonging to groups K, FIC and FIIA (P < 0.05) were preferentially detected among S. sonnei. Conclusion: The prevalence of the tet(A) and tet(B) genes differed between S. sonnei and S. flexneri. Moreover, the prevalence of plasmid Inc groups in S. flexneri and S. sonnei differed. However, no relationship was found between the two phenomena

Stabilizing HIV-1 envelope glycoprotein trimers to induce neutralizing antibodies Marit van Gils, [email protected]; Rogier W Sanders, [email protected]

An effective HIV-1 vaccine probably will need to be able to induce broadly neutralizing HIV-1 antibodies (bNAbs) in order to be efficacious. The many bNAbs that have been isolated from HIV-1 infected patients illustrate that the human immune system is able to elicit this type of antibodies. The elucidation of the structure of the HIV-1 envelope glycoprotein (Env) trimer has further fueled the search for Env immunogens that induce bNAbs, but while native Env trimer mimetics are often capable of inducing strain-specific neutralizing antibodies (NAbs) against the parental virus, they have not yet induced potent bNAb responses. To improve the performance of Env trimer immunogens, researchers have studied the immune responses that Env trimers have induced in animals; they have evaluated how to best use Env trimers in various immunization regimens; and they have engineered increasingly stabilized Env trimer variants. Here, we review the different approaches that have been used to increase the stability of HIV-1 Env trimer immunogens with the aim of improving the induction of NAbs. In particular, we draw parallels between the various approaches to stabilize Env trimers and ones that have been used by nature in extremophile microorganisms in order to survive in extreme environmental conditions

Development and analysis of furazolidone-resistant Escherichia coli mutants

Revisión por [email protected] mutants were obtained from four clinical isolates of diarrhoeagenic Escherichia coli. The stability of the resistance and the frequency of mutation were established. The minimal inhibitory concentration of furazolidone, nitrofurantoin, nalidixic acid, ampicillin, chloramphenicol and tetracycline was established both in the presence and absence of the efflux pump inhibitor Phe-Arg-β-Naphtylamyde. The presence of mutations in the nitroreductase genes nfsA and nfsB was analysed by PCR; sequencing and their enzymatic activity was assessed by a spectrophotometric assay. Alterations in outer membrane proteins were studied by SDS-PAGE. The frequency of mutation ranged from <9.6 × 10-10 to 9.59 × 10-7 . Neither an effect on efflux pumps inhibited by Phe-Arg-β-Naphtylamyde nor cross-resistance with the antibiotics studied was observed. Nineteen mutants (52.94%) presented mutations in the nitroreductase-encoding genes: 17 in the nfsA gene (15 mutants with an internal stop codon, 2 with amino acid changes), 2 in the nfsB (all amino acid changes). Alterations in the outer membrane proteins OmpA and OmpW were also observed. Although more studies are necessary to find other resistance mechanisms, present data showed the low potential of selecting furazolidone-resistant mutants, together with the lack of cross-resistance with unrelated antimicrobial agents.This work was supported by the Agencia Espa~nola de Cooperaci on Internacional al Desarrollo (AECID) [grants nos. D/024648/09 and D/030509/10] and by Generalitat de Catalunya, Departament d’Universitats, Recerca i Societat de la Informaci o (2014 SGR 26). JR has a fellowship from the programme I3 of the Instituto de Salud Carlos III (ISCIII, Spain) [grant no. CES11/012]

Complete epitopes for vaccine design derived from a crystal structure of the broadly neutralizing antibodies PGT128 and 8ANC195 in complex with an HIV-1 Env trimer

The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6 Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody-gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine desig

Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics

The HIV-1 envelope glycoprotein (Env) trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env trimer mimetics, such as SOSIP trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length unmodified Env trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP trimers. Antigenically, the full-length and SOSIP trimers were comparable, but in contrast to the full-length trimer, the SOSIP trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP trimer. Furthermore, the glycan composition of full-length and SOSIP trimers was similar overall, but the SOSIP trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env trimers as immunogens

Immunogenicity in rabbits of SOSIP trimers from clades A, B and C, given individually, sequentially or in combinations

