Arthroplasty for Treating Proximal Femur Metastatic Lesions May Be Associated with Lower Mortality Rates Compared to Intramedullary Nailing within the VA Healthcare System.

Metastatic bony disease is a significant health issue, with approximately 700,000 new cases annually that tend to metastasize to bones. The proximal femur in the appendicular skeleton is commonly affected. Our study aimed to investigate mortality rates and hospital stay duration in patients with pathologic proximal femur fractures treated with either intramedullary nailing or arthroplasty within the Veterans Health Administration system. In total, 679 patients (265 arthroplasty, 414 intramedullary nails) were identified through ICD-9 and CPT codes from 30 September 2010 to 1 October 2015. Hospital stays were similar for both groups (arthroplasty: 10.5 days, intramedullary nails: 11 days, p = 0.1). Mortality was associated with increased age and Gagne comorbidity scores (p < 0.001). Arthroplasty showed a survival benefit in the log-rank test (p = 0.018), and this difference persisted in the multivariate analysis after adjusting for age and comorbidities, with a hazard ratio of 1.3. Our study reported evidence that arthroplasty is associated with increased patient survival even when accounting for age and comorbidities in treating metastatic disease of the proximal femur.

Practical strategies for robust and inexpensive imaging of aqueous-cleared tissues.

Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting complicated and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardised 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes cause minimal spherical aberration with objective numerical apertures less than 0.65. Furthermore, we describe methods for aligning and assessing the light sheets, discriminating fluorescence from autofluorescence, identifying chromatic artefacts due to differential scattering and removing streak artefacts such that they do not confound downstream 3D object segmentation analyses, with mouse embryo, liver and heart imaging as demonstrated examples.

Selective Accumulation of Near Infrared-Labeled Multivalent Quinidine Copolymers in Tumors Overexpressing P-Glycoprotein: Potential for Noninvasive Diagnostic Imaging.

P-glycoprotein (P-gp) is a promiscuous small molecule transporter whose overexpression in cancer is associated with multidrug resistance (MDR). In these instances, anticancer drugs can select for P-gp-overexpressing cells, leading to cancer recurrence with an MDR phenotype. To avoid selection for MDR cancers and inform individual patient treatment plans, it is critical to noninvasively identify P-gp-overexpressing tumors prior to administration of chemotherapy. We report the facile free radical copolymerization of quinidine, a competitive inhibitor of P-gp, and acrylic acid to generate multiplexed polymeric P-gp-targeted imaging agents with tunable quinidine content. Copolymer targeting was demonstrated in a nude mouse xenograft model. In xenografts overexpressing P-gp, copolymer distribution was enhanced over two-fold compared to the negative control of poly(acrylic acid) regardless of quinidine content. In contrast, accumulation of the copolymers in xenografts lacking P-gp was equivalent to poly(acrylic acid). This work forms the foundation for a unique approach toward the phenotype-specific noninvasive imaging of MDR tumors and is the first in vivo demonstration of copolymer accumulation through the active targeting of P-gp.

A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

Biological Nanoparticles in Vaccine Development.

Vaccines represent one of the most successful public health initiatives worldwide. However, despite the vast number of highly effective vaccines, some infectious diseases still do not have vaccines available. New technologies are needed to fully realize the potential of vaccine development for both emerging infectious diseases and diseases for which there are currently no vaccines available. As can be seen by the success of the COVID-19 mRNA vaccines, nanoscale platforms are promising delivery vectors for effective and safe vaccines. Synthetic nanoscale platforms, including liposomes and inorganic nanoparticles and microparticles, have many advantages in the vaccine market, but often require multiple doses and addition of artificial adjuvants, such as aluminum hydroxide. Biologically derived nanoparticles, on the other hand, contain native pathogen-associated molecular patterns (PAMPs), which can reduce the need for artificial adjuvants. Biological nanoparticles can be engineered to have many additional useful properties, including biodegradability, biocompatibility, and are often able to self-assemble, thereby allowing simple scale-up from benchtop to large-scale manufacturing. This review summarizes the state of the art in biologically derived nanoparticles and their capabilities as novel vaccine platforms.

Intra-Articular Administration of a Synthetic Lubricin in Canine Stifles.

