Induction of an antibody response capable of neutralizing diverse HIV-1 isolates is a critical step for an effective vaccine that can protect against HIV-1 infection. Although vaccines have thus far failed to induce broadly neutralizing antibody responses, it is reported that approximately 1%–30% of HIV-1–infected subjects eventually develop potent and broadly reactive neutralizing antibodies.1–3 Most broadly neutralizing antibodies in chronic HIV-1 sera have been shown to target the region around the CD4-binding site, the CD4-induced site, the glycan-dependent or quaternary epitopes on the gp120, and the conserved elements of V3 region.4–8 In recent years, based on new neutralizing antibody screening systems, many potent and broadly MAbs have been isolated.9–11
The CD4-binding site (CD4bs) on gp120 is essential for viral entry and is highly conserved compared with other envelope regions. The CD4bs contains the key epitopes recognized by several broadly neutralizing antibodies obtained so far. MAb b12, a CD4bs-MAb, was isolated from a phage display library and can neutralize about 40% of known HIV-1 isolates.12,13 Recently, a battery of anti-CD4bs MAbs, including VRC01/VRC03,11 3BNC117,14 PGV04,15 and NIH45-46,16 shows significantly higher potency and wider breadth compared with b12. VRC01 can neutralize ∼90% of HIV-1 isolates at a low inhibitory concentration (IC50), and structural studies show that it achieves this neutralization by precisely recognizing the initial site of CD4 attachment on HIV-1 gp120. Mutagenesis studies indicated that VRC01 contacts within the gp120 loop D, the CD4 binding loop, and the V5 region were necessary for optimal VRC01 neutralization.17,18
N-linked glycans are important for processing of gp120 that ultimately influences envelop conformation, oligomerization, receptor binding, membrane fusion and viral entry, infectivity, and antibody neutralization of HIV.19–23 In our previous work, we individually mutated the 25 PNGS of a CRF07_BC isolate to study the effect on viral infectivity and antibody-mediated neutralization finding that certain N-linked glycosylation sites are critical for virus to infect target cells and protect the virus from neutralizing by monoclonal antibodies.24 The partial deglycosylated HIV envelope protein was previously shown to enhance the immunogenicity of some epitopes.17,25,26 Some studies reported that combined PNGS elimination at specific sites in clade B HIV can increase the neutralizing sensitivity to certain nMAbs.27,28 For example, mutating N186 and N197 simultaneously on gp120 of 3 CRF01_AE clones increased sensitivity to b12, whereas the mutating N186 or N197 individually was not sufficient to increase b12 sensitivity.29 Also the combined N460 and N463 mutation on V5 region of 08_B'C viruses resulted in higher VRC01 sensitivity than each single mutation alone.30 The conserved PNGS in gp41 were also eliminated individually and in combination to test the viral replication.31 Clade 07_BC viruses, which have been one of the most predominantly circulated HIV-1 strains in China, have a somewhat different glycan arrangement than the B/AE and other clade viruses, and the elimination of combined PNGS located on different regions of the gp120 of the predominant Clade of HIV viruses in China has not been evaluated before.
To further understand the biological functions of the gp120 PNGS of the Clade 07_BC HIV-1 strain predominantly circulating in China, we constructed combined PNGS mutants based on previous single PNGS mutants to evaluate their infectivity and neutralizing sensitivity to nMAbs. We identified certain combined PNGS mutants that become highly sensitive to certain nMAbs, suggesting that the PNGS sites on these mutants likely play a critical role for shielding the virus from being recognized by these nMAbs and targeting/recognition by the host humoral responses.
Cells and Plasmid
TZM-bl cells, Env-deficient HIV-1 backbone (pSG3ΔEnv) were obtained from the US National Institutes of Health (NIH) AIDS Research and Reference Reagent Program (ARRRP), as contributed by John C. Kappes, Xiaoyun Wu, and Tranzyme (Birmingham, AL). The 293FT cells were obtained from Invitrogen (Carlsbad, CA).
