Characterization of broadly neutralizing antibody responses to HIV-1 in a cohort of long term non-progressors

Abstract

Background: Only a small fraction of HIV-1-infected patients develop broadly neutralizing antibodies (bNAbs), a process generally associated to chronic antigen stimulation. It has been described that rare aviremic HIV-1-infected patients can generate bNAbs but this issue remains controversial. To address this matter we have assessed bNAb responses in a large cohort of long-term non-progressors (LTNPs) with low or undetectable viremia.

Methods: Samples from the LTNP cohort of the Spanish AIDS Research Network (87 elite and 42 viremic controllers) and a control population of 176 viremic typical-progressors (TPs) were screened for bNAbs using Env-recombinant viruses. bNAb specificities were studied by ELISA using mutated gp120, neutralization assays with mutated viruses, and peptide competition. Epitope specificities were also elucidated from the serum pattern of neutralization against a panel of diverse HIV-1 isolates.

Results: Broadly neutralizing sera were found among 9.3% LTNPs, both elite (7%) and viremic controllers (14%). Within the broadly neutralizing sera, CD4 binding site antibodies were detected by ELISA in 4/12 LTNPs (33%), and 16/33 of TPs (48%). Anti-MPER antibodies were detected in 6/12 LTNPs (50%) and 14/33 TPs (42%) whereas glycan-dependent HIV-1 bNAbs were more frequent in LTNPs (11/12, 92%) as compared to TPs (12/33, 36%). A good concordance between standard serum mapping and neutralization-based mapping was observed.

Conclusion: LTNPs, both viremic and elite controllers, showed broad humoral immune responses against HIV-1, including activity against many major epitopes involved in bNAbs-mediated protection.

Fig 1. Serum neutralization data (ID50s) for LTNPs capable of neutralizing all the viruses from the mini-panel.

Selected sera samples from the LTNP cohort capable to neutralize (ID50≥200) the mini-panel of 4 viruses (NL4-3, VI191, 92BR025 and CM244) were screened for neutralization against 10 more viruses. Viruses of the minipanel are shaded grey. Median viral loads for the previous five years to the serum sample are shown. In the last column the percentage of neutralized viruses is shown. No significant neutralization of the VSV pseudotyped virus was observed in any of the samples. Reciprocal serum ID50 values ≥200 and <400 are highlighted in yellow, ≥400 and <1000 in orange and ≥1000 in red.

Fig 2. CD4 binding site neutralizing antibody specificity.

Sera that were ELISA reactive with resurfaced stabilized cores RSC3 and RSC3 G367R, were assayed for inhibition of neutralization using RSC3 and RSC3 P363N as inhibitors. The net percent reduction in neutralization of JRFL, RW020 or ZA012 attributed to RSC3 compared to RSC3 P363N is plotted (y axis). Each serum is represented by a different symbol. The color of the symbol depends on the virus used in the neutralization assay. A reduction greater than 30% is considered positive. RSC3 addition inhibited neutralization mediated by 2 sera from LTNPs (449326 and 597473) and no serum of typical progressors.

Fig 3. Detection of glycan-dependent HIV-1 neutralizing antibodies in sera from LTNPs and typical progressors.

Sera with V1V2 and V3 glycan-dependent HIV-1 NAbs are detected by neutralization assays with the parental JRCSF and the corresponding N160K and N332A mutants (V1V2 and V3 respectively). Sera with reactivity to glycan-dependent motives in V2 and V3 are indicated in orange and blue respectively. Percent serum neutralizing activity sensitive to the indicated residue replacement was calculated using the equation [1 − (ID50 mutant/ID50 wild type)] x 100. A sample is considered positive if there is a decrease in ID50 greater than or equal to 50% for the mutant relative to the wild-type virus. Monoclonal antibodies 2G12 and PG9 have been used as controls and are indicated by a black box. SEMs of two independent assays are shown. In this figure only samples with neutralizing antibodies directed to glycans in V1V2 and/or V3 are shown.

Fig 4. Detection of antibodies specific for the membrane-proximal region in the sera from LTNPs and typical progressors.

For the mapping of anti-MPER neutralizing antibodies, the serum samples were tested against the parental HIV-2 isolate 7312A and the 7312A chimera containing HIV-1 MPER fragments (7312A-C1). Monoclonal antibodies 2F5 and 4E10 have been used as controls and are indicated by a black box. In this figure only samples with anti-MPER neutralizing antibodies are shown.

Fig 5. Summary of experimental serum mapping of HIV-1 sera from LTNPs and TPs with a broad neutralization profile.

Data obtained from the assays used to map CD4bs, V1V2 glycans, V3 glycans and MPER regions are shown. Epitope groups are marked with a plus sign (+) if predicted by the mapping assays to be present in a given serum.

Fig 6. Delineation of serum specificities in HIV-1 sera from LTNPs and TPs with a broad neutralization profile.

(A) Predictions based on the serum neutralization pattern against a panel of diverse HIV-1 isolates are shown on the left of the figure. For each serum the predicted relative prevalence of each reference antibody cluster is presented as a heat map where stronger neutralization signals by the antibody cluster are shown as darker colors (higher fractional numbers). Breadth was measured as the percentage of viruses neutralized with an ID50>50 and potency as the geometric mean ID50 titers of sera. Sera with breadth less than 20% are greyed out since the computational predictions in those cases are less reliable (B) Concordance between neutralization-based and standard serum mapping. Sera with breadth less than 20% were excluded. The agreement between the two methods was good. In 92% of the sera at least one of top two computational neutralization-based predictions was identified by standard mapping. (C) Frequency of the epitope specificities of HIV-1 neutralizing antibodies in the sera from LTNPs and TPs with broadly neutralizing activity. Sera with breadth less than 20% were excluded.