OBJECTIVE: The aim of this study was to evaluate the functional, systemic, synovial and articular changes after intra-articular administration of a synthetic lubricin within healthy canine stifles. STUDY DESIGN: A prospective randomized blinded placebo-controlled study composed of 10 dogs equally divided into either a treatment group (intra-articular synthetic lubricin injection, n = 5) or control group (saline, n = 5). Clinical (orthopaedic examination, gait observation, gait analysis), biochemical (complete blood count and biochemistry profile) and local tissue outcomes (joint fluid analysis, joint capsule and articular cartilage histopathology) were evaluated over a time period of 3 months. RESULTS: No significant differences between the treatment group and control group were identified with regard to baseline patient parameters. No clinically significant orthopaedic examination abnormalities, gait abnormalities, biochemical alterations, joint fluid alterations or histopathological alterations were identified over the course of the study. CONCLUSION: The synthetic lubricin studied herein is both biocompatible and safe for a single administration within the canine stifle joint. Further research is necessary to evaluate the clinical efficacy of the synthetic lubricin in canine osteoarthritic joints.

Microparticle fabricated from a series of symmetrical lipids based on dihydroxyacetone form textured architectures.

We report the synthesis of a series of symmetrical lipids composed of dihydroxyacetone and even­carbon fatty acids (eight to sixteen carbons), both components of the human metabolome, and characterize their formulation into porous microparticles through spontaneous emulsification without the use of additional porogens. Lipid hydrolysis products were identified by 1H NMR to validate lipid degradation into the parent metabolic synthons. Microparticle architecture, as determined by scanning electron microscopy, was lipid-length dependent, with shorter alkyl chains forming tight structures and longer alkyl chains forming larger pores with plate-like lipid architectures. In all cases, the lipids formed organized patterns, not irregular shapes. As a demonstration of the potential use of these solid lipid-based microparticles, the release kinetics of a model drug (piroxicam) was quantified showing that release was more greatly influenced by microparticle porosity, and hence surface area, than by hydrophobicity of the lipids.

Induced fusion and aggregation of bacterial outer membrane vesicles: Experimental and theoretical analysis.

Recombinantly engineered bacterial outer membrane vesicles (OMVs) are promising vaccine delivery vehicles. The diversity of exogenous antigens delivered by OMVs can be enhanced by induced fusion of OMV populations. To date there are no reports of induced fusion of bacterial OMVs. Here we measure the pH and salt-induced aggregation and fusion of OMVs and analyze the processes against the Derjaguin-Landau-Verwey-Overbeek (DLVO) colloidal stability model. Vesicle aggregation and fusion kinetics were investigated for OMVs isolated from native E. coli (Nissle 1917) and lipopolysaccharide (LPS) modified E. coli (ClearColi) strains to evaluate the effect of lipid type on vesicle aggregation and fusion. Electrolytes and low pHs induced OMV aggregation for both native and modified LPS constructs, approaching a calculated fusion efficiency of ~25% (i.e. ~1/4 of collision events lead to fusion). However, high fusion efficiency was achieved for Nissle OMVs solely with decreased pH as opposed to a combination of low pH and increased divalent counterion concentration for ClearColi OMVs. The lipid composition of the OMVs from Nissle negatively impacted fusion in the presence of electrolytes, causing higher deviations from DLVO-predicted critical coagulation concentrations with monovalent counterions. The outcome of the work is a defined set of conditions under which investigators can induce OMVs to fuse and make various combinations of vesicle compositions.

Effect of Lubricin Mimetics on the Inhibition of Osteoarthritis in a Rat Anterior Cruciate Ligament Transection Model.

