HIV-1 virus-like particles bearing pure env trimers expose neutralizing epitopes but occlude nonneutralizing epitopes

Abstract

Hypothetically, since native HIV-1 Env trimers are exclusively recognized by neutralizing antibodies, they might induce the neutralizing antibodies in a vaccine setting. This idea has not been evaluated due to the difficulty of separating trimers from nonfunctional Env (uncleaved gp160 and gp41 stumps). The latter are immunodominant and induce nonneutralizing antibodies. We previously showed that nonfunctional Env can be selectively cleared from virus-like particle (VLP) surfaces by enzyme digests (E. T. Crooks, T. Tong(,) K. Osawa, and J. M. Binley, J.Virol. 85:5825, 2011). Here, we investigated the effects of these digests on the antigenicity of VLPs and their sensitivity to neutralization. Before digestion, WT VLPs (bearing wild-type Env) and UNC VLPs (bearing uncleaved gp160) were recognized by various Env-specific monoclonal antibodies (MAbs), irrespective of their neutralizing activity, a result which is consistent with the presence of nonfunctional Env. After digestion, only neutralizing MAbs recognized WT VLPs, consistent with selective removal of nonfunctional Env (i.e., "trimer VLPs"). Digests eliminated the binding of all MAbs to UNC VLPs, again consistent with removal of nonfunctional Env. An exception was MAb 2F5, which weakly bound to digested UNC VLPs and bald VLPs (bearing no Env), perhaps due to lipid cross-reactivity. Trimer VLPs were infectious, and their neutralization sensitivity was largely comparable to that of undigested WT VLPs. However, they were ∼100-fold more sensitive to the MAbs 4E10 and Z13e1, suggesting increased exposure of the gp41 base. Importantly, a scatterplot analysis revealed a strong correlation between MAb binding and neutralization of trimer VLPs. This suggests that trimer VLPs bear essentially pure native trimer that should allow its unfettered evaluation in a vaccine setting.

Fig 1

Antigenicity of monomeric gp120 and various VLPs analyzed by ELISA. Monomeric gp120 and various VLPs, as indicated, were coated on ELISA wells and probed by MAbs directed to various Env epitopes. Data are representative of at least 3 titrations of each MAb against each VLP. mann., mannose.

Fig 2

Effect of endo H on PG epitopes on native trimers. E168K+N189A WT VLPs were digested with endo H as indicated and then assayed for MAb recognition by VLP ELISA (A) or BN-PAGE shifts (B), using 30 μg/ml of each MAb.

Fig 3

Effects of digests on MAb binding to E168K+N189A WT VLPs by ELISA. Data shown are representative of at least 3 titrations of each MAb.

Fig 4

MAb binding to undigested and digested UNC WT VLPs was investigated by VLP ELISA. In addition, 2F5 binding to bald VLPs before and after digestion was assessed by ELISA. Data are representative of at least 3 titrations of each MAb against each VLP.

Fig 5

Effects of protease digests on VLP neutralization sensitivity. The neutralization sensitivities of undigested E168K+N189A WT VLPs and digested E168K+N189A WT trimer VLPs to various MAbs were assayed using CF2.CD4.CCR5 cells as targets. Each assay was performed in duplicate. Data are representative of at least 3 repeats.

Fig 6

Effects of enzyme digests on native PAGE shifts of E168K+N189A WT VLPs. Undigested E168K+N189A WT VLPs (A) and enzyme-digested E168K+N189A WT trimer VLPs (B) were incubated with various MAbs or sCD4 at 30 μg/ml each for 3 h at 37°C and then washed and resolved by a BN-PAGE Western blot. An ∼200-kDa band induced by the MAbs 4E10 and Z13e1 is indicated by an asterisk in panel B.

Fig 7

Soluble CD4 binding to E168K+N189A WT trimer VLPs causes gp120 shedding. The band induced by sCD4 binding to trimer VLPs (Fig. 6, lane 4) was investigated by probing separate blots of E168K+N189A trimer VLPs with anti-gp120 or anti-gp41 MAb cocktails as indicated. In lanes 3 and 6, the wash step that removes any unbound sCD4 before VLP lysis was omitted.

Fig 8

Effects of enzyme digests on native PAGE shifts of WT VLPs. Undigested WT VLPs (A), digested WT trimer VLPs (B), and SOS UNC VLPs (C) were incubated with various MAbs or sCD4 at 30 μg/ml for 3 h at 37°C and then washed and resolved by a BN-PAGE Western blot. An ∼200-kDa band induced by the MAbs 4E10 and Z13e1 is indicated by an asterisk in panel B. A high-molecular-weight species induced by the MAb CO11 is indicated by an arrow.

Fig 9

Effect of enzyme digestion on the trimer affinity of the MAbs 4E10 and Z13e1. 4E10 (A) and Z13e1 (B) MAbs were titrated against undigested or digested E168K+N189A WT VLPs as indicated. Below each gel, the density of the trimer band is shown as a histogram, determined using UN-SCAN-IT densitometry software (Silk Scientific). The band density in the absence of MAb is set at 100%, and any trimer binding/depletion is shown as a drop in trimer staining as the trimer becomes complexed with the MAb. Note that the trimer density of the digested E168K+N189A WT trimer VLPs is somewhat weaker than that of the undigested E168K+N189A WT VLP trimer (compare lanes 1 and 8 in panel A and lanes 1 and 7 in panel B). Therefore, the density of unliganded trimer in each of these control lanes was set at 100%.

Fig 10

Investigation of 4E10 and Z13e1 binding to E168K+N189A WT VLPs by a BN-PAGE Western blot. The nature of the ∼200-kDa species induced by 4E10 and Z13e1 binding to E168K+N189A WT trimer VLPs (indicated by an asterisk in Fig. 6B and 8B) was investigated by probing separate BN-PAGE Western blots with anti-gp120 and anti-gp41 cocktails (A) and by using biotinylated versions of Z13e1 and 2G12 for shifts and detecting with either the gp120/gp41 cocktail or strepavidin-alkaline phosphatase (streptavidin-AP) (B).

Fig 11

Correlation of VLP antigenicity and neutralization sensitivity. MAb neutralization IC₅₀s against undigested E168K+N189A WT VLPs (A) and digested E168K+N189A WT trimer VLPs (B) were plotted against MAb binding titers from ELISAs using the same VLPs. Neutralizing MAbs are indicated by blue symbols. Nonneutralizing MAbs are indicated by red symbols. Although Z13e1 exhibits little neutralization against undigested VLPs, it is given a blue symbol because it effectively neutralizes trimer VLPs. Furthermore, despite the fact that PGT136 is a broadly neutralizing MAb, it is classified as a nonneutralizing MAb in this instance, as it fails to neutralize JR-FL. ELISA titers were taken as the concentration at which MAb binding exhibited an optical density of 1.0 at 405 nm (Fig. 3). Any titers of >30 μg/ml are plotted at 30 μg/ml. All titers are means of at least 3 repeat assays. Linear regression and P values were calculated.