Evidence for a Continuous Drift of the HIV-1 Species towards Higher Resistance to Neutralizing Antibodies over the Course of the Epidemic

<div><p>We compared the neutralization sensitivity of early/transmitted HIV-1 variants from patients infected by subtype B viruses at 3 periods of the epidemic (1987–1991, 1996–2000, 2006–2010). Infectious pseudotyped viruses expressing envelope glycoproteins representative of the viral quasi-species infecting each patient were tested for sensitivity to neutralization by pools of sera from HIV-1 chronically infected patients and by an updated panel of 13 human monoclonal neutralizing antibodies (HuMoNAbs). A progressive significantly enhanced resistance to neutralization was observed over calendar time, by both human sera and most of the HuMoNAbs tested (b12, VRC01, VRC03, NIH45-46^G54W, PG9, PG16, PGT121, PGT128, PGT145). Despite this evolution, a combination of two HuMoNAbs (NIH45-46^G54W and PGT128) still would efficiently neutralize the most contemporary transmitted variants. In addition, we observed a significant reduction of the heterologous neutralizing activity of sera from individuals infected most recently (2003–2007) compared to patients infected earlier (1987–1991), suggesting that the increasing resistance of the HIV species to neutralization over time coincided with a decreased immunogenicity. These data provide evidence for an ongoing adaptation of the HIV-1 species to the humoral immunity of the human population, which may add an additional obstacle to the design of an efficient HIV-1 vaccine.</p></div

Decreasing immunogenicity of clade B HIV-1 variants in term of NAbs induction over the course of the epidemic.

<p>[A and B] Comparison of the frequencies of detection of NAbs [A] and of NAbs titers [B] against 6 heterologous subtype B isolates (BX08, 92BR020, QH0692.42, AC10.0.29, RHPA4259.7 and REJO4541.67) in sera from clade B chronically infected patients at the two extreme periods of the study i.e. 1987–1991 (n = 30) and 2003–2007 (n = 30). Differences in the frequency of detection and the titers of NAbs among sera from the two groups of patients were compared by Chi² or ANOVA tests after adjustment for the strain, respectively. [C] Comparison of the neutralization breadth between the sera of the two periods. Each point represents the value for one serum. [D] Comparison of the potency scores (see methods) between the sera of the two periods. Box plots show the distribution of potency scores of each group of sera; the horizontal lines represent the 10^th, median and 90^th percentiles. Differences between breadth and potency score between the two groups of sera were evaluated using a Wilcoxon signed ranked test.</p

Phylogenetic analysis of full-length<i>env</i> sequences.

<p>The 40 <i>env</i> sequences of the early/transmitted viruses included in our study were aligned with 160 <i>env</i> sequences isolated at time of primo-infection from clade B -infected patients, with documented year (1990–2009) and country (25 were from Europe, 113 from USA, 6 from Australia, 15 from Trinidad and 1 from Zambia) of infection. A neighbor-joining tree was constructed using the MEGA software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003477#ppat.1003477-Tamura2" target="_blank">[89]</a>. Open, grey filled and black filled circles identify the sequences derived from the historical, intermediate and contemporary patients of our study, respectively. Bootstrap values above 70% are indicated.</p

Relationship between resistance to HuMoNAbs and changes in envelope targeted residues.

<p>The 40 <i>env</i> sequences of viruses from historical (HP), intermediate (IP) and contemporary patients (CP) were aligned with the HXB2 reference sequence. Key residues targeted by b12, VRC01, VRC03 and/or NIH45-46 [A], PGT9, PG16 and/or PGT145 [B], and PGT121 and/or PGT128 [C] are presented. Identical residues are represented by dots. Conserved and non-conserved residues are in grey and black, respectively. Substitutions previously shown to confer neutralization resistance are in red. The neutralization sensitivity (IC₅₀) of each HIV-1 variant is indicated for each HuMoNab. IC₅₀ values are color coded with increasing darker colors used to indicate increasing neutralization sensitivity.</p

Enhanced resistance of clade B early/transmitted HIV-1 variants to neutralization by HuMoNAbs over the course of the epidemic.

<p>[A to E] Comparison of the neutralization sensitivity of Env-pseudotyped viruses derived from historical patients (HP, n = 11), intermediate patients (IP, n = 15) and contemporary patients (CP, n = 14) by b12, VRC01, VRC03 and NIH45-46G54W [A], PG9, PG16 and PGT145 [B], PGT121, PGT128, PGT135 and 2G12 [C] and 2F5 and 4E10 [D]. Box plots show the distribution of antibody titers (IC₅₀) of each HuMoNAb towards pseudotyped viruses of each period; the horizontal lines represent the 10^th, median and 90^th percentiles. Each data represents the mean value of the assay performed in duplicate. Differences of neutralization sensitivity between viruses over calendar time were evaluated using a Jonckheere-Terpstra test. [E] Comparison of the neutralization coverage of each HuMoNabs used at various concentrations (IC₅₀≤10 µg/mL, ≤1 µg/mL or ≤0.01 µg/mL) towards pseudotyped viruses from HP, IP and CP. Differences of neutralization coverage of viruses from HP to CP were evaluated using a Chi² test for trend.</p

Neutralization coverage of HIV-1 variants from contemporary patients by HuMoNAbs combinations.

<p>[A] A heatmap of the neutralizing activities (IC₅₀) of the HuMoNAbs against HIV-1 variants from contemporary patients is shown, with increasing darker colors indicating increasing neutralization sensitivity. [B and C] The neutralization coverage of viruses from contemporary patients was tested against various concentrations of a 1∶1 combination of NIH45-45^G54W and PGT128 [B] and a 1∶1 combination of VRC01 and PGT128 [C]. Solid lines show the coverage of each HuMoNAb used alone or in combinations. Dashed lines show the theoretical coverage that would be obtained if the neutralizing activities of combined antibodies were fully additive.</p

Baseline characteristics of the 987 patients from the ANRS PRIMO cohort 1999–2010.

<p>TPHA = <i>Treponema palladium</i> haemagglutination assay; VDRL = Venereal Diseases Research Laboratory test; HBV = hepatits B virus; HCV = hepatitis C virus.</p

Comparison of length and number of PNGS of gp120 sequences.

<p>The lengths [A] and numbers of PNGS [B] of gp120 and of each of its variable loops were compared for early/transmitted viruses from historical patients (HP, n = 11), intermediate patients (IP, n = 15) and contemporary patients (CP, n = 14). Each data point represents the value for one virus. The horizontal line represents the median value. Differences of length or number of PNGS between viruses over calendar time were evaluated using a Jonckheere-Terpstra test.</p

Comparison of the characteristics of patients included into clustered transmission versus non-clustered: the ANRS PRIMO cohort 1999–2010.

<p>Note: the total of patients for each variable does not always equal the total sample due to some missing values. PHI = primary HIV infection; MSM = men having sex with men; TPHA = <i>Treponema palladium</i> haemagglutination assay; VDRL = Venereal Diseases Research Laboratory test.</p>(1)<p>Urethritis, rectitis, genital herpes infection, vulvo-vaginal candidosis, condyloma and/or syphilis.</p>(2)<p>In the last 6 months preceding PHI diagnosis.</p