bluuebunny/crossref_metadata_2025_split huggingface dataset:
Design and Expression of a Dimeric Form of Human Immunodeficiency Virus Type 1 Antibody 2G12 with Increased Neutralization Potency
Authors: West Anthony P. , Galimidi Rachel P. , Foglesong Christopher P. , Gnanapragasam Priyanthi N. P. , Huey-Tubman Kathryn E. , Klein Joshua S. , Suzuki Maria D. , Tiangco Noreen E. , Vielmetter Jost , Bjorkman Pamela J. 
Published: October 2008

ABSTRACT
The antigen-binding fragment of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) antibody 2G12 has an unusual three-dimensional (3D) domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact immunoglobulin G (IgG), 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher-molecular-weight species, which showed a significant increase in neutralization potency (50- to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher-molecular-weight species corresponds to a 2G12 dimer containing four combining sites and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared to monomer could lead to a more potent reagent for gene therapy or passive immunization.
Th2 Polarization in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus (HIV)-Infected Subjects, as Activated by HIV Virus-Like Particles
Authors: Buonaguro L. , Tornesello M. L. , Gallo R. C. , Marincola Franco M. , Lewis G. K. , Buonaguro F. M. 
Published: October 2008

ABSTRACTWe have recently shown that human immunodeficiency virus type 1 (HIV-1) Pr55gagvirus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A, induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines. Furthermore, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4+T cells in an ex vivo immunization assay. In the present study, we show that similar data can be obtained directly with fresh peripheral blood mononuclear cells (PBMCs), and the HIV-1 seropositivity status, with either low or high viremia, does not significantly impair the immune activation status and the responsiveness of circulating monocyte CD14+cell populations to an immunogenic stimulus. Some HIV-1-seropositive subjects, however, show a complete lack of maturation induced by HIV-VLPs in CD14+circulating cells, which does not consistently correlate with an advanced status of HIV-1 infection. The established Th2 polarization in both HIV-seropositive groups is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern, strongly suggesting that specific Th1 adjuvants would be required for therapeutic effectiveness in HIV-1-infected subjects. These results indicate the possibility of screening PBMCs for donor susceptibility to an immunogen treatment, which would greatly simplify the identification of “responsive” vaccinees as well as the understanding of eventual failures in individuals enrolled in clinical trials.
Polymorphisms and Splice Variants Influence the Antiretroviral Activity of Human APOBEC3H
Authors: Harari Ariana , Ooms Marcel , Mulder Lubbertus C. F. , Simon Viviana 
Published: October 2008

ABSTRACT
Human APOBEC3H belongs to the APOBEC3 family of cytidine deaminases that potently inhibit exogenous and endogenous retroviruses. The impact of single nucleotide polymorphisms (SNP) and alternative splicing on the antiretroviral activity of human APOBEC3H is currently unknown. In this study, we show that APOBEC3H transcripts derived from human peripheral blood mononuclear cells are polymorphic in sequence and subject to alternative splicing. We found that APOBEC3H variants encoding a SNP cluster (G105R, K121D and E178D, hapII-RDD) restricted human immunodeficiency virus type 1 (HIV-1) more efficiently than wild-type APOBEC3H (hapI-GKE). All APOBEC3H variants tested were resistant to HIV-1 Vif, the viral protein that efficiently counteracts APOBEC3G/3F activity. Alternative splicing of APOBEC3H was common and resulted in variants with distinct C-terminal regions and variable antiretroviral activities. Splice variants of hapI-GKE displayed a wide range of antiviral activities, whereas similar splicing events in hapII-RDD resulted in proteins that uniformly and efficiently restricted viral infectivity (>20-fold). Site-directed mutagenesis identified G105R in hapI-GKE and D121K in hapII-RDD as critical substitutions leading to an average additional 10-fold increase in antiviral activity. APOBEC3H variants were catalytically active and, similarly to APOBEC3F, favored a GA dinucleotide context. HIV-1 mutagenesis as a mode of action for APOBEC3H is suggested by the decrease of restriction observed with a cytidine deaminase domain mutant and the inverse correlation between G-to-A mutations and infectivity. Thus, the anti-HIV activity of APOBEC3H seems to be regulated by a combination of genomic variation and alternative splicing. Since prevalence of hapII-RDD is high in populations of African descent, these findings raise the possibility that some individuals may harbor effective as well as HIV-1 Vif-resistant intracellular antiviral defense mechanisms.
Human Immunodeficiency Virus-Specific Gamma Interferon Enzyme-Linked Immunospot Assay Responses Targeting Specific Regions of the Proteome during Primary Subtype C Infection Are Poor Predictors of the Course of Viremia and Set Point
Authors: Gray Clive M. , Mlotshwa Mandla , Riou Catherine , Mathebula Tiyani , de Assis Rosa Debra , Mashishi Tumelo , Seoighe Cathal , Ngandu Nobubelo , van Loggerenberg Francois , Morris Lynn , Mlisana Koleka , Williamson Carolyn , Karim Salim Abdool 
Published: October 2008

