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Increased functional stability and homogeneity of viral envelope spikes through directed evolution.

Leaman DP, Zwick MB - PLoS Pathog. (2013)

Bottom Line: Combining the seven mutations generated a variant Env with superior homogeneity and stability.Heterogeneity within the functional population of hyper-stable Envs was also reduced, as evidenced by a relative decrease in a proportion of virus that is resistant to the neutralizing Ab, PG9.The latter result may reflect a change in glycans on the stabilized Envs.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
The functional HIV-1 envelope glycoprotein (Env) trimer, the target of anti-HIV-1 neutralizing antibodies (Abs), is innately labile and coexists with non-native forms of Env. This lability and heterogeneity in Env has been associated with its tendency to elicit non-neutralizing Abs. Here, we use directed evolution to overcome instability and heterogeneity of a primary Env spike. HIV-1 virions were subjected to iterative cycles of destabilization followed by replication to select for Envs with enhanced stability. Two separate pools of stable Env variants with distinct sequence changes were selected using this method. Clones isolated from these viral pools could withstand heat, denaturants and other destabilizing conditions. Seven mutations in Env were associated with increased trimer stability, primarily in the heptad repeat regions of gp41, but also in V1 of gp120. Combining the seven mutations generated a variant Env with superior homogeneity and stability. This variant spike moreover showed resistance to proteolysis and to dissociation by detergent. Heterogeneity within the functional population of hyper-stable Envs was also reduced, as evidenced by a relative decrease in a proportion of virus that is resistant to the neutralizing Ab, PG9. The latter result may reflect a change in glycans on the stabilized Envs. The stabilizing mutations also increased the proportion of secreted gp140 existing in a trimeric conformation. Finally, several Env-stabilizing substitutions could stabilize Env spikes from HIV-1 clades A, B and C. Spike stabilizing mutations may be useful in the development of Env immunogens that stably retain native, trimeric structure.

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Virus capture assay shows that stable Env spikes are resistant to sCD4-induced inactivation.Wild-type ADA, GB21-6, and HC11-1 were pre-incubated with varying concentrations of sCD4 and then captured on microwells coated with either mAb X5 (A) or hNM01 (B). Unbound virus was washed away and the infectivity of the remaining virus was assessed using overlaid TZM-bl target cells. Experiments were performed in triplicate.
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ppat-1003184-g007: Virus capture assay shows that stable Env spikes are resistant to sCD4-induced inactivation.Wild-type ADA, GB21-6, and HC11-1 were pre-incubated with varying concentrations of sCD4 and then captured on microwells coated with either mAb X5 (A) or hNM01 (B). Unbound virus was washed away and the infectivity of the remaining virus was assessed using overlaid TZM-bl target cells. Experiments were performed in triplicate.

Mentions: Certain neutralizing monoclonal Abs (mAbs) and other inhibitory ligands, such as soluble CD4 (sCD4), can destabilize and irreversibly inactivate the Env trimer upon binding [36]–[38]. We used two approaches to determine whether the stabilized clones of HIV-1 Env would resist ligand destabilization. In the first approach, we employed a modified virus capture assay (VCA) that was designed to crudely mimic Env destabilization through host receptor engagement. Thus, GB21-6 or HC11-1 virions were incubated in solution with increasing concentrations of sCD4 for 15 min, and then overlaid on microwells coated with either mAb hNM01 (anti-V3) or X5 (anti-coreceptor binding site (CoRbs)). Unbound virus was then washed away and TZM-bl target cells were overlaid to measure remaining infectivity. Binding of sCD4 to the Env trimer initially exposes the V3 loop and CoRbs in a fusion-active state [39], but after a short period the sCD4-bound trimer decays to an inactive state [36]. The VCA design provides an aggregate measure of induction of conformational changes by sCD4 and the functional stability of the sCD4-activated state. As expected, capture of infectious wild-type ADA was increased by low concentrations of sCD4 that promotes exposure of the epitopes of the capture mAbs, but decreased at higher concentrations as virions were inactivated (Figure 7). By contrast, when using the same initial concentrations of sCD4 capture efficiency of infectious virus was increased for both GB21-6 and HC11-1, and, at high concentrations of sCD4 that inactivated wild-type ADA, the infectivities of GB21-6 and HC11-1 were still intact. We note that in the absence of sCD4 mAbs X5 and hNM01 captured lower levels of GB21-6 and HC11-1 relative to wild-type ADA (Figure 7). This may be related to the BN-PAGE results showing that these viruses display more homogeneous trimers (Figure 6), as mAb X5 does not appear to bind the unliganded trimers of most primary isolates and likely captures virions via non-native Env [6].


