bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of CpG Oligodeoxynucleotides on the Immunogenicity of Pfs25, aPlasmodium falciparumTransmission-Blocking Vaccine Antigen
Authors: Coban Cevayir , Ishii Ken J. , Stowers Anthony W. , Keister David B. , Klinman Dennis M. , Kumar Nirbhay
Published: December 2003

Antibodies directed against Pfs25, a protein present on the surface of zygotes and ookinetes ofPlasmodium falciparum, completely block pathogen transmission. We evaluated the immunomodulatory effect of CpG oligodeoxynucleotides (ODN) on the immunogenicity of recombinant Pfs25 (rPfs25) formulated in alum (Al). Immunization of mice with rPfs25 plus CpG ODN improved both the antibody titer (a 30-fold-higher antibody response than that with rPfs25-Al alone) and avidity. Coadministration of CpG ODN dramatically enhanced the titer of immunoglobulin G2A (IgG2a) compared to the titer of the IgG1-dominant response caused by rPfs25-Al alone, and the sera from the CpG ODN-coadministered group completely blocked the transmission ofP. falciparumparasites to mosquitoes, as determined by membrane feeding assays. However, transmission-blocking experiments revealed that blocking efficacy was dependent on high-titer antibody levels, independent of isotypes. These results suggest that CpG ODN can be used as an adjuvant to enhance the immunogenicity of rPfs25 as a malaria transmission-blocking vaccine.
Inhibition of CD1 Expression in Human Dendritic Cells during Intracellular Infection withLeishmania donovani
Authors: Amprey Joseph L. , Späth Gerald F. , Porcelli Steven A.
Published: December 2003

Dendritic cells can initiate antimicrobial responses by CD1-mediated presentation of pathogen-derived glycolipids. We show that the protozoanLeishmania donovaniinhibits CD1 expression and prevents activation of CD1-restricted T cells by dendritic cells. Evasion of presentation by CD1 may represent aLeishmaniasurvival strategy to avoid recognition of abundant parasite glycolipids.
Autotransporter Genes
pic
and
tsh
Are Associated with
Escherichia coli
Strains That Cause Acute Pyelonephritis and Are Expressed during Urinary Tract Infection
Authors: Heimer Susan R. , Rasko David A. , Lockatell C. Virginia , Johnson David E. , Mobley Harry L. T.
Published: December 2003

We have identified two chromosomal open reading frames in uropathogenic
Escherichia coli
(UPEC) strain CFT073 which are highly homologous to serine protease autotransporters Pic and Tsh. Both cloned determinants were correlated with the presence of 105- to 110-kDa proteins in the culture supernatants. Furthermore, in cellular fractionation experiments, 30-kDa polypeptides were identified in the outer membrane; we speculated that these proteins are the β-barrel portions of the autotransporter homologues. Furthermore, Pic-containing culture supernatants have serine protease activity. In reverse transcription-PCR analyses, the expression of the
pic
and
tsh
genes in
E. coli
CFT073 was higher in broth cultures grown at 37°C than at 25°C. Moreover,
pic
and
tsh
were expressed by bacteria isolated from urine of transurethrally infected mice. The
tsh
determinant was identified in 63% of our clinical UPEC strain isolates (
n
= 87) and in 33% of fecal strains (
n
= 27), whereas
pic
was present in 31% of the pyelonephritis (
n
= 67) and 7% of the fecal strains. There was no significant correlation between cystitis strains (
n
= 20) and the
pic
determinant.
Relationship between
Candida albicans
Virulence during Experimental Hematogenously Disseminated Infection and Endothelial Cell Damage In Vitro
Authors: Sanchez Angela A. , Johnston Douglas A. , Myers Carter , Edwards John E. , Mitchell Aaron P. , Filler Scott G.
Published: December 2003

Candida albicans
must penetrate the endothelial cell lining of the vasculature to invade the deep tissues during a hematogenously disseminated infection. We compared 27
C. albicans
mutants with their wild-type parent for their capacity to damage endothelial cells in vitro and cause a lethal infection in mice following tail vein inoculation. Of 10 mutants with significantly impaired capacity to damage endothelial cells, all had attenuated virulence. Therefore, the endothelial cell damage assay can be used as a screen to identify some virulence factors relevant to hematogenously disseminated candidiasis.
Protection against Anthrax Toxemia by Hexa-
d
-Arginine In Vitro and In Vivo
Authors: Sarac Miroslav S. , Peinado Juan R. , Leppla Stephen H. , Lindberg Iris
Published: December 2003

The anthrax toxin protective antigen precursor is activated by proteolytic cleavage by furin or a furin-like protease. We present here data demonstrating that the small stable furin inhibitor hexa-
d
-arginine amide delays anthrax toxin-induced toxemia both in cells and in live animals, suggesting that furin inhibition may represent a reasonable avenue for therapeutic intervention in anthrax.
Acquisition of Host Plasmin Activity by the Swine PathogenStreptococcus suisSerotype 2
Authors: Jobin Marie-Claude , Brassard Julie , Quessy Sylvain , Gottschalk Marcelo , Grenier Daniel
Published: December 2003