ABSTRACT

It is unknown whether patterns of human immunodeficiency virus (HIV)-specific T-cell responses during acute infection may influence the viral set point and the course of disease. We wished to establish whether the magnitude and breadth of HIV type 1 (HIV-1)-specific T-cell responses at 3 months postinfection were correlated with the viral-load set point at 12 months and hypothesized that the magnitude and breadth of HIV-specific T-cell responses during primary infection would predict the set point. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses across the complete proteome were measured in 47 subtype C HIV-1-infected participants at a median of 12 weeks postinfection. When corrected for amino acid length and individuals responding to each region, the order of recognition was as follows: Nef > Gag > Pol > Rev > Vpr > Env > Vpu > Vif > Tat. Nef responses were significantly (
P
< 0.05) dominant, targeted six epitopic regions, and were unrelated to the course of viremia. There was no significant difference in the magnitude and breadth of responses for each protein region with disease progression, although there was a trend of increased breadth (mean, four to seven pools) in rapid progressors. Correlation of the magnitude and breadth of IFN-γ responses with the viral set point at 12 months revealed almost zero association for each protein region. Taken together, these data demonstrate that the magnitude and breadth of IFN-γ ELISPOT assay responses at 3 months postinfection are unrelated to the course of disease in the first year of infection and are not associated with, and have low predictive power for, the viral set point at 12 months.
Control of the Papillomavirus Early-to-Late Switch by Differentially Expressed SRp20
Authors: Jia Rong , Liu Xuefeng , Tao Mingfang , Kruhlak Michael , Guo Ming , Meyers Craig , Baker Carl C. , Zheng Zhi-Ming 
Published: October 2008

ABSTRACT
The viral early-to-late switch of papillomavirus infection is tightly linked to keratinocyte differentiation and is mediated in part by alternative mRNA splicing. Here, we report that SRp20, a cellular splicing factor, controls the early-to-late switch via interactions with A/C-rich RNA elements. An A/C-rich SE4 element regulates the selection of a bovine papillomavirus type 1 (BPV-1) late-specific splice site, and binding of SRp20 to SE4 suppresses this selection. Expression of late BPV-1 L1 or human papillomavirus (HPV) L1, the major capsid protein, inversely correlates with SRp20 levels in the terminally differentiated keratinocytes. In HPV type 16, a similar SRp20-interacting element also controls the viral early-to-late switch. Keratinocytes in raft cultures, which support L1 expression, make considerably less SRp20 than keratinocytes in monolayer cultures, which do not support L1 expression. Conversely, abundant SRp20 in cancer cells or undifferentiated keratinocytes is important for the expression of the viral early E6 and E7 by promoting the expression of cellular transcription factor SP1 for transactivation of viral early promoters.
Brain Trauma Enhances Transient Cytomegalovirus Invasion of the Brain Only in Mice That Are Immunodeficient
Authors: van den Pol Anthony N. 
Published: October 2008

