National Institutes of Allergy and Infectious Diseases

Bethesda, MD, United States

National Institutes of Allergy and Infectious Diseases

Bethesda, MD, United States
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Harris A.K.,U.S. National Cancer Institute | Meyerson J.R.,U.S. National Cancer Institute | Meyerson J.R.,Mitochondrial Biology Unit | Matsuoka Y.,Laboratories of Infectious Diseases | And 10 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2013

Rapid antigenic variation of HA, the major virion surface protein of influenza A virus, remains the principal challenge to the development of broader and more effective vaccines. Some regions of HA, such as the stem region proximal to the viral membrane, are nevertheless highly conserved across strains and among most subtypes. A fundamental question in vaccine design is the extent to which HA stem regions on the surface of the virus are accessible to broadly neutralizing antibodies. Here we report 3D structures derived from cryoelectron tomography of HA on intact 2009 H1N1 pandemic virions in the presence and absence of the antibody C179, which neutralizes viruses expressing a broad range of HA subtypes, including H1, H2, H5, H6, and H9. By fitting previously derived crystallographic structures of trimeric HA into the density maps, we deduced the locations of the molecular surfaces of HA involved in interaction with C179. Using computational methods to distinguish individual unliganded HA trimers from those that have bound C179 antibody, we demonstrate that ~75% of HA trimers on the surface of the virus have C179 bound to the stem domain. Thus, despite their close packing on the viral membrane, the majority of HA trimers on intact virions are available to bind anti-stem antibodies that target conserved HA epitopes, establishing the feasibility of universal influenza vaccines that elicit such antibodies.

Hill B.J.,National Institutes of Allergy and Infectious Diseases | Skerry J.C.,USAMRIID | Smith T.J.,USAMRIID | Douek D.C.,National Institutes of Allergy and Infectious Diseases
BMC Microbiology | Year: 2010

Background. Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA)-accepted assay to detect and type botulinum neurotoxins (BoNTs) is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR) technology to determine the specific serotype of the neurotoxin. Results. We assayed purified C. botulinum DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD50required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies. Conclusions. While other studies have reported conventional or quantitative PCR-based assays for the detection of C. botulinum genes, our procedure's high-throughput capability and its portability allows most laboratories to quickly assess the possible presence of BoNTs either in food processing samples or in suspected cases of botulism. Thus, this assay provides rapid and specific detection of BoNT and toxin complex genes and would enable the targeting of appropriate therapeutics to infected individuals in a timely manner. © 2010 Hill et al; licensee BioMed Central Ltd.

Smith D.S.,University of Colorado at Denver | Guo K.,University of Colorado at Denver | Barrett B.S.,University of Colorado at Denver | Heilman K.J.,University of Colorado at Denver | And 4 more authors.
PLoS Pathogens | Year: 2011

Members of the APOBEC3 family of deoxycytidine deaminases counteract a broad range of retroviruses in vitro through an indirect mechanism that requires virion incorporation and inhibition of reverse transcription and/or hypermutation of minus strand transcripts in the next target cell. The selective advantage to the host of this indirect restriction mechanism remains unclear, but valuable insights may be gained by studying APOBEC3 function in vivo. Apobec3 was previously shown to encode Rfv3, a classical resistance gene that controls the recovery of mice from pathogenic Friend retrovirus (FV) infection by promoting a more potent neutralizing antibody (NAb) response. The underlying mechanism does not involve a direct effect of Apobec3 on B cell function. Here we show that while Apobec3 decreased titers of infectious virus during acute FV infection, plasma viral RNA loads were maintained, indicating substantial release of noninfectious particles in vivo. The lack of plasma virion infectivity was associated with a significant post-entry block during early reverse transcription rather than G-to-A hypermutation. The Apobec3-dependent NAb response correlated with IgG binding titers against native, but not detergent-lysed virions. These findings indicate that innate Apobec3 restriction promotes NAb responses by maintaining high concentrations of virions with native B cell epitopes, but in the context of low virion infectivity. Finally, Apobec3 restriction was found to be saturable in vivo, since increasing FV inoculum doses resulted in decreased Apobec3 inhibition. By analogy, maximizing the release of noninfectious particles by modulating APOBEC3 expression may improve humoral immunity against pathogenic human retroviral infections.

