34 results on '"Neidleman J"'
Search Results
2. Novel anionic microparticles are a potent adjuvant for the induction of cytotoxic T lymphocytes against recombinant p55 gag from HIV-1
- Author
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Kazzaz, J, Neidleman, J, Singh, M, Ott, G, and O’Hagan, D.T
- Published
- 2000
- Full Text
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3. Long COVID manifests with T cell dysregulation, inflammation and an uncoordinated adaptive immune response to SARS-CoV-2.
- Author
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Yin K, Peluso MJ, Luo X, Thomas R, Shin MG, Neidleman J, Andrew A, Young KC, Ma T, Hoh R, Anglin K, Huang B, Argueta U, Lopez M, Valdivieso D, Asare K, Deveau TM, Munter SE, Ibrahim R, Ständker L, Lu S, Goldberg SA, Lee SA, Lynch KL, Kelly JD, Martin JN, Münch J, Deeks SG, Henrich TJ, and Roan NR
- Subjects
- Female, Male, Humans, Post-Acute COVID-19 Syndrome, CD8-Positive T-Lymphocytes, Immunity, Humoral, Antibodies, Viral, Inflammation, SARS-CoV-2, COVID-19
- Abstract
Long COVID (LC) occurs after at least 10% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, yet its etiology remains poorly understood. We used 'omic" assays and serology to deeply characterize the global and SARS-CoV-2-specific immunity in the blood of individuals with clear LC and non-LC clinical trajectories, 8 months postinfection. We found that LC individuals exhibited systemic inflammation and immune dysregulation. This was evidenced by global differences in T cell subset distribution implying ongoing immune responses, as well as by sex-specific perturbations in cytolytic subsets. LC individuals displayed increased frequencies of CD4
+ T cells poised to migrate to inflamed tissues and exhausted SARS-CoV-2-specific CD8+ T cells, higher levels of SARS-CoV-2 antibodies and a mis-coordination between their SARS-CoV-2-specific T and B cell responses. Our analysis suggested an improper crosstalk between the cellular and humoral adaptive immunity in LC, which can lead to immune dysregulation, inflammation and clinical symptoms associated with this debilitating condition., (© 2024. The Author(s).)- Published
- 2024
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4. Long COVID manifests with T cell dysregulation, inflammation, and an uncoordinated adaptive immune response to SARS-CoV-2.
- Author
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Yin K, Peluso MJ, Luo X, Thomas R, Shin MG, Neidleman J, Andrew A, Young K, Ma T, Hoh R, Anglin K, Huang B, Argueta U, Lopez M, Valdivieso D, Asare K, Deveau TM, Munter SE, Ibrahim R, Ständker L, Lu S, Goldberg SA, Lee SA, Lynch KL, Kelly JD, Martin JN, Münch J, Deeks SG, Henrich TJ, and Roan NR
- Abstract
Long COVID (LC), a type of post-acute sequelae of SARS-CoV-2 infection (PASC), occurs after at least 10% of SARS-CoV-2 infections, yet its etiology remains poorly understood. Here, we used multiple "omics" assays (CyTOF, RNAseq/scRNAseq, Olink) and serology to deeply characterize both global and SARS-CoV-2-specific immunity from blood of individuals with clear LC and non-LC clinical trajectories, 8 months following infection and prior to receipt of any SARS-CoV-2 vaccine. Our analysis focused on deep phenotyping of T cells, which play important roles in immunity against SARS-CoV-2 yet may also contribute to COVID-19 pathogenesis. Our findings demonstrate that individuals with LC exhibit systemic inflammation and immune dysregulation. This is evidenced by global differences in T cell subset distribution in ways that imply ongoing immune responses, as well as by sex-specific perturbations in cytolytic subsets. Individuals with LC harbored increased frequencies of CD4+ T cells poised to migrate to inflamed tissues, and exhausted SARS-CoV-2-specific CD8+ T cells. They also harbored significantly higher levels of SARS-CoV-2 antibodies, and in contrast to non-LC individuals, exhibited a mis-coordination between their SARS-CoV-2-specific T and B cell responses. RNAseq/scRNAseq and Olink analyses similarly revealed immune dysregulatory mechanisms, along with non-immune associated perturbations, in individuals with LC. Collectively, our data suggest that proper crosstalk between the humoral and cellular arms of adaptive immunity has broken down in LC, and that this, perhaps in the context of persistent virus, leads to the immune dysregulation, inflammation, and clinical symptoms associated with this debilitating condition., Competing Interests: CONFLICTS OF INTERESTS MJP reports consulting fees from Gilead Sciences and AstraZeneca, outside the submitted work. SGD reports grants and/or personal fees from Gilead Sciences, Merck & Co., Viiv, AbbVie, Eli Lilly, ByroLogyx, and Enochian Biosciences, outside the submitted work. TJH receives grant support from Merck and consults for Roche. All other authors report no potential conflicts.
- Published
- 2023
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5. Female Genital Fibroblasts Diminish the In Vitro Efficacy of PrEP against HIV.
- Author
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George AF, McGregor M, Gingrich D, Neidleman J, Marquez RS, Young KC, Thanigaivelan KL, Greene WC, Tien PC, Deitchman AN, Spitzer TL, and Roan NR
- Subjects
- Female, Fibroblasts, Homosexuality, Male, Humans, Male, Vagina, Anti-HIV Agents pharmacology, Anti-HIV Agents therapeutic use, HIV Infections drug therapy, HIV Infections prevention & control, Sexual and Gender Minorities
- Abstract
The efficacy of HIV pre-exposure prophylaxis (PrEP) is high in men who have sex with men, but much more variable in women, in a manner largely attributed to low adherence. This reduced efficacy, however, could also reflect biological factors. Transmission to women is typically via the female reproductive tract (FRT), and vaginal dysbiosis, genital inflammation, and other factors specific to the FRT mucosa can all increase transmission risk. We have demonstrated that mucosal fibroblasts from the lower and upper FRT can markedly enhance HIV infection of CD4+ T cells. Given the current testing of tenofovir disoproxil fumarate, cabotegravir, and dapivirine regimens as candidate PrEP agents for women, we set out to determine using in vitro assays whether endometrial stromal fibroblasts (eSF) isolated from the FRT can affect the anti-HIV activity of these PrEP drugs. We found that PrEP drugs exhibit significantly reduced antiviral efficacy in the presence of eSFs, not because of decreased PrEP drug availability, but rather of eSF-mediated enhancement of HIV infection. These findings suggest that drug combinations that target both the virus and infection-promoting factors in the FRT-such as mucosal fibroblasts-may be more effective than PrEP alone at preventing sexual transmission of HIV to women.
- Published
- 2022
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6. Subsets of Tissue CD4 T Cells Display Different Susceptibilities to HIV Infection and Death: Analysis by CyTOF and Single Cell RNA-seq.