ABSTRACTCytomegalovirus (CMV) is one of the most common viral pathogens leading to neurological dysfunction in individuals with depressed immune systems. How CMV enters the brain remains an open question. The hypothesis that brain injury may enhance the entrance of CMV into the brain was tested. Insertion of a sterile needle into the brain caused a dramatic increase in mouse CMV in the brains of immunodeficient SCID mice inoculated peripherally within an hour of injury and examined 1 week later; peripheral inoculation 48 h after injury and a 1-week survival resulted in only a modest infection at the site of injury. In contrast, uninjured SCID mice, as well as injured immunocompetent control mice, showed little sign of viral infection at the same time intervals. Direct inoculation of the brain resulted in widespread dispersal and enhanced replication of mCMV in SCID brains tested 1 week later but not in parallel control brains. Differential viremia was unlikely to account for the greater viral load in the SCID brain, since increased mCMV in the blood of SCID compared to controls was not detected until a longer interval. These data suggest that brain injury enhances CMV invasion of the brain, but only when the adaptive immune system is compromised, and that the brain's ability to resist viral infection recovers rapidly after injury.
Evidence for Persistent Low-Level Viremia in Individuals Who Control Human Immunodeficiency Virus in the Absence of Antiretroviral Therapy
Authors: Hatano Hiroyu , Delwart Eric L. , Norris Philip J. , Lee Tzong-Hae , Dunn-Williams Joan , Hunt Peter W. , Hoh Rebecca , Stramer Susan L. , Linnen Jeffrey M. , McCune Joseph M. , Martin Jeffrey N. , Busch Michael P. , Deeks Steven G. 
Published: October 2008

ABSTRACT

A subset of antiretroviral-untreated, human immunodeficiency virus (HIV)-infected individuals are able to maintain undetectable plasma HIV RNA levels in the absence of antiretroviral therapy. These “elite” controllers are of high interest as they may provide novel insights regarding host mechanisms of virus control. The degree to which these individuals have residual plasma viremia has not been well defined. We performed a longitudinal study of 46 elite controllers, defined as HIV-seropositive, antiretroviral-untreated individuals with plasma HIV RNA levels of <50 to 75 copies/ml. The median duration of HIV diagnosis was 13 years, the median baseline CD4
+
T-cell count was 753 cells/mm
3
, and the median duration of follow-up was 16 months. Plasma and cellular HIV RNA levels were measured using the transcription-mediated amplification (TMA) assay (estimated limit of detection of <3.5 copies RNA/ml). A total of 1,117 TMA assays were performed (median of five time points/subject and four replicates/time point). All but one subject had detectable plasma HIV RNA on at least one time point, and 15 (33%) subjects had detectable RNA at all time points. The majority of controllers also had detectable cell-associated RNA and proviral DNA. A mixed-effect linear model showed no strong evidence of change in plasma RNA levels over time. In conclusion, the vast majority (98%) of elite controllers had measurable plasma HIV RNA, often at levels higher than that observed in antiretroviral-treated patients. This confirms the failure to eradicate the virus, even in these unique individuals who are able to reduce plasma viremia to very low levels without antiretroviral therapy.
Evaluation of Live Attenuated Influenza A Virus H6 Vaccines in Mice and Ferrets
Authors: Chen Zhongying , Santos Celia , Aspelund Amy , Gillim-Ross Laura , Jin Hong , Kemble George , Subbarao Kanta 
Published: October 2008

