Antiretroviral therapy fails to cure HIV-1 infection because latent proviruses persist in resting CD4+ T cells. T cell activation reverses latency, but <1% of proviruses are induced to release ...infectious virus after maximum in vitro activation. The noninduced proviruses are generally considered defective but have not been characterized. Analysis of 213 noninduced proviral clones from treated patients showed 88.3% with identifiable defects but 11.7% with intact genomes and normal long terminal repeat (LTR) function. Using direct sequencing and genome synthesis, we reconstructed full-length intact noninduced proviral clones and demonstrated growth kinetics comparable to reconstructed induced proviruses from the same patients. Noninduced proviruses have unmethylated promoters and are integrated into active transcription units. Thus, it cannot be excluded that they may become activated in vivo. The identification of replication-competent noninduced proviruses indicates that the size of the latent reservoir—and, hence, the barrier to cure—may be up to 60-fold greater than previously estimated.
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•A total of 1.7% of noninduced HIV-1 proviruses have intact genomes and LTR function•Reconstructed intact noninduced proviruses are replication competent•They are integrated into transcription units and have no CpG methylation in the LTR•The size of the latent reservoir for HIV-1 may be underestimated by ∼60-fold
The number of proviruses that are intact and capable of replication if induced in vivo is substantially greater than previously estimated by as much as ∼60-fold. These revised estimates will impact calculations for new therapies aimed at eradicating HIV and AIDS.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Highly active antiretroviral therapy (HAART) suppresses HIV-1 replication but cannot eliminate the virus because HIV-1 establishes latent infection. Interruption of HAART leads to a rapid rebound of ...viremia, so life-long treatment is required. Efforts to purge the latent reservoir have focused on reactivating latent proviruses without inducing global T cell activation. However, the killing of the infected cells after virus reactivation, which is essential for elimination of the reservoir, has not been assessed. Here we show that after reversal of latency in an in vitro model, infected resting CD4+ T cells survived despite viral cytopathic effects, even in the presence of autologous cytolytic T lymphocytes (CTLs) from most patients on HAART. Antigen-specific stimulation of patient CTLs led to efficient killing of infected cells. These results demonstrate that stimulating HIV-1-specific CTLs prior to reactivating latent HIV-1 may be essential for successful eradication efforts and should be considered in future clinical trials.
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► Resting CD4+ T cells latently infected with HIV-1 do not die after virus reactivation ► Viral CPEs or patient CTLs do not cause death of HIV-1-infected resting CD4+ T cells ► Stimulated patient CTLs kill infected resting CD4+ T cells after reversal of latency ► Autologous viruses recovered from latent reservoir do not escape from patient CTLs
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Recent studies have shown T cell cross-recognition of SARS-CoV-2 and common cold coronavirus spike proteins. However, the effect of SARS-CoV-2 vaccines on T cell responses to common cold ...coronaviruses (CCCs) remains unknown. In this study, we analyzed CD4+ T cell responses to spike peptides from SARS-CoV-2 and 3 CCCs (HCoV-229E, HCoV-NL63, and HCoV-OC43) before and after study participants received Pfizer-BioNTech (BNT162b2) or Moderna (mRNA-1273) mRNA-based COVID-19 vaccines. Vaccine recipients showed broad T cell responses to the SARS-CoV-2 spike protein, and we identified 23 distinct targeted peptides in 9 participants, including 1 peptide that was targeted in 6 individuals. Only 4 of these 23 targeted peptides would potentially be affected by mutations in the UK (B.1.1.7) and South African (B.1.351) variants, and CD4+ T cells from vaccine recipients recognized the 2 variant spike proteins as effectively as they recognized the spike protein from the ancestral virus. Interestingly, we observed a 3-fold increase in the CD4+ T cell responses to HCoV-NL63 spike peptides after vaccination. Our results suggest that T cell responses elicited or enhanced by SARS-CoV-2 mRNA vaccines may be able to control SARS-CoV-2 variants and lead to cross-protection against some endemic coronaviruses.