In this study, the plasminogen-binding activity ofStreptococcus suisserotype 2 was investigated. Bound human plasminogen was activated by purified streptokinase, urokinase, orStreptococcus dysgalactiaesubsp.equisimilisculture supernatant. Both human and porcine plasminogen were bound byS. suis. Binding was inhibited by ε-aminocaproic acid, and the plasminogen receptor was heat and sodium dodecyl sulfate resistant. One of the receptors was identified as glyceraldehyde-3-phosphate dehydrogenase.S. suis-associated plasmin activity was capable of activating free plasminogen, which in turn could contribute to degradation of fibronectin. This is the first report on the plasminogen-binding activity ofS. suis. Further studies may reveal a contribution of this activity to the virulence ofS. suis.
Activation of
Serratia marcescens
Hemolysin through a Conformational Change
Authors: Walker Georg , Hertle Ralf , Braun Volkmar
Published: December 2003

For
Serratia marcescens
, secreted hemolysin/cytotoxin is not only secreted but also activated by an outer membrane protein. Excluding posttranslational processing by mass spectrometry, the conformation of active and inactive ShlA derivatives strongly differed in electrophoretic mobilities, gel permeation chromatography, sensitivity to trypsin, circular dichroism, and intrinsic fluorescence. We concluded that ShlB interacts with ShlA during secretion and imposes a conformational change in ShlA to form the active hemolysin.
Antibody-Mediated Neutralization of Pertussis Toxin-Induced Mitogenicity of Human Peripheral Blood Mononuclear Cells
Authors: Millen Scott H. , Bernstein David I. , Connelly Beverly , Ward Joel I. , Chang Swei-Ju , Weiss Alison A.
Published: December 2003

Antibody-mediated neutralization of pertussis toxin-induced proliferation of human peripheral blood mononuclear cells (PBMC) was assessed using alamarBlue and compared with results from the Chinese hamster ovary (CHO) cell assay using sera from vaccinated adults and convalescent children. Neutralization values for the CHO assay were similar for vaccinated and convalescent subjects; however. the convalescent group had higher titers in the PBMC assay. Results for pertussis toxin neutralization with the CHO assay appear to be distinct from those with the PBMC assay.
Mycobacterium bovis
BCG Vaccination as Prophylaxis against
Mycobacterium ulcerans
Osteomyelitis in Buruli Ulcer Disease
Authors: Portaels F. , Aguiar J. , Debacker M. , Guédénon A. , Steunou C. , Zinsou C. , Meyers W. M.
Published: December 2003

Mycobacterium ulcerans
disease, or Buruli ulcer (BU), causes significant morbidity in West Africa. Clinically, the disease presents in the skin as either nonulcerative or ulcerative forms and often invades bones either subjacent to the skin lesion (contiguous osteomyelitis) or remote from the skin lesion (metastatic osteomyelitis). Osteomyelitis represents a severe form of the disease that often requires numerous surgical interventions, even amputations. Surgery is accepted as the present definitive treatment for BU. In the absence of an effective drug treatment, the need for the development of preventive and control strategies becomes paramount. No specific vaccine, however, is presently available for BU. Of 372 consecutive patients in Benin presenting with BU (confirmed by microbiological and histopathological analyses) whose
Mycobacterium bovis
BCG scar statuses were known, 196 children (<15 years old) and 108 adults had neonatal BCG vaccination scars. Of 196 children with BCG scars, 17 (8.7%) had osteomyelitis, while 7 of 28 children without BCG scars (25.0%) had osteomyelitis. Of 108 adults with BCG scars, 17 (15.7%) had osteomyelitis, while 14 of 40 adults without BCG scars (35.0%) had osteomyelitis. Our results show that effective BCG vaccination at birth provides significant protection against the development of
M. ulcerans
osteomyelitis in children and adults. Therefore, health authorities should give attention to the enhancement of neonatal BCG vaccination coverage in all countries of Africa where BU is endemic. Protection against severe forms of BU and childhood tuberculosis would likewise be improved by this intervention.
Development of an Intranasal Vaccine To Prevent Urinary Tract Infection byProteus mirabilis
Authors: Li Xin , Lockatell C. Virginia , Johnson David E. , Lane M. Chelsea , Warren John W. , Mobley Harry L. T.
Published: December 2003

Proteus mirabiliscommonly infects the complicated urinary tract and is associated with urolithiasis. Stone formation is caused by bacterial urease, which hydrolyzes urea to ammonia, causing local pH to rise, and leads to the subsequent precipitation of magnesium ammonium phosphate (struvite) and calcium phosphate (apatite) crystals. To prevent these infections, we vaccinated CBA mice with formalin-killed bacteria or purified mannose-resistant,Proteus-like (MR/P) fimbriae, a surface antigen expressed byP. mirabilisduring experimental urinary tract infection, via four routes of immunization: subcutaneous, intranasal, transurethral, and oral. We assessed the efficacy of vaccination using the CBA mouse model of ascending urinary tract infection. Subcutaneous or intranasal immunization with formalin-killed bacteria and intranasal or transurethral immunization with purified MR/P fimbriae significantly protected CBA mice from ascending urinary tract infection byP. mirabilis(P< 0.05). To investigate the potential of MrpH, the MR/P fimbrial tip adhesin, as a vaccine, the mature MrpH peptide (residues 23 to 275, excluding the signal peptide), and the N-terminal receptor-binding domain of MrpH (residues 23 to 157) were overexpressed as C-terminal fusions to maltose-binding protein (MBP) and purified on amylose resins. Intranasal immunization of CBA mice with MBP-MrpH (residues 23 to 157) conferred effective protection against urinary tract infection byP. mirabilis(P< 0.002).
Ability of Pneumococcal Serotypes and Clones To Cause Acute Otitis Media: Implications for the Prevention of Otitis Media by Conjugate Vaccines
Authors: Hanage William P. , Auranen Kari , Syrjänen Ritva , Herva Elja , Mäkelä P. Helena , Kilpi Terhi , Spratt Brian G.
Published: December 2003

