Publications by authors named "Rienk E Jeeninga"

32 Publications

First-in-human administration of a live-attenuated RSV vaccine lacking the G-protein assessing safety, tolerability, shedding and immunogenicity: a randomized controlled trial.

Vaccine 2020 09 25;38(39):6088-6095. Epub 2020 Jul 25.

Centre for Human Drug Research, Leiden, The Netherlands; Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands. Electronic address:

Background: Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in early infancy and in elderly. A pediatric vaccine against RSV would not only prevent morbidity and mortality amongst infants and young children but could also reduce transmission to elderly. The RSVΔG vaccine consists of a live-attenuated RSV that lacks the G attachment protein. RSVΔG is severely impaired in binding to host cells and exhibits reduced infectivity in preclinical studies. Intranasal immunization of cotton rats with RSVΔG vaccine protected against replication of wildtype RSV, without inducing enhanced disease.

Methods: We performed a first-in-human trial with primary objective to evaluate safety and shedding of RSVΔG (6.5 log CCID) after intranasal administration. Healthy adults aged between 18 and 50, with RSV neutralizing serum titers below 9.6 log, received a single dose of either vaccine or placebo (n = 48, ratio 3:1). In addition to safety and tolerability, nasal viral load, and systemic and humoral immune responses were assessed at selected time points until 4 weeks after immunization.

Results: Intranasal administration of RSVΔG was well tolerated with no findings of clinical concern. No infectious virus was detected in nasal wash samples. Similar to other live-attenuated RSV vaccines, neutralizing antibody response following inoculation was limited in seropositive adults.

Conclusions: A single dose of 6.5 log CCID of RSVΔG was safe and well-tolerated in seropositive healthy adults. RSVΔG was sufficiently attenuated but there were no signs of induction of antibodies. Safety and immunogenicity can now be explored in children and eventually in seronegative infants. Clinical trial register: NTR7173/EudraCT number 2016-002437-30.
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http://dx.doi.org/10.1016/j.vaccine.2020.07.029DOI Listing
September 2020

Prevalent levels of RSV serum neutralizing antibodies in healthy adults outside the RSV-season.

Hum Vaccin Immunother 2020 06 20;16(6):1322-1326. Epub 2019 Dec 20.

Centre for Human Drug Research , Leiden, The Netherlands.

One of the main challenges in early clinical research with respiratory syncytial virus (RSV) live-attenuated vaccines (LAVs) is to assess immunogenicity in healthy adults. Healthy adults will have preexisting levels of serum neutralizing antibodies that could prematurely neutralize the LAV and underestimate the potential effect of the vaccine on the immune system. Data on prevalence and distribution of virus neutralizing titers (VNTs) in healthy adults is limited and there is no absolute threshold for protection against RSV-infection that can serve as an eligibility criterion in early phase trials. We assessed the RSV-specific serum VNT in healthy adults outside the Dutch RSV-Season in two clinical studies performed in 2017 (exploratory study, n = 100) and 2018 (first-in-human LAV-study, n = 190) using the same neutralizing assay. Our findings show that the prevalence and distribution of serum VNT was overall consistent in the two clinical studies. Log VNTs were normally distributed, distributions of VNTs were similar and there was no statistical difference in mean log VNT for both studies ( = .3). Serum VNTs were comparable during the 6 months of screening in the FIH LAV-study. Our findings will help to determine a cutoff serum VNT to be used as an eligibility criterion in future early phase clinical trials.
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http://dx.doi.org/10.1080/21645515.2019.1688040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7482755PMC
June 2020

Genetic diversity and host adaptation of avian H5N1 influenza viruses during human infection.

Emerg Microbes Infect 2019 ;8(1):262-271

a Department of Medical Microbiology , Academic Medical Center , Amsterdam , Netherlands.

The continuing pandemic threat posed by avian influenza A/H5N1 viruses calls for improved insights into their evolution during human infection. We performed whole genome deep sequencing of respiratory specimens from 44 H5N1-infected individuals from Indonesia and found substantial within-host viral diversity. At nearly 30% of genome positions multiple amino acids were observed within or across samples, including positions implicated in aerosol transmission between ferrets. Amino acid variants detected our cohort were often found more frequently in available H5N1 sequences of human than avian isolates. We additionally identified previously unreported amino acid variants and multiple variants that increased in proportion over time in available sequential samples. Given the importance of the polymerase complex for host adaptation, we tested 121 amino acid variants found in the PB2, PB1 and PA subunits for their effects on polymerase activity in human cells. We identified multiple single amino acid variants in all three polymerase subunits that substantially increase polymerase activity including some with effects comparable to that of the widely recognized adaption and virulence marker PB2-E627 K. These results indicate highly dynamic evolutionary processes during human H5N1 virus infection and the potential existence of previously undocumented adaptive pathways.
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http://dx.doi.org/10.1080/22221751.2019.1575700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455201PMC
July 2019

ViroSpot microneutralization assay for antigenic characterization of human influenza viruses.