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Luo X, Frouard J, Zhang G, Neidleman J, Xie G, Sheedy E, Roan NR, and Greene WC
- Subjects
- CD4-Positive T-Lymphocytes, Humans, RNA-Seq, T-Lymphocyte Subsets, HIV Infections, HIV-1 physiology
- Abstract
CD4 T lymphocytes belong to diverse cellular subsets whose sensitivity or resistance to HIV-associated killing remains to be defined. Working with lymphoid cells from human tonsils, we characterized the HIV-associated depletion of various CD4 T cell subsets using mass cytometry and single-cell RNA-seq. CD4 T cell subsets preferentially killed by HIV are phenotypically distinct from those resistant to HIV-associated cell death, in a manner not fully accounted for by their susceptibility to productive infection. Preferentially-killed subsets express CXCR5 and CXCR4 while preferentially-infected subsets exhibit an activated and exhausted effector memory cell phenotype. Single-cell RNA-seq analysis reveals that the subsets of preferentially-killed cells express genes favoring abortive infection and pyroptosis. These studies emphasize a complex interplay between HIV and distinct tissue-based CD4 T cell subsets, and the important contribution of abortive infection and inflammatory programmed cell death to the overall depletion of CD4 T cells that accompanies untreated HIV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Luo, Frouard, Zhang, Neidleman, Xie, Sheedy, Roan and Greene.)
- Published
- 2022
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7. Deep Phenotypic Analysis of Blood and Lymphoid T and NK Cells From HIV+ Controllers and ART-Suppressed Individuals.
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George AF, Luo X, Neidleman J, Hoh R, Vohra P, Thomas R, Shin MG, Lee MJ, Blish CA, Deeks SG, Greene WC, Lee SA, and Roan NR
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- Adult, Aged, Anti-Retroviral Agents therapeutic use, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Female, HIV Infections drug therapy, HIV-1 immunology, Humans, Killer Cells, Natural immunology, Leukocytes immunology, Lymphocytes classification, Male, Middle Aged, HIV Infections immunology, Leukocytes classification, Lymphocytes immunology, Phenotype, Sustained Virologic Response
- Abstract
T and natural killer (NK) cells are effector cells with key roles in anti-HIV immunity, including in lymphoid tissues, the major site of HIV persistence. However, little is known about the features of these effector cells from people living with HIV (PLWH), particularly from those who initiated antiretroviral therapy (ART) during acute infection. Our study design was to use 42-parameter CyTOF to conduct deep phenotyping of paired blood- and lymph node (LN)-derived T and NK cells from three groups of HIV+ aviremic individuals: elite controllers (N = 5), and ART-suppressed individuals who had started therapy during chronic (N = 6) vs. acute infection (N = 8), the latter of which is associated with better outcomes. We found that acute-treated individuals are enriched for specific subsets of T and NK cells, including blood-derived CD56-CD16+ NK cells previously associated with HIV control, and LN-derived CD4+ T follicular helper cells with heightened expansion potential. An in-depth comparison of the features of the cells from blood vs. LNs of individuals from our cohort revealed that T cells from blood were more activated than those from LNs. By contrast, LNs were enriched for follicle-homing CXCR5+ CD8+ T cells, which expressed increased levels of inhibitory receptors and markers of survival and proliferation as compared to their CXCR5- counterparts. In addition, a subset of memory-like CD56
bright TCF1+ NK cells was enriched in LNs relative to blood. These results together suggest unique T and NK cell features in acute-treated individuals, and highlight the importance of examining effector cells not only in blood but also the lymphoid tissue compartment, where the reservoir mostly persists, and where these cells take on distinct phenotypic features., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 George, Luo, Neidleman, Hoh, Vohra, Thomas, Shin, Lee, Blish, Deeks, Greene, Lee and Roan.)- Published
- 2022
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8. mRNA vaccine-induced T cells respond identically to SARS-CoV-2 variants of concern but differ in longevity and homing properties depending on prior infection status.
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Neidleman J, Luo X, McGregor M, Xie G, Murray V, Greene WC, Lee SA, and Roan NR
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- Adult, Aged, COVID-19 prevention & control, COVID-19 virology, Female, Humans, Immunization, Secondary, Longitudinal Studies, Male, Middle Aged, Spike Glycoprotein, Coronavirus immunology, Vaccination, Young Adult, mRNA Vaccines, COVID-19 immunology, COVID-19 Vaccines pharmacology, SARS-CoV-2 immunology, T-Lymphocytes drug effects, Vaccines, Synthetic pharmacology
- Abstract
While mRNA vaccines are proving highly efficacious against SARS-CoV-2, it is important to determine how booster doses and prior infection influence the immune defense they elicit, and whether they protect against variants. Focusing on the T cell response, we conducted a longitudinal study of infection-naïve and COVID-19 convalescent donors before vaccination and after their first and second vaccine doses, using a high-parameter CyTOF analysis to phenotype their SARS-CoV-2-specific T cells. Vaccine-elicited spike-specific T cells responded similarly to stimulation by spike epitopes from the ancestral, B.1.1.7 and B.1.351 variant strains, both in terms of cell numbers and phenotypes. In infection-naïve individuals, the second dose boosted the quantity and altered the phenotypic properties of SARS-CoV-2-specific T cells, while in convalescents the second dose changed neither. Spike-specific T cells from convalescent vaccinees differed strikingly from those of infection-naïve vaccinees, with phenotypic features suggesting superior long-term persistence and ability to home to the respiratory tract including the nasopharynx. These results provide reassurance that vaccine-elicited T cells respond robustly to emerging viral variants, confirm that convalescents may not need a second vaccine dose, and suggest that vaccinated convalescents may have more persistent nasopharynx-homing SARS-CoV-2-specific T cells compared to their infection-naïve counterparts., Competing Interests: JN, XL, MM, GX, VM, WG, SL, NR No competing interests declared, (© 2021, Neidleman et al.)
- Published
- 2021
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9. Protracted yet Coordinated Differentiation of Long-Lived SARS-CoV-2-Specific CD8 + T Cells during Convalescence.
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Ma T, Ryu H, McGregor M, Babcock B, Neidleman J, Xie G, George AF, Frouard J, Murray V, Gill G, Ghosn E, Newell EW, Lee SA, and Roan NR
- Subjects
- CD8-Positive T-Lymphocytes pathology, Humans, Longitudinal Studies, CD8-Positive T-Lymphocytes immunology, COVID-19 immunology, Convalescence, SARS-CoV-2 immunology
- Abstract
CD8
+ T cells can potentiate long-lived immunity against COVID-19. We screened longitudinally-sampled convalescent human donors against SARS-CoV-2 tetramers and identified a participant with an immunodominant response against residues 322 to 311 of nucleocapsid (Nuc322-331 ), a peptide conserved in all variants of concern reported to date. We conducted 38-parameter cytometry by time of flight on tetramer-identified Nuc322-331 -specific CD8+ T cells and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins, and took 32 serological measurements. We discovered a coordination of the Nuc322-331 -specific CD8+ T response with both the CD4+ T cell and Ab pillars of adaptive immunity. Over the approximately six month period of convalescence monitored, we observed a slow and progressive decrease in the activation state and polyfunctionality of Nuc322-331 -specific CD8+ T cells, accompanied by an increase in their lymph node-homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence into a state characteristic of long-lived, self-renewing memory., (Copyright © 2021 by The American Association of Immunologists, Inc.)- Published
- 2021
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10. Hyaluronic acid is a negative regulator of mucosal fibroblast-mediated enhancement of HIV infection.