MAbs and Positive Serum
MAbs 2F5, 4E10, 2G12, and b12 were obtained from the International AIDS Vaccine Initiative (IAVI, New York, NY); MAbs 2F5 and 4E10 were contributed by Hermann Katinger (Institute of Applied Microbiology, Vienna, Austria), and b12 was contributed by Dennis Burton and Carlos Barbas (The Scripps Research Institute, La Jolla, CA); MAbs VRC01, VRC03, PG9, and PG16 were obtained from the ARRRP (NIH).
For use in the study, 20 subtype BC HIV-1–infected positive sera were obtained from chronically HIV-1–infected blood donors in main HIV-1 epidemic regions of China. Individual samples were coded based on the region of China. These samples were collected from Beijing (bj20, bj22), Sichuan (sc59r), Guangdong (gd64), Yunnan (yn99r, yn148r), Guangxi (gx66, gx75, gx76, gx77, gx78, gx79, gx82, gx85, gx87, gx93, gx94, gx95) province and Xinjiang autonomous region (xj16, xj50). Samples were stored at −70°C, and freezing/thawing cycles were avoided. All serum samples were heat-inactivated at 56°C for 1 hour before use.
Elimination of PNGS by Mutagenesis
The motif for an N-linked glycosylation site is Asn-X-Thr/Ser, where X can be any amino acid except proline.32 Elimination of PNGS was performed using site-directed mutagenesis, by changing an asparagine (N) to a glutamine (Q) or aspartate (D). The WT env gene was inserted into pcDNA 3.1D/V5-His-TOPO (Invitrogen) as a template for mutagenesis. Mutagenesis was performed as described previously.24 Standard polymerase chain reaction and cloning procedure were used to obtain the mutant clones. The entire env gene of each mutant was sequenced to confirm mutation.
Pseudovirus Preparation, Infectivity, Titration and Neutralization Assays
Pseudoviruses were produced by cotransfection of 293FT cells (>90% confluency in a 25 cm2 rectangular canted neck cell culture flask, Corning, NY) with 5.3 μg pSG3ΔEnv plasmid and 2.7 μg Env-expressing plasmids using the Lipofectamine 2000 reagent (Invitrogen). Supernatants were harvested 48 hours after transfection, filtered (0.45-μm pore size), and stored at −80°C. The concentration of HIV-1 Gag p24 antigen in viral supernatants was measured by enzyme-linked immunosorbent assay (Vironostika HIV-1 antigen microenzyme-linked immunosorbent assay system; bioMérieux, Boxtel, the Netherlands).
A fixed amount of pseudovirus (equivalent to 1.0 ng p24 antigen) was added to TZM-bl cells at 70%–80% confluency in a 96-well plate in the presence of 15 μg/mL DEAE-dextran in a total volume of 200 μL. Forty-eight hours after infection, the luciferase activity in infected cells was measured using the Bright-Glo luciferase assay system (Promega, Madison, WI). Relative infectivity was calculated by dividing the Log10 (RLU of mutant) by Log10 (RLU of WT).
The 50% tissue culture infectious dose (TCID50) of a single infectious pseudovirus batch was determined in TZM-bl cells, as described previously.33 Neutralization was measured as a reduction in luciferase expression after a single-round infection of TZM-bl cells with pseudoviruses according to previously published method.34
The full-length HIV FE gp120 was generated using the homology modeling software Modeller 9.13.35 The gp120 pdb structures 4nco, 2ny7, 3ngb, and 3se8 were used as templates for modeling and glycans were modeled from 4nco. The interfacial residues that make up the defined epitope/paratope for the gp120 and protein ligands where calculated using PDBe PISA v1.48 server “Protein interfaces, surfaces, and assemblies” service PISA at the European Bioinformatics Institute (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html).36
Construction of the Combined PNGS Mutants and Viral Infectivity
In the previous study, the asparagine residue in all 25 PNGS on the wild-type gp120/41 of the HIV strain FE were mutated individually to glutamine or aspartate at the following positions: 88 (on C1 of gp120); 133, 142, 156, 160 (on V1); 181 (on V2); 197, 234, 241, 262, 289 (on C2); 301 (on V3); 339, 355 (on C3); 392, 408, 411 (on V4 loop); 442, 448 (on C4); 463, 466 in V5; 611, 616, 625, 637 (on gp41) (residue positions on gp120/41 are based on HXB2 numbering; see Figure S1, Supplemental Digital Content, https://links.lww.com/QAI/A657).
