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Waldenstrom macroglobulinemia (WM) is an indolent, IgM-producing lymphoproliferative disorder that represents 1–2% of all non-Hodgkin lymphomas (NHL) [1]. High-risk patients, as defined by the inte...

Developing new influenza vaccines with improved performance and easier administration routes hinges on defining correlates of protection. Vaccine-elicited cellular correlates of protection for influenza in humans have not yet been demonstrated. A phase-2 double-blind randomized placebo and active (inactivated influenza vaccine) controlled study provides evidence that a human-adenovirus-5-based oral influenza vaccine tablet (VXA-A1.1) can protect from H1N1 virus challenge in humans. Mass cytometry characterization of vaccine-elicited cellular immune responses identified shared and vaccine-type-specific responses across B and T cells. For VXA-A1.1, the abundance of hemagglutinin-specific plasmablasts and plasmablasts positive for integrin α4β7, phosphorylated STAT5, or lacking expression of CD62L at day 8 were significantly correlated with protection from developing viral shedding following virus challenge at day 90 and contributed to an effective machine learning model of protection. These findings reveal the characteristics of vaccine-elicited cellular correlates of protection for an oral influenza vaccine.

Introduction: Plamotamab (XmAb13676) is a humanized bispecific antibody that binds both CD20 and CD3 to recruit cytotoxic T cells to kill CD20 expressing malignant cells. Interim results of an ongoing first-in-human (FIH), dose-escalation study (XmAb13676-01; NCT02924402) in subjects with relapsed/refractory (R/R) non-Hodgkin's lymphoma (NHL) are reported. Methods: The study is an FIH, multi-center, open-label, phase 1, dose-escalation study in subjects with R/R NHL with a standard 3 + 3 design. The primary objectives are to determine safety, tolerability, and the maximum tolerated dose (MTD) or recommended dose of plamotamab. Secondary objectives include preliminary anti-tumor activity and pharmacokinetics/pharmacodynamics of plamotamab. This study has 3 Parts: Part A, escalating dose cohorts that establish an initial priming dose as part of repeated weekly infusions at a fixed, weight-based dose in a 28-day cycle; Part B, with a dosing schedule consisting of a priming dose on Cycle 1 Day 1, established in Part A, followed by step-up dosing (SUD) on subsequent weeks; and Part C is an SUD regimen with flat and less frequent dosing. Cytokine release syndrome (CRS) prophylaxis with dexamethasone, antihistamine, and acetaminophen was mandated prior to each administration of plamotamab. Treatment was continued for 2 cycles or longer if there was evidence of therapeutic benefit. Results: At data cut-off (01Jul2021), 80 subjects with NHL have been treated. Subjects had a median age of 62 years (range 32-89), a median of 4 prior therapies (range 1-10), and had been diagnosed a median of 28 months (range 6-353) prior to treatment in the study. The most common treatment-related adverse event was CRS. Overall, 50 subjects (62.5%) experienced CRS, with 4 (5.0%) experiencing Grade ≥ 3 CRS. No related neurotoxicity >Grade 2 has been observed. Treatment responses for NHL were assessed by the Lugano criteria or International Working Group criteria for Waldenström Macroglobulinemia. There have been 23 objective responses (43.4% overall response rate [ORR]) and 13 (38.2%) at doses of ≥ 80 µg/kg or flat dosing in all evaluable subjects with NHL and diffuse large cell B-cell lymphoma, respectively. In the efficacy-evaluable, follicular lymphoma population, at doses of 80-360 µg/kg or flat dosing, objective responses were observed in 8/10 (80%) subjects. After implementation of flat dosing, as opposed to weight-based dosing, a 50% ORR (4/8 evaluable subjects) has been observed in NHL subjects in that cohort. An MTD has not been reached, and dose escalation is ongoing. Overall, 4 out of 16 (25%) evaluable subjects with prior CAR-T therapy responded to plamotamab. Conclusions: Plamotamab demonstrated evidence of clinical activity in heavily pretreated subjects with R/R NHL. CRS was generally manageable with premedication. The study is ongoing with further optimization of dose and schedule. Figure 1 Figure 1. Disclosures Patel: Kite Pharma: Consultancy, Speakers Bureau; Genentech: Consultancy; Morphosys: Consultancy; Bristol Myers Squibb: Consultancy, Speakers Bureau; BeiGene: Consultancy; Janssen: Consultancy; AstraZeneca: Consultancy, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy; Abbvie: Consultancy; TG Therapeutics: Consultancy, Speakers Bureau; MEI Pharma: Consultancy; ADC Therapeutics: Consultancy; Lilly: Consultancy. Michot: GSK: Honoraria; Celgene: Honoraria; MSD: Consultancy, Honoraria; Innate Pharma: Research Funding; Incyte: Research Funding; H3 biomedecine: Research Funding; GSK: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; Gamamabs: Research Funding; Forma: Research Funding; Exelixis: Research Funding; Eos: Research Funding; Eisai: Research Funding; Debiopharm: Research Funding; Daiichi Sankyo,: Research Funding; Clovis: Research Funding; Chugai: Research Funding; Boeringer Ingelheim: Research Funding; Celgene: Research Funding; Blueprint: Research Funding; Beigene: Research Funding; Bayer: Research Funding; Argen-x: Research Funding; Amgen: Research Funding; Agios: Research Funding; Aduro: Research Funding; Abbvie: Research Funding; ASTEX: Research Funding, Speakers Bureau; Astra Zeneca: Honoraria, Research Funding; Roche: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Chanan-Khan: Alpha2 Pharmaceuticals, NonoDev, Starton: Current holder of stock options in a privately-held company; Ascentage: Research Funding; Cellectar: Current equity holder in publicly-traded company; Alpha2 Pharmaceuticals: Patents & Royalties: Tabi; Ascentage, Starton, Cellectar, NonoDev, Alpha2 Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BeiGene, Jansen, Ascentage: Honoraria; BieGene, Jansen, Ascentage: Consultancy. Ghesquieres: Janssen: Honoraria; Mundipharma: Consultancy, Honoraria; Roche: Consultancy; Celgene: Consultancy, Honoraria; Gilead Science: Consultancy, Honoraria. Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees. Cartron: Roche, Celgene-BMS: Consultancy; Danofi, Gilead, Novartis, Jansen, Roche, Celgene-BMS, Abbvie, Takeda: Honoraria. Portell: Acerta/AstraZeneca: Research Funding; Xencor: Research Funding; SeaGen: Research Funding; Targeted Oncology: Honoraria; Aptitude Health: Honoraria; Abbvie: Research Funding; Pharmacyclics: Honoraria; Kite: Honoraria, Research Funding; Merck: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Morphosys: Honoraria; TG Therapeutics: Honoraria, Research Funding; Genentech: Research Funding; VelosBio: Research Funding. Solh: Jazz Pharmaceuticals: Consultancy; Partner Therapeutics: Research Funding; BMS: Consultancy; ADCT Therapeutics: Consultancy, Research Funding. Wierda: Loxo Oncology, Inc.: Research Funding; GSK/Novartis: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Miragen: Research Funding; Sunesis: Research Funding; Gilead Sciences: Research Funding; KITE Pharma: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Juno Therapeutics: Research Funding; Acerta Pharma Inc.: Research Funding; Xencor: Research Funding; AstraZeneca: Research Funding; Cyclacel: Research Funding; Oncternal Therapeutics, Inc.: Research Funding; Karyopharm: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding. Johnson: Xencor: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Garcha: Xencor: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company; Promedim: Ended employment in the past 24 months. Ribrag: AstraZeneca: Membership on an entity's Board of Directors or advisory committees; GSK: Research Funding; MSD Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Infinity Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argen-X: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Astex Pharmaceuticals: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; PharmaMar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Epizyme: Honoraria, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees. Liebowitz: Xencor: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Phillips: Incyte: Consultancy, Other: received travel expenses from Incyte, Research Funding; ADCT, BeiGene, Bristol Myers Squibb, Cardinal Health, Incyte, Karyopharm, Morphosys, Pharmacyclics, Seattle Genetics: Consultancy; AbbVie: Consultancy, Research Funding; AstraZeneca: Consultancy; BMS: Consultancy, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding.

