Four different qPCR probes covering the packaging signal (PS), group-specific antigen (primer/probes detected 72%, 83%, 94%, and 92% of 578 intact clade B sequences in the Los Alamos HIV sequence database (Figs. sequence database (Figs. 1 A and S1; Stadhouders et al., 2010; Lefever et al., 2013; Rutsaert et al., 2018). All genomes scored positive with at least one of the four primer/probe sets. Notably, the large majority (99%) of genomes were positive for at least one of the many combinations of two primer/probe sets. However, any single two-probe combination was at best 86% sensitive ((blue), (yellow), and (red) regions. Signal prediction for each individual proviral IOX4 sequence is represented by the presence of the color of the respective primer/probe set. Sequences containing polymorphisms that prevent signal detection are represented by the absence of color. The percentage indicates the fraction of detected sequences for individual primer/probe sets or combinations of two primer/probe sets (brackets). (B) Horizontal bars represent the predicted detection of 401 intact and 977 defective NFL genomes from nine individuals (Lu et al., 2018; Mendoza et al., 2018). The same primer/probe sets and color scheme are used as described above. The group of defective sequences includes NFL genomes that carry small insertions, deletions, and defects in the packaging site and/or MSD. The length of the scale bar represents 10 proviral sequences. To test whether these primer/probe sets can discriminate between intact and defective proviruses, we also performed the same in silico analysis on 1,378 intact and defective IOX4 HIV-1 sequences from nine individuals that received a combination of two broadly neutralizing monoclonal antibodies during treatment interruption (Lu et al., 2018). In six out of nine patients, we observed HIV-1 sequence polymorphisms that cause a predicted loss of signal for at least one of the primer/probe sets (Fig. 1 B). For example, intact IOX4 viruses in individuals 9241, 9242, 9244, 9246, and 9255 are predicted to be negative for the PS primer/probe set. In addition to the problem of IOX4 sensitivity, two probe combinations also have a potential problem with specificity, since a number of defective viruses were predicted to be positive for several of the two probe combinations tested. The potential magnitude of this problem varies with the probe combination and the individual analyzed. For example, in 9252, of the 61 viruses Rabbit Polyclonal to ZC3H11A detected with the PS+combination, 80% are defective (49 defective vs. 12 intact), whereas in 9243, it is 35% (Fig. 1 B). Thus, the in silico data suggest that any single combination of two probes would not be sufficient for sensitive and specific reservoir measurements due to HIV-1 sequence polymorphisms within and between individuals. Quadruplex qPCR (Q4PCR) To accommodate HIV-1 sequence diversity, we developed a multiplex qPCR strategy for simultaneous detection of four probes: PS, (Q4PCR). The new method enables relatively high-throughput measurements of the latent HIV-1 reservoir with real-time detection of DNA amplification, the exclusion of gel electrophoresis, and the use of a 384-well format. Using this approach, we analyzed samples from two separate time points from six individuals enrolled in a clinical trial that involved analytical treatment interruption after infusion of a combination of two broadly neutralizing monoclonal antibodies (Lu et al., 2018; Mendoza et al., 2018). Proviral genomes were amplified from DNA extracted from purified CD4+ T cells obtained 2 wk before and 12 wk after treatment interruption. To determine overall HIV-1 proviral frequency, genomic DNA from CD4+ T cells was assayed for by qPCR. We found per reaction and assayed by Q4PCR (Fig. 2). Open in a separate window Figure 2. Q4PCR approach. Schematic representation of the Q4PCR protocol. Genomic DNA from CD4+ T cells was subjected to limiting dilution qPCR with a qPCR. An aliquot of the resulting amplicons was assayed by Q4PCR using a combination of primer/probe sets covering PS, and LTR primers (Li et al., 2007; Ho et al., 2013). Individual amplicons were then tested for reactivity with each of the four qPCR probes. Participant 9254 was excluded from the quantitative analysis because of.
Categories