Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

Temporally integrated single cell RNA sequencing analysis of PBMC from experimental and natural primary human DENV-1 infections.

Dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To determine the kinetic transcriptional signature associated with experimental primary DENV-1 infection and to assess how closely this profile correlates with the transcriptional signature accompanying natural primary DENV-1 infection, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both experimental and natural primary DENV-1 infection resulted in overlapping patterns of inflammatory gene upregulation, natural primary DENV-1 infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by experimental and natural primary DENV infection are similar, but that natural primary DENV-1 infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state.

Clinical acceptance of single-unit crowns and its association with impression and tissue displacement techniques: Findings from the National Dental Practice-Based Research Network.

STATEMENT OF PROBLEM: The definitive impression for a single-unit crown involves many material and technique factors that may affect the success of the crown. PURPOSE: The purpose of this prospective cohort study was to determine whether impression technique (tray selection), impression material, or tissue displacement technique are associated with the clinical acceptability of the crown (CAC). MATERIAL AND METHODS: Dentists in the National Dental Practice-Based Research Network documented details of the preparation, impression, and delivery of 3730 consecutive single-unit crowns. Mixed-effects logistic regression analyses were performed to evaluate associations between impression techniques and materials and the CAC and to assess associations between the presence of a subgingival margin with the displacement technique and the outcome variables CAC and number of impressions required. RESULTS: Of the 3730 crowns, 3589 (96.2%) were deemed clinically acceptable. A significant difference in the CAC was found with different impression techniques (P<.001) and different impression materials (P<.001). The percentage of the CAC for digital scans was 99.5%, 95.8% for dual-arch trays, 95.2% for quadrant trays, and 94.0% for complete-arch impression trays. Although no statistically significant difference was found in the CAC produced with dual-arch trays without both mesial and distal contacts, crowns fabricated under these conditions were less likely to achieve excellent occlusion. The percentage of the CAC for digital scans was 99.5%, 97.0% for polyether impressions, 95.5% for polyvinyl siloxane impressions, and 90.5% for other impression materials. Accounting for the location of the margin, the use of a dual-cord displacement technique was significantly associated with lower rates of requiring more than 1 impression (P=.015, odds ratio=1.43). CONCLUSIONS: Dual-arch trays produced clinically acceptable crowns; however, if the prepared tooth was unbounded, the occlusal fit was more likely to have been compromised. Digital scans produced a slightly higher rate of CAC than conventional impression materials. The use of a dual-cord technique was associated with a decreased need to remake impressions when the margins were subgingival.

Laboratory Technician Assessment of the Quality of Single-Unit Crown Preparations and Impressions as Predictors of the Clinical Acceptability of Crowns as Determined by the Treating Dentist: Findings from the National Dental Practice-Based Research Network.

PURPOSE: In-laboratory assessment by laboratory technicians may offer insight to increase clinical success of dental crowns, and research in this area is lacking. MATERIALS AND METHODS: Dentists in the National Dental Practice-Based Research Network enrolled patients in a study about single-unit crowns; laboratory technicians evaluated the quality of tooth preparations and impressions. The primary outcome for each crown was clinical acceptability (CAC), as judged by the treating dentist. A secondary outcome was "Goodness of Fit (GOF)," a composite score of several aspects of clinical fit, also judged by the study dentist. A mixed-effects logistic regression was used to analyze associations between laboratory technician ratings and the CAC and GOF. RESULTS: Dentists (n = 205) evaluated 3731 crowns. Technicians ranked the marginal detail of impressions as good or excellent in 92% of cases; other aspects of the impression were ranked good or excellent 88% of the time. Regarding tooth preparation, about 90% of preparations were considered adequate (neither excessive nor inadequate reduction). Factors associated with higher CAC were more preparation taper, and use of optical imaging. Factors associated with better GOF were higher impression quality, greater occlusal reduction, more preparation taper, and optical imaging. CONCLUSIONS: Overall quality of preparations and impressions was very high, as evaluated by laboratory technicians. Several clinical parameters were associated with higher CAC and GOF. Clinicians who struggle with crown remakes might consider less conservative tooth preparation, as well as using digital impression technology.

Impact of prior flavivirus immunity on Zika virus infection in rhesus macaques.