Summary Background Influenza is an important public health problem and existing vaccines are not completely protective. New vaccines that protect by alternative mechanisms are needed to improve efficacy of influenza vaccines. In 2015, we did a phase 1 trial of an oral influenza vaccine, VXA-A1.1. A favourable safety profile and robust immunogenicity results in that trial supported progression of the vaccine to the current phase 2 trial. The aim of this study was to evaluate efficacy of the vaccine in a human influenza challenge model. Methods We did a single-site, placebo-controlled and active-controlled, phase 2 study at WCCT Global, Costa Mesa, CA, USA. Eligible individuals had an initial A/California/H1N1 haemagglutination inhibition titre of less than 20 and were aged 18–49 years and in good health. Individuals were randomly assigned (2:2:1) to receive a single immunisation of either 1011 infectious units of VXA-A1.1 (a monovalent tablet vaccine) orally, a full human dose of quadrivalent inactivated influenza vaccine (IIV) via intramuscular injection, or matched placebo. Randomisation was done by computer-generated assignments with block size of five. An unmasked pharmacist provided the appropriate vaccines and placebos to the administrating nurse. Individuals receiving the treatments, investigators, and staff were all masked to group assignments. 90 days after immunisation, individuals without clinically significant symptoms or signs of influenza, an oral temperature of higher than 37·9°C, a positive result for respiratory viral shedding on a Biofire test, and any investigator-assessed contraindications were challenged intranasally with 0·5 mL wild-type A/CA/like(H1N1)pdm09 influenza virus. The primary outcomes were safety, which was assessed in all immunised participants through 365 days, and influenza-positive illness after viral challenge, which was assessed in individuals that received the viral challenge and the required number of assessments post viral challenge. This trial is registered with ClinicalTrials.gov , number NCT02918006 . Results Between Aug 31, 2016, and Jan 23, 2017, 374 individuals were assessed for eligibility, of whom 179 were randomly assigned to receive either VXA-A1.1 (n=71 [one individual did not provide a diary card, thus the solicited events were assessed in 70 individuals]), IIV (n=72), or placebo (n=36). Between Dec 2, 2016, and April 26, 2017, 143 eligible individuals (58 in the VXA-A1.1 group, 54 in the IIV group, and 31 in the placebo group) were challenged with influenza virus. VXA-A1.1 was well tolerated with no serious or medically significant adverse events. The most prevalent solicited adverse events for each of the treatment groups after immunisation were headache in the VXA-A1.1 (in five [7%] of 70 participants) and placebo (in seven [19%] of 36 participants) groups and tenderness at injection site in the IIV group (in 19 [26%] of 72 participants) Influenza-positive illness after challenge was detected in 17 (29%) of 58 individuals in the VXA-A1.1 group, 19 (35%) of 54 in the IIV group, and 15 (48%) of 31 in the placebo group. Interpretation Orally administered VXA-A1.1 was well tolerated and generated protective immunity against virus shedding, similar to a licensed intramuscular IIV. These results represent a major step forward in developing a safe and effective oral influenza vaccine. Funding Department of Health and Human Services, Office of the Assistant Secretary for Preparedness and Response, and Biomedical Advanced Research and Development Authority.

BACKGROUND. Noroviruses are the leading cause of epidemic acute gastroenteritis and foodborne diarrheal disease in humans. However, there are no approved vaccines for noroviruses. Potential correlates of protection identified through human challenge studies include mucosal IgA, memory B cells, and serum-blocking antibody titers (BT50). METHODS. We conducted a single-site, randomized, double-blind, placebo-controlled clinical trial of an oral norovirus vaccine to determine safety and immunogenicity. This tablet vaccine is comprised of a nonreplicating adenovirus-based vector expressing the VP1 gene from the GI.1 norovirus strain and a double-stranded RNA adjuvant. Sixty-six adult subjects meeting inclusion/exclusion criteria were randomized 2:1 to receive a single vaccine dose or placebo, respectively. Immunogenicity was primarily assessed by serum BT50. Additional outcomes included serum ELISA titers, fecal and saliva antibody titers, memory and antibody-secreting cell (ASC) frequency, and B cell phenotyping. RESULTS. The vaccine was well-tolerated, with no dose-limiting toxicities. Adverse events were mild or moderate. The primary immunological endpoint (increase in BT50 titers) was met in the high-dose group (P = 0.0003), with 78% showing a ≥2-fold rise in titers after a single immunization. Vaccine recipients also developed mucosally primed VP1-specific circulating ASCs, IgA+ memory B cells expressing gut-homing receptor (α4β7), and fecal IgA, indicating substantial and local responses potentially relevant to prevent norovirus infection. CONCLUSION. This oral norovirus vaccine was well-tolerated and generated substantial immune responses, including systemic and mucosal antibodies as well as memory IgA/IgG. These results are a major step forward for the development of a safe and immunogenic oral norovirus vaccine. TRIAL REGISTRATION. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02868073","term_id":"NCT02868073"}}NCT02868073. FUNDING. Vaxart.

Background Oral vaccines delivered as tablets offer several advantages over traditional injection-based vaccines including ease of distribution and administration as well as temperature-stable formulation options. Oral vaccination is also advantageous because it directly induces a strong mucosal response, which is thought to be critical for preventing future infections. Here we present results from a phase II clinical challenge study comparing efficacy of an oral recombinant adenovirus-based vaccine expressing hemagluttinin (HA) from A/California 04/09 to that of a commercial injectable quadrivalent (QIV) influenza vaccine. Methods In this 2016–2017 clinical trial (NCT02918006), subjects were immunized with either oral vaccine, QIV, or placebo and then challenged 90 days post-immunization with wildtype influenza A H1 virus to measure vaccine efficacy and durability. Protection was assessed by measuring changes in HAI titres, microneutralization, and IgA/IgG ASC assays. Additionally, exploratory flow cytometry evaluated quantitative and qualitative aspects of immunogenicity including markers of activation and mucosal homing on B cells. Analysis was performed on days 0 and 7 post-immunization and 0 and 6 days post-viral challenge. Plasmablasts sorted from PBMCs were then isolated for genomic DNA and sequenced for heavy chain receptor sequencing using NGS analysis. Results Of the subjects immunized with Vaxart’s oral tablet vaccine, 48% were protected. QIV, by comparison, protected 38% of immunized individuals. Only 37% of Vaxart subjects developed influenza infection compared with 44% of QIV subjects and 71% of placebo subjects. While both vaccines induced a humoral immune response, FACS analysis and NGS revealed that Vaxart subjects had more activated plasmablasts expressing surface mucosal homing markers and a more diverse B cell population than QIV subjects. Conclusion Vaxart’s oral influenza tablet vaccine protected against influenza infection as well or better than injectable QIV. However, the mechanism of protection appears to be unique to the route of immunization; oral immunization allows for specific homing of influenza specific B cells to sites of infection and produces a more diverse antibody repertoire. Disclosures N. Kolhatkar, Vaxart, Inc.: Employee, Salary. K. Gottlieb, Vaxart, Inc.: Employee, Salary. K. Kasparek, Vaxart, Inc.: Employee, Salary. K. Hodgson, Vaxart, Inc.: Employee, Salary. S. Tucker, Vaxart, Inc: Employee, Salary. D. Liebowitz, Vaxart, Inc.: Employee and Investigator, Salary.