The relative abilities of pneumococcal serotypes and strains (clones) to cause acute otitis media (AOM) were investigated by comparing the serotypes and genotypes of pneumococci recovered from cases of AOM (
n
= 149) in children <2 years of age with those from nasopharyngeal carriage (
n
= 288) in age-matched controls from the same region. The odds ratio (OR) for association of pooled vaccine serotypes with AOM was found to be slightly elevated over unity, although this was not significantly different from that of pooled nonvaccine or vaccine-related serotypes. Comparing individual serotypes, 19F and 23F had 2- to 2.5-fold higher ORs, although these were not markedly different from the ORs of nonvaccine serotypes. None of the major clones had an OR that was significantly greater than the average, and the differences in ORs among serotypes and clones were much less than those for invasive disease, suggesting little variation in their ability to cause AOM. We conclude that serotype replacement may reduce the long-term efficacy of these vaccines against AOM.
Tissue Inhibitor of Metalloproteinase 1 Activates Normal Human Granulocytes, Protects Them from Apoptosis, and Blocks Their Transmigration during Inflammation
Authors: Chromek Milan , Tullus Kjell , Lundahl Joachim , Brauner Annelie
Published: December 2003

Urinary levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) higher than those of matrix metalloproteinase 9 (MMP-9) during acute pyelonephritis have previously been associated with a higher degree of acute inflammation and of postinfective renal scarring. The aim of the present study was to evaluate possible mechanisms by which TIMP-1 could affect the scarring process already during the acute phase of inflammation. The growth ofEscherichia coli, bactericidal activity of fresh human blood, and respiratory burst, spontaneous apoptosis, and trans-basement membrane migration of normal human granulocytes were studied in vitro in the presence of different concentrations of recombinant human TIMP-1. To imitate the “normal” environment during inflammation in the kidney, granulocytes were also incubated with a conditioned medium fromE. coli-stimulated renal epithelial cells. In order to compare our data with the in vivo situation, blood and urinary leukocyte levels were analyzed for 40 children with acute pyelonephritis, together with urinary MMP-9 and TIMP-1 levels. TIMP-1 at a concentration of 500 ng/ml increased the bactericidal activity of blood, increased the respiratory burst of granulocytes, decreased phosphatidylserine exposure and caspase 3 activity, which are features of spontaneous apoptosis, and inhibited granulocyte transmigration. Moreover, in the patients with pyelonephritis, MMP-9/TIMP-1 ratios in urine correlated with the degree of leukocyte transmigration. Thus, our data suggest that TIMP-1 specifically blocks the transmigration of granulocytes into urine. Entrapped and activated granulocytes, protected from apoptosis, might excessively destroy renal parenchyma and thus contribute to the pathogenesis of renal scarring following acute pyelonephritis.
Selective Induction of Tumor Necrosis Receptor Factor 6/Decoy Receptor 3 Release by Bacterial Antigens in Human Monocytes and Myeloid Dendritic Cells
Authors: Kim Sunghee , McAuliffe William J. , Zaritskaya Liubov S. , Moore Paul A. , Zhang Lurong , Nardelli Bernardetta
Published: December 2003

Tumor necrosis factor (TNF) receptor 6/decoy receptor 3 (TR6/DcR3) is an antiapoptosis soluble receptor of the TNF family produced by tumor cells. In this study, TR6 expression in human immune cells was investigated. TR6 mRNA and protein were detectable in selected antigen-presenting cells. Monocytes and myeloid-derived dendritic cells (MDC) released the protein exclusively following stimulation of Toll-like receptor 2 (TLR2) and TLR4 by gram-positive and gram-negative bacterial antigens. Plasmacytoid dendritic cells, activated by bacterial antigens via TLR9 or by viral infection, did not produce the protein. Similarly, activated T cells did not release TR6. The release of TR6 by MDC was dependent on the activation of p42/p44 mitogen-activated protein kinases, Src-like protein tyrosine kinases, and phosphatidylinositol 3-kinase, signaling pathways important for MDC maturation and survival. In agreement with the in vitro data, TR6 levels in serum were significantly elevated in patients with bacterial infections. Overall, these data suggest a novel role for TR6 in immune responses to bacteria.
Role of the C-Terminal Lysine Residues of Streptococcal Surface Enolase in Glu- and Lys-Plasminogen-Binding Activities of Group A Streptococci
Authors: Derbise Anne , Song Youngmia P. , Parikh Sonia , Fischetti Vincent A. , Pancholi Vijay
Published: December 2003