ABSTRACT

Avian influenza A virus A/teal/HK/W312/97 (H6N1) possesses seven gene segments that are highly homologous to those of highly pathogenic human influenza H5N1 viruses, suggesting that a W312-like H6N1 virus might have been involved in the generation of the A/HK/97 H5N1 viruses. The continuous circulation and reassortment of influenza H6 subtype viruses in birds highlight the need to develop an H6 vaccine to prevent potential influenza pandemics caused by the H6 viruses. Based on the serum antibody cross-reactivity data obtained from 14 different H6 viruses from Eurasian and North American lineages, A/duck/HK/182/77, A/teal/HK/W312/97, and A/mallard/Alberta/89/85 were selected to produce live attenuated H6 candidate vaccines. Each of the H6 vaccine strains is a 6:2 reassortant
ca
virus containing HA and NA gene segments from an H6 virus and the six internal gene segments from cold-adapted A/Ann Arbor/6/60 (AA
ca
), the master donor virus that is used to make live attenuated influenza virus FluMist (intranasal) vaccine. All three H6 vaccine candidates exhibited phenotypic properties of temperature sensitivity (
ts
),
ca
, and attenuation (
att
) conferred by the internal gene segments from AA
ca
. Intranasal administration of a single dose of the three H6
ca
vaccine viruses induced neutralizing antibodies in mice and ferrets and fully protected mice and ferrets from homologous wild-type (wt) virus challenge. Among the three H6 vaccine candidates, the A/teal/HK/W312/97
ca
virus provided the broadest cross-protection against challenge with three antigenically distinct H6 wt viruses. These data support the rationale for further evaluating the A/teal/HK/W312/97
ca
vaccine in humans.
Transactivator IE1 Is Required for Baculovirus Early Replication Events That Trigger Apoptosis in Permissive and Nonpermissive Cells
Authors: Schultz Kimberly L. W. , Wetter Justin A. , Fiore Diccon C. , Friesen Paul D. 
Published: October 2008

ABSTRACT

Immediate early viral protein IE1 is a potent transcriptional activator encoded by baculoviruses. Although the requirement of IE1 for multiplication of
Autographa californica
multicapsid nucleopolyhedrovirus (Ac
M
NPV) is well established, the functional roles of IE1 during infection are unclear. Here, we used RNA interference to ablate IE1, plus its splice variant IE0, and thereby define in vivo activities of these early proteins, including gene-specific regulation and induction of host cell apoptosis. Confirming an essential replicative role, simultaneous ablation of IE1 and IE0 by gene-specific double-stranded RNAs inhibited Ac
M
NPV late gene expression, reduced yields of budded virus by more than 1,000-fold, and blocked production of occluded virus particles. Depletion of IE1 and IE0 had no effect on early expression of the envelope fusion protein gene
gp64
but abolished early expression of the caspase inhibitor gene
p35
, which is required for prevention of virus-induced apoptosis. Thus, IE1 is a positive, gene-specific transactivator. Whereas an Ac
M
NPV
p35
deletion mutant caused widespread apoptosis in permissive
Spodoptera frugiperda
cells, ablation of IE1 and IE0 prevented this apoptosis. Silencing of
ie-1
also prevented Ac
M
NPV-induced apoptosis in nonpermissive
Drosophila melanogaster
cells. Thus, de novo synthesis of IE1 is required for virus-induced apoptosis. We concluded that IE1 causes apoptosis directly or contributes indirectly by promoting virus replication events that subsequently trigger cell death. This study reveals that IE1 is a gene-selective transcriptional activator which is required not only for expedition of virus multiplication but also for blocking of its own proapoptotic activity by upregulation of baculovirus apoptotic suppressors.
Deletions of the Hypervariable Region (HVR) in Open Reading Frame 1 of Hepatitis E Virus Do Not Abolish Virus Infectivity: Evidence for Attenuation of HVR Deletion Mutants In Vivo
Authors: Pudupakam R. S. , Huang Y. W. , Opriessnig T. , Halbur P. G. , Pierson F. W. , Meng X. J. 
Published: October 2008