Recombinant soluble HIV-1 envelope glycoprotein (Env) SOSIP trimers are a design platform for inducing broadly neutralizing antibodies (bNAbs) by vaccination. To date, these and alternative designs of native-like trimers given singly or in pairs, have not induced bNAbs in test animals such as rabbits or macaques. Here, we have evaluated whether trivalent and tetravalent combinations of SOSIP trimers from clades A, B and C, delivered simultaneously or sequentially, induce better neutralizing antibody responses in rabbits than when given alone. None of the tested formulations led to the induction of bNAbs. We found that BG505 clade A trimers dominated the autologous NAb responses induced by combinations, which probably relates to the presence of immunodominant glycan holes on the BG505 trimer. Furthermore, autologous NAb responses to all individual trimers were reduced when they were delivered in combinations compared with when delivered alone, suggesting that immunogen interference had occurred. Finally, in a sequential regimen, a heterologous clade C trimer cross-boosted NAb responses that were primed by earlier immunizations with clade A and B trimers. Taken together, these findings should allow us to improve the design of immunization regimens based on native-like HIV-1 Env trimers.IMPORTANCE A successful HIV-1 vaccine most probably requires a trimeric envelope glycoprotein (Env) component, as Env is the only viral protein on the surface of the virus and therefore the only target for neutralizing antibodies. Native-like Env trimers can induce strain-specific neutralizing antibodies, but not yet broadly neutralizing antibodies. To try to broaden the antibody response, we immunized rabbits with soluble native-like Env trimers from three different clades using monovalent, multivalent and sequential regimens. We found that the neutralizing antibody response against each immunogen was reduced when the immunogens were delivered in combination or sequentially compared to the monovalent regimen. In contrast, when the Env trimers from different clades were delivered sequentially, the neutralizing antibody response could be cross-boosted. Although the combination of native-like Env trimers from different clades did not induce broadly neutralizing antibodies, the results provide clues on how to use native-like trimers in vaccination experiment

Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics

The HIV-1 envelope glycoprotein (Env) trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env trimer mimetics, such as SOSIP trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length unmodified Env trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP trimers. Antigenically, the full-length and SOSIP trimers were comparable, but in contrast to the full-length trimer, the SOSIP trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP trimer. Furthermore, the glycan composition of full-length and SOSIP trimers was similar overall, but the SOSIP trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env trimers as immunogens.</p

Differences in tetracycline resistance determinant carriage among Shigella flexneri and Shigella sonnei are not related to different plasmid Inc-type carriage

Objectives: The aim of this study was to establish the prevalence of the most common molecular mechanisms involved in tetracycline resistance as well as their relationship with plasmid incompatibility (Inc) groups in a collection of Shigella spp. causing traveller’s diarrhoea. Methods: Tetracycline susceptibility was established in 187 Shigella spp. (74 Shigella flexneri and 113 Shigella sonnei), of which 153 isolates were recovered as a confirmed cause of traveller’s diarrhoea. The prevalence of the tet(A), tet(B) and tet(G) genes was analysed by PCR. Eighteen plasmid Inc groups was determined in a subset of 59 isolates. Results: Among 154 tetracycline-resistant isolates, 122 (79.2%) harboured at least tet(A) or tet(B). The tet(B) gene was the most frequently detected, being present in 70 isolates (45.5%), whilst tet(A) was detected in 57 isolates (37.0%). The tet(G) gene was present in only 11 (7.2%) isolates. Moreover, the tet(A) gene was more frequent in S. sonnei (P = 0.0007), whilst the tet(B) gene was more frequent in S. flexneri (P < 0.0001). Plasmids belonging to Inc group B (P < 0.05) were significantly more frequent among S. flexneri, whilst those belonging to groups K, FIC and FIIA (P < 0.05) were preferentially detected among S. sonnei. Conclusion: The prevalence of the tet(A) and tet(B) genes differed between S. sonnei and S. flexneri. Moreover, the prevalence of plasmid Inc groups in S. flexneri and S. sonnei differed. However, no relationship was found between the two phenomena

Stabilization of the V2 loop improves the presentation of V2 loop–associated broadly neutralizing antibody epitopes on HIV-1 envelope trimers

This research was originally published in the Journal of Biological Chemistry. de Taeye, Steven W. ; Go, Eden P. ; Sliepen, Kwinten; de la Peña, Alba Torrents ; Badal, Kimberly; Medina-Ramírez, Max; Lee, Wen-Hsin; Desaire, Heather; Wilson, Ian A. ; Moore, John P. ; Ward, Andrew B. ; Sanders, Rogier W. Stabilization of the V2 loop improves the presentation of V2 loop–associated broadly neutralizing antibody epitopes on HIV-1 envelope trimers. J Biol Chem. 2019; Vol294:5616-5631. © the American Society for Biochemistry and Molecular Biology. This work is licensed under a Creative Commons Attribution 4.0 International License.A successful HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that target the envelope glycoprotein (Env) spike on the virus. Native-like recombinant Env trimers of the SOSIP design now serve as a platform for achieving this challenging goal. However, SOSIP trimers usually do not bind efficiently to the inferred germline precursors of bNAbs (gl-bNAbs). We hypothesized that the inherent flexibilities of the V1 and V2 variable loops in the Env trimer contribute to the poor recognition of gl-bNAb epitopes at the trimer apex that extensively involve V2 residues. To reduce local V2 flexibility and improve the binding of V2-dependent bNAbs and gl-bNAbs, we designed BG505 SOSIP.664 trimer variants containing newly created disulfide bonds intended to stabilize the V2 loop in an optimally antigenic configuration. The first variant, I184C/E190C, contained a new disulfide bond within the V2 loop, whereas the second variant, E153C/R178C, had a new disulfide bond that cross-linked V2 and V1. The resulting engineered native-like trimer variants were both more reactive with and were neutralized by V2 bNAbs and gl-bNAbs, a finding that may be valuable in the design of germline targeting and boosting trimer immunogens to create an antigenic conformation optimal for HIV vaccine development