Streptococcal surface enolase (SEN) is a major plasminogen-binding protein of group A streptococci. Our earlier biochemical studies have suggested that the region responsible for this property is likely located at the C-terminal end of the SEN molecule. In the present study, the gene encoding SEN was cloned from group A streptococci M6 isolate D471. A series of mutations in the
sen
gene corresponding to the C-terminal region (
428
KSFYNLKK
435
) of the SEN molecule were created by either deleting one or more terminal lysine residues or replacing them with leucine. All purified recombinant SEN proteins with altered C-terminal ends were found to be enzymatically active and were analyzed for their Glu- and Lys-plasminogen-binding activities. Wild-type SEN bound to Lys-plasminogen with almost three times more affinity than to Glu-plasminogen. However, the recombinant mutant SEN proteins with a deletion of Lys434-435 or with K435L and K434-435L replacements showed a significant decrease in Glu- and Lys-plasminogen-binding activities. Accordingly, a streptococcal mutant expressing SEN-K434-435L showed a significant decrease in Glu- and Lys-plasminogen-binding activities. Biochemical and functional analyses of the isogenic mutant strain revealed a significant decrease in its abilities to cleave a chromogenic tripeptide substrate, acquire plasminogen from human plasma, and penetrate the extracellular matrix. Together, these data indicate that the last two C-terminal lysine residues of surface-exposed SEN contribute significantly to the plasminogen-binding activity of intact group A streptococci and hence to their ability to exploit host properties to their own advantage in tissue invasion.
Capsid-Dependent and -Independent Postentry Restriction of Primate Lentivirus Tropism in Rodent Cells
Authors: Hatziioannou Theodora , Cowan Simone , Bieniasz Paul D.
Published: December 2003

Retrovirus tropism can be restricted by cellular factors such as Fv1, Ref1, and Lv1 that inhibit infection by targeting the incoming viral capsid. Here, we show that rodent cells exhibit differential sensitivity to infection by vesicular stomatitis virus G-pseudotyped lentiviruses and that differences between human immunodeficiency virus type 1 and simian immunodeficiency virus (SIVmac) infectivity are sometimes, but not always, governed by determinants in capsid-p2. In at least one case, resistance to SIVmac infection could be eliminated by saturation of target cells with noninfectious SIVmac particles. However, cross-saturation experiments and analysis of
Fv1
-null cells engineered to express natural or artificial Fv1 proteins revealed that lentivirus restriction in mouse cells is independent of Fv1. Overall, these findings indicate that novel restriction factors in rodents can modulate sensitivity to specific primate lentiviruses.
High Fidelity of Yellow Fever Virus RNA Polymerase
Authors: Pugachev Konstantin V. , Guirakhoo Farshad , Ocran Simeon W. , Mitchell Fred , Parsons Megan , Penal Caroline , Girakhoo Soheila , Pougatcheva Svetlana O. , Arroyo Juan , Trent Dennis W. , Monath Thomas P.
Published: December 2003

Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 × 10
−4
per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 × 10
−7
to 2.3 × 10
−7
. Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.
Identification of an Envelope Protein from the FRD Family of Human Endogenous Retroviruses (HERV-FRD) Conferring Infectivity and Functional Conservation among Simians
Authors: Blaise Sandra , Ruggieri Alessia , Dewannieux Marie , Cosset François-Loic , Heidmann Thierry
Published: December 2003

A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians—from New World monkeys to humans—are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation—located in the TM subunit—was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution.
Inhibition of Human Immunodeficiency Virus Type 1 Entry in Cells Expressing gp41-Derived Peptides
Authors: Egelhofer Marc , Brandenburg Gunda , Martinius Holger , Schult-Dietrich Patricia , Melikyan Gregory , Kunert Renate , Baum Christopher , Choi Ingrid , Alexandrov Alexander , von Laer Dorothee
Published: December 2003

As the limitations of antiretroviral drug therapy, such as toxicity and resistance, become evident, interest in alternative therapeutic approaches for human immunodeficiency virus (HIV) infection is growing. We developed the first gene therapeutic strategy targeting entry of a broad range of HIV type 1 (HIV-1) variants. Infection was inhibited at the level of membrane fusion by retroviral expression of a membrane-anchored peptide derived from the second heptad repeat of the HIV-1 gp41 transmembrane glycoprotein. To achieve maximal expression and antiviral activity, the peptide itself, the scaffold for presentation of the peptide on the cell surface, and the retroviral vector backbone were optimized. This optimized construct effectively inhibited virus replication in cell lines and primary blood lymphocytes. The membrane-anchored C-peptide was also shown to bind to free gp41 N peptides, suggesting that membrane-anchored antiviral C peptides have a mode of action similar to that of free gp41 C peptides. Preclinical toxicity and efficacy studies of this antiviral vector have been completed, and clinical trials are in preparation.
CXC Chemokine Ligand 10 Controls Viral Infection in the Central Nervous System: Evidence for a Role in Innate Immune Response through Recruitment and Activation of Natural Killer Cells
Authors: Trifilo Matthew J. , Montalto-Morrison Cynthia , Stiles Linda N. , Hurst Kelley R. , Hardison Jenny L. , Manning Jerry E. , Masters Paul S. , Lane Thomas E.
Published: December 2003