Vaccine 2017 01 26;35(1):46-52. Epub 2016 Nov 26.

Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands; WHO National Influenza Center (NIC), Rotterdam, The Netherlands.

The hemagglutination inhibition (HI) assay has been used for the antigenic characterization of influenza viruses for decades. However, the majority of recent seasonal influenza A viruses of the H3N2 subtype has lost the capacity to agglutinate erythrocytes of various species. The hemagglutination (HA) activity of other A(H3N2) strains is generally sensitive to the action of the neuraminidase inhibitor oseltamivir, which indicates that the neuraminidase and not the hemagglutinin is responsible for the HA activity. These findings complicate the antigenic characterization and selection of A(H3N2) vaccine strains, calling for alternative antigenic characterization assays. Here we describe the development and use of the ViroSpot microneutralization (MN) assay as a reliable and robust alternative for the HI assay. Serum neutralization of influenza A(H3N2) reference virus strains and epidemic isolates was determined by automated readout of immunostained cell monolayers, in a format designed to minimize the influence of infectious virus doses on serum neutralization titers. Neutralization of infection was largely independent from rates of viral replication and cell-to-cell transmission, facilitating the comparison of different virus isolates. Other advantages of the ViroSpot MN assay include its relative insensitivity to variation in test dose of infectious virus, automated capture and analyses of residual infection patterns, and compatibility with standardized large scale analyses. Using this assay, a number of epidemic influenza A(H3N2) strains that failed to agglutinate erythrocytes, were readily characterized antigenically.
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http://dx.doi.org/10.1016/j.vaccine.2016.11.060DOI Listing
January 2017

Dendritic cell type-specific HIV-1 activation in effector T cells: implications for latent HIV-1 reservoir establishment.

AIDS 2015 Jun;29(9):1003-14

aLaboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity Amsterdam (CINIMA) bDepartment of Cell Biology and Histology, Center for Immunology Amsterdam (CIA) cDepartment of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands dDepartment of Microbiology and Immunology, Weill Medical College of Cornell University, New York, USA.

Background: Latent HIV type I (HIV-1) infections can frequently occur in short-lived proliferating effector T lymphocytes. These latently infected cells could revert into resting T lymphocytes and thereby contribute to the establishment of the long-lived viral reservoir. Monocyte-derived dendritic cells can revert latency in effector T cells in vitro.

Methods: Here we investigated the latency activation properties of tissue-specific immune cells, including a large panel of dendritic cell subsets, to explore in which body compartments effector T cells are most likely to maintain latent HIV-1 provirus and thus potentially contribute to the long-lived reservoir.

Results: Our results demonstrate that blood or genital tract dendritic cells do not activate latent provirus in effector T cells, whereas gut or lymphoid dendritic cells induce virus production from latently infected effector T cells in our in-vitro model for latency. Toll-like receptor 3-induced interferon production by myeloid dendritic cells abolished the dendritic cells' ability to induce viral gene expression.

Conclusions: In this study, we show that HIV-1 provirus residing in effector T cells is activated from latency by tissue-specific dendritic cell subsets and other immune cells with remarkably different efficiencies.Our new assay system points to an important, neglected aspect of HIV-1 research: the ability of other immune cells, especially dendritic cells, to differentially affect latency establishment as well as virus reactivation.
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http://dx.doi.org/10.1097/QAD.0000000000000637DOI Listing
June 2015

Improved detection of artifactual viral minority variants in high-throughput sequencing data.

Front Microbiol 2014 22;5:804. Epub 2015 Jan 22.

Department of Medical Microbiology, Academic Medical Centre Amsterdam, Netherlands.

High-throughput sequencing (HTS) of viral samples provides important information on the presence of viral minority variants. However, detection and accurate quantification is limited by the capacity to distinguish biological from artificial variation. In this study, errors related to the Illumina HiSeq2000 library generation and HTS process were investigated by determining minority variant frequencies in an influenza A/WSN/1933(H1N1) virus reverse-genetics plasmid pool. Errors related to amplification and sequencing were determined using the same plasmid pool, by generation of infectious virus using reverse genetics followed by in duplo reverse-transcriptase PCR (RT-PCR) amplification and HTS in the same sequence run. Results showed that after "best practice" quality control (QC), within the plasmid pool, one minority variant with a frequency >0.5% was identified, while 84 and 139 were identified in the RT-PCR amplified samples, indicating RT-PCR amplification artificially increased variation. Detailed analysis showed that artifactual minority variants could be identified by two major technical characteristics: their predominant presence in a single read orientation and uneven distribution of mismatches over the length of the reads. We demonstrate that by addition of two QC steps 95% of the artifactual minority variants could be identified. When our analysis approach was applied to three clinical samples 68% of the initially identified minority variants were identified as artifacts. Our study clearly demonstrated that, without additional QC steps, overestimation of viral minority variants is very likely to occur, mainly as a consequence of the required RT-PCR amplification step. The improved ability to detect and correct for artifactual minority variants, increases data resolution and could aid both past and future studies incorporating HTS. The source code has been made available through Sourceforge (https://sourceforge.net/projects/mva-ngs).
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http://dx.doi.org/10.3389/fmicb.2014.00804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302989PMC
February 2015

Candidate prioritization for low-abundant differentially expressed proteins in 2D-DIGE datasets.