ABSTRACT
Hepatitis E virus (HEV) is an important human pathogen, although little is known about its biology and replication. Comparative sequence analysis revealed a hypervariable region (HVR) with extensive sequence variations in open reading frame 1 of HEV. To elucidate the role of the HVR in HEV replication, we first constructed two HVR deletion mutants, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a genotype 1 human HEV replicon. Evidence of HEV replication was detected in Huh7 cells transfected with RNA transcripts from mutant hHVRd2, as evidenced by expression of enhanced green fluorescent protein. To confirm the in vitro results, we constructed three avian HEV mutants with various HVR deletions: mutants aHVRd1, with deletion of aa 557 to 585 (Δ557-585); aHVRd2 (Δ612-641); and aHVRd3 (Δ557-641). Chickens intrahepatically inoculated with capped RNA transcripts from mutants aHVRd1 and aHVRd2 developed active viral infection, as evidenced by seroconversion, viremia, and fecal virus shedding, although mutant aHVRd3, with complete HVR deletion, was apparently attenuated in chickens. To further verify the results, we constructed four additional HVR deletion mutants using the genotype 3 swine HEV as the backbone. Mutants sHVRd2 (Δ722-781), sHVRd3 (Δ735-765), and sHVRd4 (Δ712-765) were shown to tolerate deletions and were infectious in pigs intrahepatically inoculated with capped RNA transcripts from the mutants, whereas mutant sHVRd1 (Δ712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype in infected pigs. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion.
Insertional Gene Activation by Lentiviral and Gammaretroviral Vectors
Authors: Bokhoven Marieke , Stephen Sam L. , Knight Sean , Gevers Evelien F. , Robinson Iain C. , Takeuchi Yasuhiro , Collins Mary K. 
Published: October 2008

ABSTRACT

Gammaretroviral and lentiviral vectors are promising tools for gene therapy, but they can be oncogenic. The development of safer vectors depends on a quantitative assay for insertional mutagenesis. Here we report a rapid, inexpensive, and reproducible assay which uses a murine cell line to measure the frequency of interleukin-3 (IL-3)-independent mutants. Lentiviral and gammaretroviral vectors cause insertional mutagenesis at similar frequencies; however, they use different mechanisms. Human immunodeficiency virus (HIV)-based vectors generate mutants by insertion only into the growth hormone receptor (
Ghr
) locus. The HIV enhancer/promoter is active in the absence of the HIV Tat protein in this locus, and an HIV/
Ghr
spliced transcript expresses GHR and cells respond to GH. Deletion of the enhancer/promoter in a self-inactivating HIV-based vector prevents this mechanism of insertional mutagenesis. In contrast, gammaretroviral vectors insert into other loci, including IL-3 and genes identified as common insertion sites in the Retroviral Tagged Cancer Gene Database (RTCGD).
Effect of Neutralizing Sera on Factor X-Mediated Adenovirus Serotype 5 Gene Transfer
Authors: Parker Alan L. , Waddington Simon N. , Buckley Suzanne M. K. , Custers Jerome , Havenga Menzo J. E. , van Rooijen Nico , Goudsmit Jaap , McVey John H. , Nicklin Stuart A. , Baker Andrew H. 
Published: October 2008

ABSTRACT
The deployment of adenovirus serotype 5 (Ad5)-based vectors is hampered by preexisting immunity. When such vectors are delivered intravenously, hepatocyte transduction is mediated by the hexon-coagulation factor X (FX) interaction. Here, we demonstrate that human sera efficiently block FX-mediated cellular binding and transduction of Ad5-based vectors in vitro. Neutralizing activity correlated well with the ability to inhibit Ad5-mediated liver transduction, suggesting that prescreening patient sera in this manner accurately predicts the efficacy of Ad5-based gene therapies. Neutralization in vitro can be partially bypassed by pseudotyping with Ad45 fiber protein, indicating that a proportion of neutralizing antibodies are directed against the Ad5 fiber.
Functional Consequences of Human Immunodeficiency Virus Escape from an HLA-B*13-Restricted CD8
+
T-Cell Epitope in p1 Gag Protein
Authors: Prado Julia G. , Honeyborne Isobella , Brierley Ian , Puertas Maria Carmen , Martinez-Picado Javier , Goulder Philip J. R. 
Published: October 2008

ABSTRACT

The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI
429-437
(RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness.