Modest de novo Reactivation of Single HIV-1 Proviruses in Peripheral CD4+ T Cells by Romidepsin

A cure for human immunodeficiency virus (HIV-1) is restricted by the continued presence of a latent reservoir of memory CD4+ T cells with proviruses integrated into their DNA despite suppressive antiretroviral therapy (ART). A predominant strategy currently pursued in HIV-1 cure-related research is the “kick and kill” approach, where latency reversal agents (LRAs) are used to reactivate transcription from integrated proviruses. The premise of this approach is that “kicking” latent virus out of hiding allows the host immune system to recognize and kill infected cells. Clinical trials investigating the efficacy of LRAs, such as romidepsin, have shown that these interventions do induce transient spikes in viral RNA in HIV-1-infected individuals. However, since these trials failed to significantly reduce viral reservoir size or significantly delay time to viral rebound during analytical treatment interruptions, it is questioned how much each individual latent provirus is actually “kicked” to produce viral transcripts and/or proteins by the LRA. Here, we developed sensitive and specific digital droplet PCR-based assays with single-provirus level resolution. Combining these assays allowed us to interrogate the level of viral RNA transcripts from single proviruses in individuals on suppressive ART with or without concomitant romidepsin treatment. Small numbers of proviruses in peripheral blood memory CD4+ T cells were triggered to become marginally transcriptionally active upon romidepsin treatment. These novel assays can be applied retrospectively and prospectively in HIV-1 cure-related clinical trials to gain crucial insights into LRA efficacy at the single provirus level

Inhibition of p53-Dependent, but Not p53-Independent, Cell Death by U19 Protein from Human Herpesvirus 6B

Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein

Administration of broadly neutralizing anti-HIV-1 antibodies at ART initiation maintains long-term CD8+ T cell immunity

In simian-human immunodeficiency virus (SHIV)-infected non-human primates, broadly neutralizing antibodies (bNAbs) against the virus appear to stimulate T cell immunity. To determine whether this phenomenon also occurs in humans we measured HIV-1-specific cellular immunity longitudinally in individuals with HIV-1 starting antiviral therapy (ART) with or without adjunctive bNAb 3BNC117 treatment. Using the activation-induced marker (AIM) assay and interferon-γ release, we observe that frequencies of Pol- and Gag-specific CD8+ T cells, as well as Gag-induced interferon-γ responses, are significantly higher among individuals that received adjunctive 3BNC117 compared to ART-alone at 3 and 12 months after starting ART. The observed changes in cellular immunity were directly correlated to pre-treatment 3BNC117-sensitivity. Notably, increased HIV-1-specific immunity is associated with partial or complete ART-free virologic control during treatment interruption for up to 4 years. Our findings suggest that bNAb treatment at the time of ART initiation maintains HIV-1-specific CD8+ T cell responses that are associated with ART-free virologic control

Direct Repeat 6 from Human Herpesvirus-6B Encodes a Nuclear Protein that Forms a Complex with the Viral DNA Processivity Factor p41

The SalI-L fragment from human herpesvirus 6A (HHV-6A) encodes a protein DR7 that has been reported to produce fibrosarcomas when injected into nude mice, to transform NIH3T3 cells, and to interact with and inhibit the function of p53. The homologous gene in HHV-6B is dr6. Since p53 is deregulated in both HHV-6A and -6B, we characterized the expression of dr6 mRNA and the localization of the translated protein during HHV-6B infection of HCT116 cells. Expression of mRNA from dr6 was inhibited by cycloheximide and partly by phosphonoacetic acid, a known characteristic of herpesvirus early/late genes. DR6 could be detected as a nuclear protein at 24 hpi and accumulated to high levels at 48 and 72 hpi. DR6 located in dots resembling viral replication compartments. Furthermore, a novel interaction between DR6 and the viral DNA processivity factor, p41, could be detected by confocal microscopy and by co-immunoprecipitation analysis. In contrast, DR6 and p53 were found at distinct subcellular locations. Together, our data imply a novel function of DR6 during HHV-6B replication

Impact of a TLR9 agonist and broadly neutralizing antibodies on HIV-1 persistence:the randomized phase 2a TITAN trial