The effects of these individual PNGS mutants on nMAbs-mediated neutralization have been previously examined.24 Here, we generated 12 combined PNGS mutants that contain different combinations of the selected PNGS point mutations to evaluate their influence on infectivity and neutralization of the resulting mutant viruses. The 12 combined PNGS mutants constructed in this study were shown in Table S1 (see Supplemental Digital Content, https://links.lww.com/QAI/A657). Eleven of the 12 mutants (except for M46) contain the N197D mutation, and 8 of them contain N197D/N463Q mutations. All mutants were confirmed by sequencing.
Among all the combined mutants studied here, only 197M.1 (N197D/N301Q) has completely lost infectivity. Other multiple mutants showed no significant reduction of viral infectivity when compared with the 2 single point mutants, N197D or N301Q (Fig. 1).
Effect of Combined PNGS Mutations on Neutralization by nMAbs
The noninfectious mutant 197M.1 (N197D/N301Q) was excluded from the further neutralization assay study. All other mutant viruses on Table S1 (see Supplemental Digital Content, https://links.lww.com/QAI/A657), together with some single mutants, were examined for neutralization by nMAbs. In previous work, the single N197D mutant showed an obvious increase in susceptibility to neutralization by b12 (∼17-fold increase) and VRC03 (∼37-fold increase), as well as a modest 2-fold increase to VRC01 when compared with wild-type.24 However, the neutralization result for the 8 combined PNGS mutants in this study showed a much more dramatic increase in susceptibility to neutralization by VRC01/VRC03 compared with the single N197D or to wild-type (Table 1, Fig. 2). For example, mutants 197M.3 (N197D/N463Q), 197M.4 (N197D/N463Q/N442Q), 197M.5 (N197D/N463Q/N625Q), 197M.8 (N197D/N463Q/N625Q/N442Q/N339Q/N448), and 197M.9 (N197D/N463Q/N625Q/N442Q/N339Q) showed a 867-fold increase in susceptibility to neutralization by VRC03 when compared with wild-type, whereas mutants 197M.10 (N197D/N463Q/N625Q/N442Q/N339Q/N466Q) and 197M.11 (N197D/N463Q/N625Q/N442Q/N339Q) showed a ∼1300-fold increase, and 197M.2 (N197D/N625Q), 197M.6 (N197D/N625Q/N442Q), and 197M.7 (N197D/N625Q/N442Q/N339Q) showed a ∼100-fold increase (Table 1, Fig. 2). As far the neutralization by another nMAb VRC01, mutants 197M.3, 197M.4, 197M.5, 197M.8, 197M.9, 197M.10, and 197M.11 showed a ∼275- to 420-fold increase, whereas 197M.2, 197M.6, and 197M.7 had only ∼2-fold increase in neutralizing sensitivity compared with wild-type. M46 (N625Q/N463Q) showed no effect on VRC01/VRC03 mediated neutralization. However, all other combined PNGS mutants containing N197D mutation showed little changes of sensitivity to b12 or other nMAbs (PG9, PG16, 2F5, 4E10, and 2G12) when compared with the N197D single point mutant (Table 1, Fig. 2).
