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. 2020 Sep 15;94(19):e00814-20.
doi: 10.1128/JVI.00814-20. Print 2020 Sep 15.

A Rare Mutation in an Infant-Derived HIV-1 Envelope Glycoprotein Alters Interprotomer Stability and Susceptibility to Broadly Neutralizing Antibodies Targeting the Trimer Apex

Nitesh Mishra  1 , Shaifali Sharma  1 , Ayushman Dobhal  1 , Sanjeev Kumar  1 , Himanshi Chawla  1 , Ravinder Singh  2 , Bimal Kumar Das  2 , Sushil Kumar Kabra  3 , Rakesh Lodha  3 , Kalpana Luthra  4
Affiliations

A Rare Mutation in an Infant-Derived HIV-1 Envelope Glycoprotein Alters Interprotomer Stability and Susceptibility to Broadly Neutralizing Antibodies Targeting the Trimer Apex

Nitesh Mishra et al. J Virol. .

Abstract

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is the sole target of broadly neutralizing antibodies (bnAbs). Several mechanisms, such as the acquisition of mutations, variability of the loop length, and alterations in the glycan pattern, are employed by the virus to shield neutralizing epitopes on Env to sustain survival and infectivity within the host. The identification of mutations that lead to viral evasion of the host immune response is essential for the optimization and engineering of Env-based trimeric immunogens. Here, we report a rare leucine-to-phenylalanine escape mutation (L184F) at the base of hypervariable loop 2 (population frequency of 0.0045%) in a 9-month-old perinatally HIV-1-infected infant broad neutralizer. The L184F mutation altered the trimer conformation by modulating intramolecular interactions stabilizing the trimer apex and led to viral escape from autologous plasma bnAbs and known N160 glycan-targeted bnAbs. The L184F amino acid change led to the acquisition of a relatively open trimeric conformation, often associated with tier 1 HIV-1 isolates and increased susceptibility to neutralization by polyclonal plasma antibodies of weak neutralizers. While there was no impact of the L184F mutation on free virus transmission, a reduction in cell-to-cell transmission was observed. In conclusion, we report a naturally selected viral mutation, L184F, that influenced a change in the conformation of the Env trimer apex as a mechanism of escape from contemporaneous plasma V2 apex-targeted nAbs. Further studies should be undertaken to define viral mutations acquired during natural infection, to escape selection pressure exerted by bnAbs, to inform vaccine design and bnAb-based therapeutic strategies.IMPORTANCE The design of HIV-1 envelope-based immunogens capable of eliciting broadly neutralizing antibodies (bnAbs) is currently under active research. Some of the most potent bnAbs target the quaternary epitope at the V2 apex of the HIV-1 Env trimer. By studying naturally circulating viruses from a perinatally HIV-1-infected infant with plasma neutralizing antibodies targeted to the V2 apex, we identified a rare leucine-to-phenylalanine substitution, in two out of six functional viral clones, that destabilized the trimer apex. This single-amino-acid alteration impaired the interprotomeric interactions that stabilize the trimer apex, resulting in an open trimer conformation and escape from broadly neutralizing autologous plasma antibodies and known V2 apex-directed bnAbs, thereby favoring viral evasion of the early bnAb response of the infected host. Defining the mechanisms by which naturally occurring viral mutations influence the sensitivity of HIV-1 to bnAbs will provide information for the development of vaccines and bnAbs as anti-HIV-1 reagents.

Keywords: HIV-1; V2 apex bnAbs; infants; interprotomer interactions; rare mutation.

