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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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Stable HIV-1 Env mutants are more fully neutralized than wild-type ADA by mAbs PG9 and PG16.Viruses were pre-incubated with PG9 (A) or PG16 (B) for one hour, and then the mixture was overlaid onto TZM-bl target cells. Infectivity was measured 48 hours later. A representative experiment performed in triplicate is shown. Maximal percent inhibition (MPI) of the mAbs against each virus was calculated by non-linear regression of the neutralization curves.
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ppat-1003184-g009: Stable HIV-1 Env mutants are more fully neutralized than wild-type ADA by mAbs PG9 and PG16.Viruses were pre-incubated with PG9 (A) or PG16 (B) for one hour, and then the mixture was overlaid onto TZM-bl target cells. Infectivity was measured 48 hours later. A representative experiment performed in triplicate is shown. Maximal percent inhibition (MPI) of the mAbs against each virus was calculated by non-linear regression of the neutralization curves.

Mentions: It has been shown that mAbs PG9 and PG16 can neutralize ∼92% of HIV-1 isolates in a large multi-clade viral panel but can occasionally give rise to inhibition curves that plateau below 100% neutralization for certain sensitive isolates [10], [41]. The latter phenomenon is thought to be caused by heterogeneity in glycosylation on the Env trimer [10]. We observed such a phenomenon with wild-type ADA in which PG9 and PG16 exhibited a maximal percent inhibition (MPI) of 74% and 90%, respectively (Figure 9). Interestingly, both GB21-6 and HC11-1 exhibited higher MPIs against both mAbs; PG9 and PG16 had 90% and 96% MPI, respectively. Thus, not only do these two mutants of ADA exhibit decreased levels of heterogeneity and “cleaner” trimer bands on BN-PAGE, but they also show less heterogeneity within the pool of functional trimers as probed by mAbs PG9/16.


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

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

Stable HIV-1 Env mutants are more fully neutralized than wild-type ADA by mAbs PG9 and PG16.Viruses were pre-incubated with PG9 (A) or PG16 (B) for one hour, and then the mixture was overlaid onto TZM-bl target cells. Infectivity was measured 48 hours later. A representative experiment performed in triplicate is shown. Maximal percent inhibition (MPI) of the mAbs against each virus was calculated by non-linear regression of the neutralization curves.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003184-g009: Stable HIV-1 Env mutants are more fully neutralized than wild-type ADA by mAbs PG9 and PG16.Viruses were pre-incubated with PG9 (A) or PG16 (B) for one hour, and then the mixture was overlaid onto TZM-bl target cells. Infectivity was measured 48 hours later. A representative experiment performed in triplicate is shown. Maximal percent inhibition (MPI) of the mAbs against each virus was calculated by non-linear regression of the neutralization curves.
Mentions: It has been shown that mAbs PG9 and PG16 can neutralize ∼92% of HIV-1 isolates in a large multi-clade viral panel but can occasionally give rise to inhibition curves that plateau below 100% neutralization for certain sensitive isolates [10], [41]. The latter phenomenon is thought to be caused by heterogeneity in glycosylation on the Env trimer [10]. We observed such a phenomenon with wild-type ADA in which PG9 and PG16 exhibited a maximal percent inhibition (MPI) of 74% and 90%, respectively (Figure 9). Interestingly, both GB21-6 and HC11-1 exhibited higher MPIs against both mAbs; PG9 and PG16 had 90% and 96% MPI, respectively. Thus, not only do these two mutants of ADA exhibit decreased levels of heterogeneity and “cleaner” trimer bands on BN-PAGE, but they also show less heterogeneity within the pool of functional trimers as probed by mAbs PG9/16.

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