BMC Bioinformatics 2015 Jan 28;16:25. Epub 2015 Jan 28.

Bioinformatics Laboratory, Academic Medical Center, University of Amsterdam, PO Box 22700, DE Amsterdam, 1100, The Netherlands.

Background: Two-dimensional differential gel electrophoresis (2D-DIGE) provides a powerful technique to separate proteins on their isoelectric point and apparent molecular mass and quantify changes in protein expression. Abundantly available proteins in spots can be identified using mass spectrometry-based approaches. However, identification is often not possible for low-abundant proteins.

Results: We present a novel computational approach to prioritize candidate proteins for unidentified spots. Our approach exploits noisy information on the isoelectric point and apparent molecular mass of a protein spot in combination with functional similarities of candidate proteins to already identified proteins to select and rank candidates. We evaluated our method on a 2D-DIGE dataset comparing protein expression in uninfected and HIV-1 infected T-cells. Using leave-one-out cross-validation, we show that the true-positive rate for the top-5 ranked proteins is 43.8%.

Conclusions: Our approach shows good performance on a 2D-DIGE dataset comparing protein expression in uninfected and HIV-1 infected T-cells. We expect our method to be highly useful in (re-)mining other 2D-DIGE experiments in which especially the low-abundant protein spots remain to be identified.
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http://dx.doi.org/10.1186/s12859-015-0455-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384356PMC
January 2015

Culturing of respiratory viruses in well-differentiated pseudostratified human airway epithelium as a tool to detect unknown viruses.

Influenza Other Respir Viruses 2015 Jan 8;9(1):51-7. Epub 2014 Dec 8.

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Tehran University of Medical Sciences, Tehran, Iran.

Background: Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state-of-the-art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses.

Method: Three respiratory clinical samples were tested on well-differentiated pseudostratified tracheobronchial human airway epithelial (HAE) cultures grown at an air-liquid interface, which resemble the airway epithelium. Cells were stained with convalescent serum of the patients to identify infected cells and apical washes were analyzed by VIDISCA-454, a next-generation sequencing virus discovery technique.

Results: Infected cells were observed for all three samples. Sequencing subsequently indicated that the cells were infected by either human coronavirus OC43, influenzavirus B, or influenzavirus A. The sequence reads covered a large part of the genome (52%, 82%, and 57%, respectively).

Conclusion: We present here a new method for virus discovery that requires a virus culture on primary cells and an antibody detection. The virus in the harvest can be used to characterize the viral genome sequence and cell tropism, but also provides progeny virus to initiate experiments to fulfill the Koch's postulates.
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http://dx.doi.org/10.1111/irv.12297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280819PMC
January 2015

Interplay between viral Tat protein and c-Jun transcription factor in controlling LTR promoter activity in different human immunodeficiency virus type I subtypes.

J Gen Virol 2014 Apr 21;95(Pt 4):968-979. Epub 2014 Jan 21.

Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity Amsterdam (CINIMA), Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

HIV-1 transcription depends on cellular transcription factors that bind to sequences in the long-terminal repeat (LTR) promoter. Each HIV-1 subtype has a specific LTR promoter configuration, and minor sequence changes in transcription factor binding sites (TFBSs) or their arrangement can influence transcriptional activity, virus replication and latency properties. Previously, we investigated the proviral latency properties of different HIV-1 subtypes in the SupT1 T cell line. Here, subtype-specific latency and replication properties were studied in primary PHA-activated T lymphocytes. No major differences in latency and replication capacity were measured among the HIV-1 subtypes. Subtype B and AE LTRs were studied in more detail with regard to a putative AP-1 binding site using luciferase reporter constructs. c-Jun, a member of the AP-1 transcription factor family, can activate both subtype B and AE LTRs, but the latter showed a stronger response, reflecting a closer match with the consensus AP-1 binding site. c-Jun overexpression enhanced Tat-mediated transcription of the viral LTR, but in the absence of Tat inhibited basal promoter activity. Thus, c-Jun can exert a positive or negative effect via the AP-1 binding site in the HIV-1 LTR promoter, depending on the presence or absence of Tat.
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http://dx.doi.org/10.1099/vir.0.059642-0DOI Listing
April 2014

Emergence of the virulence-associated PB2 E627K substitution in a fatal human case of highly pathogenic avian influenza virus A(H7N7) infection as determined by Illumina ultra-deep sequencing.

J Virol 2014 Feb 20;88(3):1694-702. Epub 2013 Nov 20.

Department of Virology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.

