Identification of amino acid substitutions associated with neutralization phenotype in the human immunodeficiency virus type-1 subtype C gp120

Abstract

Neutralizing antibodies (Nabs) are thought to play an important role in prevention and control of HIV-1 infection and should be targeted by an AIDS vaccine. It is critical to understand how HIV-1 induces Nabs by analyzing viral sequences in both tested viruses and sera. Neutralization susceptibility to antibodies in autologous and heterologous plasma was determined for multiple Envs (3-6) from each of 15 subtype-C-infected individuals. Heterologous neutralization was divided into two distinct groups: plasma with strong, cross-reactive neutralization (n=9) and plasma with weak neutralization (n=6). Plasma with cross-reactive heterologous Nabs also more potently neutralized contemporaneous autologous viruses. Analysis of Env sequences in plasma from both groups revealed a three-amino-acid substitution pattern in the V4 region that was associated with greater neutralization potency and breadth. Identification of such potential neutralization signatures may have important implications for the development of HIV-1 vaccines capable of inducing Nabs to subtype C HIV-1.

Figure 1

Phylogenetic tree analysis of newly characterized full-length env gene sequences. An unrooted phylogenetic tree was constructed with complete env gene sequences using the neighbor-joining method and the Kimura two-parameter model. Viruses with two lineages are indicated by underline; monophyletic env sequences are indicated by plain text. The branch lengths are drawn to scale (the scale bar represents 0.02 nucleotide substitutions per site).

Figure 2

Phylogenetic tree analysis of clonal expansion env gene sequences. Midpoint-rooted phylogenetic trees were constructed with all env sequences from each individual who harbored clonal expansion viruses using the neighbor-joining method and the Kimura two-parameter model. Horizontal branch lengths are drawn to scale (the scale bar represents 0.005 nucleotide substitutions per site); vertical separation is for clarity only. Asterisks indicate bootstrap values in which the cluster to the right is supported in 85% or more replicates (out of 1,000). Patterns of identity or near identity are marked with black dots at the terminal leaves.

Figure 3

Infectivity of Env pseudoviruses. The infectivity of 474 pseudoviruses from 37 individuals was determined in TZM-bl cells. The env genes were considered positive when the luciferase activity RLU values were 10-fold greater than those from SG3Δenv backbone control. The dotted line indicates this cutoff.

Figure 4

Correlation between viral load and infectivity of Env pseudovirsuses. Geometric means of the average luciferase activity of multiple Env pseudoviruses from each HIV-1 infected individual were plotted and analyzed by the Kendall’s tau rank correlation coefficient method.

Figure 5

Western blot analysis of HIV-1 protein expression in transfected cells. The 293T cells transfected with pPCR products and pSG3Δenv were lysed 48 hrs after transfection. The viral proteins were separated on a 4–12% gradient reducing gel, transferred to nitrocellulose, and were reacted with an HIV-1 positive serum and a mouse mAb 13D5 (Gao et al., 2009) to the Env protein. Viral proteins were visualized with secondary antibodies IRDye800 conjugated goat anti-human and Alexa-Fluor680 goat anti-mouse using a LiCor Odyssey Infrared Imaging system. The expression levels of Env proteins are expressed as Env (gp160+gp120):P24 ratios. The infectivity of the each Env-pseudovirus is shown as relative light unit (RLU). Asterisks indicate the smaller Envs due to premature stop codons or non-inframe deletions.

Figure 6

Maximum likelihood phylogenetic tree of the env sequences. All SGA env sequences from 15 HIV-1 infected individuals were included for analysis. Those used for neutralization assays are indicated with colored marks at the tip of the tree branches. Envelopes were selected to be representative of the diversity in the sample. The sequences from samples with low diversity (monophyletic) are indicated in black; the sequences from samples with two distinctive phylogenetic clusters are colored so that those from one cluster are red and those from the other are blue; and sequences that represent recombinants between the two clusters are purple.

Figure 7

Hierarchical clustering of viruses and sera based on neutralization titers. Sera are clustered according to their ability to neutralize. Autologous responses are indicated in black boxes along the diagonal. The env SGA numbers are colored according to diversity: low diversity (black), two distinct groups (red and blue), and recombinant (purple). For ease of visualization, the heatmap is organized such that the rows (Env pseudovirues) are arranged according to individuals rather than hierarchical clustering, where hierarchical clustering patterns of the Env pseudoviruses are shown as a dendrogram to the right of the figure. Clustering of high and low neutralization plasma was statistically supported, with a probability of 0.96 that the distinctive low-neutralization cluster was robust, using the approximately unbiased multistep-multiscale bootstrap re-sampling method developed by Shimodaira (Shimodaira, 2004). To illustrate this grouping, the columns are presented according to like-responses to the pseudoviruses based on the clustering hierarchy shown at the top of the figure. The sets of plasma with low (L) or high (H) Nab titers were grouped. Clustering patterns on the right represent significant unbiased multistep multi-scale bootstrap re-sampling values 95 or greater.

Figure 8

The signature amino acid sequences associated with high levels of neutralization activity in plasma. H represents sequences in the group of plasma samples with high neutralizing activity against heterologous viruses, whereas L represents sequences that exhibited weak neutralizing activity. Numbers are used to show corresponding locations in the HXB2 reference strain. Dashes indicate the identical amino acids present in the reference sequence. Periods are used to designate gaps to maintain the alignment. Signature sites associated with potent neutralization are shown in red. Non-signature amino acids in key positions are shown in blue.

Figure 9

Crystal structure of ligated gp120 with signature amino acids. The figure shows three signature sites, 393 (orange), 397 (magneta) and 413 (red) on a crystal structure of liganded gp120 (PDB:2B4C) (Huang et al., 2005). The V4 loop and alpha-2 helix are marked in different colors for clarification. Positions in C3 that exhibit significant contact with these signature sites on V4 are also marked with the same color as the signature sites. Three-dimensional images were generated using VMD (Humphrey et al., 1996). Location of marked sites shown on this B clade template for visualization do not show any significant differences if the recently determined X-ray C clade gp120 from CAP210 (Diskin et al., 2010) is used as a template. This is expected since rigorous evaluation of root mean square deviation (RMSD) between the CD4 bound truncated gp120 structures from B and C clades showed that both structures are similar (RMSD ~ 1.0Angstroms, S. Gnanakaran, unpublished analysis).

Figure 10

Comparison of autologous neutralizing activity between low and high heterologous neutralizing plasma samples. Nab titers from low heterologous neutralization plasmas (n=6) or high heterologous neutralization plasmas (n=9) were compared. Values at Y-axis are the reciprocal plasma dilutions at which luciferase activity (RLU) was reduced 50% relative to virus control wells by autologous plasma.