Antibody response against selected epitopes in the HIV-1 envelope gp41 ectodomain contributes to reduce viral burden in HIV-1 infected patients

Abstract

The ectodomain of gp41 is the target of potent binding and neutralizing antibodies (NAbs) and is being explored in new strategies for antibody-based HIV vaccines. Previous studies have suggested that the W164A-3S (3S) and EC26-2A4 (EC26) peptides located in the gp41 ectodomain may be potential HIV vaccine candidates. We assessed 3S- and EC26-specific binding antibody responses and related neutralizing activity in a large panel of chronic HIV-1-infected Portuguese individuals on ART. A similar proportion of participants had antibodies binding to 3S (9.6%) and EC26 (9.9%) peptides but the level of reactivity against 3S was significantly higher compared to EC26, except in the rare patients with double peptide reactivity. The higher antigenicity of 3S was unrelated with disease stage, as assessed by CD4⁺ T cell counts, but it was directly related with plasma viral load. Most patients that were tested (89.9%, N = 268) showed tier 1 neutralizing activity, the potency being inversely associated with plasma viral load. In the subset of patients that were tested for neutralization of tier 2 isolates, neutralization breadth was inversely correlated with plasma viral load and directly correlated with CD4⁺ T cell counts. These results are consistent with a role for neutralizing antibodies in controlling viral replication and preventing the decline of CD4⁺ T lymphocytes. Importantly, in patients with 3S-specific antibodies, neutralizing titers were inversely correlated with viral RNA levels and proviral DNA levels. Moreover, patients with 3S and/or EC26-specific antibodies showed a 1.9-fold higher tier 2 neutralization score than patients without antibodies suggesting that 3S and/or EC26-specific antibodies contribute to neutralization breadth and potency in HIV-1 infected patients. Overall, these results suggest that antibodies targeting the S3 and EC26 epitopes may contribute to reduce viral burden and provide further support for the inclusion of 3S and EC26 epitopes in HIV-1 vaccine candidates.

Figure 1

3S and EC26-specific antibody frequency and reactivity in plasma from HIV-1-infected Portuguese. (A) Peptide-specific antibody reactivity levels in all patients; (B) Proportion of HIV-1 plasma samples reacting with the peptides in viraemic and aviraemic patients; C and D) Antibody frequency according to HIV RNA and CD4⁺ T cells counts; (E) Plasma reactivity levels against both peptides in viraemic and aviraemic patients. Lines denote the median and interquartile range. S/CO (OD sample/OD cut-off ratio), values ≥ 1 (dotted line) indicate reactive samples. P values refer to the comparison between groups and were determined using the Mann Whitney test. In all figures, white symbols represent 3S-specific responses and black symbols EC26-specific responses.

Figure 2

Box and whiskers plot showing the relationship between reactivity to 3S and EC26 and viral load (A) and proviral load (B). Lines denote median, maximum and minimum values. Median values were compared using the Mann–Whitney test.

Figure 3

Level of reactivity against the 3S and EC26 peptides in plasma samples from patients showing single (S) or double (D) reactivity against 3S and EC26. Lines denote median and interquartile range. Statistical significance was determined using the Mann–Whitney test.

Figure 4

Neutralizing antibody response in relation with viral burden and the presence of 3S or EC26-binding antibodies. (A) Percent neutralization of HIV-1 strain NL4-3 (Tier 1a virus) using plasmas from patients with detectable and undetectable viral load. Lines denote the median and interquartile range; (B) Box and whiskers plot of viral load in patients with and without neutralizing antibodies. Lines denote median, maximum and minimum values; (C) Correlation of neutralizing titers with RNA and DNA load in patients containing 3S-specific antibodies (N = 31); (D) Correlation of neutralizing titers with RNA and DNA load in patients containing EC26-specific antibodies (N = 28). The linear regression lines, goodness of fit r² and P values are indicated.

Figure 5

Viral load and CD4⁺ T cell counts in patients with or without broadly neutralizing antibodies as determined by neutralizing score. (A) Regression analysis; (B) Box and whiskers plot. Statistical significance was determined using the Mann–Whitney test. Lines denote median, maximum and minimum.

Figure 6

Neutralizing scores in patients with and without 3S or EC26 antibodies. Lines denote median and interquartile range. Statistical significance was determined using the Mann–Whitney test.

Figure 7

Sequence and location of W164A-3S and EC26-2A4 peptides in the transmembrane glycoprotein of HIV-1 (gp41, HXB2 reference strain numbering, GenBank: AF033819.3). Epitope sequences are indicated in underlined letters^{, ,} . The W614A mutation in peptide W164A-3S is shown in red. The amino acid sequence of the heptad repeat 2 (HR2) region is indicated in bold letters (aa 628–665). TMD- transmembrane domain; FP- fusion peptide; HR1- heptad repeat 1. The membrane proximal external region (MPER) is shown in italic letters (aa 660–683)^, . The epitope of the broadly neutralizing antibody 2F5 is boxed. In gp120, V1-V5 are hipervariable regions and C1-C5 are conserved regions.