Studies have demonstrated cross-reactivity of anti-dengue virus (DENV) antibodies in human sera against Zika virus (ZIKV), promoting increased ZIKV infection in vitro. However, the correlation between in vitro and in vivo findings is not well characterized. Thus, we evaluated the impact of heterotypic flavivirus immunity on ZIKV titers in biofluids of rhesus macaques. Animals previously infected (≥420 days) with DENV2, DENV4, or yellow fever virus were compared to flavivirus-naïve animals following infection with a Brazilian ZIKV strain. Sera from DENV-immune macaques demonstrated cross-reactivity with ZIKV by antibody-binding and neutralization assays prior to ZIKV infection, and promoted increased ZIKV infection in cell culture assays. Despite these findings, no significant differences between flavivirus-naïve and immune animals were observed in viral titers, neutralizing antibody levels, or immune cell kinetics following ZIKV infection. These results indicate that prior infection with heterologous flaviviruses neither conferred protection nor increased observed ZIKV titers in this non-human primate ZIKV infection model.

Remake Rates for Single-Unit Crowns in Clinical Practice: Findings from The National Dental Practice-Based Research Network.

PURPOSE: Some crowns returned from the laboratory are clinically unacceptable, and dentists must remake them. The objectives of this study were to: (1) quantify the remake rate of single-unit crowns; and (2) identify factors significantly associated with crown remakes and intraoral fit. MATERIALS AND METHODS: Dentists participating in the National Dental Practice-Based Research Network recruited patients needing crowns and documented fabrication techniques, patient characteristics, and outcomes. Crowns were considered clinically acceptable or rejected. Also, various aspects of the clinical fit of the crown were graded and categorized as 'Goodness of Fit (GOF).' Dentist and patient characteristics were tested statistically for associations with crown acceptability and GOF. RESULTS: More than 200 dentists participated in this study (N = 205) and evaluated 3750 single-unit crowns. The mean age (years) of patients receiving a crown was 55. The remake rate for crowns was 3.8%. The range of rejection rates among individual practitioners was 0% to 42%. Most clinicians (118, or 58%) did not reject any crowns; all rejections came from 42% of the clinicians (n = 87). The most common reasons for rejections were proximal misfit, marginal errors, and esthetic failures. Fewer years in practice was significantly associated with lower crown success rates and lower fit scores. GOF was also associated with practice busyness and patient insurance status, patient gender (dentists reported better fit for female patients), and patient ethnicity. CONCLUSIONS: The crown remake rate in this study was about 4%. Remakes and crown GOF were associated with certain dentist and practice characteristics.

Impression Techniques Used for Single-Unit Crowns: Findings from the National Dental Practice-Based Research Network.

PURPOSE: To: (1) determine which impression and gingival displacement techniques practitioners use for single-unit crowns on natural teeth; and (2) test whether certain dentist and practice characteristics are significantly associated with the use of these techniques. MATERIALS AND METHODS: Dentists participating in the National Dental Practice-Based Research Network were eligible for this survey study. The study used a questionnaire developed by clinicians, statisticians, laboratory technicians, and survey experts. The questionnaire was pretested via cognitive interviewing with a regionally diverse group of practitioners. The survey included questions regarding gingival displacement and impression techniques. Survey responses were compared by dentist and practice characteristics using ANOVA. RESULTS: The response rate was 1777 of 2132 eligible dentists (83%). Regarding gingival displacement, most clinicians reported using either a single cord (35%) or dual cord (35%) technique. About 16% of respondents preferred an injectable retraction technique. For making impressions, the most frequently used techniques and materials are: poly(vinyl siloxane), 77%; polyether, 12%; optical/digital, 9%. A dental auxiliary or assistant made the final impression 2% of the time. Regarding dual-arch impression trays, 23% of practitioners report they typically use a metal frame tray, 60% use a plastic frame, and 16% do not use a dual-arch tray. Clinicians using optical impression techniques were more likely to be private practice owners or associates. CONCLUSIONS: This study documents current techniques for gingival displacement and making impressions for crowns. Certain dentist and practice characteristics are significantly associated with these techniques.

Preparation Techniques Used to Make Single-Unit Crowns: Findings from The National Dental Practice-Based Research Network.

