Limits...
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.

Show MeSH

Related in: MedlinePlus

Comb-mut stabilizing mutations increase the proportion of trimers of gp140s secreted from 293S cells and increase mAb PG9 recognition.(A) The oligomeric state of ADA and comb-mut gp140 secreted from 293S (GnTI−/−) cells was analyzed by BN-PAGE using an anti-gp120 mAb cocktail. The gp140 trimer band was verified by size comparison to KNH1144 SOSIP and JRFL-foldon gp140 trimers. Other (non-trimeric) bands are postulated to correspond to gp140 dimer, gp140 monomer, and gp120 monomer, respectively, based on size, but this has not been confirmed. The blot shown is a representative example of ten separate BN-PAGE analyses of Env from three independent transfections. (B) The percentage of total density present in either the Env trimer band or in the non-trimeric Env bands combined was quantified for all BN-PAGE analyses and the differences between ADA and comb-mut were compared. (C) Data from panel B presented in numerical format. (D) Binding of a panel of mAbs to ADA wild-type and comb-mut gp140s from cell culture supernatant was determined using an ELISA. The fold change in binding EC50 to comb-mut relative to ADA (i.e. ADA EC50/comb-mut EC50) was calculated and plotted. For PG9, the difference in EC50 between ADA and comb-mut was statistically significant (p = 0.0012, n = 8). No significant difference in binding was found for the other mAbs. (E) ADA and comb-mut soluble gp140 samples were subjected to a heat gradient for 1 hour and then visualized using BN-PAGE. The experiment was performed three times and a representative Western blot is shown. (F) The intensity of the Env trimer bands from each lane in panel E was quantified and plotted as the percent of the trimer band remaining relative to the corresponding band of the sample incubated on ice. The results are the average of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585149&req=5

ppat-1003184-g014: Comb-mut stabilizing mutations increase the proportion of trimers of gp140s secreted from 293S cells and increase mAb PG9 recognition.(A) The oligomeric state of ADA and comb-mut gp140 secreted from 293S (GnTI−/−) cells was analyzed by BN-PAGE using an anti-gp120 mAb cocktail. The gp140 trimer band was verified by size comparison to KNH1144 SOSIP and JRFL-foldon gp140 trimers. Other (non-trimeric) bands are postulated to correspond to gp140 dimer, gp140 monomer, and gp120 monomer, respectively, based on size, but this has not been confirmed. The blot shown is a representative example of ten separate BN-PAGE analyses of Env from three independent transfections. (B) The percentage of total density present in either the Env trimer band or in the non-trimeric Env bands combined was quantified for all BN-PAGE analyses and the differences between ADA and comb-mut were compared. (C) Data from panel B presented in numerical format. (D) Binding of a panel of mAbs to ADA wild-type and comb-mut gp140s from cell culture supernatant was determined using an ELISA. The fold change in binding EC50 to comb-mut relative to ADA (i.e. ADA EC50/comb-mut EC50) was calculated and plotted. For PG9, the difference in EC50 between ADA and comb-mut was statistically significant (p = 0.0012, n = 8). No significant difference in binding was found for the other mAbs. (E) ADA and comb-mut soluble gp140 samples were subjected to a heat gradient for 1 hour and then visualized using BN-PAGE. The experiment was performed three times and a representative Western blot is shown. (F) The intensity of the Env trimer bands from each lane in panel E was quantified and plotted as the percent of the trimer band remaining relative to the corresponding band of the sample incubated on ice. The results are the average of three independent experiments.

Mentions: When the oligomeric states of secreted comb-mut and ADA gp140s were compared using BN-PAGE, we observed a statistically significant increase over wild-type in the proportion of comb-mut Env that spontaneously formed trimers (51% trimer for comb-mut and 25% trimer for ADA, p = <0.0001, n = 10), which was accompanied by a corresponding decrease in bands corresponding to non-trimeric Env (Figure 14A, B, and C). To rule out the possibility that this apparent increase in the trimeric population could be an artifact of BN-PAGE/Western blots, we used the same Env preparations in ELISA to analyze binding to PG9 - that has a strong preference for trimeric Env [41] - along with several control mAbs. With the control mAbs, we observed strong binding by both neutralizing (e.g. 2G12 and b12) and non-neutralizing (e.g. b6 and 7B2) control mAbs, with no change in binding between the two Envs with these or other mAbs (Figure 14D and data not shown). However, much weaker but highly reproducible binding was observed using PG9 against both ADA and comb-mut soluble Env, and, consistent with the BN-PAGE data, there was a statistically significant two-fold increase in PG9 binding to comb-mut relative to ADA (Figure 14D). Thus, the secreted Env is comprised of Env trimers that either lack certain antigenic features of native Env, or those Env trimers with native antigenicity would have to be a relatively minor constituent of the total Env population. However, in addition to slightly enhancing trimerization of soluble gp140, the stabilizing mutations in comb-mut also cause a small but significant increase in the proportion of PG9-reactive molecules, which are both sought after features in Env immunogen design.