BACKGROUND: Lubricin, a mucinous glycoprotein, plays a chondroprotective role as a constituent of synovial fluid. Structural analogs have been synthesized to mimic the structure and function of native lubricin in an effort to recapitulate this effect with the goal of delaying progression of osteoarthritis (OA). PURPOSE: To investigate the efficacy of intra-articular injections of lubricin mimetics in slowing or preventing the progression of posttraumatic OA by using a rat anterior cruciate ligament transection model. STUDY DESIGN: Controlled laboratory design. METHODS: Four lubricin mimetics were investigated, differing from one another in their binding orientations and steric interactions. Eighty skeletally mature Sprague-Dawley rats underwent bilateral anterior cruciate ligament transections and were randomly allocated to receive intra-articular injections (50 µL/injection) of 1 of the 4 mimetics in the right knee and equal volumes of saline injection in the contralateral knee (control). All rats were euthanized 8 weeks postoperatively and assessed via biomechanical analysis, which evaluated comparative friction coefficients across the 4 groups, and histological evaluation of articular cartilage, osteophytes, and synovitis. The Osteoarthritis Research Society International (OARSI) histopathological assessment system was used to evaluate the degree of articular cartilage degeneration and osteophytes, while synovitis was assessed through a semiquantitative scoring system. Binding efficacy of the 4 mimetics was assessed in vitro and in vivo through the immunohistochemical localization of polyethylene glycol. Articular cartilage degeneration and synovitis scoring data analyses were performed with generalized estimating equation modeling. RESULTS: Injection of the group 3 mimetic (random 24 + 400 + 30) directly correlated with improved OARSI scores for femoral articular cartilage degeneration when compared with saline-injected contralateral control knees (P = .0410). No lubricin mimetic group demonstrated statistically significant differences in OARSI scores for tibial articular cartilage degeneration. Injection of the group 4 mimetic (AB 24 + 400 + 30) led to a statistically significant difference in osteophyte OARSI score (P = .0019). None of the 4 lubricin mimetics injections incited an additive synovial inflammatory response. Immunohistochemical staining substantiated the binding capacity of all 4 mimetics, while in vivo experimentation revealed that the group 1 and 3 mimetics were still retained within the joint 4 weeks after injection. There were no differences in friction coefficients between any pair of groups and no significant trends based on lubricin mimetic structure. CONCLUSION: We demonstrated that the tribosupplementation of a traumatically injured knee with a specific lubricin structural analog may attenuate the natural progression of OA. CLINICAL RELEVANCE: The current lack of efficacious clinical options to counter the onset and subsequent development of OA suggests that further investigation into the synthesis and behavior of lubricin analogs could yield novel translational applications.

The Utility of Obtaining Postmobilization Imaging in Nonsurgical Pelvic Ring Injuries.

INTRODUCTION: Pelvic fractures are diverse injuries with varying degrees of severity. Treatment recommendations are determined by the associated instability. For likely stable patterns, postmobilization imaging is used to assess for occult instability. This study assesses the utility of postmobilization images and determines how often they alter the recommendations for treatment. METHODS: Records at a single level 1 trauma center from January 2007 through December 2014 were reviewed, and patients with Current Procedural Terminology codes and International Classification of Diseases, Ninth Revision codes for pelvic and acetabular fractures were identified. For those chosen for nonsurgical treatment at presentation, a detailed chart review was performed to identify patients who had postmobilization radiographs and to determine whether this imaging led to a change in treatment recommendations. RESULTS: Inclusion criteria were met by 762 patients whose average age was 50 years. Of 331 patients planned for nonsurgical treatment at presentation, 168 (51%) had postmobilization images. The postmobilization radiographs did not alter treatment recommendations in any of these patients; however, three of these patients underwent surgical stabilization based on the patients' report of pain with attempted mobilization. DISCUSSION: Routine postmobilization imaging has limited value for patients with pelvic injuries and a low likelihood for instability, such as those with incomplete sacral fractures. Eliminating this step would reduce cost and decrease radiation exposure. The need for change in treatment plan or further imaging should be based on the patient's clinical progress with weight bearing. LEVEL OF EVIDENCE: Level 4.

Influence of Block Length on Articular Cartilage Lubrication with a Diblock Bottle-Brush Copolymer.

We report how the tribological properties of a class of diblock copolymers with architecture and function inspired by the lubricating glycoprotein lubricin correlate to chemical composition. This class of diblock copolymers, consisting of a cationic cartilage-binding block and a brush-lubricating block, demonstrates that boundary lubrication of articular cartilage more strongly depends on the cartilage-binding block than the lubrication block. Specifically, the cartilage-binding functional groups (tertiary or quaternary amines) and cartilage-binding block length significantly influence the degree of lubrication under boundary mode experimental conditions. An optimal number (∼24 in this case) of cartilage-binding groups led to the lowest coefficient of friction, and an increase or decrease in the number of cations in the binding block led to partial (>24, and between 12 and 24) or complete (=12) loss of lubricating ability. The length of the lubricating block (DP = 200 or 400) chosen in this study had no effect on the degree of lubrication. These results are put into context in terms of binding affinity to the cartilage and the spatial packing density of the polymer on the cartilage surface and can serve as a useful guide for future designs of synthetic lubricants that rival the efficacy of natural lubricants.

Enabling P-glycoprotein inhibition in multidrug resistant cancer through the reverse targeting of a quinidine-PEG conjugate.