PURPOSE: To: (1) determine which preparation techniques clinicians use in routine clinical practice for single-unit crown restorations; (2) test whether certain practice, dentist, and patient characteristics are significantly associated with these techniques. MATERIALS AND METHODS: Dentists in the National Dental Practice-Based Research Network participated in a questionnaire regarding preparation techniques, dental equipment used for single-unit crown preparations, scheduled chair time, occlusal clearance determination, location of finish lines, magnification during preparation, supplemental lighting, shade selection, use of intraoral photographs, and trimming dies. Survey responses were compared by dentist and practice characteristics using ANOVA. RESULTS: Of the 2132 eligible dentists, 1777 (83%) responded to the survey. The top two margin configuration choices for single-unit crown preparation for posterior crowns were chamfer/heavy chamfer (65%) and shoulder (23%). For anterior crowns, the most prevalent choices were the chamfer (54%) and the shoulder (37%) configurations. Regarding shade selection, a combination of dentist, assistant, and patient input was used to select anterior shades 59% of the time. Photographs are used to communicate shade selection with the laboratory in about half of esthetically demanding cases. The ideal finish line was located at the crest of gingival tissue for 49% of respondents; 29% preferred 1 mm below the crest; and 22% preferred the finish line above the crest of tissue. Average chair time scheduled for a crown preparation appointment was 76 ± 21 minutes. Practice and dentist characteristics were significantly associated with margin choice including practice type (p < 0.001), region (p < 0.001), and years since graduation (p < 0.001). CONCLUSIONS: Network dentists prefer chamfer/heavy chamfer margin designs, followed by shoulder preparations. These choices were related to practice and dentist characteristics.

Tear Trough Incision for External Dacryocystorhinostomy.

PURPOSE: Scar formation is a frequently cited complication of external dacryocystorhinostomy (exDCR). The purpose of this study is to evaluate scar appearance after exDCR with the skin incision placed in the tear trough. METHODS: Multicenter, prospective, noncomparative interventional study was approved by the University of Colorado Institutional Review Board. Patients undergoing exDCR from February 2013 to January 2014 were included in the study, and surgeries were performed by all authors. The incision site for all patients started just under the medial canthal tendon and extended inferolaterally into the tear trough for 10 mm to 15 mm. External dacryocystorhinostomy was performed in the usual manner, and the incision was closed according to the surgeon's preference. At 3 months postop, all patients were asked to rate their scar on the basis of the following grading scale: 0, invisible incision; 1, minimally visible incision; 2, moderately visible incision; and 3, very visible incision. Functional success of the surgery was also determined by asking the patients if their symptoms resolved, improved, or did not change. External photographs taken at 3 months after surgery were graded by 3 independent oculofacial and facial plastic surgeons using the same grading scale. RESULTS: Seventy-two surgeries were performed in 68 consecutive exDCR patients with nasolacrimal duct obstruction during the study period. Sixty-nine out of 72 patients reported improved or resolved symptoms (95.8%). The average patient scar grade was 0.21, while the average surgeon scar grade was 0.99 (p < 0.001). Sixty out of the 72 patients graded the scar as invisible (83.3%), and only 3 patients graded the scar as moderately visible (4.2%). No patients graded the scar as very visible. Of the 216 surgeon grades, 55 scars were graded as invisible (25.5%), while 8 were graded as very visible (3.7%). CONCLUSIONS: Scar appearance after exDCR with the incision placed in the tear trough is minimally visible to surgeons, and more importantly, nearly invisible to patients.

Efficacy and immunogenicity following dengue virus-1 human challenge after a tetravalent prime-boost dengue vaccine regimen: an open-label, phase 1 trial.