Avian influenza viruses are capable of crossing the species barrier and infecting humans. Although evidence of human-to-human transmission of avian influenza viruses to date is limited, evolution of variants toward more-efficient human-to-human transmission could result in a new influenza virus pandemic. In both the avian influenza A(H5N1) and the recently emerging avian influenza A(H7N9) viruses, the polymerase basic 2 protein (PB2) E627K mutation appears to be of key importance for human adaptation. During a large influenza A(H7N7) virus outbreak in the Netherlands in 2003, the A(H7N7) virus isolated from a fatal human case contained the PB2 E627K mutation as well as a hemagglutinin (HA) K416R mutation. In this study, we aimed to investigate whether these mutations occurred in the avian or the human host by Illumina Ultra-Deep sequencing of three previously uninvestigated clinical samples obtained from the fatal case. In addition, we investigated three chicken samples, two of which were obtained from the source farm. Results showed that the PB2 E627K mutation was not present in any of the chicken samples tested. Surprisingly, the avian samples were characterized by the presence of influenza virus defective RNA segments, suggestive for the synthesis of defective interfering viruses during infection in poultry. In the human samples, the PB2 E627K mutation was identified with increasing frequency during infection. Our results strongly suggest that human adaptation marker PB2 E627K has emerged during virus infection of a single human host, emphasizing the importance of reducing human exposure to avian influenza viruses to reduce the likelihood of viral adaptation to humans.
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http://dx.doi.org/10.1128/JVI.02044-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911586PMC
February 2014

Subclinical avian influenza A(H5N1) virus infection in human, Vietnam.

Emerg Infect Dis 2013 Oct;19(10):1674-7

Laboratory-confirmed cases of subclinical infection with avian influenza A(H5N1) virus in humans are rare, and the true number of these cases is unknown. We describe the identification of a laboratory-confirmed subclinical case in a woman during an influenza A(H5N1) contact investigation in northern Vietnam.
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http://dx.doi.org/10.3201/eid1910.130730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3810763PMC
October 2013

Establishment and molecular mechanisms of HIV-1 latency in T cells.

Curr Opin Virol 2013 Dec 13;3(6):700-6. Epub 2013 Aug 13.

Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity Amsterdam (CINIMA), Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Treatment of an HIV infected individual with antiretroviral drugs is a successful way to suppress the plasma viral RNA load below the limit of detection (50 copies HIV RNA/ml plasma). This can provide lifelong protection against virus-induced pathogenesis in drug-adherent patients. Unfortunately, even after many years of continuous treatment, the virus persists and the plasma viral load will rebound rapidly when therapy is interrupted. The reason for this rapid rebound is the presence of a long-lived reservoir of latent HIV-1 proviruses that can be reactivated in resting memory T cells. Attempts to eliminate these proviruses have thus far not been successful and this long-lived latent reservoir is therefore considered a major obstacle toward a cure for HIV-1. A detailed understanding of the molecular mechanisms causing HIV latency and knowledge on the establishment of this reservoir may give us clues for future strategies aiming at the eradication of this reservoir.
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http://dx.doi.org/10.1016/j.coviro.2013.07.006DOI Listing
December 2013

Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections.

mBio 2013 Jun 18;4(3):e00231-13. Epub 2013 Jun 18.

Oxford University Clinical Research Unit, Wellcome Trust Major Overseas Programme, South East Asia Infectious Diseases Clinical Research Network, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.

Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology remains unknown in a large proportion of cases. We identified and characterized the full genome of a novel cyclovirus (tentatively named cyclovirus-Vietnam [CyCV-VN]) in cerebrospinal fluid (CSF) specimens of two Vietnamese patients with CNS infections of unknown etiology. CyCV-VN was subsequently detected in 4% of 642 CSF specimens from Vietnamese patients with suspected CNS infections and none of 122 CSFs from patients with noninfectious neurological disorders. Detection rates were similar in patients with CNS infections of unknown etiology and those in whom other pathogens were detected. A similar detection rate in feces from healthy children suggested food-borne or orofecal transmission routes, while high detection rates in feces from pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further research is needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.
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http://dx.doi.org/10.1128/mBio.00231-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684831PMC
June 2013

Dendritic cell-induced activation of latent HIV-1 provirus in actively proliferating primary T lymphocytes.

PLoS Pathog 2013 Mar 21;9(3):e1003259. Epub 2013 Mar 21.

Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity Amsterdam, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.

HIV-1 latency remains a formidable barrier towards virus eradication as therapeutic attempts to purge these reservoirs are so far unsuccessful. The pool of transcriptionally silent proviruses is established early in infection and persists for a lifetime, even when viral loads are suppressed below detection levels using anti-retroviral therapy. Upon therapy interruption the reservoir can re-establish systemic infection. Different cellular reservoirs that harbor latent provirus have been described. In this study we demonstrate that HIV-1 can also establish a silent integration in actively proliferating primary T lymphocytes. Co-culturing of these proliferating T lymphocytes with dendritic cells (DCs) activated the provirus from latency. Activation did not involve DC-mediated C-type lectin DC-SIGN signaling or TCR-stimulation but was mediated by DC-secreted component(s) and cell-cell interaction between DC and T lymphocyte that could be inhibited by blocking ICAM-1 dependent adhesion. These results imply that circulating DCs could purge HIV-1 from latency and re-initiate virus replication. Moreover, our data show that viral latency can be established early after infection and supports the idea that actively proliferating T lymphocytes with an effector phenotype contribute to the latent viral reservoir. Unraveling this physiologically relevant purging mechanism could provide useful information for the development of new therapeutic strategies that aim at the eradication of HIV-1 reservoirs.
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http://dx.doi.org/10.1371/journal.ppat.1003259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605277PMC
March 2013

An AP-1 binding site in the enhancer/core element of the HIV-1 promoter controls the ability of HIV-1 to establish latent infection.

J Virol 2013 Feb 12;87(4):2264-77. Epub 2012 Dec 12.

Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Following integration, HIV-1 in most cases produces active infection events; however, in some rare instances, latent infection events are established. The latter have major clinical implications, as latent infection allows the virus to persist despite antiretroviral therapy. Both the cellular factors and the viral elements that potentially determine whether HIV-1 establishes active or latent infection events remain largely elusive. We detail here the contribution of different long terminal repeat (LTR) sequences for the establishment of latent HIV-1 infection. Using a panel of full-length replication-competent virus constructs that reflect naturally occurring differences of HIV-1 subtype-specific LTRs and targeted LTR mutants, we found the primary ability of HIV-1 to establish latent infection in this system to be controlled by a four-nucleotide (nt) AP-1 element just upstream of the NF-κB element in the viral promoter. Deletion of this AP-1 site mostly deprived HIV-1 of the ability to establish latent HIV-1 infection. Extension of this site to a 7-nt AP-1 sequence massively promoted latency establishment, suggesting that this promoter region represents a latency establishment element (LEE). Given that these minimal changes in a transcription factor binding site affect latency establishment to such large extent, our data support the notion that HIV-1 latency is a transcription factor restriction phenomenon.
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http://dx.doi.org/10.1128/JVI.01594-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571467PMC
February 2013

Quantitation of HIV-1 DNA with a sensitive TaqMan assay that has broad subtype specificity.

J Virol Methods 2013 Jan 8;187(1):94-102. Epub 2012 Oct 8.

Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity Amsterdam, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, Amsterdam, The Netherlands.

The increasing diversity of HIV-1 isolates makes virus quantitation challenging, especially when diverse isolates co-circulate in a geographical area. Measuring the HIV-1 DNA levels in cells has become a valuable practical tool for fundamental and clinical research. A quantitative HIV-1 DNA assay was developed based on TaqMan(®) technology. Primers that target the highly conserved LTR region were designed to detect a broad array of HIV-1 variants, including viral isolates from many subtypes, with high sensitivity. Introduction of a pre-amplification step prior to the TaqMan(®) reaction allowed the specific amplification of fully reverse transcribed viral DNA. Execution of the pre-amplification step with a second primer set enables for the exclusive quantitation of the 2-LTR circular HIV-1 DNA form.
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http://dx.doi.org/10.1016/j.jviromet.2012.09.019DOI Listing
January 2013

Proteomic analysis of HIV-T cell interaction: an update.

Front Microbiol 2012 4;3:240. Epub 2012 Jul 4.

Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands.

This mini-review summarizes techniques applied in, and results obtained with, proteomic studies of human immunodeficiency virus type 1 (HIV-1)-T cell interaction. Our group previously reported on the use of two-dimensional differential gel electrophoresis (2D-DIGE) coupled to matrix assisted laser-desorption time of flight peptide mass fingerprint analysis, to study T cell responses upon HIV-1 infection. Only one in three differentially expressed proteins could be identified using this experimental setup. Here we report on our latest efforts to test models generated by this data set and extend its analysis by using novel bioinformatic algorithms. The 2D-DIGE results are compared with other studies including a pilot study using one-dimensional peptide separation coupled to MS(E), a novel mass spectrometric approach. It can be concluded that although the latter method detects fewer proteins, it is much faster and less labor intensive. Last but not least, recent developments and remaining challenges in the field of proteomic studies of HIV-1 infection and proteomics in general are discussed.
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http://dx.doi.org/10.3389/fmicb.2012.00240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3389432PMC
October 2012

Inhibition of HIV-1 replication with stable RNAi-mediated knockdown of autophagy factors.

Virol J 2012 Mar 16;9:69. Epub 2012 Mar 16.