Feng S.,University of California at San Francisco | Ekong U.D.,Northwestern University | Lobritto S.J.,Morgan Stanley | Demetris A.J.,University of Pittsburgh | And 10 more authors.
JAMA - Journal of the American Medical Association | Year: 2012

Context: Although life-saving, liver transplantation burdens children with lifelong immunosuppression and substantial potential for morbidity and mortality. Objective: To establish the feasibility of immunosuppression withdrawal in pediatric living donor liver transplant recipients. Design, Setting, and Patients: Prospective, multicenter, open-label, single-group pilot trial conducted in 20 stable pediatric recipients (11 male; 55%) of parental living donor liver transplants for diseases other than viral hepatitis or an autoimmune disease who underwent immunosuppression withdrawal. Their median age was 6.9 months (interquartile range [IQR], 5.5-9.1 months) at transplant and 8 years 6 months (IQR, 6 years 5 months to 10 years 9 months) at study enrollment. Additional entry requirements included stable allograft function while taking a single immunosuppressive drug and no evidence of acute or chronic rejection or significant fibrosis on liver biopsy. Gradual immunosuppression withdrawal over a minimum of 36 weeks was instituted at 1 of 3 transplant centers between June 5, 2006, and November 18, 2009. Recipients were followed up for a median of 32.9 months (IQR, 1.0-49.9 months). Main Outcome Measures: The primary end point was the proportion of operationally tolerant patients, defined as patients who remained off immunosuppression therapy for at least 1 year with normal graft function. Secondary clinical end points included the durability of operational tolerance, and the incidence, timing, severity, and reversibility of rejection. Results: Of 20 pediatric patients, 12 (60%; 95% CI, 36.1%-80.9%) met the primary end point, maintaining normal allograft function for a median of 35.7 months (IQR, 28.1-39.7 months) after discontinuing immunosuppression therapy. Follow-up biopsies obtained more than 2 years after completing withdrawal showed no significant change compared with baseline biopsies. Eight patients did not meet the primary end point secondary to an exclusion criteria violation (n=1), acute rejection (n=2), or indeterminate rejection (n=5). Seven patients were treated with increased or reinitiation of immunosuppression therapy; all returned to baseline allograft function. Patients with operational tolerance compared with patients without operational tolerance initiated immunosuppression withdrawal later after transplantation (median of 100.6 months [IQR, 71.8-123.5] vs 73.0 months [IQR, 57.6-74.9], respectively; P=.03), had less portal inflammation (91.7% [95% CI, 61.5%-99.8%] vs 42.9% [95% CI, 9.9%-81.6%] with no inflammation; P=.04), and had lower total C4d scores on the screening liver biopsy (median of 6.1 [IQR, 5.1-9.3] vs 12.5 [IQR, 9.3-16.8]; P=.03). Conclusion: In this pilot study, 60% of pediatric recipients of parental living donor liver transplants remained off immunosuppression therapy for at least 1 year with normal graft function and stable allograft histology. ©2012 American Medical Association. All rights reserved.

Kirkpatrick B.D.,University of Vermont | Whitehead S.S.,National Institutes of Allergy and Infectious Diseases | Pierce K.K.,University of Vermont | Tibery C.M.,Center for Immunization Research | And 10 more authors.
Science Translational Medicine | Year: 2016

