Transcriptional effects of superinfection in HIV chronically infected T cells: Studies in dually infected clones

We had previously shown that chronically infected ACH-2 cells (HIV(LAI) could be superinfected with HIV(RF), that the frequency of superinfection increased with time, and that the transcription of the superinfecting virus exceeded that of the host HIV(LAI) provirus. In contrast, ACH-2 cells superinfected with a nef-substituted neomycin-resistant (proNEO) provirus were not detectable by DNA polymerase chain reaction (PCR) until geneticin (G418) was added, suggesting that the ability to propagate progressively in culture may be HIV strain specific. Clonal populations of ACH-2 superinfected with proNEO did not demonstrate preferential transcription of the superinfecting virus. However, clones of ACH-2 superinfected with HIVRf (ACH2/RF) showed a preponderance of HIV(RF), transcripts similar to that seen in bulk populations. Induction of the superinfecting virus by phorbol ester (PMA) occurred more rapidly than the host provirus and did not equalize transcriptional activity. PCR-derived long terminal repeat (LTR) fragments and Tat cDNAs from A3.01 cells acutely infected with HIVRF or from ACH-2 cells were sequenced and tested for transactivation. The HIV(LAI) LTR was two to three times more Tat-responsive than the HIV(RF) LTR. Tat(RF), was two to three times more transcriptionally active on either LTR than Tat(LAI). Demethylation with 5-azacytidine did not significantly affect HIV expression from the HIV(LAI) host provirus of superinfected ACH2/RF cell clones. These data suggest that the mechanism of preferential transcription in HIV(RF) superinfected ACH2/RF may be attributed to the Tat/TAR axis and the effect of the specific locus of host proviral integration

Neutralization sensitivity of a novel HIV-1 CRF01_AE panel of infectious molecular clones

BACKGROUND: HIV-1 CRF01_AE is dominant in Thailand where RV144 vaccine trial was conducted. To study immune correlates of protection in ongoing trials, CRF01_AE derived reagents are essential. Here we present a panel of 14 HIV-1 infectious molecular clones (IMC) identified from different stages of infection, and characterization of their neutralization sensitivity using two standard assays. METHODS: One full-length IMC was constructed using a transmitted-founder virus to express Renilla luciferase (LucR) reporter gene and full-length envelopes (envs) of exogenous HIV-1. A panel of IMCs was generated, expressing envs of viruses from acute (Fiebig stages I/II and I-IV) and chronic (>Febig VI) infection. Neutralization assays were performed using TZM-bl or A3R5 cell lines, and sera or monoclonal antibodies (mAbs). Wilcoxon matched-paired test was used to assess neutralization differences between assays and reagents; correlation coefficients were evaluated by linear regression. RESULTS: Neutralization potency observed was significantly higher in the A3R5 assay when testing mAbs and serum pools (p<0.0001); the stage of infection from which env was derived did not associate with IMC neutralization sensitivity. Neutralization values from A3R5 and TZM-bl assays were strongly correlated when mAbs were tested (R=0.7, p<0.0001), but a weaker association was seen with serum pools (R=0.17, p=0.03). CONCLUSIONS: This novel panel of CRF01_AE reporter-IMC is useful for assessing vaccine-induced neutralizing antibodies in multiple assays, including those utilizing primary cell targets. The significant differences in TZM-bl and A3R5 neutralization sensitivity, as well as the poor association when using polyclonal sera indicates the need for caution in choosing one specific platform

Transcriptomic signatures of NK cells suggest impaired responsiveness in HIV-1 infection and increased activity post-vaccination

Natural killer (NK) cells are important for eliminating cells under stress or infected by virus, and may have a function in anti-HIV immunity. Here the authors show that different NK-activating stimuli induce distinct transcriptional fingerprints in human NK cells that are analogous to changes caused by HIV vaccination or chronic infection