Introduction: XmAb13676 is a humanized bispecific antibody that binds both CD20 and CD3 in order to recruit cytotoxic T cells to kill CD20 expressing malignant cells. Interim results of an ongoing first-in-human, dose-escalation study (XmAb13676-01; NCT02924402) in subjects with relapsed/refractory (R/R) non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL) are reported here. Methods: The study is a first-in-human, multi-center, open-label, phase 1, dose-escalation study in subjects with R/R NHL and R/R CLL with a standard 3 + 3 design. The primary objectives are to determine safety, tolerability, and the maximum tolerated dose (MTD) or recommended dose of XmAb13676. Secondary objectives include preliminary anti-tumor activity and PK/PD of XmAb13676. This study is designed in two parts: Part A, escalating dose cohorts that establish an initial priming dose as part of repeated weekly infusions at a fixed dose in a 28-day cycle; and Part B, with a dosing schedule consisting of a priming dose on C1D1 , established in Part A, followed by escalated dose(s) on subsequent weeks. Cytokine Release Syndrome (CRS) prophylaxis with dexamethasone was mandated prior to each administration of XmAb13676. Treatment was continued for 2 cycles or longer if there was evidence of therapeutic benefit. Results: At data cut-off, 44 subjects have been treated, 36 with NHL and 8 with CLL. NHL: Subjects with R/R NHL had a median age of 61.5 years (range 32-89), a median of 3.5 prior therapies (range 1-9) and had been diagnosed a median of 24.6 months (range 6.3-181.2) prior to treatment in the study. Treatment-emergent adverse events (TEAEs) related to treatment occurring in ˃ 3 subjects are shown in Table 1A. Nine treatment-related serious adverse events (SAE) occurred in 6 subjects. The most common treatment-related SAE was CRS, which occurred in 4 (11.1%) subjects with 1 of the events being Grade 4 and the other events being ≤ Grade 2. Treatment responses were assessed by the Lugano criteria or International Working Group criteria for Waldenström Macroglobulinemia (WM). There have been 7 objective responses: 2 complete responses (CR; DLBCL), 1 Very Good PR (VGPR; WM), and 4 partial responses (PR; 1FL, 3 DLBCL) at doses of 20-125 µg/kg. In the efficacy-evaluable population, at doses of 80-125 µg/kg, objective responses were observed in 6/18 patients. A priming dose of 45 µg/kg has been chosen for Part B. An MTD has not been reached and dose escalation is ongoing in Part B in NHL. CLL: Subjects with R/R CLL had a median age of 76 years (range 62-81), a median of 4.5 prior therapies (range 2-6) and had been diagnosed a median of 76.1 months (range 17.5-328.9) prior to treatment in the study. Treatment-related TEAEs occurring in ˃ 1 subject are shown in Table 1B. Three treatment-related serious adverse events (SAE) occurred in 2 subjects. The treatment related SAEs were CRS (Grade 3), hepatocellular injury (Grade 3), and jaundice cholestatic (Grade 2), each of which occurred in 1 (12.5%) subject. There has been 1 CR reported (Richter transformation) in 5 subjects at 20 µg/kg, the highest dose administered thus far. The treatment response was assessed by the Lugano criteria. An MTD has not been reached and dose escalation is ongoing in Part A in CLL. Conclusions: XmAb13676 demonstrated evidence of clinical activity in heavily pretreated subjects with R/R NHL and R/R CLL treated at doses between 20 and 125 µg/kg. CRS was generally manageable with premedication. The study is ongoing with further optimization of dose and schedule. Disclosures Patel: AstraZeneca: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics/Janssen: Consultancy, Speakers Bureau; Sunesis: Consultancy. Chanan-Khan:AbbVie: Research Funding; Xencor: Research Funding; Pharmacyclics: Research Funding; Merck: Research Funding; Jansen: Research Funding; Mayo Clinic: Employment; Ascentage: Research Funding; Millennium: Research Funding. Salles:Novartis, Servier, AbbVie, Karyopharm, Kite, MorphoSys: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Epizyme: Consultancy, Honoraria; Roche, Janssen, Gilead, Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Other: Educational events; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; BMS: Honoraria. Cartron:Gilead: Honoraria; Jansen: Honoraria; Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Sanofi: Honoraria. Thomas:Celgene: Research Funding; Amgen: Research Funding; Xencor: Research Funding; BMS: Research Funding. Wierda:KITE pharma: Research Funding; Sunesis: Research Funding; Miragen: Research Funding; Gilead Sciences: Research Funding; Acerta Pharma Inc: Research Funding; GSK/Novartis: Research Funding; Cyclcel: Research Funding; Loxo Oncology Inc.: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Juno Therapeutics: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Pharmacyclics LLC: Research Funding; Xencor: Research Funding; Janssen: Research Funding. Liebowitz:Xencor: Employment, Equity Ownership. Pagel:AstraZeneca: Consultancy; Gilead Sciences: Consultancy; Pharmacyclics: Consultancy. Ribrag:MSD: Membership on an entity's Board of Directors or advisory committees; Roche: Other: Travel, accommodations, and expenses ; Nanostring: Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy, Research Funding; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; AZ: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, and expenses ; Incyte: Membership on an entity's Board of Directors or advisory committees; ArgenX: Research Funding. Saville:Xencor: Employment, Equity Ownership. Johnson:Xencor: Employment, Equity Ownership. Ly:Xencor: Employment, Equity Ownership. Phillips:Pharmacyclics: Consultancy, Research Funding; Bayer: Consultancy; Abbvie: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees.

Influenza is one of the leading causes of morbidity and mortality, with 36,000 deaths and 226,000 hospitalizations occurring annually in the USA, primarily in the elderly. The currently licensed influenza vaccines, trivalent inactivated influenza vaccine and live, attenuated influenza vaccine, although effective in many respects, need to be more efficacious for the elderly and the very young. They can also be improved upon to induce broader immunity and cross-protection against drifted or variant strains. Additionally, there is room for improvement in manufacturing technologies. Increased antigen dose, adjuvants, virus-like particles and virosomes, novel live, attenuated influenza vaccines, and universal vaccines are all being developed or have been developed to address the unmet need in the elderly. Many of these approaches may provide incremental improvements in efficacy, where transformative improvement is necessary.