A dengue human challenge model can be an important tool to identify candidate dengue vaccines that should be further evaluated in large efficacy trials in endemic areas. Dengue is responsible for about 390 million infections annually. Protective efficacy results for the most advanced dengue vaccine candidate (CYD) were disappointing despite its ability to induce neutralizing antibodies against all four dengue virus (DENV) serotypes. TV003 is a live attenuated tetravalent DENV vaccine currently in phase 2 evaluation. To better assess the protective efficacy of TV003, a randomized double-blind, placebo-controlled trial in which recipients of TV003 or placebo were challenged 6 months later with a DENV-2 strain, rDEN2D30, was conducted. The primary endpoint of the trial was protection against dengue infection, defined as rDEN2D30 viremia. Secondary endpoints were protection against rash and neutropenia. All 21 recipients of TV003 who were challenged with rDEN2D30 were protected from infection with rDEN2D30. None developed viremia, rash, or neutropenia after challenge. In contrast, 100% of the 20 placebo recipients who were challenged with rDEN2D30 developed viremia, 80% developed rash, and 20% developed neutropenia. TV003 induced complete protection against challenge with rDEN2D30 administered 6months after vaccination. TV003 will be further evaluated in dengue-endemic areas. The controlled dengue human challenge model can accelerate vaccine development by evaluating the protection afforded by the vaccine, thereby eliminating poor candidates from further consideration before the initiation of large efficacy trials.

Callacondo D.,Cayetano Heredia Peruvian University | Garcia H.H.,Cayetano Heredia Peruvian University | Garcia H.H.,Instituto Nacional Of Ciencias Neurologicas | Gonzales I.,Instituto Nacional Of Ciencias Neurologicas | And 2 more authors.
Neurology | Year: 2012

Objective: To determine the frequency of spinal neurocysticercosis (NCC) in patients with basal subarachnoid NCC compared with that in individuals with viable limited intraparenchymal NCC (≤20 live cysts in the brain). Methods: We performed a prospective observational case-control study of patients with NCC involving the basal cisterns or patients with only limited intraparenchymal NCC. All patients underwent MRI examinations of the brain and the entire spinal cord to assess spinal involvement. Results: Twenty-seven patients with limited intraparenchymal NCC, and 28 patients with basal subarachnoid NCC were included in the study. Spinal involvement was found in 17 patients with basal subarachnoid NCC and in only one patient with limited intraparenchymal NCC (odds ratio 40.18, 95% confidence interval 4.74-340.31; p < 0.0001). All patients had extramedullary (intradural) spinal NCC, and the lumbosacral region was the most frequently involved (89%). Patients with extensive spinal NCC more frequently had ventriculoperitoneal shunt placement (7 of 7 vs 3 of 11; p = 0.004) and tended to have a longer duration of neurologic symptoms than those with regional involvement (72 months vs 24 months; p = 0.062). Conclusions: The spinal subarachnoid space is commonly involved in patients with basal subarachnoid NCC, compared with those with only intraparenchymal brain cysts. Spinal cord involvement probably explains serious late complications including chronic meningitis and gait disorders that were described before the introduction of antiparasitic therapy. MRI of the spine should be performed in basal subarachnoid disease to document spinal involvement, prevent complications, and monitor for recurrent disease. Copyright © 2012 by AAN Enterprises, Inc.

Researchers from the College of Veterinary Medicine, Northwest A&F University in Shaanxi, China claim they have successfully genetically modified cows to be resistant to bovine tuberculosis. Bovine tuberculosis is an infectious disease caused by the Mycobacterium bovis bacteria. It can also spread to and affect other mammals, including deer, goats, pigs, cats, dogs, and humans. In cattle, bovine tuberculosis has the characteristics of a respiratory disease, causing weight loss, cough, and fever in severe cases. It is mostly asymptomatic, although evidence of infection can be seen in the lymph glands, throat, or lungs of the animal. Bovine TB can spread from cattle to cattle through exposure to breath or discharges from the infected animal's mouth or nose, consumption of infected milk, before birth through the placenta, and indirectly via environmental contamination. Using an advanced technique called clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper), or more specifically, the CRISPR-Cas9 system, a genome editing tool, the scientists inserted a gene linked to tuberculosis resistance into 20 cattle. Results show that 11 of the genetically modified cows lived beyond the age of 3 months and were more resistant to the disease compared to their non-genetically modified counterparts. The researchers didn't note any side effects in the animals as a consequence of genetic modification. The full study was published online on Jan. 31 in the journal Genome Biology. With this new development and the use of the CRISPR/Cas9 system, the authors are hopeful that their study will be valuable for agricultural applications. Many developing countries have resorted to slaughtering thousands of infected cattle annually to put an end to bovine TB, but to no avail. More than 26,000 cattle were reportedly slaughtered in the UK back in 2013, costing taxpayers at least £100 million or more than $120 million. "I think this is a very neat study that demonstrates the feasibility of introducing a desired gene of interest via a potentially safer way," Suk See De Ravin, a researcher with the Laboratory of Host Defenses, under the U.S. National Institutes of Allergy and Infectious Diseases, remarked. De Ravin, who is not part of the study, also noted that this new information may be the key to raising animals with a robust resistance against diseases and may potentially pave the way to reducing or even eliminating the excessive use of antibiotics in livestock, which has its negative effects on human health, too. However, some experts think otherwise. "Although it is a thorough and novel paper on using gene technology in transgenic cattle at this stage I doubt if the research will have any application to prevention of TB in cattle using transgenic technology," Ian McConnell, emeritus professor of veterinary science at the University of Cambridge, told the BBC, adding that TB in cattle is more complicated than we can imagine. © 2017 Tech Times, All rights reserved. Do not reproduce without permission.