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

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

Comb-mut stabilizing mutations increase the proportion of trimers of gp140s secreted from 293S cells and increase mAb PG9 recognition.(A) The oligomeric state of ADA and comb-mut gp140 secreted from 293S (GnTI−/−) cells was analyzed by BN-PAGE using an anti-gp120 mAb cocktail. The gp140 trimer band was verified by size comparison to KNH1144 SOSIP and JRFL-foldon gp140 trimers. Other (non-trimeric) bands are postulated to correspond to gp140 dimer, gp140 monomer, and gp120 monomer, respectively, based on size, but this has not been confirmed. The blot shown is a representative example of ten separate BN-PAGE analyses of Env from three independent transfections. (B) The percentage of total density present in either the Env trimer band or in the non-trimeric Env bands combined was quantified for all BN-PAGE analyses and the differences between ADA and comb-mut were compared. (C) Data from panel B presented in numerical format. (D) Binding of a panel of mAbs to ADA wild-type and comb-mut gp140s from cell culture supernatant was determined using an ELISA. The fold change in binding EC50 to comb-mut relative to ADA (i.e. ADA EC50/comb-mut EC50) was calculated and plotted. For PG9, the difference in EC50 between ADA and comb-mut was statistically significant (p = 0.0012, n = 8). No significant difference in binding was found for the other mAbs. (E) ADA and comb-mut soluble gp140 samples were subjected to a heat gradient for 1 hour and then visualized using BN-PAGE. The experiment was performed three times and a representative Western blot is shown. (F) The intensity of the Env trimer bands from each lane in panel E was quantified and plotted as the percent of the trimer band remaining relative to the corresponding band of the sample incubated on ice. The results are the average of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003184-g014: Comb-mut stabilizing mutations increase the proportion of trimers of gp140s secreted from 293S cells and increase mAb PG9 recognition.(A) The oligomeric state of ADA and comb-mut gp140 secreted from 293S (GnTI−/−) cells was analyzed by BN-PAGE using an anti-gp120 mAb cocktail. The gp140 trimer band was verified by size comparison to KNH1144 SOSIP and JRFL-foldon gp140 trimers. Other (non-trimeric) bands are postulated to correspond to gp140 dimer, gp140 monomer, and gp120 monomer, respectively, based on size, but this has not been confirmed. The blot shown is a representative example of ten separate BN-PAGE analyses of Env from three independent transfections. (B) The percentage of total density present in either the Env trimer band or in the non-trimeric Env bands combined was quantified for all BN-PAGE analyses and the differences between ADA and comb-mut were compared. (C) Data from panel B presented in numerical format. (D) Binding of a panel of mAbs to ADA wild-type and comb-mut gp140s from cell culture supernatant was determined using an ELISA. The fold change in binding EC50 to comb-mut relative to ADA (i.e. ADA EC50/comb-mut EC50) was calculated and plotted. For PG9, the difference in EC50 between ADA and comb-mut was statistically significant (p = 0.0012, n = 8). No significant difference in binding was found for the other mAbs. (E) ADA and comb-mut soluble gp140 samples were subjected to a heat gradient for 1 hour and then visualized using BN-PAGE. The experiment was performed three times and a representative Western blot is shown. (F) The intensity of the Env trimer bands from each lane in panel E was quantified and plotted as the percent of the trimer band remaining relative to the corresponding band of the sample incubated on ice. The results are the average of three independent experiments.
Mentions: When the oligomeric states of secreted comb-mut and ADA gp140s were compared using BN-PAGE, we observed a statistically significant increase over wild-type in the proportion of comb-mut Env that spontaneously formed trimers (51% trimer for comb-mut and 25% trimer for ADA, p = <0.0001, n = 10), which was accompanied by a corresponding decrease in bands corresponding to non-trimeric Env (Figure 14A, B, and C). To rule out the possibility that this apparent increase in the trimeric population could be an artifact of BN-PAGE/Western blots, we used the same Env preparations in ELISA to analyze binding to PG9 - that has a strong preference for trimeric Env [41] - along with several control mAbs. With the control mAbs, we observed strong binding by both neutralizing (e.g. 2G12 and b12) and non-neutralizing (e.g. b6 and 7B2) control mAbs, with no change in binding between the two Envs with these or other mAbs (Figure 14D and data not shown). However, much weaker but highly reproducible binding was observed using PG9 against both ADA and comb-mut soluble Env, and, consistent with the BN-PAGE data, there was a statistically significant two-fold increase in PG9 binding to comb-mut relative to ADA (Figure 14D). Thus, the secreted Env is comprised of Env trimers that either lack certain antigenic features of native Env, or those Env trimers with native antigenicity would have to be a relatively minor constituent of the total Env population. However, in addition to slightly enhancing trimerization of soluble gp140, the stabilizing mutations in comb-mut also cause a small but significant increase in the proportion of PG9-reactive molecules, which are both sought after features in Env immunogen design.

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