BACKGROUND: Dengue human infection models (DHIMs) are important tools to down-select dengue vaccine candidates and establish tetravalent efficacy before advanced clinical field trials. We aimed to provide data for the safety and immunogenicity of DHIM and evaluate dengue vaccine efficacy. METHODS: We performed an open-label, phase 1 trial at the University of Maryland (Baltimore, MD, USA). Eligible participants were healthy individuals aged 18-50 years who either previously received a tetravalent dengue purified inactivated vaccine prime followed by a live-attenuated vaccine boost (ie, the vaccinee group), or were unvaccinated flavivirus-naive participants (ie, the control group). Participants in the vaccinee group with detectable pre-challenge dengue virus-1 neutralising antibody titres and flavivirus-naive participants in the control group were inoculated with dengue virus-1 strain 45AZ5 in the deltoid region, 27-65 months following booster dosing. These participants were followed-up from days 4-16 following dengue virus-1 live virus human challenge, with daily real-time quantitative PCR specific to dengue virus-1 RNA detection, and dengue virus-1 solicited local and systemic adverse events were recorded. The primary outcomes were safety (ie, solicited local and systemic adverse events) and vaccine efficacy (ie, dengue virus-1 RNAaemia) following dengue challenge. This study is registered with ClinicalTrials.gov, number NCT04786457. FINDINGS: In January 2021, ten eligible participants were enrolled; of whom, six (60%) were in the vaccinee group and four (40%) were in the control group. Daily quantitative PCR detected dengue virus-1 RNA in nine (90%) of ten participants (five [83%] of six in the vaccinee group and all four [100%] in the control group). The mean onset of RNAaemia occurred on day 5 (SD 1·0) in the vaccinee group versus day 8 (1·5) in the control group (95% CI 1·1-4·9; p=0·007), with a trend towards reduced RNAaemia duration in the vaccinee group compared with the control group (8·2 days vs 10·5 days; 95% CI -0·08 to 4·68; p=0·056). Mild-to-moderate symptoms (nine [90%] of ten), leukopenia (eight [89%] of nine), and elevated aminotransferases (seven [78%] of nine) were commonly observed. Severe adverse events were detected only in the vaccinee group (fever ≥38·9°C in three [50%] of six, headache in one [17%], and transient grade 4 aspartate aminotransferase elevation in one [17%]). No deaths were reported. INTERPRETATION: Participants who had tetravalent dengue purified inactivated vaccine prime and live-attenuated vaccine boost were unprotected against dengue virus-1 infection and further showed increased clinical, immunological, and transcriptomic evidence for inflammation potentially mediated by pre-existing infection-enhancing antibodies. This study highlights the impact of small cohort, human challenge models studying dengue pathogenesis and downstream vaccine development. FUNDING: Military Infectious Disease Research Program and Medical Technology Enterprise Consortium and Advanced Technology International.

Development of a transmission model for dengue virus.

BACKGROUND: Dengue virus (DENV) research has historically been hampered by the lack of a susceptible vertebrate transmission model. Recently, there has been progress towards such models using several varieties of knockout mice, particularly those deficient in type I and II interferon receptors. Based on the critical nature of the type I interferon response in limiting DENV infection establishment, we assessed the permissiveness of a mouse strain with a blunted type I interferon response via gene deficiencies in interferon regulatory factors 3 and 7 (IRF3/7 -/- -/-) with regards to DENV transmission success. We investigated the possibility of transmission to the mouse by needle and infectious mosquito, and subsequent transmission back to mosquito from an infected animal during its viremic period. METHODS: Mice were inoculated subcutaneously with non-mouse adapted DENV-2 strain 1232 and serum was tested for viral load and cytokine production each day. Additionally, mosquitoes were orally challenged with the same DENV-2 strain via artificial membrane feeder, and then allowed to forage or naïve mice. Subsequently, we determined acquisition potential by allowing naïve mosquitoes on forage on exposed mice during their viremic period. RESULTS: Both needle inoculation and infectious mosquito bite(s) resulted in 100% infection. Significant differences between these groups in viremia on the two days leading to peak viremia were observed, though no significant difference in cytokine production was seen. Through our determination of transmission and acquisition potentials, the transmission cycle (mouse-to mosquito-to mouse) was completed. We confirmed that the IRF3/7 -/- -/- mouse supports DENV replication and is competent for transmission experiments, with the ability to use a non-mouse adapted DENV-2 strain. A significant finding of this study was that this IRF3/7 -/- -/- mouse strain was able to be infected by and transmit virus to mosquitoes, thus providing means to replicate the natural transmission cycle of DENV. CONCLUSION: As there is currently no approved vaccine for DENV, public health monitoring and a greater understanding of transmission dynamics leading to outbreak events are critical. The further characterization of DENV using this model will expand knowledge of key entomological, virological and immunological components of infection establishment and transmission events.