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Autophagy is a cellular process leading to the degradation of cytoplasmic components such as organelles and intracellular pathogens. It has been shown that HIV-1 relies on several components of the autophagy pathway for its replication, but the virus also blocks late steps of autophagy to prevent its degradation. We generated stable knockdown T cell lines for 12 autophagy factors and analyzed the impact on HIV-1 replication. RNAi-mediated knockdown of 5 autophagy factors resulted in inhibition of HIV-1 replication. Autophagy analysis confirmed a specific defect in the autophagy pathway for 4 of these 5 factors. We also scored the impact on cell viability, but no gross effects were observed. Upon simultaneous knockdown of 2 autophagy factors (Atg16 and Atg5), an additive inhibitory effect was scored on HIV-1 replication. Stable knockdown of several autophagy factors inhibit HIV-1 replication without any apparent cytotoxicity. We therefore propose that targeting of the autophagy pathway can be a novel therapeutic approach against HIV-1.
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http://dx.doi.org/10.1186/1743-422X-9-69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342116PMC
March 2012

Latency profiles of full length HIV-1 molecular clone variants with a subtype specific promoter.

Retrovirology 2011 Sep 16;8:73. Epub 2011 Sep 16.

Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity Amsterdam, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands.

Background: HIV-1 transcription initiation depends on cellular transcription factors that bind to promoter sequences in the Long Terminal Repeat (LTR). Each HIV-1 subtype has a specific LTR promoter configuration and even minor sequence changes in the transcription factor binding sites (TFBS) or their arrangement can impact transcriptional activity. Most latency studies have focused on HIV-1 subtype B strains, and the degree to which LTR promoter variation contributes to differences in proviral latency is therefore largely unknown. Latency differences may influence establishment and size of viral reservoirs as well as the possibility to clear the virus by therapeutic intervention.

Results: We investigated the proviral transcriptional latency properties of different HIV-1 subtypes as their LTRs have unique assemblies of transcription factor binding sites. We constructed recombinant viral genomes with the subtype-specific promoters inserted in the common backbone of the subtype B LAI isolate. The recombinant viruses are isogenic, except for the core promoter region that encodes all major TFBS, including NFκB and Sp1 sites. We developed and optimized an assay to investigate HIV-1 proviral latency in T cell lines. Our data show that the majority of HIV-1 infected T cells only start viral gene expression after TNFα activation.

Conclusions: There were no gross differences among the subtypes, both in the initial latency level and the activation response, except for subtype AE that combines an increased level of basal transcription with a reduced TNFα response. This subtype AE property is related to the presence of a GABP instead of NFκB binding site in the LTR.
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http://dx.doi.org/10.1186/1742-4690-8-73DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182984PMC
September 2011

Long-term inhibition of HIV-1 replication with RNA interference against cellular co-factors.

Antiviral Res 2011 Jan 18;89(1):43-53. Epub 2010 Nov 18.

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center of University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

In this study we tested whether HIV-1 replication could be inhibited by stable RNAi-mediated knockdown of cellular co-factors. Cell lines capable of expressing shRNAs against 30 candidate co-factors implicated at different steps of the viral replication cycle were generated and analyzed for effects on cell viability and inhibition of HIV-1 replication. For half of these candidate co-factors we obtained knockdown cell lines that are less susceptible to virus replication. For three co-factors (ALIX, ATG16 and TRBP) the cell lines were resistant to HIV-1 replication for up to 2 months. With these cells we could test the hypothesis that HIV-1 is not able to escape from RNAi-mediated suppression of cellular co-factors, which was indeed not detected.
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http://dx.doi.org/10.1016/j.antiviral.2010.11.005DOI Listing
January 2011

Possible applications for replicating HIV 1 vectors.

HIV Ther 2010 May;4(3):361-369

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection & Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Since its discovery some 25 years ago, much has been learned about HIV type 1 and the molecular details of its replication cycle. This insight has been used to develop lentiviral vector systems that have advantages over conventional retroviral vector systems. For safety reasons, the lentiviral vector systems are replication incompetent and the risk of generating a replication competent virus has been minimized. Nevertheless, there may be certain applications for replication competent HIV based vector systems, and we will review our activities in this particular field. This includes the generation of a conditionally replicating HIV 1 variant as a safe live attenuated virus vaccine, the construction of mini HIV variants as cancer selective viruses for virotherapy against leukemia, and the use of a conditionally live anti HIV gene therapy vector. Although safety concerns will undoubtedly remain for the use of replication competent HIV based vector systems, some of the results in cell culture systems are very promising and warrant further testing in appropriate animal models.
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http://dx.doi.org/10.2217/hiv.10.20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889699PMC
May 2010

HIV-1 latency in actively dividing human T cell lines.

Retrovirology 2008 Apr 25;5:37. Epub 2008 Apr 25.

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, The Netherlands.

Background: Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication.

Results: We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox)-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNFalpha treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings.

Conclusion: We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus.
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http://dx.doi.org/10.1186/1742-4690-5-37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2387167PMC
April 2008

Proteomic studies reveal coordinated changes in T-cell expression patterns upon infection with human immunodeficiency virus type 1.

