Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans.

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

Breadth and Functionality of Varicella-Zoster Virus Glycoprotein-Specific Antibodies Identified after Zostavax Vaccination in Humans.

Herpes zoster (HZ) (shingles) is the clinical manifestation of varicella-zoster virus (VZV) reactivation. HZ typically develops as people age, due to decreased cell-mediated immunity. However, the importance of antibodies for immunity against HZ prevention remains to be understood. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated HZ vaccine (Zostavax). Direct enumeration of VZV-specific antibody-secreting cells (ASCs) via enzyme-linked immunosorbent spot assay (ELISPOT assay) showed that Zostavax can induce both IgG and IgA ASCs 7 days after vaccination but not IgM ASCs. The VZV-specific ASCs range from 33 to 55% of the total IgG ASCs. Twenty-five human VZV-specific monoclonal antibodies (MAbs) were cloned and characterized from single-cell-sorted ASCs of five subjects (>60 years old) who received Zostavax. These MAbs had an average of ∼20 somatic hypermutations per VH gene, similar to those seen after seasonal influenza vaccination. Fifteen of the 25 MAbs were gE specific, whereas the remaining MAbs were gB, gH, or gI specific. The most potent neutralizing antibodies were gH specific and were also able to inhibit cell-to-cell spread of the virus in vitro Most gE-specific MAbs were able to neutralize VZV, but they required the presence of complement and were unable to block cell-to-cell spread. These data indicate that Zostavax induces a memory B cell recall response characterized by anti-gE > anti-gI > anti-gB > anti-gH antibodies. While antibodies to gH could be involved in limiting the spread of VZV upon reactivation, the contribution of anti-gE antibodies toward protective immunity after Zostavax needs further evaluation.IMPORTANCE Varicella-zoster virus (VZV) is the causative agent of chickenpox and shingles. Following infection with VZV, the virus becomes latent and resides in nerve cells. Age-related declines in immunity/immunosuppression can result in reactivation of this latent virus, causing shingles. It has been shown that waning T cell immunity correlates with an increased incidence of VZV reactivation. Interestingly, serum with high levels of VZV-specific antibodies (VariZIG; IV immunoglobulin) has been administered to high-risk populations, e.g., immunocompromised children, newborns, and pregnant women, after exposure to VZV and has shown some protection against chickenpox. However, the relative contribution of antibodies against individual surface glycoproteins toward protection from shingles in elderly/immunocompromised individuals has not been established. Here, we examined the breadth and functionality of VZV-specific antibodies after vaccination with the live attenuated VZV vaccine Zostavax in humans. This study will add to our understanding of the role of antibodies in protection against shingles.

Induction of broadly cross-reactive antibody responses to the influenza HA stem region following H5N1 vaccination in humans.

The emergence of pandemic influenza viruses poses a major public health threat. Therefore, there is a need for a vaccine that can induce broadly cross-reactive antibodies that protect against seasonal as well as pandemic influenza strains. Human broadly neutralizing antibodies directed against highly conserved epitopes in the stem region of influenza virus HA have been recently characterized. However, it remains unknown what the baseline levels are of antibodies and memory B cells that are directed against these conserved epitopes. More importantly, it is also not known to what extent anti-HA stem B-cell responses get boosted in humans after seasonal influenza vaccination. In this study, we have addressed these two outstanding questions. Our data show that: (i) antibodies and memory B cells directed against the conserved HA stem region are prevalent in humans, but their levels are much lower than B-cell responses directed to variable epitopes in the HA head; (ii) current seasonal influenza vaccines are efficient in inducing B-cell responses to the variable HA head region but they fail to boost responses to the conserved HA stem region; and (iii) in striking contrast, immunization of humans with the avian influenza virus H5N1 induced broadly cross-reactive HA stem-specific antibodies. Taken together, our findings provide a potential vaccination strategy where heterologous influenza immunization could be used for increasing the levels of broadly neutralizing antibodies and for priming the human population to respond quickly to emerging pandemic influenza threats.

Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite.

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding 'higher' Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H2 metabolism, CO2-reductive acetogenesis and N2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-microl environment can be.

Development of a pharmacist-psychiatrist collaborative medication therapy management clinic.

OBJECTIVE: To describe a needs assessment, practice description, practice innovation and reimbursement of a psychiatric pharmacist medication therapy management (MTM) clinic with related challenges and opportunities. SETTING: An MTM clinic established in collaboration with the Outpatient Psychiatric Services (OPS) at the University of California San Diego (UCSD), under contract with the San Diego County Health and Human Services Agency Adult and Older Adult Mental Health Services (A/OAMHS). PRACTICE DESCRIPTION: Two board-certified psychiatric pharmacists provided direct patient care using a collaborative practice protocol 3 days per week. Clinical services included pharmacotherapy management, laboratory monitoring, medication counseling, and psychosocial referrals to other providers. PRACTICE INNOVATION: Payment to UCSD OPS for clinical services was based on a contract between the San Diego County A/OAMHS and the clinic. Two pharmacists co-managed mental health patients and billed for medication management based on face-to-face contact time (medication minutes) and documentation time with each patient. MAIN OUTCOME MEASURES: Number of patients comanaged, dropout rates, visit duration, and billed minutes. RESULTS: From May 2009 to December 2010, two pharmacists comanaged 68 patients, mean (± SD) age 48.6 ± 11.6 years, diagnosed with major depressive, schizophrenic, schizoaffective, and/or bipolar disorder. A total of 56 (82.3%) patients were clinically stable and remained on the pharmacist caseload, but 12 (17.6%) patients were lost to follow-up (10 lost contact, 1 moved, 1 expired). On average, patients had 7.7 patient visits , for 491 total visits (with an average of 26 minutes per visit) that were billed at a rate of $4.82 per minute for medication minutes, translating to $84,542.80. CONCLUSION: With provider education and appropriate physician champions, pharmacists are able to work collaboratively with psychiatrists in a mental health clinic.

