A Mechanistic Understanding of Allosteric Immune Escape Pathways in the HIV-1 Envelope Glycoprotein

<div><p>The HIV-1 envelope (Env) spike, which consists of a compact, heterodimeric trimer of the glycoproteins gp120 and gp41, is the target of neutralizing antibodies. However, the high mutation rate of HIV-1 and plasticity of Env facilitates viral evasion from neutralizing antibodies through various mechanisms. Mutations that are distant from the antibody binding site can lead to escape, probably by changing the conformation or dynamics of Env; however, these changes are difficult to identify and define mechanistically. Here we describe a network analysis-based approach to identify potential allosteric immune evasion mechanisms using three known HIV-1 Env gp120 protein structures from two different clades, B and C. First, correlation and principal component analyses of molecular dynamics (MD) simulations identified a high degree of long-distance coupled motions that exist between functionally distant regions within the intrinsic dynamics of the gp120 core, supporting the presence of long-distance communication in the protein. Then, by integrating MD simulations with network theory, we identified the optimal and suboptimal communication pathways and modules within the gp120 core. The results unveil both strain-dependent and -independent characteristics of the communication pathways in gp120. We show that within the context of three structurally homologous gp120 cores, the optimal pathway for communication is sequence sensitive, i.e. a suboptimal pathway in one strain becomes the optimal pathway in another strain. Yet the identification of conserved elements within these communication pathways, termed inter-modular hotspots, could present a new opportunity for immunogen design, as this could be an additional mechanism that HIV-1 uses to shield vulnerable antibody targets in Env that induce neutralizing antibody breadth.</p> </div

Data_Sheet_1_Calcification of the abdominal aorta is an under-appreciated cardiovascular disease risk factor in the general population.pdf

Calcification of large arteries is a high-risk factor in the development of cardiovascular diseases, however, due to the lack of routine monitoring, the pathology remains severely under-diagnosed and prevalence in the general population is not known. We have developed a set of machine learning methods to quantitate levels of abdominal aortic calcification (AAC) in the UK Biobank imaging cohort and carried out the largest to-date analysis of genetic, biochemical, and epidemiological risk factors associated with the pathology. In a genetic association study, we identified three novel loci associated with AAC (FGF9, NAV9, and APOE), and replicated a previously reported association at the TWIST1/HDAC9 locus. We find that AAC is a highly prevalent pathology, with ~ 1 in 10 adults above the age of 40 showing significant levels of hydroxyapatite build-up (Kauppila score > 3). Presentation of AAC was strongly predictive of future cardiovascular events including stenosis of precerebral arteries (HR~1.5), myocardial infarction (HR~1.3), ischemic heart disease (HR~1.3), as well as other diseases such as chronic obstructive pulmonary disease (HR~1.3). Significantly, we find that the risk for myocardial infarction from elevated AAC (HR ~1.4) was comparable to the risk of hypercholesterolemia (HR~1.4), yet most people who develop AAC are not hypercholesterolemic. Furthermore, the overwhelming majority (98%) of individuals who develop pathology do so in the absence of known pre-existing risk conditions such as chronic kidney disease and diabetes (0.6% and 2.7% respectively). Our findings indicate that despite the high cardiovascular risk, calcification of large arteries remains a largely under-diagnosed lethal condition, and there is a clear need for increased awareness and monitoring of the pathology in the general population.</p

Data_Sheet_2_Calcification of the abdominal aorta is an under-appreciated cardiovascular disease risk factor in the general population.PDF