Summary Background Most influenza vaccines are manufactured in eggs, and the inactivated virus is purified for injection. For a seasonal influenza product, manufacturing, distribution, and perhaps even vaccine coverage, would be greatly improved with an oral tablet alternative made in cell culture. We aimed to assess the safety and immunogenicity of an oral tablet vaccine against influenza A H1N1 in healthy adults. Methods At a single site, we did a randomised, double-blind, placebo-controlled trial of a monovalent influenza A H1N1 vaccine to establish the safety and immunogenicity of a recombinant, non-replicating, adenovirus vector expressing haemagglutinin and double-stranded RNA adjuvant delivered orally by tablets. Participants had to have an initial haemagglutination inhibition titre of at most 1/20, be aged between 18 and 49 years, and be in good health. We randomly assigned (1:1) participants to receive either a single oral dose of vaccine or placebo. Randomisation was done by computer-generated assignment, and study drug was distributed with concealed identity to the masked staff by an unmasked pharmacist. Investigative site staff, people directly involved with immunological assays or the assessment of clinical safety, and participants were masked to treatment assignments. Solicited symptoms of reactogenicity were assessed, and all safety assessments were reported through the active phase of the study (day 28). Immunogenicity was assessed by haemagglutination inhibition titres, the percentage of participants that seroconverted, microneutralisation titres, and the number of antibody secreting cells. Descriptive statistics were used for continuous variables and t -tests or Fisher's exact tests were used to compare treatment groups. The study is registered at ClinicalTrials.gov, number NCT01688297. Findings 24 participants were enrolled in the study at WCCT Global between Dec 2, 2013, and April 15, 2014. Adverse events were mild in nature, and occurred with similar frequency in vaccine (four events) and placebo recipients (four events). After immunisation, 11 (92%) of 12 vaccine-treated participants had a four-fold increase in haemagglutination inhibition titres (group geometric mean fold rise of 7·7) and microneutralisation titres (group geometric mean fold rise of 29). No participants in the placebo group had a four-fold increase in haemagglutination inhibition titres (group geometric mean fold rise of 1·1) or microneutralisation titres (group geometric mean fold rise of 1·0). Neutralising antibody responses to influenza were not hindered by pre-existing immunity to the vector. Interpretation An oral recombinant adenovirus vaccine to influenza was well tolerated and can elicit neutralising antibody responses to influenza virus in human beings. These data are a step forward in making oral influenza vaccination possible. Funding Vaxart Inc.

Epstein-Barr virus (EBV) is a human DNA tumor virus with an extraordinarily diverse oncogenic potential. No other virus, human or otherwise, has shown the ability to contribute to malignant transformation in such a variety of cell backgrounds. Studies of EBV related malignancies in immunodeficiency states (posttransplant and AIDS) have provided insight into the significance in vivo of EBV-specific T-cell immunity, which has been studied so elegantly in vitro, in controlling primary and persistent EBV infection. Recent studies on tumor tissue from the EBV diseases have suggested that viral signaling, especially through the LMP1 protein, may directly contribute to the development and maintenance of the malignant phenotype. Novel strategies aimed at restoring antiviral T-cell immunity have shown promise in the postbone marrow transplant setting in controlling EBV-related lymphoproliferations. Future effort into better characterizing the role of EBV in T-lymphocyte malignancies and in non-lymphoid malignancies (Nasopharyngeal carcinoma (NPC) and smooth muscle tumors) will likely identify new pathogenic mechanisms utilized by this versatile virus that have yet to be appreciated in the better-studied EBV-related B-lymphoproliferative diseases.

The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B-cell lymphoma, a low-grade lymphoma of mucosa-associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29-cM region within the chromosomal band 18q21. Using nine of these YACs in single- and/or dual-color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6-Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8-cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a

Costimulation is critical for induction of full T-cell effector function, and thus represents an attractive immunotherapeutic approach for the treatment of cancer. This review examines these approaches, including ex vivo T-cell expansion, systemic "delivery" of constimulation, tumors transduced or transfected with costimulatory ligands, and vaccine strategies using coimmunization with the genes for costimulatory ligands. Impressive results in animal models have been demonstrated and a wide range of human clinical trials are underway.

Epstein-Barr virus (EBV) is associated with various malignant and benign lymphoproliferative disorders. It also efficiently transforms human B lymphocytes in vitro. The latent membrane protein 1 (LMP1) of EBV-infected cells plays a central part in this process by mimicking members of the family of tumor necrosis factor (TNF) receptors, thereby transmitting growth signals from the cell membrane to the nucleus through cytoplasmic TNF-receptor-associated factors (TRAFs). I sought evidence of LMP1-mediated signal transduction through TRAFs in tumor tissue from patients with post-transplantation lymphoproliferative disease and non-Hodgkin's lymphomas related to the acquired immunodeficiency syndrome (AIDS).The association of LMP1 with TRAF-1 or TRAF-3 in tumor tissue was studied with double-immunofluorescence microscopy and immunoprecipitation assays. Evidence of LMP1-TRAF signaling was sought with an electrophoretic mobility shift assay for the nuclear factor-kappaB (NF-kappaB) transcription factor.Tumors from eight patients with post-transplantation lymphoproliferative disease, two patients with AIDS-associated non-Hodgkin's lymphoma, and three patients with endemic Burkitt's lymphoma were analyzed. Tumors from six of the patients with post-transplantation lymphoproliferative disease were positive for EBV and expressed LMP1; two samples were EBV-negative. Tumors from both patients with AIDS-associated non-Hodgkin's lymphoma were EBV-positive and expressed LMP1, whereas tumors from all three patients with Burkitt's tumors were positive for EBV but negative for LMP1. Double-immunofluorescence microscopy showed that LMP1 localized with and immunoprecipitated with TRAF-1 and TRAF-3 in all eight of the EBV-positive, LMP1-positive samples. An electrophoretic mobility shift assay revealed activated NF-kappaB in all eight EBV-positive, LMP1-positive samples as well, but not in either of the EBV-negative, LMP1-negative samples or in the three EBV-positive, LMP1-negative samples.LMP1-mediated signaling through the TRAF system has a role in the pathogenesis of the EBV-positive lymphomas that arise in immunosuppressed patients.