The results shown above indicate that whenever a combined mutant of the HIV FE strain (a BC clade virus) contains N197Q together with N463Q, a dramatic increase of the neutralizing sensitivity to VRC01/VRC03 is observed. To investigate whether the combination of N197Q and N463Q mutation has the same effect in other clade viruses to these CD4bs MAbs neutralization, a clade B virus B05 and a clade AE virus GX74.20 were tested. The N197Q mutation alone in the 2 viruses showed a ∼2- to 4-fold increase in susceptibility to neutralization by VRC01/VRC03, whereas the N463Q mutation alone showed no effect (see Figure S2, Supplemental Digital Content, https://links.lww.com/QAI/A657). The combination of N197Q and N463Q showed a less dramatic, but still significant increase in susceptibility to neutralization by VRC01/VRC03, ranging between 8- and 20-fold increase. For the sensitivity to b12, similar to the behavior of the FE strain mutants, the combined N197Q and N463Q mutations in these 2 clade viruses showed no obvious changes when compared with the N197Q single mutant (see Figure S2, Supplemental Digital Content, https://links.lww.com/QAI/A657).
Effect of PNGS Mutations on Neutralization by Serum Antibodies
Here, we used serum collected from 20 subjects infected with subtype BC isolates to test the single and combined PNGS mutants for neutralization phenotype. The ID50 for neutralization is shown in Table 2. Among the single PNGS mutants, N197D had a 2- to 3-fold ID50 increase in the 7 sera of the serum panel; N301Q showed a 2- to 5-fold ID50 increase in 8 of the serum panel; N442Q showed a 2- to 5-fold ID50 increase in 7 of the serum panel; and the N625Q made a 2- to 4-fold ID50 increase in 7 of the serum panel. In addition, the N611Q mutant was neutralized by serum gx66, gx76 with a titer increase of over 3-fold compared with the wild-type virus, and the N637Q mutant was neutralized by serum yn148r with a 5-fold titer increase compared with the wild-type virus. A few PNGS mutations also decrease the neutralizing sensitivity to HIV-1 positive serum. The N197D mutation resulted a 2-fold reduction in serum xj50 neutralization; this neutralization reduction also happened in the N611Q mutation to serum yn99r, N289D to serum bj22 and gx75, N448Q to serum sc59r, and N625Q to serum yn99r. These data suggest that the presence of the PNGS in the C2 (N197), V3 (N301), C4 (N442) regions, and gp41 (N625) protect FE from antibody-mediated neutralization by the serum panel.
Interestingly, compared with the single point mutants, most of the combined PNGS mutants displayed higher neutralizing sensitivity to the serum. All the 11 combined PNGS mutants (except for M46) showed a 2- to 34-fold ID50 increase in 7–15 of the serum panels, with the mutant 197M.11 (N197D/N463Q/N625Q/N442Q/N339Q) showing 2- to 34-fold increase in 15 tested serum samples compared with the respective single PNGS mutants and wild-type virus. 197M.11 also showed a ∼9-fold and ∼4-fold increase in neutralization sensitivity to serum gx66 and gx76, compared with the other mutants (Table 2). Another interesting observation is that, while some the single PNGS mutants showed no effect on neutralizing sensitivity by serum, combining such single PNGS mutants somehow displayed increased neutralizing sensitivity to serum antibodies, as shown by the sensitivity increase of mutants 197M.6 (N197D/N625Q/N442Q) and 197M.7 (N197D/N625Q/N442Q/N339Q) to serum sc59R, and mutants 197M.8 (N197D/N463Q/N625Q/N442Q/N339Q/N448), 197M.9 (N197D/N463Q/N625Q/N442Q/N339Q), and 197M.10 (N197D/N463Q/N625Q/N442Q/N339Q/N466Q) to serum gx93 (Table 2).