Pan S.,Murdoch University | Thompson R.C.A.,Murdoch University | Grigg M.E.,National Institutes of Allergy and Infectious Diseases | Sundar N.,National Institutes of Allergy and Infectious Diseases | And 2 more authors.
PLoS ONE | Year: 2012

Five different organs from 16 asymptomatic free-ranging marsupial macropods (Macropus rufus, M. fuliginosus, and M. robustus) from inland Western Australia were tested for infection with Toxoplasma gondii by multi-locus PCR-DNA sequencing. All macropods were infected with T. gondii, and 13 had parasite DNA in at least 2 organs. In total, 45 distinct T. gondii genotypes were detected. Fourteen of the 16 macropods were multiply infected with genetically distinct T. gondii genotypes that often partitioned between different organs. The presence of multiple T. gondii infections in macropods suggests that native mammals have the potential to promote regular cycles of sexual reproduction in the definitive felid host in this environment. © 2012 Pan et al.

Boularan C.,National Institutes of Allergy and Infectious Diseases | Kamenyeva O.,National Institutes of Allergy and Infectious Diseases | Cho H.,National Institutes of Allergy and Infectious Diseases | Kehrl J.H.,National Institutes of Allergy and Infectious Diseases
PLoS ONE | Year: 2014

Resistance to inhibitors of cholinesterase (Ric)-8A is a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13, which is implicated in cell signaling and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes. Ric-8A, Gαi subunits, and their regulators are localized at the midbody prior to abscission and linked to the final stages of cell division. Here, we identify a molecular mechanism by which Ric-8A affects cytokinesis and abscission by controlling Vps34 activity. We showed that Ric-8A protein expression is post-transcriptionally controlled during the cell cycle reaching its maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy) revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis, Ric-8A co-localized with Vps34 at the midbody along with Gαi and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity.

Murphy B.R.,National Institutes of Allergy and Infectious Diseases | Whitehead S.S.,National Institutes of Allergy and Infectious Diseases
Annual Review of Immunology | Year: 2011

Dengue virus (DENV) is a mosquito-borne member of the Flavivirus genus and includes four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), each of which is capable of causing dengue fever and dengue hemorrhagic fever/dengue shock syndrome. Serious disease can be seen during primary infection but is more frequent following second infection with a serotype different from that of a previous infection. Infection with wild-type DENV induces high-titered neutralizing antibody that can provide long-term immunity to the homotypic virus and can provide short-term immunity (only several months duration) to a heterotypic DENV. The high level of virus replication seen during both secondary infection with a heterotypic virus and during primary DENV infection in late infancy is a direct consequence of antibody-dependent enhancement of replication. This enhanced virus replication is mediated primarily by preexisting, nonneutralizing, or subneutralizing antibodies to the virion surface antigens that enhance access of the virion-antibody complex to FcγR-bearing cells. Vaccines will need to provide long-term protection against each of the four DENV serotypes by inducing neutralizing antibodies, and live, attenuated and various nonliving virus vaccines are in development. © 2011 by Annual Reviews. All rights reserved.

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