How chemokines shape the immune response to viral infection of the central nervous system (CNS) has largely been considered within the context of recruitment and activation of antigen-specific lymphocytes. However, chemokines are expressed early following viral infection, suggesting an important role in coordinating innate immune responses. Herein, we evaluated the contributions of CXC chemokine ligand 10 (CXCL10) in promoting innate defense mechanisms following coronavirus infection of the CNS. Intracerebral infection ofRAG1−/−mice with a recombinant CXCL10-expressing murine coronavirus (mouse hepatitis virus) resulted in protection from disease and increased survival that correlated with a significant increase in recruitment and activation of natural killer (NK) cells within the CNS. Accumulation of NK cells resulted in a reduction in viral titers that was dependent on gamma interferon secretion. These results indicate that CXCL10 expression plays a pivotal role in defense following coronavirus infection of the CNS by enhancing innate immune responses.
Persistent Recognition of Autologous Virus by High-Avidity CD8 T Cells in Chronic, Progressive Human Immunodeficiency Virus Type 1 Infection
Authors: Draenert R. , Verrill C. L. , Tang Y. , Allen T. M. , Wurcel A. G. , Boczanowski M. , Lechner A. , Kim A. Y. , Suscovich T. , Brown N. V. , Addo M. M. , Walker B. D.
Published: December 2003

CD8 T-cell responses are thought to be crucial for control of viremia in human immunodeficiency virus (HIV) infection but ultimately fail to control viremia in most infected persons. Studies in acute infection have demonstrated strong CD8-mediated selection pressure and evolution of mutations conferring escape from recognition, but the ability of CD8 T-cell responses that persist in late-stage infection to recognize viruses present in vivo has not been determined. Therefore, we studied 24 subjects with advanced HIV disease (median viral load = 142,000 copies/ml; median CD4 count = 71/μl) and determined HIV-1-specific CD8 T-cell responses to all expressed viral proteins using overlapping peptides by gamma interferon Elispot assay. Chronic-stage virus was sequenced to evaluate autologous sequences within Gag epitopes, and functional avidity of detected responses was determined. In these subjects, the median number of epitopic regions targeted was 13 (range, 2 to 39) and the median cumulative magnitude of CD8 T-cell responses was 5,760 spot-forming cells/106peripheral blood mononuclear cells (range, 185 to 24,700). On average six (range, one to 8) proteins were targeted. For 89% of evaluated CD8 T-cell responses, the autologous viral sequence was predicted to be well recognized by these responses and the majority of analyzed optimal epitopes were recognized with medium to high functional avidity by the contemporary CD8 T cells. Withdrawal of antigen by highly active antiretroviral therapy led to a significant decline both in breadth (P= 0.032) and magnitude (P= 0.0098) of these CD8 T-cell responses, providing further evidence that these responses had been driven by recognition of autologous virus. These results indicate that strong, broadly directed, and high-avidity gamma-interferon-positive CD8 T-cells directed at autologous virus persist in late disease stages, and the absence of mutations within viral epitopes indicates a lack of strong selection pressure mediated by these responses. These data imply functional impairment of CD8 T-cell responses in late-stage infection that may not be reflected by gamma interferon-based screening techniques.
Effects of Altering the Transcription Termination Signals of Respiratory Syncytial Virus on Viral Gene Expression and Growth In Vitro and In Vivo
Authors: Tran Kim C. , Collins Peter L. , Teng Michael N.
Published: December 2003

Nonsegmented negative-sense RNA viruses (mononegaviruses) control viral gene expression largely through a transcription gradient such that promoter-proximal genes are transcribed more abundantly than downstream genes. For some paramyxoviruses, naturally occurring differences in the levels of efficiency of transcription termination by various gene end (GE) signals provide an additional level of regulation of gene expression. The first two genes (NS1 and NS2) of respiratory syncytial virus (RSV) are particularly inefficient in termination. We investigated whether altering the termination efficiency (TE) of these two genes in infectious recombinant virus would affect transcription of promoter-proximal and promoter-distal genes, production of viral proteins, and viral replication in cell culture and in the respiratory tract of mice. Recombinant RSVs were constructed with mutations that increased or decreased the TE of the NS1 GE signal, increased that of the NS2 GE signal, or increased that of both signals. Increasing the TE of either or both GE signals resulted in decreased production of the related polycistronic readthrough mRNAs, which normally arise due to the failure of the viral polymerase to recognize the GE signal. This was accompanied by a small increase in the levels of monocistronic NS1 and NS2 mRNAs. Conversely, decreasing the TE of the NS1 GE increased the production of readthrough mRNAs concomitant with a decrease of monocistronic NS1 and NS2 mRNA levels. These changes were reflected in the levels of NS1 and NS2 protein. All of the mutant viruses displayed growth kinetics and virus yields similar to wild-type recombinant RSV (rA2) in both HEp-2 and Vero cells. In addition, all mutants grew similarly to rA2 in the upper- and lower-respiratory tract of BALB/c mice, though some of the mutants displayed slightly decreased replication. These data suggest that the natural inefficiencies of transcription termination by the NS1 and NS2 GE signals do not play important roles in controlling the magnitude of RSV gene expression or the efficiency of virus replication. Furthermore, while changes in the TE of a GE signal clearly can affect the transcription of its gene as well as that of the one immediately downstream, these changes did not have a significant effect on the overall transcriptional gradient.
Nucleocapsid-RNA Interactions Are Essential to Structural Stability but Not to Assembly of Retroviruses
Authors: Wang Shainn-Wei , Noonan Kristin , Aldovini Anna
Published: December 2003

The process of RNA incorporation into nascent virions is thought to be critical for efficient retroviral particle assembly and production. Here we show that human immunodeficiency virus type 1 mutant particles (which are highly unstable and break down soon after release from the cell) lacking nucleocapsid (NC) core protein-mediated RNA incorporation are produced efficiently and can be recovered at the normal density when viral protease function is abolished. These results demonstrate that RNA binding by Gag is not necessary for retroviral particle assembly. Rather, the RNA interaction with NC is critical for retroviral particle structural stability subsequent to release from the membrane and protease-mediated Gag cleavage. Thus, the NC-RNA interaction, and not simply the presence of RNA, provides the virus with a structural function that is critical for stable retroviral particle architecture.
Dual Stimulation of Epstein-Barr Virus (EBV)-Specific CD4+- and CD8+-T-Cell Responses by a Chimeric Antigen Construct: Potential Therapeutic Vaccine for EBV-Positive Nasopharyngeal Carcinoma
Authors: Taylor G. S. , Haigh T. A. , Gudgeon N. H. , Phelps R. J. , Lee S. P. , Steven N. M. , Rickinson A. B.
Published: December 2003

Virus-associated malignancies are potential targets for immunotherapeutic vaccines aiming to stimulate T-cell responses against viral antigens expressed in tumor cells. Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma, a high-incidence tumor in southern China, expresses a limited set of EBV proteins, including the nuclear antigen EBNA1, an abundant source of HLA class II-restricted CD4+T-cell epitopes, and the latent membrane protein LMP2, a source of subdominant CD8+T-cell epitopes presented by HLA class I alleles common in the Chinese population. We used appropriately modified gene sequences from a Chinese EBV strain to generate a modified vaccinia virus Ankara recombinant, MVA-EL, expressing the CD4 epitope-rich C-terminal domain of EBNA1 fused to full-length LMP2. The endogenously expressed fusion protein EL is efficiently processed via the HLA class I pathway, and MVA-EL-infected dendritic cells selectively reactivate LMP2-specific CD8+memory T-cell responses from immune donors in vitro. Surprisingly, endogenously expressed EL also directly accesses the HLA class II presentation pathway and, unlike endogenously expressed EBNA1 itself, efficiently reactivates CD4+memory T-cell responses in vitro. This unscheduled access to the HLA class II pathway is coincident with EL-mediated redirection of the EBNA1 domain from its native nuclear location to dense cytoplasmic patches. Given its immunogenicity to both CD4+and CD8+T cells, MVA-EL has potential as a therapeutic vaccine in the context of nasopharyngeal carcinoma.
Gamma Interferon-Mediated Inflammation Is Associated with Lack of Protection from Intravaginal Simian Immunodeficiency Virus SIVmac239 Challenge in Simian-Human Immunodeficiency Virus 89.6-Immunized Rhesus Macaques
Authors: Abel Kristina , La Franco-Scheuch Lisa , Rourke Tracy , Ma Zhong-Min , de Silva Veronique , Fallert Beth , Beckett Laurel , Reinhart Todd A. , Miller Christopher J.
Published: December 2003

Although gamma interferon (IFN-γ) is a key mediator of antiviral defenses, it is also a mediator of inflammation. As inflammation can drive lentiviral replication, we sought to determine the relationship between IFN-γ-related host immune responses and challenge virus replication in lymphoid tissues of simian-human immunodeficiency virus 89.6 (SHIV89.6)-vaccinated and unvaccinated rhesus macaques 6 months after challenge with simian immunodeficiency virus SIVmac239. Vaccinated-protected monkeys had low tissue viral RNA (vRNA) levels, vaccinated-unprotected animals had moderate tissue vRNA levels, and unvaccinated animals had high tissue vRNA levels. The long-term challenge outcome in vaccinated monkeys was correlated with the relative balance between SIV-specific IFN-γ T-cell responses and nonspecific IFN-γ-driven inflammation. Vaccinated-protected monkeys had slightly increased tissue IFN-γ mRNA levels and a high frequency of IFN-γ-secreting T cells responding to in vitro SIVgag peptide stimulation; thus, it is likely that they could develop effective anti-SIV cytotoxic T lymphocytes in vivo. In contrast, both high tissue IFN-γ mRNA levels and strong in vitro SIV-specific IFN-γ T-cell responses were detected in lymphoid tissues of vaccinated-unprotected monkeys. Unvaccinated monkeys had increased tissue IFN-γ mRNA levels but weak in vitro anti-SIV IFN-γ T-cell responses. In addition, in lymphoid tissues of vaccinated-unprotected and unvaccinated monkeys, the increased IFN-γ mRNA levels were associated with increased Mig/CXCL9, IP-10/CXCL10, and CXCR3 mRNA levels, suggesting that increased Mig/CXCL9 and IP-10/CXCL10 expression resulted in recruitment of CXCR3+activated T cells. Thus, IFN-γ-driven inflammation promotes SIV replication in vaccinated-unprotected and unvaccinated monkeys. Unlike all unvaccinated monkeys, most monkeys vaccinated with SHIV89.6 did not develop IFN-γ-driven inflammation, but they did develop effective antiviral CD8+-T-cell responses.
Potential Selection of LMP1 Variants in Nasopharyngeal Carcinoma
Authors: Edwards Rachel H. , Sitki-Green Diane , Moore Dominic T. , Raab-Traub Nancy
Published: December 2003