- Author
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Egedal JH, Xie G, Packard TA, Laustsen A, Neidleman J, Georgiou K, Pillai SK, Greene WC, Jakobsen MR, and Roan NR
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- Biomarkers, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes metabolism, CD4-Positive T-Lymphocytes virology, Disease Susceptibility immunology, Extracellular Matrix metabolism, Fibroblasts drug effects, Gene Expression Regulation, Gene Knockdown Techniques, HIV Infections transmission, HIV-1, Host-Pathogen Interactions genetics, Host-Pathogen Interactions immunology, Humans, Hyaluronan Synthases genetics, Hyaluronic Acid pharmacology, Mucous Membrane virology, Fibroblasts metabolism, HIV Infections immunology, HIV Infections metabolism, HIV Infections virology, Hyaluronic Acid metabolism, Mucous Membrane immunology, Mucous Membrane metabolism
- Abstract
The majority of HIV infections are established through the genital or rectal mucosa. Fibroblasts are abundant in these tissues, and although not susceptible to infection, can potently enhance HIV infection of CD4+ T cells. Hyaluronic acid (HA) is a major component of the extracellular matrix of fibroblasts, and its levels are influenced by the inflammatory state of the tissue. Since inflammation is known to facilitate HIV sexual transmission, we investigated the role of HA in genital mucosal fibroblast-mediated enhancement of HIV infection. Depletion of HA by CRISPR-Cas9 in primary foreskin fibroblasts augmented the ability of the fibroblasts to increase HIV infection of CD4+ T cells. This amplified enhancement required direct contact between the fibroblasts and CD4+ T cells, and could be attributed to both increased rates of trans-infection and the increased ability of HA-deficient fibroblasts to push CD4+ T cells into a state of higher permissivity to infection. This HIV-permissive state was characterized by differential expression of genes associated with regulation of cell metabolism and death. Our results suggest that conditions resulting in diminished cell-surface HA on fibroblasts, such as genital inflammation, can promote HIV transmission by conditioning CD4+ T cells toward a state more vulnerable to infection by HIV., (© 2021. The Author(s).)
- Published
- 2021
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11. Distinctive features of SARS-CoV-2-specific T cells predict recovery from severe COVID-19.
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Neidleman J, Luo X, George AF, McGregor M, Yang J, Yun C, Murray V, Gill G, Greene WC, Vasquez J, Lee SA, Ghosn E, Lynch KL, and Roan NR
- Abstract
Although T cells are likely players in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity, little is known about the phenotypic features of SARS-CoV-2-specific T cells associated with recovery from severe coronavirus disease 2019 (COVID-19). We analyze T cells from 34 individuals with COVID-19 with severity ranging from mild (outpatient) to critical, culminating in death. Relative to individuals who succumbed, individuals who recovered from severe COVID-19 harbor elevated and increasing numbers of SARS-CoV-2-specific T cells capable of homeostatic proliferation. In contrast, fatal COVID-19 cases display elevated numbers of SARS-CoV-2-specific regulatory T cells and a time-dependent escalation in activated bystander CXCR4
+ T cells, as assessed by longitudinal sampling. Together with the demonstration of increased proportions of inflammatory CXCR4+ T cells in the lungs of individuals with severe COVID-19, these results support a model where lung-homing T cells activated through bystander effects contribute to immunopathology, whereas a robust, non-suppressive SARS-CoV-2-specific T cell response limits pathogenesis and promotes recovery from severe COVID-19., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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12. Protracted yet coordinated differentiation of long-lived SARS-CoV-2-specific CD8+ T cells during COVID-19 convalescence.
- Author
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Ma T, Ryu H, McGregor M, Babcock B, Neidleman J, Xie G, George AF, Frouard J, Murray V, Gill G, Ghosn E, Newell E, Lee S, and Roan NR
- Abstract
CD8+ T cells are important antiviral effectors that can potentiate long-lived immunity against COVID-19, but a detailed characterization of these cells has been hampered by technical challenges. We screened 21 well-characterized, longitudinally-sampled convalescent donors that recovered from mild COVID-19 against a collection of SARS-CoV-2 tetramers, and identified one participant with an immunodominant response against Nuc
322-331 , a peptide that is conserved in all the SARS-CoV-2 variants-of-concern reported to date. We conducted 38-parameter CyTOF phenotyping on tetramer-identified Nuc322-331 -specific CD8+ T cells, and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins from SARS-CoV-2, and took 32 serological measurements on longitudinal specimens from this participant. We discovered a coordination of the Nuc322-331 -specific CD8+ T response with both the CD4+ T cell and antibody pillars of adaptive immunity. Nuc322-331 -specific CD8+ T cells were predominantly central memory T cells, but continually evolved over a ~6-month period of convalescence. We observed a slow and progressive decrease in the activation state and polyfunctionality of the Nuc322-331 -specific CD8+ T cells, accompanied by an increase in their lymph-node homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence, into a state characteristic of long-lived, self-renewing memory., Competing Interests: COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.- Published
- 2021
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13. Characterization of HIV-induced remodeling reveals differences in infection susceptibility of memory CD4 + T cell subsets in vivo.
- Author
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Xie G, Luo X, Ma T, Frouard J, Neidleman J, Hoh R, Deeks SG, Greene WC, and Roan NR
- Subjects
- Humans, CD4-Positive T-Lymphocytes metabolism, HIV-1 genetics, Immunologic Memory genetics, T-Lymphocyte Subsets metabolism
- Abstract
Relatively little is known about features of T cells targeted by HIV in vivo. By applying bioinformatics analysis to mass cytometry (CyTOF)-phenotyped specimens from individuals with viremia and in-vitro-infected cells from uninfected donors, we provide an atlas of the phenotypes of in vivo and in vitro HIV-susceptible cells. T helper 17 (Th17) and α4β1
+ cells are preferentially targeted in vivo, whereas T effector memory (Tem), T transitional memory (Ttm), Th1, and Th1/Th17 subsets are targeted in vitro. Multiple proteins-including chemokine and cytokine receptors-are remodeled by HIV in vivo, and these changes are mostly recapitulated in vitro. HIV remodels cells to a T follicular helper (Tfh) phenotype. Using clustering, we uncover a subset of CD29-expressing, Tem-like cells that are highly susceptible to infection in vivo and in vitro and experimentally confirm that susceptibility. These studies provide an in-depth look at features of HIV-susceptible cells in individuals with viremia and demonstrate that some-but not all-HIV-susceptible cells identified in vitro effectively model in vivo susceptibility., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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14. Phenotypic analysis of the unstimulated in vivo HIV CD4 T cell reservoir.
