Multiplexed tat-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape

HIV-1 cure strategy by means of proviral knock-out using CRISPR-Cas9 has been hampered by the emergence of viral resistance against the targeting guide RNA (gRNA). Here, we proposed multiple, concentrated gRNA attacks against HIV-1 regulatory genes to block viral escape. The T cell line were transduced with single and multiple gRNAs targeting HIV-1 tat and rev using lentiviral-based CRISPR-Cas9, followed by replicative HIV-1NL4-3 challenge in vitro. Viral p24 rebound was observed for almost all gRNAs, but multiplexing three tat-targeting gRNAs maintained p24 suppression and cell viability, indicating the inhibition of viral escape. Multiplexed tat gRNAs inhibited acute viral replication in the 2nd round of infection, abolished cell-associated transmission to unprotected T cells, and maintained protection through 45 days, post-infection (dpi) after a higher dose of HIV-1 infection. Finally, we describe here for the first time the assembly of all-in-one lentiviral vectors containing three and six gRNAs targeting tat and rev. A single-vector tat-targeting construct shows non-inferiority to the tat-targeting multi-vector in low-dose HIV-1 infection. We conclude that Cas9-induced, DNA repair-mediated mutations in tat are sufficiently deleterious and deplete HIV-1 fitness, and multiplexed disruption of tat further limits the possibility of an escape mutant arising, thus elevating the potential of CRISPR-Cas9 to achieve a long-term HIV-1 cure.

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Preferential and persistent impact of acute HIV-1 infection on CD4+ iNKT cells in colonic mucosa

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2104721118 ◽

2021 ◽

Vol 118 (46) ◽

pp. e2104721118

Author(s):

Dominic Paquin-Proulx ◽

Kerri G. Lal ◽

Yuwadee Phuang-Ngern ◽

Matthew Creegan ◽

Andrey Tokarev ◽

...

Keyword(s):

T Cells ◽

Peripheral Blood ◽

Colonic Mucosa ◽

Inkt Cells ◽

Elevated Expression ◽

Acute Hiv ◽

The Impact ◽

Hiv 1

Acute HIV-1 infection (AHI) results in the widespread depletion of CD4+ T cells in peripheral blood and gut mucosal tissue. However, the impact on the predominantly CD4+ immunoregulatory invariant natural killer T (iNKT) cells during AHI remains unknown. Here, iNKT cells from peripheral blood and colonic mucosa were investigated during treated and untreated AHI. iNKT cells in blood were activated and rapidly depleted in untreated AHI. At the time of peak HIV-1 viral load, these cells showed the elevated expression of cell death–associated transcripts compared to preinfection. Residual peripheral iNKT cells suffered a diminished responsiveness to in vitro stimulation early into chronic infection. Additionally, HIV-1 DNA, as well as spliced and unspliced viral RNA, were detected in iNKT cells isolated from blood, indicating the active infection of these cells in vivo. The loss of iNKT cells occurred from Fiebig stage III in the colonic mucosa, and these cells were not restored to normal levels after initiation of ART during AHI. CD4+ iNKT cells were depleted faster and more profoundly than conventional CD4+ T cells, and the preferential infection of CD4+ iNKT cells over conventional CD4+ T cells was confirmed by in vitro infection experiments. In vitro data also provided evidence of latent infection in iNKT cells. Strikingly, preinfection levels of peripheral blood CD4+ iNKT cells correlated directly with the peak HIV-1 load. These findings support a model in which iNKT cells are early targets for HIV-1 infection, driving their rapid loss from circulation and colonic mucosa.

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Role of Escape Mutant-Specific T Cells in Suppression of HIV-1 Replication and Coevolution with HIV-1

Journal of Virology ◽

10.1128/jvi.01151-20 ◽

2020 ◽

Vol 94 (19) ◽

Author(s):

Yu Zhang ◽

Nozomi Kuse ◽

Tomohiro Akahoshi ◽

Takayuki Chikata ◽

Hiroyuki Gatanaga ◽

...