Epstein–Barr virus (EBV) is a member of the human herpesvirus family and, like other herpesviruses, infects and establishes a persistent infection in the host. Clinically, primary infection with EB...

We explored the feasibility and toxicity of administering escalating doses of anti-CD3/CD28 ex vivo costimulated T cells as a therapeutic adjunct for patients with relapsed, refractory, or chemotherapy-resistant, aggressive non-Hodgkin lymphoma (NHL) following high-dose chemotherapy and CD34+-selected hematopoietic cell transplantation (HCT). Sixteen patients had infusions on day 14 after HCT of autologous T cells that had been stimulated using beads coated with anti-CD3 and anti-CD28 monoclonal antibodies. At baseline, the subjects had severe quantitative and functional T-cell impairments. The culture procedure partially reversed impaired cytokine responsiveness in T cells in vitro and in vivo. Transient dose-dependent infusion toxicities were observed. There was a rapid reconstitution of lymphocytes; however, there were persistent defects in CD4 T cells. Most interestingly, 5 patients had a delayed lymphocytosis between day 30 and day 120 after HCT. Maximal clinical responses included 5 patients with a complete response (CR), 7 patients with a partial response (PR), and 4 patients with stable disease. At a median follow-up of 33 months (range, 26-60 months), 5 patients are alive with stable or relapsed disease and 3 patients remain in CR. In conclusion, this phase 1 trial demonstrates that adoptive transfer of autologous costimulated T cells (1) is feasible in heavily pretreated patients with advanced NHL, (2) is associated with a rapid recovery of lymphocyte counts, (3) reverses cytokine activation deficits in vitro, and (4) is associated with delayed lymphocytosis in a subset of patients.

The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5′ promoter. Culture of highly purified human CD34+ bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3–10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20–50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34+ cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.

Purpose: To determine the in vitro effects of flavopiridol on bladder cancer cell lines, immortalized urothelial cell lines, and normal urothelial cells well characterized for defects in p53, pRb, and p16. Methods: Growth inhibition was assessed via an MTT assay and apoptosis via DAPI nuclear staining. Cell cycle analysis was performed via propidium iodide staining and fluorescent activated cell sorting (FACS). Multidrug-resistant cells were generated by continuous exposure to doxorubicin. Results: Growth inhibition was not correlated with inactivation of p53, pRb, or p16. All cells experienced G2/M arrest within 24 h of flavopiridol exposure. Modest apoptosis was observed but required 72 h of continuous drug exposure to become evident. There was no obvious synergistic or antagonistic toxicity when flavopiridol was combined with radiotherapy or cisplatin dosed at the IC50 despite the observation that radiotherapy and flavopiridol led to more profound G2/M arrest than either agent alone. Doxorubicin-resistant cells, demonstrated to overexpress the MDR1 multidrug-resistance protein were equally as sensitive to flavopiridol as the parental cells. Conclusions: Flavopiridol is a novel cell cycle inhibitor that may be a useful agent in bladder cancers with tumor suppressor gene alterations and/or multidrug resistance.

