Chronic HIV-1 infection frequently fails to protect against superinfection

Abstract

Reports of HIV-1 superinfection (re-infection) have demonstrated that the immune response generated against one strain of HIV-1 does not always protect against other strains. However, studies to determine the incidence of HIV-1 superinfection have yielded conflicting results. Furthermore, few studies have attempted to identify superinfection cases occurring more than a year after initial infection, a time when HIV-1-specific immune responses would be most likely to have developed. We screened a cohort of high-risk Kenyan women for HIV-1 superinfection by comparing partial gag and envelope sequences over a 5-y period beginning at primary infection. Among 36 individuals, we detected seven cases of superinfection, including cases in which both viruses belonged to the same HIV-1 subtype, subtype A. In five of these cases, the superinfecting strain was detected in only one of the two genome regions examined, suggesting that recombination frequently occurs following HIV-1 superinfection. In addition, we found that superinfection occurred throughout the course of the first infection: during acute infection in two cases, between 1-2 y after infection in three cases, and as late as 5 y after infection in two cases. Our results indicate that superinfection commonly occurs after the immune response against the initial infection has had time to develop and mature. Implications from HIV-1 superinfection cases, in which natural re-exposure leads to re-infection, will need to be considered in developing strategies for eliciting protective immunity to HIV-1.

Figure 1. Maximum-Likelihood Phylogenetic Trees of env (A) and gag (B) Sequences from 36 Individuals

Each tree contains two initial and two chronic sequences from each individual (unlabeled branches) that were selected to represent both strains in presumed superinfection cases and were selected randomly for other cases. Reference sequences representing different subtypes from the LANL database are also included (labeled branches). Cases that demonstrate separate clustering on either or both trees are highlighted in color. In most cases, separate clustering is evident in only env or only gag. Sequences from each of these presumed superinfection cases are labeled with the case number and either “initial” or “chronic” on the tree(s) that demonstrates separate clustering. Green = Case 1, orange = Case 2, blue = Case 3, purple = Case 4, red = Case 5, yellow = Case 6, light blue = Case 7. Bootstrap values were omitted for clarity; however, except for the superinfection cases, sequences from each individual form a monophyletic cluster with ≥82% bootstrap support (most have 100% bootstrap support).

Figure 2. Maximum Divergence from the MRCA for Chronic env (A) and gag (B) Sequences

Comparisons within individuals (black circles) and between subtype A–infected individuals (gray triangles) are arbitrarily distributed along the x-axis. Superinfection cases are labeled in both plots. For most cases, the high divergence indicative of superinfection is apparent in one region of the genome but not the other, consistent with Figure 1.

Figure 3. Case 1 (QB726): Intrasubtype Superinfection (Subtype A) between 749 and 1,031 DPI Detected in env and gag

(A) Plot of viral load (log RNA copies/mL plasma) versus DPI. Sequences were obtained by single-copy PCR from time points marked by circles. Arrows indicate the initial and chronic time points used in the analysis in Figure 1, and the box indicates the first time point at which the superinfecting strain was detected. The number of sequences of each strain at each time point is indicated in a table below the graph. ND, not done. (B) Maximum-likelihood tree of env sequences from all time points. Sequence names from QB726 are comprised of the sample DPI and a unique identifier. The cluster of strain A contains sequences from all time points except 1,633 and 2,876 DPI. The cluster of strain A* contains sequences from 1,031, 1,165, 1,381, and 2,002 DPI. Sequences from three other individuals (QA520, QA268, and QA750), as well as subtype reference sequences from the LANL database, are also included. Bootstrap values greater than 70% are shown. (C) Maximum-likelihood tree of gag sequences from all time points. Sequence names from QB726 are comprised of the sample DPI and a unique identifier. The cluster of strain D contains sequences from all time points except 2,876 DPI. The cluster of strain A* contains sequences from 1,031 and 2,876 DPI. Sequences from four other individuals (QC036, QA255, QD121, and QC805), as well as subtype reference sequences from the LANL database, are also included. Bootstrap values greater than 70% are shown. (D) Amplification of 50 HIV-1 genome copies from the indicated time points using env strain–specific primers (top panel) or env common primers (bottom panel). For each time point, each lane contains an estimated ten HIV-1 genome copies of template input. As a control, we also amplified 10³ copies of a cloned A sequence (two lanes) and one copy of a cloned A* sequence (four lanes). (E) Amplification of 50 HIV-1 genome copies from the indicated time points using gag strain–specific primers (top panel) or gag common primers (bottom panel). For each time point, each lane contains an estimated ten HIV-1 genome copies of template input. We also amplified 10³ copies of a cloned D sequence (two lanes) and one copy of a cloned A* sequence (four lanes). (F) Summary of the strains detected in each genome region at the initial time point and during chronic infection (all other time points). Light gray = subtype A; dark gray = A*; black = subtype D.