Keyword(s):

T Cells ◽

Wild Type Virus ◽

Escape Mutant ◽

Type Virus ◽

Wild Type ◽

B 52 ◽

Hiv 1

ABSTRACT The accumulation of HIV-1 escape mutations affects HIV-1 control by HIV-1-specific T cells. Some of these mutations can elicit escape mutant-specific T cells, but it still remains unclear whether they can suppress the replication of HIV-1 mutants. It is known that HLA-B*52:01-restricted RI8 (Gag 275 to 282; RMYSPTSI) is a protective T cell epitope in HIV-1 subtype B-infected Japanese individuals, though 3 Gag280A/S/V mutations are found in 26% of them. Gag280S and Gag280A were HLA-B*52:01-associated mutations, whereas Gag280V was not, implying a different mechanism for the accumulation of Gag280 mutations. In this study, we investigated the coevolution of HIV-1 with RI8-specific T cells and suppression of HIV-1 replication by its escape mutant-specific T cells both in vitro and in vivo. HLA-B*52:01+ individuals infected with Gag280A/S mutant viruses failed to elicit these mutant epitope-specific T cells, whereas those with the Gag280V mutant one effectively elicited RI8-6V mutant-specific T cells. These RI8-6V-specific T cells suppressed the replication of Gag280V virus and selected wild-type virus, suggesting a mechanism affording no accumulation of the Gag280V mutation in the HLA-B*52:01+ individuals. The responders to wild-type (RI8-6T) and RI8-6V mutant peptides had significantly higher CD4 counts than nonresponders, indicating that the existence of not only RI8-6T-specific T cells but also RI8-6V-specific ones was associated with a good clinical outcome. The present study clarified the role of escape mutant-specific T cells in HIV-1 evolution and in the control of HIV-1. IMPORTANCE Escape mutant-specific CD8+ T cells were elicited in some individuals infected with escape mutants, but it is still unknown whether these CD8+ T cells can suppress HIV-1 replication. We clarified that Gag280V mutation were selected by HLA-B*52:01-restricted CD8+ T cells specific for the GagRI8 protective epitope, whereas the Gag280V virus could frequently elicit GagRI8-6V mutant-specific CD8+ T cells. GagRI8-6V mutant-specific T cells had a strong ability to suppress the replication of the Gag280V mutant virus both in vitro and in vivo. In addition, these T cells contributed to the selection of wild-type virus in HLA-B*52:01+ Japanese individuals. We for the first time demonstrated that escape mutant-specific CD8+ T cells can suppress HIV-1 replication and play an important role in the coevolution with HIV-1. Thus, the present study highlighted an important role of escape mutant-specific T cells in the control of HIV-1 and coevolution with HIV-1.

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In Vitro Priming Recapitulates In Vivo HIV-1 Specific T Cell Responses, Revealing Rapid Loss of Virus Reactive CD4+ T Cells in Acute HIV-1 Infection

PLoS ONE ◽

10.1371/journal.pone.0004256 ◽

2009 ◽

Vol 4 (1) ◽

pp. e4256 ◽

Cited By ~ 31

Author(s):

Rachel Lubong Sabado ◽

Daniel G. Kavanagh ◽

Daniel E. Kaufmann ◽

Karlhans Fru ◽

Ethan Babcock ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

T Cell Responses ◽

Rapid Loss ◽

Cell Responses ◽

Acute Hiv ◽

Hiv 1

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Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.593077 ◽

2021 ◽

Vol 11 ◽

Author(s):

Cheng-Han Chung ◽

Alexander G. Allen ◽

Andrew Atkins ◽

Robert W. Link ◽

Michael R. Nonnemacher ◽

...

Keyword(s):

Computational Design ◽

The Other ◽

Viral Escape ◽

Sequence Variants ◽

Guide Rna ◽

Functional Regions ◽

Computational Evaluation ◽

Hiv 1

Clustered regularly interspaced short palindromic repeats (CRISPR)-based HIV-1 genome editing has shown promising outcomes in in vitro and in vivo viral infection models. However, existing HIV-1 sequence variants have been shown to reduce CRISPR-mediated efficiency and induce viral escape. Two metrics, global patient coverage and global subtype coverage, were used to identify guide RNA (gRNA) sequences that account for this viral diversity from the perspectives of cross-patient and cross-subtype gRNA design, respectively. Computational evaluation using these parameters and over 3.6 million possible 20-bp sequences resulted in nine lead gRNAs, two of which were previously published. This analysis revealed the benefit and necessity of considering all sequence variants for gRNA design. Of the other seven identified novel gRNAs, two were of note as they targeted interesting functional regions. One was a gRNA predicted to induce structural disruption in the nucleocapsid binding site (Ψ), which holds the potential to stop HIV-1 replication during the viral genome packaging process. The other was a reverse transcriptase (RT)-targeting gRNA that was predicted to cleave the subdomain responsible for dNTP incorporation. CRISPR-mediated sequence edits were predicted to occur on critical residues where HIV-1 has been shown to develop resistance against antiretroviral therapy (ART), which may provide additional evolutionary pressure at the DNA level. Given these observations, consideration of broad-spectrum gRNAs and cross-subtype diversity for gRNA design is not only required for the development of generalizable CRISPR-based HIV-1 therapy, but also helps identify optimal target sites.