This paper describes a technique for enhancing certain features in grayscale images. Of particular interest are the class of objects that are reasonably large in extent, but are only faintly darker or lighter than the background. An example of such objects is the mandibular canal which appears in panoramic dental X-ray images. Identification of this canal is required in some dental and orthodontal investigations. Traditional image segmentation techniques often fail to detect the full extent of the canal due to the large amount of structural noise in the image. We propose a new method for the enhancement of this class of objects and the subsequent segmentation task. The flow of a fluid is simulated over the image topology, allowing fluid to settle in local minima and, by application of a difference image, enhancing the visibility of features that are characterized by a significant spatially-distributed local minimum. The procedure is similar to the watershed algorithm in concept, visualizing the movement of fluid over the image surface to draw conclusions about significant local minima. Our approach is different however since it is not aimed at segmenting the image, but enhancing distributed local minima. We consider the flow of fluid from high lying to lower lying areas under gravity. This is analogous to the rain fall method of filling the catchment basins in watershed segmentation. Various models of flow, based on co- operative networks are presented and discussed. Post processing is applied to reduce the amount of false outputs. We demonstrate that our proposed method is more suitable than simple edge detection or the watershed algorithms for the enhancement and segmentation of the mandibular canal.

CD34 + cells were isolated from G-CSF mobilized normal volunteers and cultured for 4 days in serum-free medium (X-VIVO 15) in the presence of SCF (20 ng/ml) + TPO (50 ng/ml) + FLT-3L (50 ng/ml). The phenotype (CD38, Thy-1, c-kit, HLA-DR) and cell division history (CFSE labeling) of CD34 + cells were examined. The frequency of LTC-IC and SCID repopulating cells (SRC) was measured by limiting dilution assays. After 4 days, the total number of CD34 + cells was maintained but a 25% reduction in the frequency of CFU and a 5-fold increase in LTC-IC were observed. The percentage of CD34 + CD38 − and CD34 + DR − cells increased 57 and 5 fold, respectively, as > 85% of the CD38 − and DR − cells divided 5 to 6 times from Day 0 to Day 4. The percentage of CD34 + /c-kit + was maintained after 4 days of culture even though > 90% of the cells had divided 2 to 5 times. In contrast, there was a 6-fold reduction in Thy-1 + cells while 60% of the Thy-1 + cells detected at Day 4 had undergone 0 to 2 divisions. Cultures of sorted CD34 + Thy-1 + cells indicated that 3 or more cell divisions resulted in a gradual loss of Thy-1 expression. This data indicates that the combination of SCF+TPO+FLT-3L preferentially expands CD34 + CD38 − and DR − subsets. Data correlating CD34 + subsets, number of cell divisions and frequency of LTC-IC and SRC will be presented.

PURPOSE: Flavopiridol is the first cyclin-dependent kinase (cdk) inhibitor to enter clinical trials. Serum levels of flavopiridol obtained during phase I studies were sufficient to inhibit in vitro cancer cell growth. Because responses were observed in kidney cancer patients in the phase I trials, we performed a phase II trial of flavopiridol in this patient population. PATIENTS AND METHODS: Thirty-five minimally pretreated patients were accrued using a standard two-step mechanism. Flavopiridol (50 mg/m2/d) was administered by continuous infusion for 72 hours every 2 weeks, and response was evaluated every 8 weeks. Peripheral blood mononuclear cells (PBMCs) were collected at baseline, at completion of drug infusion, and on day 7 of the first therapy cycle, and cell cycle parameters after phytohemagglutinin and interleukin-2 stimulation were assessed. RESULTS: There were two objective responses (response rate = 6%, 95% confidence interval, 1% to 20%). The most common toxicities were asthenia, occurring in 83% of patients (grade 3 or 4 in 9%), and diarrhea, occurring in 77% of patients (grade 3 or 4 in 20%). Also, nine patients (26%) experienced grade 3 or 4 vascular thrombotic events, including one myocardial infarction, two transient neurologic ischemic attacks, four deep venous thrombosis, and two pulmonary emboli. Cell cycle studies did not reveal any effect of flavopiridol on stimulated PBMCs. CONCLUSION: Flavopiridol, at the dose and schedule administered in this trial, is ineffective in metastatic renal cancer. In addition to the diarrhea observed in phase I studies, we also observed a higher incidence of asthenia and serious vascular thrombotic events than expected.

Epstein-Barr virus expresses a cytoplasmic and plasma membrane protein (LMP) in latently infected growth transformed lymphocytes. The gene specifying LMP has now been expressed in NIH3T3 and Rat-1 cells. Expression of the gene in these cells resulted in altered cell morphology and some resistance to the growth inhibiting effect of medium containing low serum. In Rat-1 cells, LMP expression often led to loss of contact inhibition and anchorage-independent growth in soft agar. Rat-1 cells expressing LMP were uniformly tumorigenic in nude mice. Thus, LMP is a transforming gene which is likely to account for many aspects of EBV induced cell transformations. This is the first demonstration of a transforming gene in Epstein-Barr virus, a ubiquitous human pathogen associated with neoplasia.