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Figures

FIG 1
FIG 1
Plasma bnAbs from infant broad neutralizer AIIMS731 target the V2 apex of HIV-1 Env. (A) Heat map representing HIV-1-specific neutralization titers (inverse plasma dilution) of plasma bnAbs from infant broad neutralizer AIIMS731 against the 12-virus global panel. ID50 values are color-coded per the key given, with darker colors implying higher ID50 titers. MuLV, murine leukemia virus; NA, not applicable. (B) Epitope mapping of AIIMS731 plasma bnAbs showing ID50 fold changes against the 25710_2_43, 16055_2_3, CAP45_G3, and BG505_W6M_C2 wild-type pseudoviruses and their N160A mutants as well as pseudoviruses grown in the presence of the glycosidase inhibitors kifunensine and swainsonine.
FIG 2
FIG 2
Limited diversity in the circulating viral variants of infant AIIMS731. (A and B) Highlighter plots with maximum likelihood trees of 40 SGA env sequences from infant AIIMS731 showing limited variability in the circulating viral variants and the presence of 6 strains circulating at a population frequency of >5%. In the highlighter plot, mutations compared to the consensus sequence are represented by green for adenine, blue for cytosine, orange for guanine, and red for thymine. In the case of 73105h, both of the adenine mutations were silent (green bars). (C) Donut plot of the distribution of 40 SGA Env amplicons showing viral variants 73106f and 73105h to be the dominant strains.
FIG 3
FIG 3
A rare mutation provided neutralization escape from autologous plasma bnAbs in infant AIIMS731. (A) The neutralization susceptibility of circulating viral variants from infant AIIMS731 to contemporaneous autologous plasma bnAbs was assessed via neutralization assays based on TZM-bl cells. Even though four of the circulating viral variants were susceptible to plasma bnAbs, the maximum percent neutralization ranged from 83 to 91%. Viral variants 73105h and 73106f were resistant to autologous plasma bnAbs (maximum percent neutralization values of 24% and 25%, respectively). (B) AIIMS731 viral strains were arranged based on neutralization sensitivity to contemporaneous plasma nAbs (ID50 values). V2 loop sequences (positions 156 to 199 [HXB2 numbering]) of viral variants resistant to neutralization by autologous plasma nAbs showed a leucine-to-phenylalanine mutation at position 184. (C) Complete amino acid sequence comparison among all six viral variants showing a single mutation (L184F) in 73106f (relative to sensitive strain 73105b) that led to neutralization escape from autologous plasma bnAbs. While 73105c also had a rare N229Y mutation, no difference in susceptibility to autologous plasma bnAbs was visible. (D) Amino acid frequency plot at position 184 in all reported HIV-1 Env sequences (7,094 sequences) showing an abundance of leucine or isoleucine, whereas phenylalanine at position 184 occurred at a population frequency of 0.0045% (32/7,094 sequences).
FIG 4
FIG 4
Neutralization curves of AIIMS731 viral variants against V2 apex bnAbs. The neutralization susceptibilities of all six viral variants to the V2 apex bnAbs (PG9, PG16, PGT145, PGDM1400, CAP256.25, and CH01) were assessed via neutralization assays based on TZM-bl cells. Of note, except for PGDM1400 and CAP256.25, none of the V2 apex bnAbs reached 100% neutralization for the AIIMS731 autologous plasma bnAb-sensitive viral cluster (73105b, 73105c, 73105d, and 73105e), while with the L184F mutant clones 73105h and 73106f (autologous plasma bnAb-resistant cluster), all V2 apex bnAbs showed markedly lower neutralization efficiencies. For 73106f, maximum neutralizations of 57% and 53% were reached with PG16 and CAP256.25, respectively. Neutralization assays were repeated three times, and curves were drawn with means ± standard deviations.
FIG 5
FIG 5
Neutralization of AIIMS731 viral variants by non-nAbs targeting the V3 loop and CD4-induced epitopes. The neutralization susceptibilities of all six viral variants to the V3 loop-targeting non-nAbs (447-52D and 19b) and CD4-induced non-nAbs (17b, A32, 48d, and b6) were assessed via neutralization assays based on TZM-bl cells. Viral variants 73105h and 73106f showed moderate neutralization by 447-52D, 19b, 17b, and 48d. Neutralization assays were repeated three times, curves were drawn with means ± standard deviations, and MPN values were calculated based on average neutralization.
FIG 6
FIG 6
Viral variant 73106f is highly susceptible to subtype-matched heterologous plasma antibodies. (A and B) Violin plot and heat map representing the neutralization susceptibilities of AIIMS731 circulating viral variants against plasma antibodies from HIV-1-infected pediatric individuals in chronic stages of disease. Distinct neutralization profiles were seen for AIIMS731 autologous plasma bnAb-sensitive (73105b, 73105c, 73105d, and 73105e) and -resistant (73105h and 73106f) viral clusters. The plasma panel contained well-characterized HIV-1 clade C-infected pediatric donors whose plasma antibodies showed varied neutralization activities against the 12-virus global panel. Plasma samples were categorized as strong or weak based on their breadth and potency against the 12-virus global panel (see Materials and Methods). A comparison is shown for 73105b and 73106f, although similar patterns were observed by comparing sensitive versus resistant clusters. (C) Viruses belonging to the sensitive cluster were primarily neutralized by plasma samples that were categorized as strong, while viruses belonging to the resistant cluster showed considerable neutralization by plasma samples categorized as weak. P values are given by asterisks, where ** implies a P value of <0.01, *** implies a P value of <0.001, and **** implies a P value of <0.0001.
FIG 7
FIG 7
The L184F mutation has no impact on Env trimer stability. (A to C) AIIMS731 viral variants were tested for stability by assessing decay in infectivity as a function of temperature (thermostability) and time (spontaneous decay). Similar infectivity decay curves were observed for all viral variants, and no significant change in the T90 value (temperature at which 90% of infectivity was lost) or half-life of Env decay was observed. Infectivity decay assays were repeated three times in triplicates, and the curves were drawn using means ± standard deviations. For thermostability, infectivity at 37°C was taken as 100% infectivity. For spontaneous decay, infectivity at 0 h was taken as 100% infectivity.
FIG 8
FIG 8
The L184F mutation leads to reduced cell-to-cell transmission. (A) Pseudoviruses were titrated after normalization, and replicate titration curves were used to calculate the area under the curve (AUC) values. Each experiment was repeated three times in triplicates, providing a total of 9 reference values. (B) Fusogenicity in cocultures of Tat/Env-cotransfected 293T and TZM-bl cells was used as a measure of cell-to-cell transmission ability. The fusion of AIIMS731 Env in relation to the fusion observed with the well-characterized Env of HIV-1 isolate MW965.26 was calculated. Each experiment was repeated three times in triplicates, providing a total of 9 reference values. Two-tailed Student’s t test was used for comparison (*** implies a P value of <0.001). A comparison is shown for 73105b and 73106f, although similar patterns were observed by comparing sensitive versus resistant clusters.
FIG 9
FIG 9
Critical role of L184 in modulating interprotomer interactions at the trimer apex. Shown are interprotomer interactions between R165 (protomer A) and L184 (protomer B). The dot meshes surrounding R165A (orange) and L184B and F184B (yellow) represent the solvent-accessible surfaces (SASs) (van der Waals surfaces expanded by the water molecule radius). In panel A, interprotomer contacts (lipophilic) between R165A and L184B can be seen by overlapping SASs. In the case of F184B, no interprotomer contacts can be seen, evident by the lack of SAS overlap. The HIV-1 Env protomeric backbones are represented by two distinct colors (protomer A in green and protomer B in cornflower blue). The illustration was generated from data under PDB accession number 4ZMJ. L165 and I184 were rotamerized to the respective residues in 73105b (R165 and L184) and 73106f (R165 and F184).
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