Calcification of large arteries is a high-risk factor in the development of cardiovascular diseases, however, due to the lack of routine monitoring, the pathology remains severely under-diagnosed and prevalence in the general population is not known. We have developed a set of machine learning methods to quantitate levels of abdominal aortic calcification (AAC) in the UK Biobank imaging cohort and carried out the largest to-date analysis of genetic, biochemical, and epidemiological risk factors associated with the pathology. In a genetic association study, we identified three novel loci associated with AAC (FGF9, NAV9, and APOE), and replicated a previously reported association at the TWIST1/HDAC9 locus. We find that AAC is a highly prevalent pathology, with ~ 1 in 10 adults above the age of 40 showing significant levels of hydroxyapatite build-up (Kauppila score > 3). Presentation of AAC was strongly predictive of future cardiovascular events including stenosis of precerebral arteries (HR~1.5), myocardial infarction (HR~1.3), ischemic heart disease (HR~1.3), as well as other diseases such as chronic obstructive pulmonary disease (HR~1.3). Significantly, we find that the risk for myocardial infarction from elevated AAC (HR ~1.4) was comparable to the risk of hypercholesterolemia (HR~1.4), yet most people who develop AAC are not hypercholesterolemic. Furthermore, the overwhelming majority (98%) of individuals who develop pathology do so in the absence of known pre-existing risk conditions such as chronic kidney disease and diabetes (0.6% and 2.7% respectively). Our findings indicate that despite the high cardiovascular risk, calcification of large arteries remains a largely under-diagnosed lethal condition, and there is a clear need for increased awareness and monitoring of the pathology in the general population.</p

Determination of the earliest R880F nAb escape signatures in Env.

<p>Suspected 2-month nAb escape mutations from four different amino acid positions were introduced by site-directed mutagenesis into the transmitted/founder Env 0-B24 or escape Env 2-A3, which differed from the founder only at the site of mutation introduction. The transmitted/founder and wild-type 2-month Envs (solid lines) along with site-directed mutant Envs (dashed lines) were pseudotyped and assayed with 2-month plasma to determine if substitutions at C2 295 (<b>A</b>), alpha2 helix 338 (<b>B</b>), alpha2 helix 341 (<b>C</b>), or V5 456 (<b>D</b>) (HXB2 residues 293, 337, 340, or 460) could individually confer resistance. Percent viral infectivity, as adjusted against wells containing no test plasma, is depicted on the vertical axis; reciprocal plasma dilutions are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-plasma combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells (0-month Envs = circles, 2-month Envs and representative point mutants = triangles). Colored lines (2-A9/2-A13/0-B24 I295R in magenta, 2-B31/0-B24 I295T in red, and 2-B12/0-B24 D341N in cyan) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs.</p

Homology model of R880F gp120.

<p>A 3-dimensional gp120 monomer (blue) based on the R880F transmitted/founder Env 0-A6/B24 sequence was homology modeled from existing gp120 structures (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003173#s4" target="_blank">Materials and Methods</a>) and spatially oriented using MacPyMOL software to illuminate the region targeted by the earliest nAbs in this subject. Functional domains such as the alpha2 helix (green) and V3 (magenta) are delineated, and subject-specific amino acid numbering indicates positions that mutated at 2-months post-seroconversion to confer nAb escape. These residues (295, 338, and 341 in red) nest together in a putative epitope.</p

Amino acid alignment of R880F immunoglobulin heavy and light chain variable domains and neutralization by R880F mAbs 19.3H-L1 and 19.3H-L3.

<p>Germline heavy and light chain gene segment utilization was determined by SoDA, a somatic diversification analysis program <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003173#ppat.1003173-Volpe1" target="_blank">[32]</a>, and amino acid sequences were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software. Dashes represent conserved positions. Complementarity-determining regions (CDRs) are highlighted in gray. The two R880F mAbs share a common heavy chain, 19.3H-HC (<b>A</b>), which utilizes V3-30*02, D1-7*01, and J4*02 gene families, while the somatically related light chains 19.3H-L1 and 19.3H-L3 (<b>B</b>) employ V2-14*01 and J2*01 gene families and differ from each other at five positions in and just downstream of CDR1. Heavy chain 19.3H-HC, when paired with either 19.3H-L1 (<b>C</b>) or 19.3H-L3 (<b>D</b>), was evaluated for neutralization against pseudotyped R880F wild-type (solid lines) and site-directed mutant Envs (dashed lines). Percent viral infectivity, as adjusted against wells containing no mAb, is depicted on the vertical axis; mAb concentrations (in µg/ml) are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-mAb combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells (0-month Envs = circles, 2-month Envs and representative point mutants = triangles, 5-month Envs and a representative point mutant = inverted triangles, 7-month Envs = squares, 10-month Envs = diamonds). Colored lines (2-A9/2-A13/0-B24 I295R in magenta, 2-B31/0-B24 I295T in red, 2-B12/0-B24 D341N in cyan, and 5-B52/2-A3 K338G D341N in green) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs.</p