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Neidleman J, Luo X, Frouard J, Xie G, Hsiao F, Ma T, Morcilla V, Lee A, Telwatte S, Thomas R, Tamaki W, Wheeler B, Hoh R, Somsouk M, Vohra P, Milush J, James KS, Archin NM, Hunt PW, Deeks SG, Yukl SA, Palmer S, Greene WC, and Roan NR
- Subjects
- Cell Separation, Humans, Immunophenotyping, Mass Spectrometry, Proviruses, CD4-Positive T-Lymphocytes cytology, CD4-Positive T-Lymphocytes virology, HIV Infections immunology, HIV Infections virology, HIV-1 immunology, HIV-1 pathogenicity, Virus Latency immunology
- Abstract
The latent reservoir is a major barrier to HIV cure. As latently infected cells cannot be phenotyped directly, the features of the in vivo reservoir have remained elusive. Here, we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently infected cells reactivated ex vivo to their original pre-activation states. Our results suggest that, contrary to common assumptions, the reservoir is not randomly distributed among cell subsets, and is remarkably conserved between individuals. However, reservoir composition differs between tissues and blood, as do cells successfully reactivated by different latency reversing agents. By selecting 8-10 of our 39 original CyTOF markers, we were able to isolate highly purified populations of unstimulated in vivo latent cells. These purified populations were highly enriched for replication-competent and intact provirus, transcribed HIV, and displayed clonal expansion. The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies on HIV persistence., Competing Interests: JN, XL, JF, GX, FH, TM, VM, AL, ST, RT, WT, BW, RH, MS, PV, JM, KJ, NA, PH, SD, SY, SP, WG, NR No competing interests declared, (© 2020, Neidleman et al.)
- Published
- 2020
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15. SARS-CoV-2-Specific T Cells Exhibit Phenotypic Features of Helper Function, Lack of Terminal Differentiation, and High Proliferation Potential.
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Neidleman J, Luo X, Frouard J, Xie G, Gill G, Stein ES, McGregor M, Ma T, George AF, Kosters A, Greene WC, Vasquez J, Ghosn E, Lee S, and Roan NR
- Abstract
Convalescing coronavirus disease 2019 (COVID-19) patients mount robust T cell responses against SARS-CoV-2, suggesting an important role of T cells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific T cells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ T cells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ T cells were exclusively Th1 cells and predominantly Tcm cells with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ T cells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific T cells express CD127, can proliferate homeostatically, and can persist for over 2 months. Our results suggest that long-lived and robust T cell immunity is generated following natural SARS-CoV-2 infection and support an important role of SARS-CoV-2-specific T cells in host control of COVID-19., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)
- Published
- 2020
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16. SARS-CoV-2-specific T cells exhibit unique features reflecting robust helper function, lack of terminal differentiation, and high proliferative potential.
- Author
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Neidleman J, Luo X, Frouard J, Xie G, Gill G, Stein ES, McGregor M, Ma T, George A, Kosters A, Greene WC, Vasquez J, Ghosn E, Lee S, and Roan NR
- Abstract
Convalescing COVID-19 patients mount robust T cell responses against SARS-CoV-2, suggesting an important role for T cells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific T cells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ T cells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ T cells were exclusively Th1 cells, and predominantly Tcm with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ T cells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific T cells express CD127, can homeostatically proliferate, and can persist for over two months. Our results suggest that long-lived and robust T cell immunity is generated following natural SARS-CoV-2 infection, and support an important role for SARS-CoV-2-specific T cells in host control of COVID-19.
- Published
- 2020
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17. Seminal Plasma-Derived Extracellular-Vesicle Fractions from HIV-Infected Men Exhibit Unique MicroRNA Signatures and Induce a Proinflammatory Response in Cells Isolated from the Female Reproductive Tract.
- Author
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Marques de Menezes EG, Jang K, George AF, Nyegaard M, Neidleman J, Inglis HC, Danesh A, Deng X, Afshari A, Kim YH, Billaud JN, Marson K, Pilcher CD, Pillai SK, Norris PJ, and Roan NR
- Subjects
- Adult, Cohort Studies, Cytokines metabolism, Extracellular Vesicles metabolism, Female, Genitalia, Female, HIV Infections immunology, HIV-1 physiology, Humans, Male, Sexual Behavior, Transcriptome genetics, Extracellular Vesicles genetics, MicroRNAs genetics, Semen metabolism
- Abstract
The continuing spread of HIV/AIDS is predominantly fueled by sexual exposure to HIV-contaminated semen. Seminal plasma (SP), the liquid portion of semen, harbors a variety of factors that may favor HIV transmission by facilitating viral entry into host cells, eliciting the production of proinflammatory cytokines, and enhancing the translocation of HIV across the genital epithelium. One important and abundant class of factors in SP is extracellular vesicles (EVs), which, in general, are important intercellular signal transducers. Although numerous studies have characterized blood plasma-derived EVs from both uninfected and HIV-infected individuals, little is known about the properties of EVs from the semen of HIV-infected individuals. We report here that fractionated SP enriched for EVs from HIV-infected men induces potent transcriptional responses in epithelial and stromal cells that interface with the luminal contents of the female reproductive tract. Semen EV fractions from acutely infected individuals induced a more proinflammatory signature than those from uninfected individuals. This was not associated with any observable differences in the surface phenotypes of the vesicles. However, microRNA (miRNA) expression profiling analysis revealed that EV fractions from infected individuals exhibit a broader and more diverse profile than those from uninfected individuals. Taken together, our data suggest that SP EVs from HIV-infected individuals exhibit unique miRNA signatures and exert potent proinflammatory transcriptional changes in cells of the female reproductive tract, which may facilitate HIV transmission. IMPORTANCE Seminal plasma (SP), the major vehicle for HIV, can modulate HIV transmission risk through a variety of mechanisms. Extracellular vesicles (EVs) are extremely abundant in semen, and because they play a key role in intercellular communication pathways and immune regulation, they may impact the likelihood of HIV transmission. However, little is known about the properties and signaling effects of SP-derived EVs in the context of HIV transmission. Here, we conduct a phenotypic, transcriptomic, and functional characterization of SP and SP-derived EVs from uninfected and HIV-infected men. We find that both SP and its associated EVs elicit potent proinflammatory transcriptional responses in cells that line the genital tract. EVs from HIV-infected men exhibit a more diverse repertoire of miRNAs than EVs from uninfected men. Our findings suggest that EVs from the semen of HIV-infected men may significantly impact the likelihood of HIV transmission through multiple mechanisms., (Copyright © 2020 American Society for Microbiology.)