Strength comparison of four techniques to secure implant attachment housings to complete dentures.

STATEMENT OF PROBLEM: Implant overdentures become thinner and weaker after direct transfer of implant attachment housings. While much has been published on denture repair, these data do not directly apply to implant overdentures because the introduction of a metal housing changes the character of the repair. It is desirable to make a strong repair to avoid prosthesis fracture. PURPOSE: The purpose of this study was to compare the flexural strengths of 4 different methods for chairside direct transfer of implant attachment housings. MATERIAL AND METHODS: Eighty 11.5 × 9.1 × 39 mm heat-polymerized acrylic resin blocks were processed, assessed for porosities, and polished. An 8.5 mm diameter hole was drilled to a depth of 5 mm in the center of each block. Attachment housings were set into the bases with 4 different repair materials: autopolymerized acrylic resin (APAR), light-polymerized acrylic resin (LPAR), autopolymerized acrylic resin with silanated attachment housings (APSAH), and light-polymerized acrylic resin with silanated attachment housings (LPSAH). Blocks were immersed in water for 30 days in an incubator. A 3-point bend test was done in a universal testing machine, and load to fracture was recorded (MPa). Results were compared with 2-way ANOVA (α=.05). RESULTS: APSAH had the highest mean flexural strength at fracture (863.1 ±87 MPa) as compared to APAR (678.4 ±72.4 MPa), LPAR (550.9 ±119.3 MPa), and LPSAH (543.2 ±100.8 MPa). A comparison among the 4 groups showed that there were significant differences in maximum flexural strength (P<.001). The mean maximum strength of autopolymerized acrylic resin groups was significantly higher than light-polymerized acrylic resin groups. Silanation increased strength significantly compared to nonsilanated groups. CONCLUSIONS: The flexural strength of autopolymerized acrylic resin with silanated attachment housings was significantly higher than autopolymerized acrylic resin alone, light-polymerized acrylic resin alone, or light-polymerized acrylic resin with silanated attachment housings. Autopolymerized acrylic resin produced stronger constructs than light-polymerized materials.

Humoral immune responses associated with control of SARS-CoV-2 breakthrough infections in a vaccinated US military population.

BACKGROUND: COVID-19 vaccines have been critical for protection against severe disease following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but gaps remain in our understanding of the immune responses that contribute to controlling subclinical and mild infections. METHODS: Vaccinated, active-duty US military service members were enrolled in a non-interventional, minimal-risk, observational study starting in May, 2021. Clinical data, serum, and saliva samples were collected from study participants and were used to characterise the humoral immune responses to vaccination and to assess its impact on clinical and subclinical infections, as well as virologic outcomes of breakthrough infections (BTI) including viral load and infection duration. FINDINGS: The majority of VIRAMP participants had received the Pfizer COVID-19 vaccine and by January, 2022, N = 149 had a BTI. The median BTI duration (PCR+ days) was 4 days and the interquartile range was 1-8 days. Participants that were nucleocapsid seropositive prior to their BTI had significantly higher levels of binding and functional antibodies to the spike protein, shorter median duration of infections, and lower median peak viral loads compared to seronegative participants. Furthermore, levels of neutralising antibody, ACE2 blocking activity, and spike-specific IgA measured prior to BTI also correlated with the duration of infection. INTERPRETATION: We extended previous findings and demonstrate that a subset of vaccine-induced humoral immune responses, along with nucleocapsid serostatus are associated with control of SARS-CoV-2 breakthrough infections in the upper airways. FUNDING: This work was funded by the DoD Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense (JPEO-CBRND) in collaboration with the Defense Health Agency (DHA) COVID-19 funding initiative for the VIRAMP study.

Zika-specific neutralizing antibodies targeting inter-dimer envelope epitopes.