Increased functional stability and homogeneity of viral envelope spikes through directed evolution.

Leaman DP, Zwick MB - PLoS Pathog. (2013)

Virus capture assay shows that stable Env spikes are resistant to sCD4-induced inactivation.Wild-type ADA, GB21-6, and HC11-1 were pre-incubated with varying concentrations of sCD4 and then captured on microwells coated with either mAb X5 (A) or hNM01 (B). Unbound virus was washed away and the infectivity of the remaining virus was assessed using overlaid TZM-bl target cells. Experiments were performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585149&req=5

ppat-1003184-g007: Virus capture assay shows that stable Env spikes are resistant to sCD4-induced inactivation.Wild-type ADA, GB21-6, and HC11-1 were pre-incubated with varying concentrations of sCD4 and then captured on microwells coated with either mAb X5 (A) or hNM01 (B). Unbound virus was washed away and the infectivity of the remaining virus was assessed using overlaid TZM-bl target cells. Experiments were performed in triplicate.
Mentions: Certain neutralizing monoclonal Abs (mAbs) and other inhibitory ligands, such as soluble CD4 (sCD4), can destabilize and irreversibly inactivate the Env trimer upon binding [36]–[38]. We used two approaches to determine whether the stabilized clones of HIV-1 Env would resist ligand destabilization. In the first approach, we employed a modified virus capture assay (VCA) that was designed to crudely mimic Env destabilization through host receptor engagement. Thus, GB21-6 or HC11-1 virions were incubated in solution with increasing concentrations of sCD4 for 15 min, and then overlaid on microwells coated with either mAb hNM01 (anti-V3) or X5 (anti-coreceptor binding site (CoRbs)). Unbound virus was then washed away and TZM-bl target cells were overlaid to measure remaining infectivity. Binding of sCD4 to the Env trimer initially exposes the V3 loop and CoRbs in a fusion-active state [39], but after a short period the sCD4-bound trimer decays to an inactive state [36]. The VCA design provides an aggregate measure of induction of conformational changes by sCD4 and the functional stability of the sCD4-activated state. As expected, capture of infectious wild-type ADA was increased by low concentrations of sCD4 that promotes exposure of the epitopes of the capture mAbs, but decreased at higher concentrations as virions were inactivated (Figure 7). By contrast, when using the same initial concentrations of sCD4 capture efficiency of infectious virus was increased for both GB21-6 and HC11-1, and, at high concentrations of sCD4 that inactivated wild-type ADA, the infectivities of GB21-6 and HC11-1 were still intact. We note that in the absence of sCD4 mAbs X5 and hNM01 captured lower levels of GB21-6 and HC11-1 relative to wild-type ADA (Figure 7). This may be related to the BN-PAGE results showing that these viruses display more homogeneous trimers (Figure 6), as mAb X5 does not appear to bind the unliganded trimers of most primary isolates and likely captures virions via non-native Env [6].

Bottom Line: Combining the seven mutations generated a variant Env with superior homogeneity and stability.Heterogeneity within the functional population of hyper-stable Envs was also reduced, as evidenced by a relative decrease in a proportion of virus that is resistant to the neutralizing Ab, PG9.The latter result may reflect a change in glycans on the stabilized Envs.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
The functional HIV-1 envelope glycoprotein (Env) trimer, the target of anti-HIV-1 neutralizing antibodies (Abs), is innately labile and coexists with non-native forms of Env. This lability and heterogeneity in Env has been associated with its tendency to elicit non-neutralizing Abs. Here, we use directed evolution to overcome instability and heterogeneity of a primary Env spike. HIV-1 virions were subjected to iterative cycles of destabilization followed by replication to select for Envs with enhanced stability. Two separate pools of stable Env variants with distinct sequence changes were selected using this method. Clones isolated from these viral pools could withstand heat, denaturants and other destabilizing conditions. Seven mutations in Env were associated with increased trimer stability, primarily in the heptad repeat regions of gp41, but also in V1 of gp120. Combining the seven mutations generated a variant Env with superior homogeneity and stability. This variant spike moreover showed resistance to proteolysis and to dissociation by detergent. Heterogeneity within the functional population of hyper-stable Envs was also reduced, as evidenced by a relative decrease in a proportion of virus that is resistant to the neutralizing Ab, PG9. The latter result may reflect a change in glycans on the stabilized Envs. The stabilizing mutations also increased the proportion of secreted gp140 existing in a trimeric conformation. Finally, several Env-stabilizing substitutions could stabilize Env spikes from HIV-1 clades A, B and C. Spike stabilizing mutations may be useful in the development of Env immunogens that stably retain native, trimeric structure.

Show MeSH
Related in: MedlinePlus