- Published
- 2020
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18. Seminal plasma promotes decidualization of endometrial stromal fibroblasts in vitro from women with and without inflammatory disorders in a manner dependent on interleukin-11 signaling.
- Author
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George AF, Jang KS, Nyegaard M, Neidleman J, Spitzer TL, Xie G, Chen JC, Herzig E, Laustsen A, Marques de Menezes EG, Houshdaran S, Pilcher CD, Norris PJ, Jakobsen MR, Greene WC, Giudice LC, and Roan NR
- Subjects
- Cross-Sectional Studies, Endometriosis, Endometrium cytology, Female, Humans, Interleukin-11 genetics, Polycystic Ovary Syndrome, Decidua physiology, Fibroblasts cytology, Interleukin-11 physiology, Semen
- Abstract
Study Question: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)?, Summary Answer: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization., What Is Known Already: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis., Study Design, Size, Duration: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization., Participants/materials, Setting, Methods: eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs., Main Results and the Role of Chance: SP promoted decidualization both in the absence and presence of steroid hormones (P < 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization., Large Scale Data: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640., Limitations, Reasons for Caution: This study is limited to in vitro analyses., Wider Implications of the Findings: Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation., Study Funding/competing Interest(s): This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)
- Published
- 2020
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19. Potent and rapid activation of tropomyosin-receptor kinase A in endometrial stromal fibroblasts by seminal plasma.
- Author
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Martin JW, Chen JC, Neidleman J, Tatsumi K, Hu J, Giudice LC, Greene WC, and Roan NR
- Subjects
- Adult, Embryo Implantation physiology, Endometrium cytology, Female, Fibroblasts cytology, Humans, Phosphorylation, Signal Transduction physiology, Endometrium metabolism, Fibroblasts metabolism, Receptor, trkA metabolism, Semen metabolism, Stromal Cells metabolism
- Abstract
Seminal plasma (SP), the liquid fraction of semen, is not mandatory for conception, but clinical studies suggest that SP improves implantation rates. Prior in vitro studies examining the effects of SP on the endometrium, the site of implantation, surprisingly revealed that SP induces transcriptional profiles associated with neurogenesis. We investigated the presence and activity of neurogenesis pathways in the endometrium, focusing on TrkA, one of the canonical receptors associated with neurotrophic signaling. We demonstrate that TrkA is expressed in the endometrium. To determine if SP activates TrkA signaling, we isolated the two most abundant endometrial cell types-endometrial epithelial cells (eEC) and endometrial stromal fibroblasts (eSF)-and examined TrkA activity in these cells after SP exposure. While SP only moderately activated TrkA in eEC, it potently and rapidly activated TrkA in eSF. This activation occurred in both non-decidualized and decidualized eSF. Blocking this pathway resulted in dysregulation of SP-induced cytokine production by eSF. Surprisingly, while the canonical TrkA agonist nerve growth factor was detected in SP, TrkA activation was principally induced by a 30-100-kDa protein whose identity remains to be established. Our results show that TrkA signaling is highly active in eSF and is rapidly induced by SP.
- Published
- 2018
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20. Semen amyloids participate in spermatozoa selection and clearance.
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Roan NR, Sandi-Monroy N, Kohgadai N, Usmani SM, Hamil KG, Neidleman J, Montano M, Ständker L, Röcker A, Cavrois M, Rosen J, Marson K, Smith JF, Pilcher CD, Gagsteiger F, Sakk O, O'Rand M, Lishko PV, Kirchhoff F, Münch J, and Greene WC
- Subjects
- Humans, Macrophages physiology, Male, Phagocytosis, Amyloid metabolism, Cell Adhesion, Semen chemistry, Semen cytology, Spermatozoa physiology
- Abstract
Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens.
- Published
- 2017
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21. Gallic Acid Is an Antagonist of Semen Amyloid Fibrils That Enhance HIV-1 Infection.
- Author
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LoRicco JG, Xu CS, Neidleman J, Bergkvist M, Greene WC, Roan NR, and Makhatadze GI
- Subjects
- Amino Acid Sequence, Amyloid chemistry, HIV Infections virology, HIV-1, Humans, Male, Microscopy methods, Amyloid drug effects, Gallic Acid pharmacology, HIV Infections physiopathology, Semen metabolism
- Abstract
Recent in vitro studies have demonstrated that amyloid fibrils found in semen from healthy and HIV-infected men, as well as semen itself, can markedly enhance HIV infection rates. Semen fibrils are made up of multiple naturally occurring peptide fragments derived from semen. The best characterized of these fibrils are SEVI (semen-derived enhancer of viral infection), made up of residues 248-286 of prostatic acidic phosphatase, and the SEM1 fibrils, made up of residues 86-107 of semenogelin 1. A small molecule screen for antagonists of semen fibrils identified four compounds that lowered semen-mediated enhancement of HIV-1 infectivity. One of the four, gallic acid, was previously reported to antagonize other amyloids and to exert anti-inflammatory effects. To better understand the mechanism by which gallic acid modifies the properties of semen amyloids, we performed biophysical measurements (atomic force microscopy, electron microscopy, confocal microscopy, thioflavin T and Congo Red fluorescence assays, zeta potential measurements) and quantitative assays on the effects of gallic acid on semen-mediated enhancement of HIV infection and inflammation. Our results demonstrate that gallic acid binds to both SEVI and SEM1 fibrils and modifies their surface electrostatics to render them less cationic. In addition, gallic acid decreased semen-mediated enhancement of HIV infection but did not decrease the inflammatory response induced by semen. Together, these observations identify gallic acid as a non-polyanionic compound that inhibits semen-mediated enhancement of HIV infection and suggest the potential utility of incorporating gallic acid into a multicomponent microbicide targeting both the HIV virus and host components that promote viral infection., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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22. Interaction of fibronectin with semen amyloids synergistically enhances HIV infection.
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Roan NR, Chu S, Liu H, Neidleman J, Witkowska HE, and Greene WC
- Subjects
- Humans, Protein Binding, Amyloid metabolism, Fibronectins metabolism, HIV-1 physiology, Semen chemistry, Semen virology, Virus Internalization
- Abstract
Semen harbors amyloids that enhance human immunodeficiency virus type 1 (HIV-1) infection. We set out to identify factors that bind these amyloids and to determine whether these factors modulate amyloid-mediated HIV-enhancing activity. Using biochemical and mass spectrometric approaches, we identified fibronectin as a consistent interaction partner. Although monomeric fibronectin did not enhance HIV infection, it synergistically increased the infectivity enhancement activity of the amyloids. Depletion of fibronectin decreased the enhancing activity of semen, suggesting that interfering with the binding interface between fibronectin and the amyloids could be an approach to developing a novel class of microbicides targeting the viral-enhancing activity of semen., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2014
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23. HIV-1 Fusion Assay.