Chronology of R880F envelope/monoclonal antibody isolation.

a<p>Viral load at this time point was ∼580,422 copies/ml in plasma.</p

IC₅₀ values for autologous plasma/mAbs with R880F wild-type and mutant Envs.

a<p>Longitudinal wild-type and mutant Envs were assayed for neutralization sensitivity to autologous plasma and mAbs 19.3H-L1 and 19.3H-L3 in Tzm-bl cells. Average IC₅₀ values for two independent experiments using duplicate wells are given in the second through fifth columns.</p>b<p>Autologous plasma samples assayed in the second column were contemporaneous with envelope isolation dates (e.g. 0-month plasma with 0-month Envs, 2-month plasma with 2-month Envs, etc.).</p>c<p>nd, not done.</p>d<p>19.3H-L1 and 19.3H-L3 were isolated from a 16-month cryopreserved R880F PBMC sample.</p>e<p>These envelopes, though technically demonstrating IC₅₀ values greater than 25 µg/ml, saw slight neutralization at the highest concentration of antibody tested.</p>f<p>The four right-most columns detail which amino acids appear at the specified Env residues (295, 335, 338, and 341). N* denotes the introduction of a potential N-linked glycosylation site, while N** marks where such a site has been shifted downstream from a previously existing site in the Env sequence.</p

Crystal structures of R880F mAbs 19.3H-L1 and 19.3H-L3.

<p>(<b>A</b>) CDR loops. A top view looking down at the antigen-binding site of 19.3H-L1 represented by ribbons. The framework regions of the light chain and heavy chain are colored cyan and green, respectively, while each CDR loop is colored separately. The side chains of the three VL CDR1 residues different between 19.3H-L1 and 19.3H-L3 are displayed. (<b>B</b>) ODA analysis of the Fab 19.3H-L1. The size/redness of each sphere is proportional to the binding strength of the region indicated. Note that the antigen-binding site is centered at VL CDR1 and VH CDR3. (<b>C</b>) The electrostatic surface potentials of the antigen-binding site of 19.3H-L1. Red and blue coloration represents the negatively and positively charged regions, respectively, while a dashed line encircles the flat surface of the antigen-binding site. (<b>D</b>) The three VL CDR1 amino acid differences, S27T, G29T, and Y32F, between 19.3H-L1 (cyan) and 19.3H-L3 (yellow).</p

Heterologous neutralization breadth in R880F.

<p>The two R880F mAbs, 19.3H-L1 (<b>A</b>) and 19.3H-L3 (<b>B</b>), in conjunction with 16-month (<b>C</b>) and 3-year (<b>D</b>) autologous plasma were evaluated for cross-neutralizing capacity against virions pseudotyped with fourteen heterologous HIV-1 Envs from three clades (A/C recombinant and subtype A Envs = lavender, subtype B Envs = coral, subtype C Envs = teal). Two 0-month Envs (0-A6/B24) representative of the transmitted/founder sequence are included in each of these panels. Percent viral infectivity, as adjusted against wells containing no mAb or test plasma, is depicted on the vertical axis; mAb concentrations (in µg/ml) or reciprocal plasma dilutions are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-mAb or Env-plasma combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells. V3/alpha2 helix amino acid sequences were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software (<b>E</b>). Dashes represent conserved positions; dots represent gaps. Significant PNGS sequons (N295, N333, N335) are highlighted in black at their points of origin.</p