J Virol 2008 May 20;82(9):4320-30. Epub 2008 Feb 20.

K1-262, Academic Medical Center, University of Amsterdam, Clinical Proteomics Group, Medical Biochemistry, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

We performed an extensive two-dimensional differential in-gel electrophoresis proteomic analysis of the cellular changes in human T cells upon human immunodeficiency virus type 1 (HIV-1) infection. We detected 2,000 protein spots, 15% of which were differentially expressed at peak infection. A total of 93 proteins that changed in relative abundance were identified. Of these, 27 were found to be significantly downregulated and 66 were upregulated at peak HIV infection. Early in infection, only a small group of proteins was changed. A clear and consistent program of metabolic rerouting could be seen, in which glycolysis was downregulated and mitochondrial oxidation enhanced. Proteins that participate in apoptotic signaling were also significantly influenced. Apart from these changes, the virus also strongly influenced levels of proteins involved in intracellular transport. These and other results are discussed in light of previous microarray and proteomic studies regarding the impact of HIV-1 infection on cellular mRNA and protein content.
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http://dx.doi.org/10.1128/JVI.01819-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2293043PMC
May 2008

Characterization of an HIV-1 group M variant that is distinct from the known subtypes.

AIDS Res Hum Retroviruses 2007 Mar;23(3):466-70

Laboratory of Experimental Virology, and Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam, the Netherlands.

We identified an HIV-1 variant that belongs to the M group, with limited similarity of short genetic regions (100-200 nt) to subtype K, but the remainder of the genome is unrelated to any established HIV-1 subtype. The isolate was obtained from an HIV-1-positive male, living in the Netherlands, who encountered the virus before 1989, most probably via heterosexual contact in Africa. We describe the full-length genome sequence of four biological clones that were obtained from two samples collected 5 years apart. At both time points all open reading frames were intact. Within the 5-year interval, the person received antiretroviral therapy with zalcitabine and zidovudine for almost 4 years. Evolution of drug-resistant variants is likely given the increase in viral RNA load to +/-10,000 copies/ml during the last year of treatment. Surprisingly, the only regular RT mutation acquired during this period was K70R, which suggests that the genetic background of this variant is perhaps not suitable for the generation of the standard 41L, 67N, and 215Y/F mutations that typically arise during prolonged, nonsuccessful, zidovudine treatment. Awaiting the discovery of at least two additional, epidemiologically unrelated patients with a phylogenetically related HIV-1 variant, we can designate this variant a new HIV-1 subtype, or a distinct branch of subtype K.
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http://dx.doi.org/10.1089/aid.2006.0184DOI Listing
March 2007

Heterogeneity of signal transducer and activator of transcription binding sites in the long-terminal repeats of distinct HIV-1 subtypes.

Open Virol J 2007 20;1:26-32. Epub 2007 Oct 20.

AIDS Immunopathogenesis, San Raffaele Scientific Institute, Milano, Italy.

HIV-1 can be subdivided into distinct subtypes; the consequences of such a genomic variability remain largely speculative. The long terminal repeats (LTR) control HIV transcription and reflect the major differences of distinct viral subtypes. Three regions in the HIV-1 subtype B LTR are close matches to the Signal Transducer and Activator of Transcription (STAT) consensus sequence. Here, we show heterogeneity in these putative STAT binding sites among HIV-1 LTR subtypes A through G. Transfection of constitutively activated STAT5 lead to transcriptional activation of HIV-1 expression in 293T cells transfected with a reporter assay driven by HIV-1 LTR subtype B. Constitutively activated STAT5 transactivated the LTR of various subtypes in U937 cells with different potency. These findings support and expand the potential relevance of STAT5 activation in HIV infection and may bear relevance for a differential regulation of latency and expression of different subtypes of HIV-1.
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http://dx.doi.org/10.2174/1874357900701010026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675545PMC
June 2009

Construction of doxycyline-dependent mini-HIV-1 variants for the development of a virotherapy against leukemias.

Retrovirology 2006 Sep 27;3:64. Epub 2006 Sep 27.

Laboratory of Experimental Virology, Department of Medical Microbiology Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk type of blood-cell cancer. We describe the improvement of a candidate therapeutic virus for virotherapy of leukemic cells. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. To improve the safety of such a virus, we constructed an HIV-1 variant that replicates exclusively in the presence of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. This HIV-rtTA virus replicates in a strictly dox-dependent manner. In this virus, additional deletions and/or inactivating mutations were introduced in the genes for accessory proteins. These proteins are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. These minimized HIV-rtTA variants contain up to 7 deletions/inactivating mutations (TAR, Tat, vif, vpR, vpU, nef and U3) and replicate efficiently in the leukemic SupT1 T cell line, but do not replicate in normal peripheral blood mononuclear cells. These virus variants are also able to efficiently remove leukemic cells from a mixed culture with untransformed cells. The therapeutic viruses use CD4 and CXCR4 for cell entry and could potentially be used against CXCR4 expressing malignancies such as T-lymphoblastic leukemia/lymphoma, NK leukemia and some myeloid leukemias.
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http://dx.doi.org/10.1186/1742-4690-3-64DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592508PMC
September 2006

Pertussis toxin B-oligomer suppresses IL-6 induced HIV-1 and chemokine expression in chronically infected U1 cells via inhibition of activator protein 1.

