Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.Seasonal influenza outbreaks across the globe cause an estimated 250,000–500,000 deaths annually (1). Current influenza vaccines need to be updated every few years because of antigenic drift (2). Despite intensive monitoring, strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past (2). Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift facilitating a potential pandemic outbreak. These concerns have expedited efforts toward developing a universal influenza vaccine.Neutralizing antibodies (nAbs) against hemagglutinin (HA) are the primary correlate for protection in humans and hence HA is an attractive target for vaccine development (3). The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the disulfide-linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit, whereas the stem domain predominantly comprises the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH-induced conformational rearrangement in the host-cell endosomes to adopt the virus–host membrane fusion-competent state (4, 5).The antigenic sites on the globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed nAbs (6). However, extensive efforts have resulted in the isolation of monoclonal antibodies (mAbs) that bind within the globular head and inhibit receptor attachment, which neutralize drifted variants of an HA subtype or heterosubtypic HA (7–16). The HA stem is targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes (17). The epitopes of these bnAbs in the HA stem are more conserved across different influenza HA subtypes compared with the antigenic sites in the HA globular head (18).During a primary infection, the immunodominant globular head domain suppresses the response toward the conserved stem. Several efforts have been made to circumvent this problem. Repeated immunizations with full-length, chimeric HAs (cHAs) in a protracted vaccination regimen have been shown to boost stem-directed responses in mice (19). Alternatively, full-length HA presented on nanoparticles (np) has been shown to elicit stem-directed nAbs (20). Attempts have also been made to steer the immune response toward the conserved HA stem by hyperglycosylating the head domain (21). Although the aforementioned strategies need to be further evaluated and provide novel alternatives, detrimental interference from the highly variable immunodominant head domain in eliciting a broad functional response cannot be completely evaded. A “headless” stem domain immunogen offers an attractive solution. However, early attempts at expressing the HA2 subunit independently in a native, prefusion conformation were unsuccessful. In the absence of the head domain, the HA2 subunit expressed in Escherichia coli spontaneously adopted the low-pH conformation (22) in which the functional epitopes of stem-directed bnAbs are disrupted. More recently, the entire HA stem region has been expressed in a prefusion, native-like conformation in both prokaryotic and eukaryotic systems adopting multiple strategies (23–26).Design of independently folding HA stem fragments which adopt the prefusion HA conformation presents another approach to elicit bnAbs against influenza (27, 28). The A helix of the HA2 subunit contributes substantial contact surface to the epitope of stem-directed bnAbs such as CR6261, F10, and others. Although multivalent display of A helix on the flock house virus as a virus-like particle platform elicited cross-reactive antibodies, it conferred only minimal protection (20%) against virus challenge in mice (29).We report the design and characterization of engineered headless HA stem immunogens based on the influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation-sensitive, stem-directed bnAbs such as CR6261 (30), F10 (31), and FI6v3 (32) with a high-affinity [equilibrium dissociation constant (K_D) of 10–50 nM]. The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both group 1 (H1 and H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD₉₀) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens confer robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous stem domain immunogens (23–25), the designed immunogens were purified from the soluble fraction in E. coli. The HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free, which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks.     

Emergency Contraceptive Knowledge and Use among Urban Women in Nigeria and Kenya

Rates of emergency contraceptive (EC) use in sub‐Saharan Africa are highest in Kenya and Nigeria, although little is known about user characteristics and use dynamics in these countries. To better meet women's emergency contraceptive needs and to contribute to the limited knowledge base regarding this method in Africa, this study examines data from a sample of EC users drawn from a large, representative household survey that included sexually experienced women in urban Kenya and Nigeria. Bivariate and multivariate analyses reveal greater knowledge of EC among these urban women than was reported in other nationally representative surveys. Recent users of EC were more likely to be in their 20s, unmarried, and more highly educated than never users or ever users of EC in both countries. Results contradict public perceptions of EC users as young adolescents and indicate the importance of strengthening EC provision in Africa, including targeting information and services to unmarried women and supporting private pharmacies in delivering quality services.     

Rolling Circle Amplification for Direct Detection of rpoB Gene Mutations in Mycobacterium tuberculosis Isolates from Clinical Specimens

Rapid and accurate detection of multidrug resistance (MDR) in Mycobacterium tuberculosis is essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) of rpoB, using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 stored M. tuberculosis clinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78 M. tuberculosis culture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistant M. tuberculosis in clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDR M. tuberculosis, particularly in high prevalence settings with limited resources.     

Appendicitis as the presenting manifestation of Kawasaki disease

In cases of Kawasaki’s disease (KD) presenting as acute surgical abdomen, rarely has the presence of acute appendicitis been found. We report two cases of histologically confirmed acute appendicitis in the presence of KD and a review of the literature as it pertains to acute abdomen and atypical presentations of KD.     