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HIV-1 Employs Multiple Mechanisms To Resist Cas9/Single Guide RNA Targeting the Viral Primer Binding Site

Journal of Virology ◽

10.1128/jvi.01135-18 ◽

2018 ◽

Vol 92 (20) ◽

Cited By ~ 11

Author(s):

Zhen Wang ◽

Wenzhou Wang ◽

Ya Cheng Cui ◽

Qinghua Pan ◽

Weijun Zhu ◽

...

Keyword(s):

Binding Site ◽

Reverse Transcription ◽

Gene Editing ◽

Viral Escape ◽

Primer Binding Site ◽

Minus Strand ◽

Guide Rna ◽

Target Dna ◽

Almost All ◽

Hiv 1

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing technology has been used to inactivate viral DNA as a new strategy to eliminate chronic viral infections, including HIV-1. This utility of CRISPR-Cas9 is challenged by the high heterogeneity of HIV-1 sequences, which requires the design of the single guide RNA (sgRNA; utilized by the CRISPR-Cas9 system to recognize the target DNA) to match a specific HIV-1 strain in an HIV patient. One solution to this challenge is to target the viral primer binding site (PBS), which HIV-1 copies from cellular tRNA3Lysin each round of reverse transcription and is thus conserved in almost all HIV-1 strains. In this study, we demonstrate that PBS-targeting sgRNA directs Cas9 to cleave the PBS DNA, which evokes deletions or insertions (indels) and strongly diminishes the production of infectious HIV-1. While HIV-1 escapes from PBS-targeting Cas9/sgRNA, unique resistance mechanisms are observed that are dependent on whether the plus or the minus strand of the PBS DNA is bound by sgRNA. Characterization of these viral escape mechanisms will inform future engineering of Cas9 variants that can more potently and persistently inhibit HIV-1 infection.IMPORTANCEThe results of this study demonstrate that the gene-editing complex Cas9/sgRNA can be programmed to target and cleave HIV-1 PBS DNA, and thus, inhibit HIV-1 infection. Given that almost all HIV-1 strains have the same PBS, which is copied from the cellular tRNA3Lysduring reverse transcription, PBS-targeting sgRNA can be used to inactivate HIV-1 DNA of different strains. We also discovered that HIV-1 uses different mechanisms to resist Cas9/sgRNAs, depending on whether they target the plus or the minus strand of PBS DNA. These findings allow us to predict that a Cas9 variant that uses the CCA sequence as the protospacer adjacent motif (PAM) should more strongly and persistently suppress HIV-1 replication. Together, these data have identified the PBS as the target DNA of Cas9/sgRNA and have predicted how to improve Cas9/sgRNA to achieve more efficient and sustainable suppression of HIV-1 infection, therefore improving the capacity of Cas9/sgRNA in curing HIV-1 infection.

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Mutational analysis of human immunodeficiency virus type 1 (HIV-1) accessory genes: requirement of a site in the nef gene for HIV-1 replication in activated CD4+ T cells in vitro and in vivo.

Journal of Virology ◽

10.1128/jvi.71.11.8456-8466.1997 ◽

1997 ◽

Vol 71 (11) ◽

pp. 8456-8466 ◽

Cited By ~ 36

Author(s):

Y Kawano ◽

Y Tanaka ◽

N Misawa ◽

R Tanaka ◽

J I Kira ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Human Immunodeficiency Virus Type ◽

Mutational Analysis ◽

Immunodeficiency Virus ◽

A Site ◽

Hiv 1

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G2-S16 Polyanionic Carbosilane Dendrimer Can Reduce HIV-1 Reservoir Formation by Inhibiting Macrophage Cell to Cell Transmission

International Journal of Molecular Sciences ◽

10.3390/ijms22168366 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8366

Author(s):

Ignacio Relaño-Rodríguez ◽

María de la Sierra Espinar-Buitrago ◽

Vanessa Martín-Cañadilla ◽

Rafael Gómez-Ramírez ◽

María Ángeles Muñoz-Fernández

Keyword(s):