Zika virus (ZIKV) is an emerging pathogen that causes devastating congenital defects. The overlapping epidemiology and immunologic cross-reactivity between ZIKV and dengue virus (DENV) pose complex challenges to vaccine design, given the potential for antibody-dependent enhancement of disease. Therefore, classification of ZIKV-specific antibody targets is of notable value. From a ZIKV-infected rhesus macaque, we identify ZIKV-reactive B cells and isolate potent neutralizing monoclonal antibodies (mAbs) with no cross-reactivity to DENV. We group these mAbs into four distinct antigenic groups targeting ZIKV-specific cross-protomer epitopes on the envelope glycoprotein. Co-crystal structures of representative mAbs in complex with ZIKV envelope glycoprotein reveal envelope-dimer epitope and unique dimer-dimer epitope targeting. All four specificities are serologically identified in convalescent humans following ZIKV infection, and representative mAbs from all four groups protect against ZIKV replication in mice. These results provide key insights into ZIKV-specific antigenicity and have implications for ZIKV vaccine, diagnostic, and therapeutic development.

A residual granuloma in association with a dental implant.

At times, dental implants are placed into sites with a history of periapical pathology. Sometimes the infection is active, and other times the tooth may have been extracted years before implant placement. In either case, the possibility exists for long-term residual cysts or infections that can negatively impact the prognosis of the implant. In this case report, an implant is placed into a healed mandibular ridge several months after extraction of the tooth. A radiolucency was noted on routine radiographic examination 2 years later. Surgical inspection and histology revealed a periapical granuloma with acute and chronic inflammatory cells. After surgical curettage of the site, the patient healed without complication. Implants may develop apical pathology as a result of a preexisting long-term residual infection.

Phage display demonstrates durable differences in serological profile by route of inoculation in primary infections of non-human primates with Dengue Virus 1.

Natural dengue virus (DENV) infections occur by mosquito bite but how the inoculation route affects the humoral immune response is unknown. We serologically profiled 20 non-human primates (NHP) from a prior study of DENV1 infection where animals were inoculated by mosquito (N = 10) or subcutaneous injection (N = 10). Using a comprehensive, densely tiled and highly redundant pan-flavivirus programmable phage library containing 91,562 overlapping 62 amino acid peptides, we produced a high-resolution map of linear peptide sequences enriched during DENV seroconversion. Profiles in mosquito-inoculated and subcutaneously-inoculated animals were similar up to 90 days after primary infection, but diverged at 1 year with differences in sero-reactivity in the Envelope (E; residues 215-406; p < 0.08), and Nonstructural-3 (NS3; residues 549-615; p < 0.05) proteins in mosquito-inoculated versus subcutaneously-inoculated animals. Within the E protein, residues 339-384 in domain III accounted for > 99% of the observed sero-reactivity difference. Antibody breadth did not vary by mode of inoculation. The differential reactivity to E domain III seen by phage display validated orthogonally by ELISA, but did not correlate with late neutralization titers. Serological profiling of humoral immune responses to DENV infection in NHP by programmable phage display demonstrated durable differences in sero-reactivity by route of inoculation.

Monomeric IgA Antagonizes IgG-Mediated Enhancement of DENV Infection.

Dengue virus (DENV) is a prevalent human pathogen, infecting approximately 400 million individuals per year and causing symptomatic disease in approximately 100 million. A distinct feature of dengue is the increased risk for severe disease in some individuals with preexisting DENV-specific immunity. One proposed mechanism for this phenomenon is antibody-dependent enhancement (ADE), in which poorly-neutralizing IgG antibodies from a prior infection opsonize DENV to increase infection of Fc gamma receptor-bearing cells. While IgM and IgG are the most commonly studied DENV-reactive antibody isotypes, our group and others have described the induction of DENV-specific serum IgA responses during dengue. We hypothesized that monomeric IgA would be able to neutralize DENV without the possibility of ADE. To test this, we synthesized IgG and IgA versions of two different DENV-reactive monoclonal antibodies. We demonstrate that isotype-switching does not affect the antigen binding and neutralization properties of the two mAbs. We show that DENV-reactive IgG, but not IgA, mediates ADE in Fc gamma receptor-positive K562 cells. Furthermore, we show that IgA potently antagonizes the ADE activity of IgG. These results suggest that levels of DENV-reactive IgA induced by DENV infection might regulate the overall IgG mediated ADE activity of DENV-immune plasma in vivo, and may serve as a predictor of disease risk.

Designed, highly expressing, thermostable dengue virus 2 envelope protein dimers elicit quaternary epitope antibodies.

