Evidence for frequent reinfection with human immunodeficiency virus type 1 of a different subtype

Abstract

A major premise underlying current human immunodeficiency virus type 1 (HIV-1) vaccine approaches is that preexisting HIV-1-specific immunity will block or reduce infection. However, the recent identification of several cases of HIV-1 reinfection suggests that the specific immune response generated for chronic HIV-1 infection may not be adequate to protect against infection by a second HIV-1 strain. It has been unclear, though, whether these individuals are representative of the global epidemic or are rare cases. Here we show that in a population of high-risk women, HIV-1 reinfection occurs almost as commonly as first infections. The study was designed to detect cases of reinfection by HIV-1 of a different subtype and thus captured cases where there was considerable diversity between the first and second strain. In each case, the second virus emerged approximately 1 year after the first infection, and in two cases, it emerged when viral levels were high, suggesting that a well-established HIV-1 infection may provide little benefit in terms of immunizing against reinfection, at least by more-divergent HIV-1 variants. Our findings indicate an urgent need for studies of larger cohorts to determine the incidence and timing of both intersubtype and intrasubtype reinfection.

FIG. 1.

Analyses of reinfection in subject QA013. (a) The graph illustrates HIV-1 plasma viral loads at various times postinfection. In this subject, both HIV-1 antibodies and RNA were detected 44 days after the subject had tested seronegative and HIV-1 RNA negative. Thus, the date of infection was estimated as the midpoint between the two visits, as described previously (14). Results of the three different methods of analysis of viral sequences are summarized below the indicated times p.i. The detection of subtype D or A sequences among clones obtained from the subject's PBMC DNA is shown on the top lines, with a + to indicate that at least one clone was present and with a − to indicate that no clones of that subtype were detected among those analyzed. Similarly, the presence or absence of a subtype-specific PCR product is shown with a + or −, respectively. NA means not available. The middle set of data is from analyses of the subject's PBMC DNA using SSP PCR, and the bottom set is from analyses of DNA from cells that were cultured to amplify virus. (Examples of these data are shown in panel c). (b) Phylogenetic analyses of sequences from different times p.i. by using a distance-based, neighbor-joining method. The numbers at the nodes represent bootstrap values for these nodes based on 100 bootstrap resamplings. Clones from the subject are designated QA013 followed by the days p.i. from which they wereobtained and a clone designation such as A1 (where A indicates the PCR and 1 was the first clone from PCR A). The two clusters of different clones from this subject are marked with braces; one cluster groups with subtype D and one with subtype A, as indicated. (c) Results of subtype-specific PCR of PBMC DNA (top) and culture DNA (bottom) using subtype-specific inner primers. The samples are from the indicated days p.i. The amount of DNA that was added to the PCR mixture was based on the HIV-1 copy number in the sample, as indicated at the top of each lane. The last four lanes in each gel were PCRs of clones obtained from the subject that were known to be of one or the other subtype, as determined by phylogenetic analyses described above for panel b. Two different clones were tested at the indicated copy numbers, which were calculated from DNA concentration. In all cases, the product from the same first-round PCR was used for the indicated second-round PCRs; the primers for the second-round PCR are indicated to the right of the gels and are as described in the supplemental material (Fig. S1).

FIG. 2.

Analyses of reinfection in subject QB008. (a) Plasma viral loads and results of HIV-1 sequence analyses at various times p.i. both HIV-1 antibodies and RNA were detected 121 days after the subject tested HIV-1 RNA negative and seronegative. Thus, the date of infection was estimated as the midpoint between the two visits. The layout for this figure is as described in the legend for Fig. 1a. (b) Bootscan analyses of representative initial (QB008.129.A3) and reinfecting clones (QB008.591.A1). The day p.i. from which the clone was obtained is indicated in the clone name, after the subject designation. The percentage of permuted trees is shown versus the sequence position. Recombination with less than 70% bootstrap support was considered to be statistically insignificant. The reference strains that were used in the analysis are indicated to the right and are given a color. A phylogenetic tree of the sequence is shown; in the case of the virus QB008.591.A1, which had a clear breakpoint in the bootscan analysis, the sequences from before and after that breakpoint were analyzed individually. The subtype designation is shown schematically at the bottom of each bootscan. (c) Results of subtype-specific PCR of PBMC DNA (top) and culture DNA (bottom). The layout is as described in the legend for Fig. 1c.

FIG. 2.

Analyses of reinfection in subject QB008. (a) Plasma viral loads and results of HIV-1 sequence analyses at various times p.i. both HIV-1 antibodies and RNA were detected 121 days after the subject tested HIV-1 RNA negative and seronegative. Thus, the date of infection was estimated as the midpoint between the two visits. The layout for this figure is as described in the legend for Fig. 1a. (b) Bootscan analyses of representative initial (QB008.129.A3) and reinfecting clones (QB008.591.A1). The day p.i. from which the clone was obtained is indicated in the clone name, after the subject designation. The percentage of permuted trees is shown versus the sequence position. Recombination with less than 70% bootstrap support was considered to be statistically insignificant. The reference strains that were used in the analysis are indicated to the right and are given a color. A phylogenetic tree of the sequence is shown; in the case of the virus QB008.591.A1, which had a clear breakpoint in the bootscan analysis, the sequences from before and after that breakpoint were analyzed individually. The subtype designation is shown schematically at the bottom of each bootscan. (c) Results of subtype-specific PCR of PBMC DNA (top) and culture DNA (bottom). The layout is as described in the legend for Fig. 1c.

FIG. 2.

Analyses of reinfection in subject QB008. (a) Plasma viral loads and results of HIV-1 sequence analyses at various times p.i. both HIV-1 antibodies and RNA were detected 121 days after the subject tested HIV-1 RNA negative and seronegative. Thus, the date of infection was estimated as the midpoint between the two visits. The layout for this figure is as described in the legend for Fig. 1a. (b) Bootscan analyses of representative initial (QB008.129.A3) and reinfecting clones (QB008.591.A1). The day p.i. from which the clone was obtained is indicated in the clone name, after the subject designation. The percentage of permuted trees is shown versus the sequence position. Recombination with less than 70% bootstrap support was considered to be statistically insignificant. The reference strains that were used in the analysis are indicated to the right and are given a color. A phylogenetic tree of the sequence is shown; in the case of the virus QB008.591.A1, which had a clear breakpoint in the bootscan analysis, the sequences from before and after that breakpoint were analyzed individually. The subtype designation is shown schematically at the bottom of each bootscan. (c) Results of subtype-specific PCR of PBMC DNA (top) and culture DNA (bottom). The layout is as described in the legend for Fig. 1c.

FIG. 3.

Analyses of reinfection in subject QB609. This subject was found to be HIV-1 seropositive 77 days after testing seronegative. HIV-1 RNA was detected in plasma at the seronegative visit; thus, the time of infection was estimated as 17 days prior to that, as described previously (14). The layout is as described in the legend for Fig. 1a.