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Durbin A.P.,Center for Immunization Research | Kirkpatrick B.D.,University of Vermont | Pierce K.K.,University of Vermont | Schmidt A.C.,National Institute of Allergy and Infectious Diseases | Whitehead S.S.,National Institute of Allergy and Infectious Diseases
Vaccine | Year: 2011

The Laboratory of Infectious Diseases at the National Institute of Allergy and Infectious Diseases, National Institutes of Health has been engaged in an effort to develop a safe, efficacious, and affordable live attenuated tetravalent dengue vaccine (LATV) for more than ten years. Numerous recombinant monovalent DENV vaccine candidates have been evaluated in the SCID-HuH-7 mouse and in rhesus macaques to identify those candidates with a suitable attenuation phenotype. In addition, the ability of these candidates to infect and disseminate in Aedes mosquitoes had also been determined. Those candidates that were suitably attenuated in SCID-HuH-7 mice, rhesus macaques, and mosquitoes were selected for further evaluation in humans. This review will describe the generation of multiple candidate vaccines directed against each DENV serotype, the preclinical and clinical evaluation of these candidates, and the process of selecting suitable candidates for inclusion in a LATV dengue vaccine. © 2011 Elsevier Ltd. Source

Durbin A.P.,Center for Immunization Research | Whitehead S.S.,National Institute of Allergy and Infectious Diseases
Viruses | Year: 2011

Dengue has become the most important arboviral infection worldwide with more than 30 million cases of dengue fever estimated to occur each year. The need for a dengue vaccine is great and several live attenuated dengue candidate vaccines are proceeding through clinical evaluation. The need to induce a balanced immune response against all four DENV serotypes with a single vaccine has been a challenge for dengue vaccine developers. A live attenuated DENV chimeric vaccine produced by Sanofi Pasteur has recently entered Phase III evaluation in numerous dengue-endemic regions of the world. Viral interference between serotypes contained in live vaccines has required up to three doses of the vaccine be given over a 12-month period of time. For this reason, novel DENV candidate vaccines are being developed with the goal of achieving a protective immune response with an immunization schedule that can be given over the course of a few months. These next-generation candidates include DNA vaccines, recombinant adenovirus vectored vaccines, alphavirus replicons, and sub-unit protein vaccines. Several of these novel candidates will be discussed. © 2011 by the authors; licensee MDPI, Basel, Switzerland. Source

Lindow J.C.,University of Vermont | Durbin A.P.,Center for Immunization Research | Whitehead S.S.,National Institute of Allergy and Infectious Diseases | Pierce K.K.,University of Vermont | And 2 more authors.
Vaccine | Year: 2013

There are currently no vaccines or therapeutics to prevent dengue disease which ranges in severity from asymptomatic infections to life-threatening illness. The National Institute of Allergy and Infectious Diseases (NIAID) Division of Intramural Research has developed live, attenuated vaccines to each of the four dengue serotypes (DENV-1-DENV-4). Two doses (10. PFU and 1000. PFU) of three monovalent vaccines were tested in human clinical trials to compare safety and immunogenicity profiles. DEN4δ30 had been tested previously at multiple doses. The three dengue vaccine candidates tested (DEN1δ30, DEN2/4δ30, and DEN3δ30/31) were very infectious, each with a human infectious dose 50%. ≤. 10. PFU. Further, infectivity rates ranged from 90 to 100% regardless of dose, excepting DEN2/4δ30 which dropped from 100% at the 1000. PFU dose to 60% at the 10. PFU dose. Mean geometric peak antibody titers did not differ significantly between doses for DEN1δ30 (92 ± 19 vs. 214 ± 97, p=. 0.08); however, significant differences were observed between the 10. PFU and 1000. PFU doses for DEN2/4δ30, 19 ± 9 vs. 102 ± 25 (p=. 0.001), and DEN3δ30/31, 119 ± 135 vs. 50 ± 50 (p=. 0.046). No differences in the incidences of rash, neutropenia, or viremia were observed between doses for any vaccines, though the mean peak titer of viremia for DEN1δ30 was higher at the 1000. PFU dose (0.5 ± 0 vs. 1.1 ± 0.1, p=. 0.007). These data demonstrate that a target dose of 1000. PFU for inclusion of each dengue serotype into a tetravalent vaccine is likely to be safe and generate a balanced immune response for all serotypes. © 2013 Elsevier Ltd. Source

Dowd K.A.,U.S. National Institutes of Health | Jost C.A.,U.S. National Institutes of Health | Durbin A.P.,Center for Immunization Research | Whitehead S.S.,U.S. National Institutes of Health | Pierson T.C.,U.S. National Institutes of Health
PLoS Pathogens | Year: 2011

Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this "multiple-hit" perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus "breathing" in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization. Source

Mukherjee S.,U.S. National Institutes of Health | Dowd K.A.,U.S. National Institutes of Health | Manhart C.J.,U.S. National Institutes of Health | Ledgerwood J.E.,U.S. National Institutes of Health | And 3 more authors.
Journal of Virology | Year: 2014

The production of neutralizing antibodies (NAbs) is a correlate of protection for many human vaccines, including currently licensed vaccines against flaviviruses. NAbs are typically measured using a plaque reduction neutralization test (PRNT). Despite its extensive use, parameters that impact the performance of the PRNT have not been investigated from a mechanistic perspective. The results of a recent phase IIb clinical trial of a tetravalent dengue virus (DENV) vaccine suggest that NAbs, as measured using a PRNT performed with Vero cells, do not correlate with protection. This surprising finding highlights the importance of understanding how well the PRNT captures the complexity of the NAb response to DENV. In this study, we demonstrated that the structural heterogeneity of flaviviruses arising from inefficient virion maturation impacts the results of neutralization assays in a cell type-dependent manner. Neutralization titers of several monoclonal antibodies were significantly reduced when assayed on Vero cells compared to Raji cells expressing DC-SIGNR. This pattern can be explained by differences in the efficiency with which partially mature flaviviruses attach to each cell type, rather than a differential capacity of antibody to block infection. Vero cells are poorly permissive to the fraction of virions that are most sensitive to neutralization. Analysis of sera from recipients of live-attenuated monovalent DENV vaccine candidates revealed a strong correlation between the sensitivity of serum antibodies to the maturation state of DENV and cell type-dependent patterns of neutralization. Cross-reactive patterns of neutralization may be underrepresented by the "gold-standard" PRNT that employs Vero cells. © 2014, American Society for Microbiology. Source

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