Improving the immunogenicity of native-like HIV-1 envelope trimers by hyperstabilization

The production of native-like recombinant versions of the HIV-1 envelope glycoprotein (Env) trimer requires overcoming the natural flexibility and instability of the complex. The engineered BG505 SOSIP.664 trimer mimics the structure and antigenicity of native Env. Here, we describe how the introduction of new disulfide bonds between the glycoprotein (gp)120 and gp41 subunits of SOSIP trimers of the BG505 and other genotypes improves their stability and antigenicity, reduces their conformational flexibility, and helps maintain them in the unliganded conformation. The resulting next-generation SOSIP.v5 trimers induce strong autologous tier-2 neutralizing antibody (NAb) responses in rabbits. In addition, the BG505 SOSIP.v6 trimers induced weak heterologous NAb responses against a subset of tier-2 viruses that were not elicited by the prototype BG505 SOSIP.664. These stabilization methods can be applied to trimers from multiple genotypes as components of multivalent vaccines aimed at inducing broadly NAbs (bNAbs)

A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors

In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAc alpha 2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAc alpha 2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor bindin

Three mutations switch H7N9 influenza to human-type receptor specificity

The avian H7N9 influenza outbreak in 2013 resulted from an unprecedented incidence of influenza transmission to humans from infected poultry. The majority of human H7N9 isolates contained a hemagglutinin (HA) mutation (Q226L) that has previously been associated with a switch in receptor specificity from avian-type (NeuAcα2-3Gal) to human-type (NeuAcα2-6Gal), as documented for the avian progenitors of the 1957 (H2N2) and 1968 (H3N2) human influenza pandemic viruses. While this raised concern that the H7N9 virus was adapting to humans, the mutation was not sufficient to switch the receptor specificity of H7N9, and has not resulted in sustained transmission in humans. To determine if the H7 HA was capable of acquiring human-type receptor specificity, we conducted mutation analyses. Remarkably, three amino acid mutations conferred a switch in specificity for human-type receptors that resembled the specificity of the 2009 human H1 pandemic virus, and promoted binding to human trachea epithelial cell

Specificity of wild type and mutant H7 HAs on glycan arrays and binding to chicken and human trachea epithelium.

<p>Glycan binding analyses of Sh2 H7N9 HA wild type and several mutants that confer human-type receptor binding: G228S, K193T G228S, V186K K193T G228S, V186G K193T G228S, with human Cal/04/09 2009 pandemic H1N1 HA as a control. (A) ELISA-like assay using sialoside polymers. The mean signal and standard error were calculated from six independent replicates; white open circles represent α2–3 linked sialylated di-LacNAc (3’SLNLN), black closed circles represent α2–6 linked sialylated di-LacNAc (6’SLNLN), and non-sialylated di-LacNAc (LNLN) are represented in asterisks. (B) The glycan array mean signal and standard error were calculated from six independent replicates; α2–3 linked sialosides are shown in white bars (glycans 11 to 79 on the x axis) and α2–6 linked sialosides in black (glycans 80 to 135). Glycans 1 to 10 are non-sialylated controls (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006390#ppat.1006390.s001" target="_blank">S1 Table</a>). (C) Tissue binding to either chicken or human tracheal sections is observed by HRP-staining. The sialoside array, ELISA-like assay, and tissue binding experiments are representative of three independent assays performed with different batches of HA proteins.</p

Melting curve of recombinant HA obtained by DSC to determine the thermostability of Sh2, Sh2 V186K K193T G228S, and A/KY/07.

<p>The raw data are depicted in the solid line, while the fitted curve, from which the <i>T</i>_m was derived, is depicted with a dotted line. (A) A summary of the peaks observed during the DSC experiments for each recombinant HA, (B) Sh2, (C) A/KY/07 and (D) Sh2 V186K K193T G228S.</p

H7 Sh2 mutant combinations that also bind to human-type receptors.

<p>Glycan binding analyses of Sh2 H7N9 mutant HAs, V186N G226S (A) and V186N N224K G228S (B). The mean signal and standard error were calculated from six independent replicates on both the PAA (left column) and the sialoside array (right column). Tissue binding to either chicken or human tracheal sections is observed by HRP-staining (right column). In the PAA array, white open circles represent α2–3 linked sialylated di-LacNAc (3’SLNLN), black closed circles represent α2–6 linked sialylated di-LacNAc (6’SLNLN), and non-sialylated di-LacNAc (LNLN) are represented in asterisks. In the sialoside array α2–3 linked sialosides are shown in white bars (glycans 11 to 79 on the x axis) and α2–6 linked sialosides in black (glycans 80 to 135). Glycans 1 to 10 are non-sialylated controls (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006390#ppat.1006390.s001" target="_blank">S1 Table</a>). The sialoside array, ELISA-like assay and tissue binding experiments shown are representative of three independent assays performed with different batches of HA proteins.</p

Avidity of Sh2 (WT) and Sh2 V186K-K193T-G228S variant HA for N-linked glycan receptors assessed by glycan ELISA.

<p>Sh2 (upper panels) binds strongly to avian-type (α2–3) receptors (left, white open shapes) with weaker binding to human-type (α2–6) receptors (right, black closed shapes). Sh2 V186K-K193T-G228S (lower panels) shows vastly reduced avidity for avian N-glycans and increased selectivity for extended glycan receptors to human receptors. Assays are conducted with biantennary, N-linked glycans (N) with one to four LacNAc (LN, Galβ1-4GlcNAc) repeats terminated with sialic acid (S) in α2–3 or α2–6 linkage (SLN_1-4-N). An asialo, mono-LacNAc (LacNAc-biotin, LN-L) was used as a negative binding control.</p

Amino acid variation in the receptor binding pocket of influenza HAs and impact of K193T mutation on receptor conformation.

<p>(A) Variation at HA positions that are known to mediate the switch in receptor binding specificity for human H1, H2 and H3 pandemic viruses and corresponding avian viruses of H1, H2, H3 and H5 subtypes in comparison with human H7N9. Red indicates amino acids involved in either human- or avian-type receptor specificity, blue indicates amino-acid positions that are mutated to the amino acids found in human H3N2 and H2N2 viruses. (B) Projection of the receptor glycan from the binding pocket. The receptor analog 6’SLNLN (α2–6 linked sialylated di-LacNAc; NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc) is modeled in the WT H7 with K193 (dark gray), and the mutant H7 with V186K K193T G228S (light gray). In the WT, K193 causes the receptor to project further away from the 190 helix. Symbols in the sugar rings are the conventions for the Symbol Nomenclature For Glycans (SNFG) where sialic acid is the purple cubic diamond, galactose is the yellow sphere and GlcNAc is the blue cube.</p