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Cavrois M, Neidleman J, and Greene WC
- Abstract
The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).
- Published
- 2014
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24. Liquefaction of semen generates and later degrades a conserved semenogelin peptide that enhances HIV infection.
- Author
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Roan NR, Liu H, Usmani SM, Neidleman J, Müller JA, Avila-Herrera A, Gawanbacht A, Zirafi O, Chu S, Dong M, Kumar ST, Smith JF, Pollard KS, Fändrich M, Kirchhoff F, Münch J, Witkowska HE, and Greene WC
- Subjects
- Amino Acid Sequence, Amyloid chemistry, Blotting, Western, Humans, Molecular Sequence Data, Phylogeny, Proteolysis, Semen chemistry, Sequence Homology, Amino Acid, Virus Internalization, Amyloid metabolism, HIV Infections metabolism, HIV Infections virology, HIV-1 physiology, Peptide Fragments metabolism, Semen metabolism, Seminal Vesicle Secretory Proteins metabolism
- Abstract
Unlabelled: Semen enhances HIV infection in vitro, but how long it retains this activity has not been carefully examined. Immediately postejaculation, semen exists as a semisolid coagulum, which then converts to a more liquid form in a process termed liquefaction. We demonstrate that early during liquefaction, semen exhibits maximal HIV-enhancing activity that gradually declines upon further incubation. The decline in HIV-enhancing activity parallels the degradation of peptide fragments derived from the semenogelins (SEMs), the major components of the coagulum that are cleaved in a site-specific and progressive manner upon initiation of liquefaction. Because amyloid fibrils generated from SEM fragments were recently demonstrated to enhance HIV infection, we set out to determine whether any of the liquefaction-generated SEM fragments associate with the presence of HIV-enhancing activity. We identify SEM1 from amino acids 86 to 107 [SEM1(86-107)] to be a short, cationic, amyloidogenic SEM peptide that is generated early in the process of liquefaction but that, conversely, is lost during prolonged liquefaction due to the activity of serine proteases. Synthetic SEM1(86-107) amyloids directly bind HIV-1 virions and are sufficient to enhance HIV infection of permissive cells. Furthermore, endogenous seminal levels of SEM1(86-107) correlate with donor-dependent variations in viral enhancement activity, and antibodies generated against SEM1(86-107) recognize endogenous amyloids in human semen. The amyloidogenic potential of SEM1(86-107) and its virus-enhancing properties are conserved among great apes, suggesting an evolutionarily conserved function. These studies identify SEM1(86-107) to be a key, HIV-enhancing amyloid species in human semen and underscore the dynamic nature of semen's HIV-enhancing activity., Importance: Semen, the most common vehicle for HIV transmission, enhances HIV infection in vitro, but how long it retains this activity has not been investigated. Semen naturally undergoes physiological changes over time, whereby it converts from a gel-like consistency to a more liquid form. This process, termed liquefaction, is characterized at the molecular level by site-specific and progressive cleavage of SEMs, the major components of the coagulum, by seminal proteases. We demonstrate that the HIV-enhancing activity of semen gradually decreases over the course of extended liquefaction and identify a naturally occurring semenogelin-derived fragment, SEM1(86-107), whose levels correlate with virus-enhancing activity over the course of liquefaction. SEM1(86-107) amyloids are naturally present in semen, and synthetic SEM1(86-107) fibrils bind virions and are sufficient to enhance HIV infection. Therefore, by characterizing dynamic changes in the HIV-enhancing activity of semen during extended liquefaction, we identified SEM1(86-107) to be a key virus-enhancing component of human semen., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
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25. Enhanced fusion and virion incorporation for HIV-1 subtype C envelope glycoproteins with compact V1/V2 domains.
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Cavrois M, Neidleman J, Santiago ML, Derdeyn CA, Hunter E, and Greene WC
- Subjects
- Base Sequence, Cloning, Molecular, DNA Primers genetics, Enzyme-Linked Immunosorbent Assay, Genetic Engineering, Humans, Molecular Sequence Data, Protein Structure, Tertiary genetics, Sequence Alignment, Sequence Analysis, DNA, Virus Integration physiology, Genetic Variation, HIV-1 genetics, Virus Integration genetics, env Gene Products, Human Immunodeficiency Virus genetics
- Abstract
In infected people, the HIV-1 envelope glycoprotein (Env) constantly evolves to escape the immune response while retaining the essential elements needed to mediate viral entry into target cells. The extensive genetic variation of Env is particularly striking in the V1/V2 hypervariable domains. In this study, we investigated the trade-off, in terms of fusion efficiency, for encoding V1/V2 domains of different lengths. We found that natural variations in V1/V2 length exert a profound impact on HIV-1 entry. Variants encoding compact V1/V2 domains mediated fusion with higher efficiencies than related Envs encoding longer V1/V2 domains. By exchanging the V1/V2 domains between Envs of the same infected person or between two persons linked by a transmission event, we further demonstrated that V1/V2 domains critically influence both Env incorporation into viral particles and fusion to primary CD4 T cells and monocyte-derived dendritic cells. Shortening the V1/V2 domains consistently increased Env incorporation and fusion, whereas lengthening the V1/V2 domains decreased Env incorporation and fusion. Given that in a new host transmitted founder viruses are distinguished by compact Envs with fewer glycosylation sites, our study points to fusion and possibly Env incorporation into virions as limiting steps for transmission of HIV-1 to a new host and suggests that the length and/or the N-glycosylation profile of the V1/V2 domain influences these early steps in the HIV life cycle.
- Published
- 2014
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26. Measuring HIV fusion mediated by envelopes from primary viral isolates.
- Author
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Cavrois M, Neidleman J, Galloway N, Derdeyn CA, Hunter E, and Greene WC
- Subjects
- Biological Assay, Cell Culture Techniques, Cells, Cultured, DNA, Viral physiology, Dendritic Cells virology, Flow Cytometry, HIV-1 genetics, HIV-1 isolation & purification, Humans, Lymphocytes virology, Proviruses genetics, Proviruses physiology, Virion genetics, Virion physiology, env Gene Products, Human Immunodeficiency Virus genetics, HIV-1 physiology, Virus Attachment, Virus Internalization, env Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
Over the course of infection, the human immunodeficiency virus type 1 (HIV-1) continuously adapts in part to evade the host's neutralizing antibody response. Antibodies often target the HIV envelope proteins that mediate HIV fusion to its cellular targets. HIV virions pseudotyped with primary envelopes have often been used to explore the fusogenic properties of these envelopes. Unfortunately, these pseudotyped virions fuse with greatly reduced efficiency to primary cells. Here, we describe a relatively simple strategy to clone primary envelopes into a provirus and increase the sensitivity of the virion-based fusion assay., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2011
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27. The cationic properties of SEVI underlie its ability to enhance human immunodeficiency virus infection.