Seven distinct sequence variants of the Epstein-Barr virus latent membrane protein 1 (LMP1) have been identified by distinguishing amino acid changes in the carboxy-terminal domain. In this study the transmembrane domains are shown to segregate identically with the distinct carboxy-terminal amino acid sequences. Since strains of LMP1 have been shown to differ in abundance between blood and throat washes, nasopharyngeal carcinomas (NPCs) from areas of endemicity and nonendemicity with matching blood were analyzed by using a heteroduplex tracking assay to distinguish LMP1 variants. Striking differences were found between the compartments with the Ch1 strain prevalent in the NPCs from areas of endemicity and nonendemicity and the B958 strain prevalent in the blood of the endemic samples, whereas multiple strains of LMP1 were prevalent in the blood of the nonendemic samples. The possible selection against the B958 strain appearing in the tumor was highly significant (P< 0.0001). Sequence analysis of the full-length LMP1 variants revealed changes in many of the known and computer-predicted HLA-restricted epitopes with changes in key positions in multiple, potential epitopes for the specific HLA of the patients. These amino acid substitutions at key positions in the LMP1 epitopes may result in a reduced cytotoxic-T-lymphocyte response. These data indicate that strains with specific variants of LMP1 are more likely to be found in NPC. The predominance of specific LMP1 variants in NPC could reflect differences in the biologic or molecular properties of the distinct forms of LMP1 or possible immune selection.
PEGylation of a Vesicular Stomatitis Virus G Pseudotyped Lentivirus Vector Prevents Inactivation in Serum
Authors: Croyle Maria A. , Callahan Shellie M. , Auricchio Alberto , Schumer Gregg , Linse Klause D. , Wilson James M. , Brunner Lane J. , Kobinger Gary P.
Published: December 2003

One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing
Escherichia coli
beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.
Small-Molecule Inhibition of Human Immunodeficiency Virus Type 1 Replication by Specific Targeting of the Final Step of Virion Maturation
Authors: Zhou Jing , Yuan Xiong , Dismuke David , Forshey Brett M. , Lundquist Christopher , Lee Kuo-Hsiung , Aiken Christopher , Chen Chin Ho
Published: December 2003

Despite the effectiveness of currently available human immunodeficiency virus type 1 (HIV-1) therapies, a continuing need exists for new drugs to treat HIV-1 infection. We investigated the mechanism by which 3-
O
-{3′,3′-dimethylsuccinyl}-betulinic acid (DSB) inhibits HIV-1 replication. DSB functions at a late stage of the virus life cycle but does not inhibit the HIV-1 protease in vitro or interfere with virus assembly or release. DSB specifically delays the cleavage of Gag between the capsid (CA) and p2, resulting in delayed formation of the mature viral core and reduced HIV-1 infectivity. Replication of simian immunodeficiency virus (SIV) was resistant to DSB; however, a chimeric SIV carrying CA-p2 sequences from HIV-1 was inhibited by the drug, indicating that susceptibility to DSB maps to the CA-p2 region of the HIV-1 Gag protein. A single point mutation at the CA-p2 cleavage site of HIV-1 conferred strong resistance to DSB, confirming the target of the drug. HIV-1 strains that are resistant to a variety of protease inhibitors were sensitive to DSB. These findings indicate that DSB specifically protects the CA-p2 cleavage site from processing by the viral protease during virion maturation, thereby revealing a novel mechanism for pharmacologic inhibition of HIV-1 replication.
Persistent Infection with Ebola Virus under Conditions of Partial Immunity
Authors: Gupta Manisha , Mahanty Siddhartha , Greer Patricia , Towner Jonathan S. , Shieh Wun-Ju , Zaki Sherif R. , Ahmed Rafi , Rollin Pierre E.
Published: December 2003

Ebola hemorrhagic fever in humans is associated with high mortality; however, some infected hosts clear the virus and recover. The mechanisms by which this occurs and the correlates of protective immunity are not well defined. Using a mouse model, we determined the role of the immune system in clearance of and protection against Ebola virus. All CD8 T-cell-deficient mice succumbed to subcutaneous infection and had high viral antigen titers in tissues, whereas mice deficient in B cells or CD4 T cells cleared infection and survived, suggesting that CD8 T cells, independent of CD4 T cells and antibodies, are critical to protection against subcutaneous Ebola virus infection. B-cell-deficient mice that survived the primary subcutaneous infection (vaccinated mice) transiently depleted or not depleted of CD4 T cells also survived lethal intraperitoneal rechallenge for ≥25 days. However, all vaccinated B-cell-deficient mice depleted of CD8 T cells had high viral antigen titers in tissues following intraperitoneal rechallenge and died within 6 days, suggesting that memory CD8 T cells by themselves can protect mice from early death. Surprisingly, vaccinated B-cell-deficient mice, after initially clearing the infection, were found to have viral antigens in tissues later (day 120 to 150 post-intraperitoneal infection). Furthermore, following intraperitoneal rechallenge, vaccinated B-cell-deficient mice that were transiently depleted of CD4 T cells had high levels of viral antigen in tissues earlier (days 50 to 70) than vaccinated undepleted mice. This demonstrates that under certain immunodeficiency conditions, Ebola virus can persist and that loss of primed CD4 T cells accelerates the course of persistent infections. These data show that CD8 T cells play an important role in protection against acute disease, while both CD4 T cells and antibodies are required for long-term protection, and they provide evidence of persistent infection by Ebola virus suggesting that under certain conditions of immunodeficiency a host can harbor virus for prolonged periods, potentially acting as a reservoir.
Continued Production of Drug-Sensitive Human Immunodeficiency Virus Type 1 in Children on Combination Antiretroviral Therapy Who Have Undetectable Viral Loads
Authors: Persaud Deborah , Siberry George K. , Ahonkhai Aima , Kajdas Joleen , Monie Daphne , Hutton Nancy , Watson Douglas C. , Quinn Thomas C. , Ray Stuart C. , Siliciano Robert F.
Published: December 2003

