scholarly journals Transient CRISPR-Cas Treatment Can Prevent Reactivation of HIV-1 Replication in a Latently Infected T-Cell Line

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2461
Author(s):  
Ye Liu ◽  
Rienk E. Jeeninga ◽  
Bep Klaver ◽  
Ben Berkhout ◽  
Atze T. Das
Keyword(s):  
T Cell ◽  
Hiv Replication ◽  
Delivery Methods ◽  
Cas9 Protein ◽  
Target Sites ◽  
Latently Infected ◽  
Novel Therapeutic Strategies ◽  
Hiv 1

Novel therapeutic strategies aiming at the permanent inactivation of the HIV-1 reservoir in infected individuals are currently being explored, including approaches based on CRISPR-Cas gene editing. Extinction of all infectious HIV provirus in infected T-cell cultures was previously achieved when cells were transduced with lentiviral vectors for the stable expression of CRISPR-Cas9 or Cas12a systems targeting HIV DNA. Because lentiviral transduction and long-term CRISPR-Cas activity are less suitable for in vivo application of this antiviral strategy, we investigated whether HIV can also be completely inactivated by transient CRISPR-Cas activity. Latently infected SupT1 T-cells were repeatedly transfected with different Cas9 and Cas12a mRNA/protein sources in combination with dual gRNAs/crRNAs targeting highly conserved viral sequences. Upon repeated Cas9 protein treatment, viral replication could no longer be reactivated. We demonstrate that this was due to complete mutational inactivation of the proviral DNA, mostly through mutations at the target sites, but also through excision or inversion of the viral DNA fragment between the two target sites. These results demonstrate that repeated transient CRISPR-Cas treatment of a latently infected T-cell culture can lead to the permanent inactivation of HIV replication, indicating that transient CRISPR-Cas delivery methods can be considered for in vivo application.

Ex Vivo Expanded Multi-Specific Cytotoxic T Lymphocytes Derived From HIV+ Patients and HIV Negative Donors Using GMP Compliant Methodologies Recognize Multiple HIV Antigens and Suppress HIV Replication

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 188-188
Author(s):  
Sharon Lam ◽  
Julia Marsh Sung ◽  
Conrad RY Cruz ◽  
Paul Castillo-Caro ◽  
Minh T. Ngo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Third Party ◽  
Hiv Patients ◽  
Specific Lysis ◽  
Latently Infected ◽  
Hiv Negative

Abstract Antiretroviral therapy (ART) does not eliminate HIV from latently infected reservoirs, has long-term toxicities and fails to fully prevent immune attenuation. Therefore there is a need for alternative therapies that will decrease dependency on ART. Previous studies have demonstrated the safety and feasibility of infusing single-epitope specific CD8 T cells or artificial T cell receptor- transduced T cells to HIV+ patients. However, these T cells were restricted to a single HLA restricted epitope and had limited persistence in vivo. Hence, we hypothesized that broadly HIV-specific T cells could be expanded from patients on ART and HIV negative individuals to effectively target HIV infection using a non-HLA restricted, GMP-compliant approach. We developed a method by which PBMCs from patients on ART were stimulated with autologous dendritic cells (DCs) pulsed with gag, pol, and nef peptide libraries (pepmixes) in the presence of IL-7, IL-12, and IL-15, followed by a second stimulation with pepmix-pulsed PHA blasts and IL-15 and a third stimulation with pepmix-pulsed PHA blasts, co-stimulatory K562 cells, and IL-2. Starting from 60 to 100 mL of blood, T cells expanded to clinically relevant numbers (Mean=1.62e8 cells, Range (3.72e7, 2.87e8 cells), n=7) using co-stimulatory K-562 cells and gas-permeable tissue culture devices in the presence of antiretrovirals to prevent possible viral spread during expansion. The majority of the expanded T cells had an effector memory phenotype (CD3+CD45RO+CD62L-) with approximately 10% suggestive of a central memory phenotype (CD3+CD45RO+CD62L+) which is important for long-term persistence of T cells in vivo. After 3 stimulations, 5 of 7 patient sample lines showed specific activity to all 3 HIV antigens in IFNY ELISPOT assays, with the remaining 2 showing specificity to 1 of 3 antigens. The T cell lines were broadly specific to gag (mean=99.33 SFC/10e5 cells), pol (mean=131.11 SFC/10e5 cells) and nef (mean=337.26 SFC/10e5 cells), and polyclonal as shown by flow-based Vbeta usage analysis (mean usage= 14.67 of the 24 Vbetas analyzed). In addition, due to the association of gag-specific T cell responses with viral control in long-term nonprogressors we also determined whether gag-specific T cells could be derived from seronegative donors for potential third party use. Using the same methodology developed for HIV+ patients, but using only the gag pepmix, gag-specific T cells were expanded from HIV seronegative individuals. These T cells released IFNγ in response to gag pepmix (163.79 SFC/1e5 cells, n=9) but not an irrelevant antigen (mean=7.0 SFC/1e5 cells). Importantly, T cells expanded from both ART patients and HIV seronegative individuals were cytotoxic, as expanded T cells lysed antigen loaded autologous PHA blasts (mean=67.55% specific lysis at 10:1 effector:target ratio) but not PHA blasts alone (mean=0.46% specific lysis at 10:1 effector target ratio) in chromium release assays. Expanded T cells from ART patients also showed a greater ability to suppress HIV outgrowth in vitro compared to unexpanded CD8 T cells when co-cultured with reactivated resting CD4+ T cells from ART-suppressed HIV+ patients, the authentic latently infected cells that define viral reservoirs in treated patients. In 5 patients on ART a statistically lower recovery of virus from resting CD4+ cells was seen in the presence of CTLs as compared to no effectors (p<0.006 by Mann Whitney), while the unexpanded autologous CD8 cells showed only a modest trend towards decreased recovery that was not statistically significant (p>0.9). Similarly, HIV-specific T cells derived from HIV seronegative individuals were able to suppress HIV replication more than unexpanded CD8 T cells when co-cultured with autologous CD4 T cells infected with HIVSF162 (HIV only condition p24=681.95 pg/mL, nonspecific CD8 T cells=448.80 pg/mL, expanded CTL=145.82 pg/mL). We have developed robust GMP-compliant methodologies for expanding functional HIV-specific T cells from both HIV+ patients and HIV negative donors for autologous and third-party use, respectively. We now plan to translate our approach to the clinical setting where we will test HIV-polyspecific T cell products as a part of a strategy to fully eradicate HIV infection. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In Vivo Analysis of Human T-Cell Leukemia Virus Type 1 Reverse Transcription Accuracy

2000 ◽  
Vol 74 (20) ◽  
pp. 9525-9531 ◽  
Author(s):  
Louis M. Mansky
Keyword(s):  
T Cell ◽  
Mutation Rate ◽  
Virus Type ◽  
Reverse Transcription ◽  
Leukemia Virus ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Hiv 1

ABSTRACT Several studies have indicated that the genetic diversity of human T-cell leukemia virus type 1 (HTLV-1), a virus associated with adult T-cell leukemia, is significantly lower than that of other retroviruses, including that of human immunodeficiency virus type 1 (HIV-1). To test whether HTLV-1 variation is lower than other retroviruses, a tractable vector system has been developed to measure reverse transcription accuracy in one round of HTLV-1 replication. This system consists of a HTLV-1 vector that contains a cassette with the neomycin phosphotransferase (neo) gene, a bacterial origin of DNA replication, and the lacZα peptide gene region (the mutational target). The vector was replicated bytrans-complementation with helper plasmids. The in vivo mutation rate for HTLV-1 was determined to be 7 × 10−6 mutations per target base pair per replication cycle. The majority of the mutations identified were base substitution mutations, namely, G-to-A and C-to-T transitions, frameshift mutations, and deletion mutations. Mutation of the methionine residue in the conserved YMDD motif of the HTLV-1 reverse transcriptase to either alanine or valine (i.e., M188A or M188V) led to a factor of two increase in the rate of mutation, indicating the role of this motif in enzyme accuracy. The HTLV-1 in vivo mutation rate is comparable to that of bovine leukemia virus (BLV), another member of the HTLV/BLV genus of retroviruses, and is about fourfold lower than that of HIV-1. These observations indicate that while the mutation rate of HTLV-1 is significantly lower than HIV-1, this lower rate alone would not explain the low diversity in HTLV-1 isolates, supporting the hypothesis that HTLV-1 replicates primarily as a provirus during cellular DNA replication rather than as a virus via reverse transcription.

scholarly journals Factors Associated With CD8+ T-Cell Activation in HIV-1–Infected Patients on Long-term Antiretroviral Therapy

2014 ◽  
Vol 67 (2) ◽  
pp. 153-160 ◽  
Author(s):  
Lu Zheng ◽  
Babafemi Taiwo ◽  
Rajesh T. Gandhi ◽  
Peter W. Hunt ◽  
Ann C. Collier ◽  
...  
Keyword(s):  
T Cell ◽  
Antiretroviral Therapy ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
Factors Associated ◽  
Hiv 1

Low CD4+ T-Cell Surface CCR5 Density as a Cause of Resistance to In Vivo HIV-1 Infection

2003 ◽  
Vol 34 (1) ◽  
pp. 114-116 ◽  
Author(s):  
Jacques Reynes ◽  
Vincent Baillat ◽  
Pierre Portales ◽  
Jacques Clot ◽  
Pierre Corbeau
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Cd4 T Cell ◽  
Hiv 1

scholarly journals TAILORED DC INDUCE PROTECTIVE HIV-1 SPECIFIC POLYFUNCTIONAL CD8+ T CELLS IN THE LYMPHOID TISSUE FROM HUMANIZED BLT MICE

2021 ◽  
Author(s):  
Marta Calvet-Mirabent ◽  
Daniel T. Claiborne ◽  
Maud Deruaz ◽  
Serah Tanno ◽  
Carla Serra ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Lymphoid Tissue ◽  
White Pulp ◽  
Blt Mice ◽  
Secondary Lymphoid Organs ◽  
The Impact ◽  
Hiv 1

Effective function of CD8+ T cells and enhanced innate activation of dendritic cells (DC) in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of TBK1-primed DC inducing protective CD8+ T cell responses in lymphoid tissue and peripheral blood and their association with reduced HIV-1 disease progression in vivo in the humanized bone marrow, liver and thymus (hBLT) mouse model. A higher proportion of hBLT-mice vaccinated with TBK1-primed DC exhibited less severe CD4+ T cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to secondary lymphoid organs and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, TBK1-primed DC might be an useful tool for subsequent vaccine studies.

scholarly journals Use of an In Vivo FTA Assay to Assess the Magnitude, Functional Avidity and Epitope Variant Cross-Reactivity of T Cell Responses Following HIV-1 Recombinant Poxvirus Vaccination

PLoS ONE ◽  
2014 ◽  
Vol 9 (8) ◽  
pp. e105366 ◽  
Author(s):  
Danushka K. Wijesundara ◽  
Charani Ranasinghe ◽  
Ronald J. Jackson ◽  
Brett A. Lidbury ◽  
Christopher R. Parish ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Responses ◽  
Cross Reactivity ◽  
Functional Avidity ◽  
Cell Responses ◽  
Epitope Variant ◽  
Hiv 1

scholarly journals Modulation of the CCR5 Receptor/Ligand Axis by Seminal Plasma and the Utility of In Vitro versus In Vivo Models

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Jennifer A. Juno ◽  
Kathleen M. Wragg ◽  
Anne B. Kristensen ◽  
Wen Shi Lee ◽  
Kevin J. Selva ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Seminal Plasma ◽  
Target Cells ◽  
Ccr5 Expression ◽  
Ccr5 Receptor ◽  
The Impact ◽  
Hiv 1