Structural Modeling and Rationalization
To understand the molecular basis for the observed increase of sensitivity to VRC01/VRC03 when N463Q mutation is added on top of N197D, we analyzed the available structural information for gp120 structures alone and its complexes with CD4 and various nMAbs.18,30,37–40 In doing so, the antibody-bound gp120 structures available to date were aligned to the FE gp120 model, and the structural basis by which specific glycosylation positions influence neutralization were examined. Of the 25 potential PNGS sites, both N197 and N463 map to the CD4 binding “face” of gp120 (Figs. 3A, B). However, N197 and N463 are not directly on top of the CD4 binding interface, neither N197 nor N463 seems to occlude CD4 binding (Figs. 3A, B), suggesting that they should not directly interfere with or participate in CD4 binding for infection. This is consistent with the observed viral infectivity for the mutants containing N197/N463 residues (Fig. 1). As for the antibody neutralization, nMAb b12 relies solely on heavy chain contacts that partially overlap with the CD4-binding site,40 and unlike the VRC antibodies, neutralization of HIV FE by b12 is not as dramatically affected by FE gp120 N197D/N463Q mutation (Fig. 2, Table 1). Based on the structural modeling, N197 and N463 of FE gp120 should not present steric hindrance to b12 antibody binding to the CD4-binding site (Figs. 3C, D). Compared with the binding interfaces for b12 and CD4, the N197 and N463 residues of FE gp120 are located directly adjacent to or within theVRC01 epitope/paratope (Figs. 3E, F), and these 2 residues are located within the VRC03 epitope/paratope (Figs. 3G, H). The degree of steric occlusion on the structural model seems to be directly related to the level of our observed sensitivity changes of neutralization by VRC01 and VRC03, where VRC03 binding to gp120 should be more hindered by glycosylation at N197/N463 compared with VRC01 binding, which is consistent with the observation that mutation at N197/N463 has even more marked increase of neutralizing sensitivity to VRC03 than VRC01. Therefore, the effects of FE gp120 glycosylation at N197 and N463 on neutralization sensitivity can be directly correlated to the structural properties that rationalize nMAb recognition and binding.
Among the 12 combined PNGS mutants of the HIV-1 gp120/gp41, 11 of them had no significant loss of infectivity, suggesting that these PNGS are not important for gp120/gp41 folding, maturation, viral assembly, and cell entry during infection. Surprisingly, 1 of the 12 mutants, the double mutant 197M.1 (N197D and N301Q) resulted in a complete loss of viral infectivity, which is rather surprising considering that as all the other combined mutants contain the N197D mutation had infectivity, and some of them having additional 3 to 7 point mutations on top of N197D. Our previous study indicated that N197D or N301Q mutation alone did not have severe impact on the infectivity,24 which is also confirmed here (Fig. 1). However, the combination of the 2 point mutations proved to be deadly for the virus, which suggests that such double mutant somehow abolish certain essential function(s) required for viral infection.
For the 11 combined PNGS mutants that showed viral infectivity, we examined their sensitivity to 7 broadly neutralizing MAbs.41,42 The neutralization ability of CD4bs MAbs VRC03 showed a marked increase for combined PNGS mutants that contained the both N197D and N463Q mutations, with a remarkable ∼800- to 1300-fold increase over WT FE. These mutants also showed a ∼300- to 400-fold increase in sensitivity to VRC01. Compared with the combined PNGS mutants, only the N197D and the N442Q among the single mutants showed relatively modest increase in neutralizing sensitivity to VRC03, with N197D showing 37-fold increase and N442Q showing 3-fold to VRC03. N197D also showed a 2-fold increase sensitivity to VRC01. However, N463Q single mutant, together with most other single mutants alone, showed little effect on neutralization by VRC01/VRC03, b12 or other nMAbs (Table 1).