Influenza vaccine-induced human bone marrow plasma cells decline within a year after vaccination.

A universal vaccine against influenza would ideally generate protective immune responses that are not only broadly reactive against multiple influenza strains but also long-lasting. Because long-term serum antibody levels are maintained by bone marrow plasma cells (BMPCs), we investigated the production and maintenance of these cells after influenza vaccination. We found increased numbers of influenza-specific BMPCs 4 weeks after immunization with the seasonal inactivated influenza vaccine, but numbers returned to near their prevaccination levels after 1 year. This decline was driven by the loss of BMPCs induced by the vaccine, whereas preexisting BMPCs were maintained. Our results suggest that most BMPCs generated by influenza vaccination in adults are short-lived. Designing strategies to enhance their persistence will be a key challenge for the next generation of influenza vaccines.

"Product on Stopper" in a Lyophilized Drug Product: Cosmetic Defect or a Product Quality Concern?

During manufacturing of a lyophilized drug product, operator errors in product handling during loading of product filled vials onto the lyophilizer can lead to a seemingly cosmetic defect which can impact certain critical quality attributes of finished product. In this study, filling of a formulated monoclonal antibody in vials was performed using a peristaltic pump filling unit, and subsequently, the product was lyophilized. After lyophilization, upon visual inspection, around 40% of vials had cosmetic defect with residual product around stopper of the vial and were categorized as "product on stopper" vials, whereas remaining 60% vials with no cosmetic defect were called "acceptable vials." Both groups of vials from 1 single batch were tested for critical quality attributes including protein concentration (ultraviolet absorbance at 280), residual moisture (Karl Fischer), sterility (membrane filtration), and container closure integrity (CCI) (blue dye ingress). Analysis of protein quality attributes such as aggregation, protein concentration, residual moisture showed no significant difference between vials with "product on stopper" and "acceptable vials." However, CCI of the "product on stopper" vials was compromised due to the presence of product around stopper of the vial. The results from this case study demonstrate the following 2 important findings: (1) that a seemingly cosmetic defect may impact product quality, compromising the integrity of the product and (2) that CCI test method can be used as an orthogonal method to sterility testing to evaluate sterility assurance of the product. The corrective action proposed to mitigate this defect is use of a larger sized vial that can potentially minimize this defect that arises because of product handling errors.

Building Bridges through Science.

Science is ideally suited to connect people from different cultures and thereby foster mutual understanding. To promote international life science collaboration, we have launched "The Science Bridge" initiative. Our current project focuses on partnership between Western and Middle Eastern neuroscience communities.

Combination antiretroviral therapy (cART) component ritonavir significantly alters docetaxel exposure.

PURPOSE: Non-AIDS-defining cancers (NADCs) now exceed rates of AIDS-defining cancers in HIV-positive patients. Treatment of NADCs may be complicated by drug-drug interactions between antiretrovirals and chemotherapy. Docetaxel is a widely used anticancer agent that is primarily metabolized by CYP3A4 enzymes and used to treat NADCs. A preclinical in vivo assessment was performed to gain a better understanding of CYP3-mediated drug-drug interactions between antiretrovirals and docetaxel, as well as to assess any alterations in gene expression with these combinations. METHODS: Docetaxel (20 mg/kg i.v.) was administered to male FVB mice in the presence and absence of dexamethasone (10 mg/kg p.o. ×4d), efavirenz (25 mg/kg p.o. ×4d), ketoconazole (50 mg/kg p.o.), or ritonavir (12.5 mg/kg p.o.). At various time points, plasma and liver tissue were harvested. Docetaxel concentrations were determined by LC/MS/MS. Pharmacokinetic parameters were calculated. Liver tissue RNA was used to evaluate alterations in Cyp3a11 and Abcb1a gene expression. RESULTS: Docetaxel exposure was altered by CYP3A4 inhibitors but not by inducers. The CYP3A4 inducers efavirenz and dexamethasone did not have a significant effect on docetaxel exposure (AUC). However, the CYP3A4 inhibitors ritonavir and ketoconazole resulted in a 6.9- and 3.1-fold increase in AUC, respectively. Alterations in gene expression did not account for the altered docetaxel exposure. CONCLUSIONS: Docetaxel exposure was significantly altered by CYP3A4 inhibitors. Until a definitive clinical trial is performed, docetaxel should be used with caution in patients on a ritonavir-containing antiretroviral regimen or an alternative antineoplastic therapy or antiretroviral regimen should be considered.

Monoclonal antibodies recognizing the Enterococcus faecalis collagen-binding MSCRAMM Ace: conditional expression and binding analysis.

Enterococci are opportunistic pathogens known to cause numerous clinical infections and complications in humans. Adhesin-mediated binding to extracellular matrix (ECM) proteins of the host is thought to be a crucial step in the pathogenesis of these bacterial infections. Adhesin of collagen from Enterococcus faecalis (Ace) is a cell-wall anchored protein of E. faecalis that has been shown to be important for bacterial binding to the ECM. In this report, we characterize the conditions for Ace expression and demonstrate Ace binding to mammalian epithelial and endothelial cells as well as to collagens found in the ECM. To further characterize Ace expression and function, we report the generation of a panel of monoclonal antibodies (mAbs) directed against this important E. faecalis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance and flow cytometry, we have characterized this panel of mAbs which may prove to be not only beneficial in studies that address the precise biological role of adhesion of E. faecalis, but may also serve as beneficial therapeutic agents against E. faecalis infections.