Inducing antiretroviral therapy (ART)-free virological control is a critical step toward a human immunodeficiency virus type 1 (HIV-1) cure. In this phase 2a, placebo-controlled, double-blinded trial, 43 people (85% males) with HIV-1 on ART were randomized to (1) placebo/placebo, (2) lefitolimod (TLR9 agonist)/placebo, (3) placebo/broadly neutralizing anti-HIV-1 antibodies (bNAbs) or (4) lefitolimod/bNAb. ART interruption (ATI) started at week 3. Lefitolimod was administered once weekly for the first 8 weeks, and bNAbs were administered twice, 1 d before and 3 weeks after ATI. The primary endpoint was time to loss of virologic control after ATI. The median delay in time to loss of virologic control compared to the placebo/placebo group was 0.5 weeks (P = 0.49), 12.5 weeks (P = 0.003) and 9.5 weeks (P = 0.004) in the lefitolimod/placebo, placebo/bNAb and lefitolimod/bNAb groups, respectively. Among secondary endpoints, viral doubling time was slower for bNAb groups compared to non-bNAb groups, and the interventions were overall safe. We observed no added benefit of lefitolimod. Despite subtherapeutic plasma bNAb levels, 36% (4/11) in the placebo/bNAb group compared to 0% (0/10) in the placebo/placebo group maintained virologic control after the 25-week ATI. Although immunotherapy with lefitolimod did not lead to ART-free HIV-1 control, bNAbs may be important components in future HIV-1 curative strategies. ClinicalTrials.gov identifier: NCT03837756 .</p

Impact of a TLR9 agonist and broadly neutralizing antibodies on HIV-1 persistence: the randomized phase 2a TITAN trial

Inducing antiretroviral therapy (ART)-free virological control is a critical step toward a human immunodeficiency virus type 1 (HIV-1) cure. In this phase 2a, placebo-controlled, double-blinded trial, 43 people (85% males) with HIV-1 on ART were randomized to (1) placebo/placebo, (2) lefitolimod (TLR9 agonist)/placebo, (3) placebo/broadly neutralizing anti-HIV-1 antibodies (bNAbs) or (4) lefitolimod/bNAb. ART interruption (ATI) started at week 3. Lefitolimod was administered once weekly for the first 8 weeks, and bNAbs were administered twice, 1 d before and 3 weeks after ATI. The primary endpoint was time to loss of virologic control after ATI. The median delay in time to loss of virologic control compared to the placebo/placebo group was 0.5 weeks (P = 0.49), 12.5 weeks (P = 0.003) and 9.5 weeks (P = 0.004) in the lefitolimod/placebo, placebo/bNAb and lefitolimod/bNAb groups, respectively. Among secondary endpoints, viral doubling time was slower for bNAb groups compared to non-bNAb groups, and the interventions were overall safe. We observed no added benefit of lefitolimod. Despite subtherapeutic plasma bNAb levels, 36% (4/11) in the placebo/bNAb group compared to 0% (0/10) in the placebo/placebo group maintained virologic control after the 25-week ATI. Although immunotherapy with lefitolimod did not lead to ART-free HIV-1 control, bNAbs may be important components in future HIV-1 curative strategies. ClinicalTrials.gov identifier: NCT03837756

Complex formation of DR6(6B) and p41 during HHV-6B infection.

<p>(A) Western blotting of input lysate before immunoprecipitation. Probed with anti-DR6. (B) Western blotting of p41 and control IgG immunoprecipitation with or without HHV-6B-infection at 48 hpi. Probed with anti-DR6. (C) Western blotting of lysates after one round of p41 and IgG immunoprecipitation. Blots were probed with anti-DR6. A representative of two experiments is shown.</p

Association of DR6(6B) and the p41 viral DNA processivity factor.

<p>(A) Confocal laser scanning microscopy of DR6(6B) (green) and p41 (red) of HCT116 cells at 48 hpi. DNA was stained with DAPI (blue). Columns with DR6/p41 and DNA/DR6/p41 indicate an overlay of the individual stainings. (B) High magnification of DR6/p41 overlay. (C) Co-localization coefficient computed by the LSM710 software. Ch1-DR6 indicates the fraction of DR6(6B) that co-localized with p41. Ch2-p41 indicates the fraction of p41 that co-localized with DR6(6B). Values are the average of measurements on 14 cells with an indication of standard deviation on top of the bars. A representative of three experiments is shown.</p

Alignments of <i>dr7(6A)</i>, <i>dr6(6A)</i>, <i>orf-1(6A)</i>, and <i>dr6(6B)</i>.

<p>(A) Schematic alignment of the <i>dr6</i> gene from HHV-6A (NC_001664, nt 4725-6720) with the <i>Sal</i>I-L fragment (X73675), the <i>orf-1</i> from the <i>Sal</i>I-L fragment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007457#pone.0007457-Thompson1" target="_blank">[14]</a>, the <i>dr6(6A)</i> and <i>dr7(6A)</i> open reading frame <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007457#pone.0007457-Gompels1" target="_blank">[9]</a>. (B) Schematic alignment of <i>dr6</i> gene of HHV-6B (NC_000898, nt 5027-7203). Arrows indicate the primers used for amplification by real-time PCR. (C) Alignment of the 3′-end of <i>dr7(6A)</i>, <i>dr6(6A)</i>, <i>orf-1(6A)</i>, and <i>dr6(6B)</i>. Nt 6664 indicates the position where a frameshift takes place in <i>dr6(6A)</i> and <i>dr7(6A)</i>. (D) Alignment of the C-terminal tail of DR7(6A), DR6(6A), ORF-1, and DR6(6B). The amino acid sequence for the antigenic peptide is indicated.</p