ABSTRACT Sexual HIV-1 transmission occurs primarily in the presence of semen. Although data from macaque studies suggest that CCR5+ CD4+ T cells are initial targets for HIV-1 infection, the impact of semen on T cell CCR5 expression and ligand production remains inconclusive. To determine if semen modulates the lymphocyte CCR5 receptor/ligand axis, primary human T cell CCR5 expression and natural killer (NK) cell anti-HIV-1 antibody-dependent beta chemokine production was assessed following seminal plasma (SP) exposure. Purified T cells produce sufficient quantities of RANTES to result in a significant decline in CCR5bright T cell frequency following 16 h of SP exposure (P = 0.03). Meanwhile, NK cells retain the capacity to produce limited amounts of MIP-1α/MIP-1β in response to anti-HIV-1 antibody-dependent stimulation (median, 9.5% MIP-1α+ and/or MIP-1β+), despite the immunosuppressive nature of SP. Although these in vitro experiments suggest that SP-induced CCR5 ligand production results in the loss of surface CCR5 expression on CD4+ T cells, the in vivo implications are unclear. We therefore vaginally exposed five pigtail macaques to SP and found that such exposure resulted in an increase in CCR5+ HIV-1 target cells in three of the animals. The in vivo data support a growing body of evidence suggesting that semen exposure recruits target cells to the vagina that are highly susceptible to HIV-1 infection, which has important implications for HIV-1 transmission and vaccine design. IMPORTANCE The majority of HIV-1 vaccine studies do not take into consideration the impact that semen exposure might have on the mucosal immune system. In this study, we demonstrate that seminal plasma (SP) exposure can alter CCR5 expression on T cells. Importantly, in vitro studies of T cells in culture cannot replicate the conditions under which immune cells might be recruited to the genital mucosa in vivo, leading to potentially erroneous conclusions about the impact of semen on mucosal HIV-1 susceptibility.

scholarly journals HIV-specific T-cell Responses and Generalized Activation in HIV-1 Infected Long-term Non-progressors and Progressors from South India

2019 ◽  
Vol 16 (4) ◽  
pp. 302-314
Author(s):  
Chinnambedu Ravichandran Swathirajan ◽  
Ramachandran Vignesh ◽  
Greer Waldrop ◽  
Uma Shanmugasundaram ◽  
Pannerselvam Nandagopal ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd4 T Cell ◽  
Cell Responses ◽  
Activation Rates ◽  
Hiv 1

Background:Anti-viral cytokine expressions by cytotoxic T-cells and lower activation rates have been reported to correlate with suppressed HIV replication in long-term non-progressors (LTNP). Immune mechanisms underlying disease non-progression in LTNP might vary with HIV-1 subtype and geographical locations.Objective:This study evaluates cytokine expression and T-cells activation in relation to disease non-progression in LTNP.Methods:HIV-1 Subtype C infected LTNP (n=20) and progressors (n=15) were enrolled and flowcytometry assays were performed to study HIV-specific CD8 T-cells expressing IL-2, IFN-&#947;, TNF-&#945; and MIP-1&#946; against gag and env peptides. CD4+ T-cell activation was evaluated by surface expression of HLADR and CD38.Results:Proportions of cytokines studied did not differ significantly between LTNP and progressors, while contrasting correlations with disease progression markers were observed in LTNP. CD4+ T-cell activation rates were significantly lower in LTNP compared to progressors which indicate the potential role of T-cell activation rates in disease non-progression in LTNP.Conclusion:LTNP and progressors showed similar CD8+ T-cell responses, but final conclusions can be drawn only by comparing multiple immune factors in larger LTNP cohort with HIV-1 infected individuals at various levels of disease progression. A possible role of HIV-1 subtype variation and ethnic differences in addition to host-genetic and viral factors cannot be ruled out.

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