Dengue virus (DENV) is a worldwide health burden, and a safe vaccine is needed. Neutralizing antibodies bind to quaternary epitopes on DENV envelope (E) protein homodimers. However, recombinantly expressed soluble E proteins are monomers under vaccination conditions and do not present these quaternary epitopes, partly explaining their limited success as vaccine antigens. Using molecular modeling, we found DENV2 E protein mutations that induce dimerization at low concentrations (<100 pM) and enhance production yield by more than 50-fold. Cross-dimer epitope antibodies bind to the stabilized dimers, and a crystal structure resembles the wild-type (WT) E protein bound to a dimer epitope antibody. Mice immunized with the stabilized dimers developed antibodies that bind to E dimers and not monomers and elicited higher levels of DENV2-neutralizing antibodies compared to mice immunized with WT E antigen. Our findings demonstrate the feasibility of using structure-based design to produce subunit vaccines for dengue and other flaviviruses.

Enhanced dengue vaccine virus replication and neutralizing antibody responses in immune primed rhesus macaques.

Antibody-dependent enhancement (ADE) is suspected to influence dengue virus (DENV) infection, but the role ADE plays in vaccination strategies incorporating live attenuated virus components is less clear. Using a heterologous prime-boost strategy in rhesus macaques, we examine the effect of priming with DENV purified inactivated vaccines (PIVs) on a tetravalent live attenuated vaccine (LAV). Sera exhibited low-level neutralizing antibodies (NAb) post PIV priming, yet moderate to high in vitro ADE activity. Following LAV administration, the PIV primed groups exhibited DENV-2 LAV peak viremias up to 1,176-fold higher than the mock primed group, and peak viremia correlated with in vitro ADE. Furthermore, PIV primed groups had more balanced and higher DENV-1-4 NAb seroconversion and titers than the mock primed group following LAV administration. These results have implications for the development of effective DENV vaccine prime-boost strategies and for our understanding of the role played by ADE in modulating DENV replication.

A randomized clinical trial to compare diagnostic casts made using plastic and metal trays.

STATEMENT OF PROBLEM: Virtually every prosthodontic rehabilitation involves making diagnostic casts for analysis and prosthesis fabrication. Frequently, these casts are produced using irreversible hydrocolloid (IH) impression materials in stock metal or plastic trays. However, it is unclear whether one technique produces a more accurate cast. PURPOSE: The purpose of this randomized clinical trial was to compare the linear accuracy of diagnostic casts produced using IH with 1 of 3 different tray types: (1) perforated metal trays, (2) stock plastic trays, and (3) directed-flow stock plastic trays. All groups were compared to casts produced with custom trays and vinyl polysiloxane (VPS) impression materials, which were considered the control. MATERIAL AND METHODS: Seven subjects participated in this trial. IH impressions were made in a random order using 1 of 3 tray types: stock plastic, perforated metal, or plastic directed flow. These were compared to VPS impressions using custom trays (control group). Each impression technique was repeated 3 times per subject, for a total of 84 observations. Impressions were disinfected and poured in a type IV stone. Linear accuracy of casts was measured using computer software analysis of scanned images of the casts at x30 magnification. Three linear measurements were made on each cast: second molar to second molar, right second molar to left first premolar, and left second molar to right first premolar. Measurements were compared among techniques using mixed-model analysis of variance to account for correlation among the multiple measurements made on each subject. Dunnett's adjustment for multiple comparisons with control was used (α=.05). RESULTS: For molar-to-molar and right second molar to the left first premolar measurements, there were no significant differences in linear dimensions between casts made from different trays. However, linear measurements from the left second molar to the right first premolar demonstrated significant differences for casts made with stock metal, directed-flow, and stock plastic trays compared to custom trays. In this group, casts produced by stock metal, directed-flow, and stock plastic trays differed from controls by 102, 68, and 71 um, respectively. Generally, casts made with plastic trays (stock plastic and directed flow) had values closer to those of custom trays than did casts made with metal trays. CONCLUSIONS: Impressions made with irreversible hydrocolloid produced casts that were significantly different in linear dimension than casts produced by custom trays and VPS impressions. These differences were not uniform, but varied by location on the cast.