J Immunol 2006 Jan;176(2):999-1006

AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Milan, Italy.

Pertussis toxin B-oligomer (PTX-B) inhibits HIV replication in T lymphocytes and monocyte-derived macrophages by interfering with multiple steps of the HIV life cycle. PTX-B prevents CCR5-dependent (R5) virus entry in a noncompetitive manner, and it also exerts suppressive effects on both R5- and CXCR4-dependent HIV expression at a less-characterized postentry level. We demonstrate in this study that PTX-B profoundly inhibits HIV expression in chronically infected promonocytic U1 cells stimulated with several cytokines and, particularly, the IL-6-mediated effect, a cytokine that triggers viral production in these cells independently of NF-kappaB activation. From U1 cells we have subcloned a cell line, named U1-CR1, with increased responsiveness to IL-6. In these cells, PTX-B neither down-regulated the IL-6R nor prevented IL-6 induced signaling in terms of STAT3 phosphorylation and DNA binding. In contrast, PTX-B inhibited AP-1 binding to target DNA and modified its composition with a proportional increases in FosB, Fra2, and ATF2. PTX-B inhibited IL-6-induced HIV-1 long-terminal repeat-driven transcription from A, C, E, and F viral subtypes, which contain functional AP-1 binding sites, but failed to inhibit transcription from subtypes B and D LTR devoid of these sites. In addition, PTX-B inhibited the secretion of IL-6-induced, AP-1-dependent genes, including urokinase-type plasminogen activator, CXCL8/IL-8, and CCL2/monocyte chemotactic protein-1. Thus, PTX-B suppression of IL-6 induced expression of HIV and cellular genes in chronically infected promonocytic cells is strongly correlated to inhibition of AP-1.
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http://dx.doi.org/10.4049/jimmunol.176.2.999DOI Listing
January 2006

Construction of a minimal HIV-1 variant that selectively replicates in leukemic derived T-cell lines: towards a new virotherapy approach.

Cancer Res 2005 Apr;65(8):3347-55

Department of Human Retrovirology, Emma Children Hospital, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands.

T-cell acute lymphoblastic leukemia is a high-risk type of blood-cell cancer. We analyzed the possibility of developing virotherapy for T-cell acute lymphoblastic leukemia. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. We constructed a minimized derivative of HIV-1, a complex lentivirus encoding multiple accessory functions that are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. This mini-HIV virus has five deletions (vif, vpR, vpU, nef, and U3) and replicated in the SupT1 cell line, but did not replicate in normal peripheral blood mononuclear cells. The stripped down mini-HIV variant was also able to efficiently remove leukemic cells from a mixed culture with untransformed control cells. In contrast to wild-type HIV-1, we did not observe bystander killing in mixed culture experiments with the mini-HIV variant. Furthermore, viral escape was not detected in long-term cultures. The mini-HIV variant that uses CD4 and CXCR4 for cell entry could potentially be used against CXCR4-expressing malignancies such as T-lymphoblastic leukemia/lymphoma, natural killer leukemia, and some myeloid leukemias.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-4280DOI Listing
April 2005

The human immunodeficiency virus type 1 promoter contains a CATA box instead of a TATA box for optimal transcription and replication.

J Virol 2004 Jul;78(13):6883-90

Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

The human immunodeficiency virus type 1 (HIV-1) transcriptional promoter contains a single polymorphism in the TATA box. Most subtypes contain the sequence TATAAGC, but subtype E and some recombinant AG strains have the sequence TAAAAGC. Based on mutagenesis studies of cellular RNA polymerase II (pol II) promoters, it has been proposed that the subtype E TATA box is nonfunctional due to the T-to-A substitution at the critical position 3. By means of transcription and virus replication assays, we demonstrate that the true TATA box motif within the viral long terminal repeat (LTR) promoter starts two nucleotides further upstream. Because of this realignment, subtype E has the sequence CATAAAA and all other subtypes have the sequence CATATAA. The polymorphism therefore has shifted from position 3 to position 5 and is no longer incompatible with efficient transcription according to rules determined for cellular pol II promoters. In addition, through sensitive competition experiments, we demonstrate that the CATA box of subtypes B and E can be improved for replication by the mutations 1T and 5T, respectively. The fact that the fitness of both subtype LTRs can be increased by specific point mutations in the CATA box suggests that the transcriptional promoter of HIV-1 is fine-tuned towards a suboptimal level of replication. However, this replication rate may be optimal in the in vivo context of an infected individual.
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http://dx.doi.org/10.1128/JVI.78.13.6883-6890.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC421681PMC
July 2004