- Author
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Roan NR, Münch J, Arhel N, Mothes W, Neidleman J, Kobayashi A, Smith-McCune K, Kirchhoff F, and Greene WC
- Subjects
- Amino Acid Substitution, CD4-Positive T-Lymphocytes virology, Cells, Cultured, Humans, Isoelectric Point, Mutagenesis, Site-Directed, Mutant Proteins genetics, Mutant Proteins metabolism, Peptides genetics, Protein Binding, HIV physiology, HIV Infections virology, Peptides chemistry, Peptides metabolism, Semen chemistry, Semen virology, Virus Attachment
- Abstract
Human semen contains peptides capable of forming amyloid fibrils termed semen-derived enhancer of viral infection (SEVI) that can greatly increase human immunodeficiency virus (HIV) infection. While SEVI appears to enhance virion attachment to target cells, its underlying mechanism of action is unknown. We now demonstrate that the intrinsic positive charges of SEVI (pI = 10.21) facilitate virion attachment to and fusion with target cells. A mutant form of SEVI in which lysines and arginines are replaced with alanines retains the ability to form amyloid fibrils but is defective in binding virions and enhancing infection. In addition, the interaction of wild-type SEVI with virions and the ability of these fibrils to increase infection are abrogated in the presence of various polyanionic compounds. These anionic polymers also decrease the enhancement of HIV infection mediated by semen. These findings suggest that SEVI enhances viral infection by serving as a polycationic bridge that neutralizes the negative charge repulsion that exists between HIV virions and target cells. Combinations of agents that neutrale SEVI action and produce HIV virucidal effects are an attractive future direction for microbicide development.
- Published
- 2009
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28. The achilles heel of the trojan horse model of HIV-1 trans-infection.
- Author
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Cavrois M, Neidleman J, and Greene WC
- Subjects
- CD4-Positive T-Lymphocytes virology, Dendritic Cells virology, Humans, Virion pathogenicity, HIV Infections transmission, HIV-1 physiology
- Abstract
To ensure their survival, microbial pathogens have evolved diverse strategies to subvert host immune defenses. The human retrovirus HIV-1 has been proposed to hijack the natural endocytic function of dendritic cells (DCs) to infect interacting CD4 T cells in a process termed trans-infection. Although DCs can be directly infected by certain strains of HIV-1, productive infection of DCs is not required during trans-infection; instead, DCs capture and internalize infectious HIV-1 virions in vesicles for later transmission to CD4 T cells via vesicular exocytosis across the infectious synapse. This model of sequential endocytosis and exocytosis of intact HIV-1 virions has been dubbed the "Trojan horse" model of HIV-1 trans-infection. While this model gained rapid favor as a strong example of how a pathogen exploits the natural properties of its cellular host, our recent studies challenge this model by showing that the vast majority of virions transmitted in trans originate from the plasma membrane rather than from intracellular vesicles. This review traces the experimental lines of evidence that have contributed to what we view as the "rise and decline" of the Trojan horse model of HIV-1 trans-infection.
- Published
- 2008
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29. In vitro derived dendritic cells trans-infect CD4 T cells primarily with surface-bound HIV-1 virions.
- Author
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Cavrois M, Neidleman J, Kreisberg JF, and Greene WC
- Subjects
- Animals, HIV Infections virology, Humans, In Vitro Techniques, CD4-Positive T-Lymphocytes virology, Dendritic Cells virology, HIV-1 pathogenicity, Virion pathogenicity
- Abstract
In the prevailing model of HIV-1 trans-infection, dendritic cells (DCs) capture and internalize intact virions and transfer these virions to interacting T cells at the virological synapse. Here, we show that HIV-1 virions transmitted in trans from in vitro derived DCs to T cells principally originate from the surface of DCs. Selective neutralization of surface-bound virions abrogated trans-infection by monocyte-derived DCs and CD34-derived Langerhans cells. Under conditions mimicking antigen recognition by the interacting T cells, most transferred virions still derived from the cell surface, although a few were transferred from an internal compartment. Our findings suggest that attachment inhibitors could neutralize trans-infection of T cells by DCs in vivo.
- Published
- 2007
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30. Human immunodeficiency virus fusion to dendritic cells declines as cells mature.
- Author
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Cavrois M, Neidleman J, Kreisberg JF, Fenard D, Callebaut C, and Greene WC
- Subjects
- Cells, Cultured, Flow Cytometry, Humans, Immunohistochemistry, Kinetics, Langerhans Cells cytology, Langerhans Cells virology, Receptors, CCR5 physiology, Receptors, CXCR4 physiology, Receptors, HIV physiology, Virus Replication, Dendritic Cells cytology, Dendritic Cells virology, HIV physiology
- Abstract
The maturation of dendritic cells (DCs) is associated with a diminished ability to support human immunodeficiency virus (HIV) replication; however, the precise step in the HIV life cycle impaired by DC maturation remains uncertain. Using an HIV virion-based fusion assay, we now show that HIV fusion to monocyte-derived DCs (MDDCs) both decreases and kinetically slows when DCs are induced to mature with poly(I:C) and tumor necrosis factor alpha. Specifically, laboratory-adapted CCR5-tropic 81A virions fused with markedly lower efficiency to mature MDDCs than immature DCs. In contrast, fusion of NL4-3, the isogenic CXCR4-tropic counterpart of 81A, was low in both immature and mature MDDCs. Fusion mediated by primary HIV envelopes, including seven CCR5- and four CXCR4-tropic envelopes, also decreased with DC maturation. The kinetics of virion fusion were also altered by both the state of DC maturation and the coreceptor utilized. Fusion of 81A and NL4-3 virions was delayed in mature compared to immature MDDCs, and NL4-3 fused more slowly than 81A in both mature and immature MDDCs. Surprisingly, primary envelopes with CXCR4 tropism mediated fusion to immature MDDCs with efficiencies similar to those of primary CCR5-tropic envelopes. This result contrasted with the marked preferential fusion of the laboratory-adapted 81A over NL4-3 in immature MDDCs and in ex vivo Langerhans cells, indicating that these laboratory-adapted HIV strains do not fully recapitulate all of the properties of primary HIV isolates. In conclusion, our results demonstrate that the defect in HIV replication observed in mature MDDCs stems at least in part from a decline in viral fusion.
- Published
- 2006
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31. HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion.
- Author
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Cavrois M, Neidleman J, Yonemoto W, Fenard D, and Greene WC
- Subjects
- Cell Line, Cytoplasm virology, Fluoresceins, Gene Products, nef deficiency, Gene Products, vpr metabolism, HIV Core Protein p24 metabolism, HIV-1 metabolism, HIV-1 pathogenicity, Humans, Substrate Specificity, Virulence, Virus Replication, beta-Lactamases, beta-Lactams, nef Gene Products, Human Immunodeficiency Virus, vpr Gene Products, Human Immunodeficiency Virus, Gene Products, nef physiology, HIV-1 physiology, Immunoenzyme Techniques methods, Lactams, Membrane Fusion
- Abstract
We recently described a sensitive and specific assay that detects the fusion of HIV-1 virions to a broad range of target cells, including primary CD4 cells. This assay involves the use of virions containing beta-lactamase-Vpr (BlaM-Vpr) and the loading of target cells with CCF2, a fluorogenic substrate of beta-lactamase. Since Vpr strongly associates with the viral core, uncoating of the viral particle might be required for effective cleavage of CCF2 by BlaM-Vpr. Here, we show that BlaM-Vpr within mature viral cores effectively cleaves CCF2, indicating that this assay measures virion fusion independently of uncoating. We also show that wildtype and Nef-deficient HIV-1 virions fuse with equivalent efficiency to HeLa-CD4 cells, SupT1 T cells, and primary CD4 T cells. Since Nef enhances cytoplasmic delivery of viral cores and increases viral infectivity, these findings indicate that Nef enhances an early post-fusion event in the multistep process of viral entry. Possible sites of Nef action include enlargement of the fusion pore, enhanced uncoating of viral particles, and more efficient passage of viral cores through the dense cortical actin network located immediately beneath the plasma membrane., (Copyright 2004 Elsevier Inc.)
- Published
- 2004
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32. Fluorescence resonance energy transfer-based HIV-1 virion fusion assay.
- Author
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Cavrois M, Neidleman J, Bigos M, and Greene WC
- Subjects
- Flow Cytometry, Fluorescent Dyes pharmacology, Gene Products, vpr metabolism, Leukocytes, Mononuclear metabolism, Models, Biological, Spectrophotometry, Ultraviolet Rays, beta-Lactamases metabolism, vpr Gene Products, Human Immunodeficiency Virus, Fluorescence Resonance Energy Transfer methods, HIV-1 metabolism, Virion
- Abstract
The fluorescence resonance energy transfer (FRET)-based HIV-1 virion fusion assay exploits the incorporation of beta-lactamase-Vpr chimeric proteins into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a marker of fusion. This transfer can be monitored by the enzymatic cleavage of the CCF2-AM dye, a fluorescent substrate of beta-lactamase (BlaM), loaded into the target cells. BlaM cleavage of the beta-lactam ring in CCF2-AM prevents the FRET between the coumarin and fluorescein moieties of the dye. This cleavage changes the fluorescence emission spectrum of CCF2-AM from green (520 nm) to blue (447 nm), and thus permits detection of fusion by fluorescence microscopy, flow cytometry, or UV photometry. This assay is simple and rapid to perform, and exhibits high sensitivity and specificity. Importantly, it can be applied to study HIV-1 virion fusion in primary cells and can be combined with immunostaining for subset discrimination in heterogeneous target cell populations. Finally, the assay can also be adapted to study fusion mediated by the envelope proteins from other viruses through the construction of HIV-1 pseudotypes.
- Published
- 2004
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33. Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression.
- Author
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Zander K, Sherman MP, Tessmer U, Bruns K, Wray V, Prechtel AT, Schubert E, Henklein P, Luban J, Neidleman J, Greene WC, and Schubert U
- Subjects
- Amino Acid Sequence, Blotting, Northern, Blotting, Western, Cyclophilin A metabolism, DNA metabolism, Disulfides, Flow Cytometry, G2 Phase, Gene Products, vpr biosynthesis, HeLa Cells, Humans, Jurkat Cells, Molecular Sequence Data, Plasmids metabolism, Proline chemistry, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Surface Plasmon Resonance, T-Lymphocytes metabolism, Time Factors, Transfection, Cyclophilin A chemistry, Gene Products, vpr chemistry
- Abstract
Viral protein R (Vpr) of human immunodeficiency virus, type 1 (HIV-1) is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the preintegration complex into the nucleus and the induction of G2 host cell cycle arrest. Our recent investigation of the conformational heterogeneity of the proline residues in the N terminus of Vpr suggested a functional interaction between Vpr and a host peptidylprolyl cis/trans isomerase (PPIase) that might regulate the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Using surface plasmon resonance spectroscopy, Far Western blot, and pulldown experiments a physical interaction of Vpr with the major host PPIase cyclophilin A (CypA) is now demonstrated. The interaction domain involves the N-terminal region of Vpr including an essential role for proline in position 35. The CypA inhibitor cyclosporin A and non-immunosuppressive PPIase inhibitors such as NIM811 and sanglifehrin A block expression of Vpr without affecting pre- or post-translational events such as transcription, intracellular transport, or virus incorporation of Vpr. Similarly to CypA inhibition, Vpr expression is also reduced in HIV-1 infected CypA-/- knock-out T cells. This study thus shows that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA is also important for the de novo synthesis of Vpr and that in the absence of CypA activity, the Vpr-mediated cell cycle arrest is completely lost in HIV-1-infected T cells.
- Published
- 2003
- Full Text
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34. Human immunodeficiency virus type 1 Gag-specific vaginal immunity and protection after local immunizations with sindbis virus-based replicon particles.
- Author
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Vajdy M, Gardner J, Neidleman J, Cuadra L, Greer C, Perri S, O'Hagan D, and Polo JM
- Subjects
- AIDS Vaccines administration & dosage, AIDS Vaccines genetics, Animals, Female, Gene Products, gag administration & dosage, Gene Products, gag genetics, Gene Products, gag metabolism, Genetic Vectors, HIV Infections immunology, HIV-1 physiology, Immunity, Mucosal, Mice, Mice, Inbred BALB C, Ovary virology, T-Lymphocytes, Cytotoxic immunology, Vaccination, Vaccines, DNA administration & dosage, Vaccines, DNA genetics, Vaccines, DNA immunology, Virus Replication, AIDS Vaccines immunology, Gene Products, gag immunology, HIV Infections prevention & control, HIV-1 immunology, Replicon, Sindbis Virus genetics, Vagina immunology
- Abstract
The majority of human immunodeficiency virus (HIV) infections occur through vaginal and rectal transmission. In seeking a safe, nonreplicating gene-delivery vector that can induce mucosal and systemic immune responses and protection, Sindbis virus-based replicon particles expressing HIV-1 Gag (SIN-Gag) were developed. In mice, after nasal or intramuscular immunization with SIN-Gag and vaginal challenge with vaccinia virus (VV) expressing HIV-1 Gag (VV-Gag), CD8(+) T cell-mediated responses were detected locally, in the vaginal mucosa and in the draining iliac lymph nodes (ILNs), and systemically, in the spleen. However, the mice were not protected against VV-Gag replication in the ovaries. In contrast, after vaginal or rectal immunization with SIN-Gag and vaginal challenge with VV-Gag, despite lower local CD8(+) T cell-mediated responses in the vaginal mucosa and ILNs, the mice were protected against VV-Gag replication in the ovaries. Therefore, local immunization with SIN-Gag induced both local mucosal cell-mediated responses and protection.
- Published
- 2001
- Full Text
- View/download PDF
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