Effect of bupivacaine on intervertebral disc cell viability

Background contextBupivacaine is a local anesthetic commonly used to relieve or control pain in interventional spine procedures. Bupivacaine has been shown to be toxic to articular cartilage, which has similarities to intervertebral disc (IVD) cartilage, raising concern over a potentially negative effect of bupivacaine on the disc.PurposeTo determine bupivacaine's effect on cell viability of IVD cells in vitro and to elucidate whether this is through apoptosis or necrosis.Study designIn vitro controlled study of bupivacaine effect on cell viability in human and rabbit IVD cells.SubjectsRabbit annulus fibrosus (AF) tissue, nucleus pulposus (NP) cells, and knee articular chondrocytes were isolated from New Zealand white rabbits. Human AF and NP cells were isolated from stage 3 to 4 degenerative disc surgical specimens.Outcome measuresCell viability was assessed after exposure to bupivacaine via trypan blue staining or flow cytometry.MethodsAnnulus fibrosus and NP cells were grown in monolayer and alginate beads, respectively, to simulate their physiologic environment. The cells were then exposed to bupivacaine or saline control at 60 and 120 minutes and examined for cell viability.ResultsRabbit NP cell death demonstrated a time and dose dependence in response to bupivacaine. In addition, cell death was greater than that observed for articular chondrocytes. Rabbit AF tissue also demonstrated increased cell death in response to bupivacaine exposure. Human NP cells demonstrated time-dependent cell death, with greater necrosis than apoptosis. Annulus fibrosus cells grown in monolayers also resulted in similar effects, with greater necrosis rather than apoptosis.ConclusionsDespite its pain relieving properties, bupivacaine decreases cell viability in rabbit and human disc cells in a time-dependent manner. In addition, the changes observed are greater than that seen for articular chondrocytes. This increase in cell death appears to be related to an increase in necrosis rather than apoptosis. Whether bupivacaine exerts similar effects in vivo or how this relates to overall clinical outcome remains to be explored.     

Rolling circle amplification and multiplex allele-specific PCR for rapid detection of katG and inhA gene mutations in Mycobacterium tuberculosis

The aim of the study was to compare a novel, rolling circle amplification (RCA) assay for detection of common isoniazid (INH) resistance mutations in Mycobacterium tuberculosis with a multiplex allele-specific PCR (MAS-PCR) and sequencing of katG and the fabG1-inhA promoter region. One or more mutations were identified by RCA, MAS-PCR, and sequencing in 21 (68%), 22 (71%), and 23 (74%), respectively, of 31 epidemiologically unrelated INH-resistant isolates, and in none of 8 INH-susceptible isolates. The RCA assay is a rapid, inexpensive, and practical screening method for INH resistance in M. tuberculosis in countries with high prevalence of INH resistance.     

A rapid and fully-automated method for the quantitation of tricyclic antidepressants in serum using turbulent-flow liquid chromatography–tandem mass spectrometry

BackgroundWe describe a fully-automated turbulent-flow liquid chromatography–tandem mass spectrometry method for the detection of tricyclic antidepressant drugs (amitriptyline, desipramine, imipramine, and nortriptyline) in serum.MethodsHuman serum and an internal standard were injected directly onto a Cyclone-P online solid-phase extraction (SPE) column (0.5 × 50 mm). Following removal of serum proteins and other components the analytes were transferred to a Hypersil Gold C-18 (50 × 3 mm) analytical column. Elution occurred with a gradient of water and acetonitrile each with 0.1% formic acid. Analytes were ionized and detected over a 3.5 min analysis time by electrospray-ionization mass spectrometry with selected reaction monitoring (SRM). Matrix effects were well-characterized and carryover, precision, linearity, recovery and limits of detection and quantitation were evaluated.ResultsThe simple and complex precision CVs for all compounds were ≤ 16%. The limits of detection and quantitation for all drugs were ≤ 3 ng/ml and < 20 ng/ml, respectively. Recoveries were between 97 and 114%. Slopes for method comparison plots were all > 0.96. Proficiency testing materials had values within 2 SDI of peer group means for all drugs.ConclusionBased on validation data, this is a specific, sensitive fully-automated method for rapid quantitation of tricyclic antidepressants in serum.     

A July Spike in Fatal Medication Errors: A Possible Effect of New Medical Residents

BACKGROUND  

Each July thousands begin medical residencies and acquire increased responsibility for patient care. Many have suggested that these new medical residents may produce errors and worsen patient outcomes—the so-called “July Effect;” however, we have found no U.S. evidence documenting this effect.