Highly active antiretroviral therapy (HAART) can suppress plasma human immunodeficiency virus type 1 (HIV-1) levels to below the detection limit of ultrasensitive clinical assays. However, HIV-1 persists in cellular reservoirs, and in adults, persistent low-level viremia is detected with more sensitive assays. The nature of this viremia is poorly understood, and it is unclear whether viremia persists in children on HAART, particularly those who start therapy shortly after birth. We therefore developed a reverse transcriptase PCR (RT-PCR) assay that allows genotyping of HIV-1 protease even when viremia is present at levels as low as 5 copies of HIV-1 RNA/ml. We demonstrated that viremia persists in children with plasma virus levels below the limit of detection of clinical assays. Viremia was detected even in children who began HAART in early infancy and maintained such strong suppression of viremia that HIV-1-specific antibody responses were absent or minimal. The low-level plasma virus lacked protease inhibitor resistance mutations despite the frequent use of nelfinavir, which has a low mutational barrier to resistance. Protease sequences resembled those of viruses in the latent reservoir in resting CD4
+
T cells. Thus, in most children on HAART with clinically undetectable viremia, there is continued virus production without evolution of resistance in the protease gene.
Sequence Motifs Involved in the Regulation of Discontinuous Coronavirus Subgenomic RNA Synthesis
Authors: Zúñiga Sonia , Sola Isabel , Alonso Sara , Enjuanes Luis
Published: December 2003

Coronavirus transcription leads to the synthesis of a nested set of mRNAs with a leader sequence derived from the 5′ end of the genome. The mRNAs are produced by a discontinuous transcription in which the leader is linked to the mRNA coding sequences. This process is regulated by transcription-regulating sequences (TRSs) preceding each mRNA, including a highly conserved core sequence (CS) with high identity to sequences present in the virus genome and at the 3′ end of the leader (TRS-L). The role of TRSs was analyzed by reverse genetics using a full-length infectious coronavirus cDNA and site-directed mutagenesis of the CS. The canonical CS-B was nonessential for the generation of subgenomic mRNAs (sgmRNAs), but its presence led to transcription levels at least 10
3
-fold higher than those in its absence. The data obtained are compatible with a transcription mechanism including three steps: (i) formation of 5′-3′ complexes in the genomic RNA, (ii) base-pairing scanning of the nascent negative RNA strand by the TRS-L, and (iii) template switching during synthesis of the negative strand to complete the negative sgRNA. This template switch takes place after copying the CS sequence and was predicted in silico based on high base-pairing score between the nascent negative RNA strand and the TRS-L and minimum Δ
G
.
Production of Novel Ebola Virus-Like Particles from cDNAs: an Alternative to Ebola Virus Generation by Reverse Genetics
Authors: Watanabe Shinji , Watanabe Tokiko , Noda Takeshi , Takada Ayato , Feldmann Heinz , Jasenosky Luke D. , Kawaoka Yoshihiro
Published: December 2003

We established a plasmid-based system for generating infectious Ebola virus-like particles (VLPs), which contain an Ebola virus-like minigenome consisting of a negative-sense copy of the green fluorescent protein gene. This system produced nearly 10
3
infectious particles per ml of supernatant, equivalent to the titer of Ebola virus generated by a reverse genetics system. Interestingly, infectious Ebola VLPs were generated, even without expression of VP24. Transmission and scanning electron microscopic analyses showed that the morphology of the Ebola VLPs was indistinguishable from that of authentic Ebola virus. Thus, this system allows us to study Ebola virus entry, replication, and assembly without biosafety level 4 containment. Furthermore, it may be useful in vaccine production against this highly pathogenic agent.
Are mobile speed cameras effective? A controlled before and after study
Authors: Christie S M , Lyons R A , Dunstan F D , Jones S J
Published: December 2003

Objective:To identify the most appropriate metric to determine the effectiveness of mobile speed cameras in reducing road traffic related injuries.

Design:Controlled before and after study which compares two methods for examining the local effectiveness of mobile speed cameras—a circular zone around the camera and a route based method to define exposure at various distances from sites.

Setting:South Wales, UK.

Subjects:Persons injured by road traffic before and after intervention.

Intervention:Use of mobile speed cameras at 101 sites.

Main outcome measures:Rate ratio of injurious crashes at intervention and control sites.

Results:Camera sites had lower than expected numbers of injurious crashes up to 300 metres using circles and up to 500 metres using routes. Routes methods indicated a larger effect than the circles method except in the 100 metres nearest sites. A 500 metre route method was used to investigate the effect within strata of time after intervention, time of day, speed limit, and type of road user injured. The number of injurious crashes after intervention was substantially reduced (rate ratio 0.49, 95% confidence interval 0.42 to 0.57) and sustained throughout two years after intervention. Significant decreases occurred in daytime and night time, on roads with speed limits of 30 and 60–70 miles/hour and for crashes that injured pedestrians, motorcycle users, and car occupants.

Conclusions:The route based method is the better method of measure effectiveness at distances up to 500 metres. This method demonstrates a 51% reduction in injurious crashes.