T Cells ◽

Developed Countries ◽

Main Role ◽

Viral Particles ◽

Carbosilane Dendrimer ◽

Science Community ◽

New Compounds ◽

Hiv 1

Human immunodeficiency virus (HIV-1) is still a major problem, not only in developing countries but is also re-emerging in several developed countries, thus the development of new compounds able to inhibit the virus, either for prophylaxis or treatment, is still needed. Nanotechnology has provided the science community with several new tools for biomedical applications. G2-S16 is a polyanionic carbosilane dendrimer capable of inhibiting HIV-1 in vitro and in vivo by interacting directly with viral particles. One of the main barriers for HIV-1 eradication is the reservoirs created in primoinfection. These reservoirs, mainly in T cells, are untargetable by actual drugs or immune system. Thus, one approach is inhibiting HIV-1 from reaching these reservoir cells. In this context, macrophages play a main role as they can deliver viral particles to T cells establishing reservoirs. We showed that G2-S16 dendrimer is capable of inhibiting the infection from infected macrophages to healthy T CD4/CD8 lymphocytes by eliminating HIV-1 infectivity inside macrophages, so they are not able to carry infectious particles to other body locations, thus preventing the reservoirs from forming.

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CD73+ CD127high Long-Term Memory CD4 T Cells Are Highly Proliferative in Response to Recall Antigens and Are Early Targets in HIV-1 Infection

International Journal of Molecular Sciences ◽

10.3390/ijms22020912 ◽

2021 ◽

Vol 22 (2) ◽

pp. 912

Author(s):

Nabila Seddiki ◽

John Zaunders ◽

Chan Phetsouphanh ◽

Vedran Brezar ◽

Yin Xu ◽

...

Keyword(s):

T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Significant Proportion ◽

Long Term Memory ◽

Ki 67 ◽

Memory Cd4 T Cells ◽

Hiv 1

HIV-1 infection rapidly leads to a loss of the proliferative response of memory CD4+ T lymphocytes, when cultured with recall antigens. We report here that CD73 expression defines a subset of resting memory CD4+ T cells in peripheral blood, which highly express the α-chain of the IL-7 receptor (CD127), but not CD38 or Ki-67, yet are highly proliferative in response to mitogen and recall antigens, and to IL-7, in vitro. These cells also preferentially express CCR5 and produce IL-2. We reasoned that CD73+ memory CD4+ T cells decrease very early in HIV-1 infection. Indeed, CD73+ memory CD4+ T cells comprised a median of 7.5% (interquartile range: 4.5–10.4%) of CD4+ T cells in peripheral blood from healthy adults, but were decreased in primary HIV-1 infection to a median of 3.7% (IQR: 2.6–6.4%; p = 0.002); and in chronic HIV-1 infection to 1.9% (IQR: 1.1–3%; p < 0.0001), and were not restored by antiretroviral therapy. Moreover, we found that a significant proportion of CD73+ memory CD4+ T cells were skewed to a gut-homing phenotype, expressing integrins α4 and β7, CXCR3, CCR6, CD161 and CD26. Accordingly, 20% of CD4+ T cells present in gut biopsies were CD73+. In HIV+ subjects, purified CD73+ resting memory CD4+ T cells in PBMC were infected with HIV-1 DNA, determined by real-time PCR, to the same level as for purified CD73-negative CD4+ T cells, both in untreated and treated subjects. Therefore, the proliferative CD73+ subset of memory CD4+ T cells is disproportionately reduced in HIV-1 infection, but, unexpectedly, their IL-7 dependent long-term resting phenotype suggests that residual infected cells in this subset may contribute significantly to the very long-lived HIV proviral DNA reservoir in treated subjects.

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Phenotypic Susceptibility to Bevirimat in Isolates from HIV-1-Infected Patients without Prior Exposure to Bevirimat

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01784-09 ◽

2010 ◽

Vol 54 (6) ◽

pp. 2345-2353 ◽

Cited By ~ 39

Author(s):

Nicolas A. Margot ◽

Craig S. Gibbs ◽

Michael D. Miller

Keyword(s):

Recombinant Viruses ◽

Gag Polyprotein ◽

New Class ◽

Major Mutation ◽

Hiv Drugs ◽

The Impact ◽

First Time ◽

Hiv 1

ABSTRACT Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.

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