Two pairs of combined mutants 197M.2/197M.5 and 197M.7/197M.9 (see Table S1, Supplemental Digital Content, https://links.lww.com/QAI/A657), which differ only in containing N463Q mutation or not, showed significant difference in VRC01/VRC03 sensitivity, with the mutants containing N463Q displaying much higher VRC01/03 sensitivity (Fig. 2, Table 1). In fact, all the 7 mutants that contain both N197D and N463Q in them showed the same dramatic increase of sensitivity to VRC01/VRC03, whereas those combined mutants with N197D but without N463Q (mutants 197M.2, 197M.6, and 197M.7) showed little effect (Table 1; see Table S1, Supplemental Digital Content, https://links.lww.com/QAI/A657). Interestingly, further mutations of the PNGS up to 5 sites on top of the N197D/N463Q mutation seem to have very minor additional effect on the sensitivity level to the 2 VRC nMAbs. Thus, N463Q mutation seems to play an important role in determining the neutralizing sensitivity to nMAbs VRC01/VRC03 in the N197D mutant background. In other words, although the effect of N463Q mutation alone did little in altering the sensitivity to nMAbs VRC01/VRC03 (with ∼1.5-fold increase), combining N463Q mutation with N197D has an unexpected synergistic effect in increasing the sensitivity to VRC01/VRC03 by a remarkable 300- to 1300-fold (Fig. 2, Table 1), and a less pronounced, but still significant change was also observed in clade B/AE virus in our experiment (see Figure S2, Supplemental Digital Content, https://links.lww.com/QAI/A657). The fact that neither single mutant of N197 and N463 exhibits a drastic change in neutralization sensitivity may be a bit confusing/confounding, but in considering the structural properties of the N-linked sugars (being rather bulky and unstructured), it seems not surprising that changes neutralization sensitivity do not appear until both N197 and N463 are mutated. First, N197 and N463 are both located on flexible loops within gp120, and so their positioning within gp120 may be quite dynamic. This property has direct implications when considering interactions with CD4bs antibodies. Second, and in addition to the structural properties of N197/463, the structural properties of the sugars (mainly mannose 5 or 9 regarding gp120) linked to glycosylated N residues should be taken into account. Such synergistic effect could be the result of the absence of glycosylation on the PNGS mutant, as suggested by the structural modeling. Alternatively, the PNGS mutation could also affect the Env conformation to increase the sensitivity to some of the nMAbs.
Very interestingly, the remarkable increase of sensitivity of mutants containing N463Q/N197D was only observed for nMAbs VRC01/VRC03 and not for other tested nMAbs (Table 1). This is rather intriguing especially considering b12 belongs to the class of CD4bs nMAbs as VCR01/03. The results further confirm that b12 neutralizes HIV-1 with mechanisms that are not identical to those used by VCR01/03, despite the fact that they all bind to the general area of CD4bs.15,17,18,39,43–45
The neutralization phenotype of combined mutants using the serum antibodies showed a much less profound impact on sensitivity to the serum antibody than to the nMAbs, with ∼20-fold as the highest increase of sensitivity for mutant 10 and 11 (197M.10 and 197M.11). Another interesting observation is that, while the combined mutants containing N197D/N463Q did not show continued increase of neutralizing sensitivity to nMAbs with additional PNGS mutations, these combined mutants displayed a general trend of cumulative mutational effect on the increase of neutralizing sensitivity to the serum antibodies (Table 2). These combined mutants also showed a broader sensitivity increase to HIV-1 positive serum than the single PNGS mutants (Table 2). However, there seems to be a general trend that the observed cumulative effect to the serum antibodies become stronger when the combined mutants contain at least the N197D and N463Q mutations, which further suggests that the neutralizing antibodies in HIV-1–positive serum targeting the CD4bs and the conserved elements of V3 region may play a dominant role in neutralizing the virus for those patient serum in our assays.
Through analyzing HIV sequences, we found that only ∼30% of 4829 sequences of HIV Env retrieved from LANL HIV databases have N463 site. Approximately 70% of them have Asn-X-Thr/Ser PNGS motif and others lost it by amino acid substitution at Thr/Ser. Thus, the glycosylation at N463 is present in small part of HIV isolates, and they maybe involved in viral evolution to escape from neutralization.
In conclusion, our results indicate that N463 plays an important role in regulating the CD4bs MAbs VRC01/VRC03 sensitivity in the genetic background of N197D mutation of gp120. We also found that combined PNGS mutations that include N197D/N463Q mutations of gp120 made the virus more readily to be neutralized by serum polyclonal antibodies from patients. We have provided a structural basis to rationalize the observed synergistic effects of this combined mutations through molecular modeling. In the future, it remains to be examined regarding the issue of whether the modified gp120 Env bearing such combined mutations can induce a stronger immune response as a more potent vaccine candidate source.
The authors thank all who provided MAbs and TZM-bl cells for these studies.
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