Figure 4. Case 2 (QB045): Intrasubtype Superinfection (Sub-Subtype A2–A1) between 1,680 and 2,048 DPI Detected in env and gag

The layout of this figure is similar to that of Figure 3, as described in the legend for Figure 3. (A) Plot of viral load (log RNA copies/mL plasma) versus DPI with the number of sequences of each strain indicated. (B) Maximum-likelihood tree of env sequences from all time points. The A2 cluster contains sequences from all time points, while the A1 cluster contains sequences from 2,048 and 2,247 DPI. (C) Maximum-likelihood tree of gag sequences from all time points. The cluster of strain D/A2 contains sequences from all time points. The cluster of strains D/A1 and A1 contains sequences from 2,048 DPI. (D) Summary of the strains detected. Light gray = sub-subtype A2; dark gray = sub-subtype A1; black = subtype D.

Figure 5. Case 3 (QB850): Intersubtype Superinfection (Subtype A–D) between 52 and 73 DPI Detected in env

The layout of this figure is similar to that of Figure 3, as described in the legend for Figure 3. (A) Plot of viral load (log RNA copies/mL plasma) versus DPI with the number of sequences of each strain indicated. (B) Maximum-likelihood tree of env sequences from all time points. Cluster A contains sequences from all time points, while cluster A/D contains sequences from 73 and 1,768 DPI. (C) Maximum-likelihood tree of gag sequences from all time points, illustrating unusually high divergence. (D) Amplification of 50 HIV-1 env genome copies from the indicated time points using strain-specific primers (top panel) and common primers (bottom panel). (E) Summary of the strains detected. Light gray = subtype A; black = subtype D.

Figure 6. Case 4 (QD022): Intersubtype Superinfection (Subtype A–C) between 1,832 and 1,957 DPI Detected in env

The layout of this figure is similar to that of Figure 3, as described in the legend for Figure 3. (A) Plot of viral load (log RNA copies/mL plasma) versus DPI with the number of sequences of each strain indicated. B) Maximum-likelihood tree of env sequences from all time points. Cluster A contains sequences from 51, 522, and 1,832 DPI, while cluster C contains sequences from 1,957 and 2,752 DPI. (C) Amplification of 50 env HIV-1 genome copies from the indicated time points using strain-specific primers (top panel) and common primers (bottom panel). (D) Summary of the strains detected. Light gray = subtype A; black = subtype C.

Figure 7. Case 5 (QA413): Intrasubtype Superinfection (Subtype A) between 714 and 1,007 DPI Detected in env

The layout of this figure is similar to that of Figure 3, as described in the legend for Figure 3. (A) Plot of viral load (log RNA copies/mL plasma) versus DPI with the number of sequences of each strain indicated. (B) Maximum-likelihood tree of env sequences from all time points. The cluster of strain A contains sequences from all time points, while the cluster of strain A* contains sequences from 1,007, 1,146, and 1,346 DPI. (C) Amplification of 50 env HIV-1 genome copies from the indicated time points using strain-specific primers (top panel) and common primers (bottom panel). (D) Summary of the strains detected. Light gray = subtype A; dark gray = A*.

Figure 8. Case 6 (QB685): Intrasubtype Superinfection (Subtype A) between 303 and 1,453 DPI Detected in gag

The layout of this figure is similar to that of Figure 3, as described in the legend for Figure 3. (A) Plot of viral load (log RNA copies/mL plasma) versus DPI with the number of sequences of each strain indicated. (B) Maximum-likelihood tree of gag sequences from all time points. The cluster of strain A contains sequences from all time points except 1,800 DPI. The cluster of strain A* contains sequences from 1,453 and 1,800 DPI. (C) Amplification of 50 gag HIV-1 genome copies from the indicated time points using strain-specific primers (top panel) and common primers (bottom panel). (D) Summary of the strains detected. Light gray = subtype A; dark gray = A*.

Figure 9. Case 7 (QC885): Intersubtype Superinfection (Subtype C–A) between 58 and 152 DPI Detected in gag

The layout of this figure is similar to that of Figure 3, as described in the legend for Figure 3. (A) Plot of viral load (log RNA copies/mL plasma) versus DPI with the number of sequences of each strain indicated. (B) Maximum-likelihood tree of gag sequences from all time points. The cluster of strain C contains sequences from all time points except 339 and 1,405 DPI. The cluster of strain A/C contains sequences from 152, 339, 635, and 1,405 DPI. (C) Amplification of 50 gag HIV-1 genome copies from the indicated time points using strain-specific primers (top panel) and common primers (bottom panel). (D) Summary of the strains detected. Light gray = subtype A; black = subtype C.