HIV-1 persists in infected individuals in a stable pool of resting CD4(+) T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of ...HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4(+) T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4(+) T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Vaccine‐induced SARS‐CoV‐2 antibody responses are attenuated in solid organ transplant recipients (SOTRs) and breakthrough infections are more common. Additional SARS‐CoV‐2 vaccine doses increase ...anti‐spike IgG in some SOTRs, but it is uncertain whether neutralization of variants of concern (VOCs) is enhanced. We tested 47 SOTRs for clinical and research anti‐spike IgG, pseudoneutralization (ACE2 blocking), and live‐virus neutralization (nAb) against VOCs before and after a third SARS‐CoV‐2 vaccine dose (70% mRNA, 30% Ad26.COV2.S) with comparison to 15 healthy controls after two mRNA vaccine doses. We used correlation analysis to compare anti‐spike IgG assays and focused on thresholds associated with neutralization. A third SARS‐CoV‐2 vaccine dose increased median total anti‐spike (1.6‐fold), pseudoneutralization against VOCs (2.5‐fold vs. Delta), and neutralizing antibodies (1.4‐fold against Delta). However, neutralization activity was significantly lower than healthy controls (p < .001); 32% of SOTRs had zero detectable nAb against Delta after third vaccination compared to 100% for controls. Correlation with nAb was seen at anti‐spike IgG >4 Log10(AU/ml) on the Euroimmun ELISA and >4 Log10(AU/ml) on the MSD research assay. These findings highlight benefits of a third vaccine dose for some SOTRs and the need for alternative strategies to improve protection in a significant subset of this population.
Neutralizing antibodies against SARS‐CoV‐2 variants of concern increase in solid organ transplant recipients after a third dose of SARS‐CoV2 vaccine but remain significantly lower iin comparison to healthy controls.
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BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A stable latent reservoir for HIV-1 in resting CD4
T cells is the principal barrier to a cure
. Curative strategies that target the reservoir are being tested
and require accurate, scalable reservoir ...assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation
. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation
may underestimate the reservoir size because one round of activation does not induce all proviruses
. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective
. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
A small percentage of people living with HIV-1 can control viral replication without antiretroviral therapy (ART). These patients are called elite controllers (ECs) if they are able to maintain viral ...suppression without initiating ART and posttreatment controllers (PTCs) if they control HIV replication after ART has been discontinued. Both types of controllers may serve as a model of a functional cure for HIV-1 but the mechanisms responsible for viral control have not been fully elucidated. In this review, we highlight key lessons that have been learned so far in the study of ECs and PTCs and their implications for HIV cure research.
Innate immune sensing of viral infection results in type I interferon (IFN) production and inflammasome activation. Type I IFNs, primarily IFN-α and IFN-β, are produced by all cell types upon virus ...infection and promote an antiviral state in surrounding cells by inducing the expression of IFN-stimulated genes. Type I IFN production is mediated by Toll-like receptor (TLR) 3 in HCV infected hepatocytes. Type I IFNs are also produced by plasmacytoid dendritic cells (pDC) after sensing of HIV and HCV through TLR7 in the absence of productive pDC infection. Inflammasomes are multi-protein cytosolic complexes that integrate several pathogen-triggered signaling cascades ultimately leading to caspase-1 activation and generation pro-inflammatory cytokines including interleukin (IL)-18 and IL-1β. Here, we demonstrate that HIV and HCV activate the inflammasome, but not Type I IFN production, in monocytes and macrophages in an infection-independent process that requires clathrin-mediated endocytosis and recognition of the virus by distinct endosomal TLRs. Knockdown of each endosomal TLR in primary monocytes by RNA interference reveals that inflammasome activation in these cells results from HIV sensing by TLR8 and HCV recognition by TLR7. Despite its critical role in type I IFN production by pDCs stimulated with HIV, TLR7 is not required for inflammasome activation by HIV. Similarly, HCV activation of the inflammasome in monocytes does not require TLR3 or its downstream signaling adaptor TICAM-1, while this pathway leads to type I IFN in infected hepatocytes. Monocytes and macrophages do not produce type I IFN upon TLR8 or TLR7 sensing of HIV or HCV, respectively. These findings reveal a novel infection-independent mechanism for chronic viral induction of key anti-viral programs and demonstrate distinct TLR utilization by different cell types for activation of the type I IFN vs. inflammasome pathways of inflammation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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