Previously identified as a key mediator of multidrug resistance, the drug efflux behavior of P-glycoprotein (P-gp) remains a prominent challenge in cancer treatment. P-gp belongs to the ATP-binding cassette transporter family of membrane proteins, and modulates the efflux of many drugs at the cell membrane, resulting in inadequate retention of chemotherapeutic drugs in cancer cells. Here, we explore the FDA-approved drug quinidine as a P-gp inhibitor. Although used clinically for the treatment of malaria, arrhythmia, and pseudobulbar effect, quinidine can induce acquired long QT syndrome and torsade de pointes through its interaction with the Purkinje fibers, which hinders its clinical application as a P-gp inhibitor. We hypothesize that the conjugation of quinidine to a polymer will permit its use as a P-gp inhibitor through mitigation of its distribution into the myocardium. Methoxypolyethylene glycol (mPEG) was conjugated to quinidine through a glycine linker, making a monovalent quinidine-polymer conjugate, which was then evaluated for its interactions with P-gp in vitro. The mPEG-glycine-quinidine conjugate retained its ability to inhibit the function of P-gp (log IC50 of 4.20 nM for quinidine and 4.61 nM for the mPEG-glycine-quinidine conjugate). Additionally, the distribution of quinidine into perfused mouse myocardium was decreased by almost an order of magnitude, strongly supporting our working hypothesis (2.28 × 10-3 µmol/g tissue for quinidine and ~4.10 × 10-4 µmol/g tissue for the conjugate). The results suggest the potential use of such polymer-drug conjugates to reverse multidrug resistance through P-gp inhibition and to mitigate the off-target pharmacologic effects that complicate their clinical use.

A lipid mixing assay to accurately quantify the fusion of outer membrane vesicles.

The intermixing of phospholipids from opposing bilayers, or membrane fusion, is a naturally occurring process that can be leveraged to produce hybrid vesicle systems. Optimizing the production of these hybrid vesicles requires accurate, sensitive, and quantitative methods of the lipid mixing that occurs during fusion. A fluorescence-based assay that uses octadecyl-rhodamine B chloride to measure lipid mixing, or R18 assay, was developed by Hoekstra to investigate viral entry almost four decades ago. However, the R18 assay has so far only been used to measure heterotypic fusion events. Consequently, the fusion efficiencies that are calculated from the R18 assay underestimate the total number of fusion events and the true efficiency of vesicle fusions. In this article, we outline the experimental format and data analysis that is necessary to perform the R18 fusion assay and to accurately and reliably measure the true total fusion efficiency of outer membrane vesicles isolated from the Nissle 1917 strain of E. coli.

Selective and Tunable Galectin Binding of Glycopolymers Synthesized by a Generalizable Conjugation Method.

Glycopolymers, conjugates of synthetic polymers with pendant carbohydrates, are becoming increasingly important to probe the role of carbohydrates in cellular processes and for applications like biosensors and drug delivery. A library of glycopolymers bearing different sugar moieties was synthesized by grafting amino-functionalized sugars to poly(acrylic acid) via DMTMM coupling. Primary amines were introduced at the anomeric (C-1) position to a number of unprotected mono-, di-, and trisaccharides using ammonium carbamate and conjugated to poly(acrylic acid) of different molecular weights, synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. This approach provides a simple and efficient route for the preparation of glycopolymers that differ only in the identity or degree of substitution of the sugar moiety on the polymer. The binding parameters (ka, kd, and KD) of these new glycopolymers to galectins-1 and -3 were quantified using surface plasmon resonance. The galectins selectively bound only to lactose-containing polymers, and the binding affinity was dependent on the galectin type, degree of sugar substitution and the molecular weight of polymer chains. Binding to both galectin-1 and -3 increased with a higher degree of sugar substitution, and higher molecular weight of the polymer backbone, reaching KD values on the order of 10-11 M.

Boundary mode lubrication of articular cartilage with a biomimetic diblock copolymer.

We report the design of a diblock copolymer with architecture and function inspired by the lubricating glycoprotein lubricin. This diblock copolymer, synthesized by sequential reversible addition-fragmentation chain-transfer polymerization, consists of a cationic cartilage-binding domain and a brush-lubricating domain. It reduces the coefficient of friction of articular cartilage under boundary mode conditions (0.088 ± 0.039) to a level equivalent to that provided by lubricin (0.093 ± 0.011). Additionally, both the EC50 (0.404 mg/mL) and cartilage-binding time constant (7.19 min) of the polymer are comparable to purified human and recombinant lubricin. Like lubricin, the tribological properties of this polymer are dependent on molecular architecture. When the same monomer composition was evaluated either as an AB diblock copolymer or as a random copolymer, the diblock effectively lubricated cartilage under boundary mode conditions whereas the random copolymer did not. Additionally, the individual polymer blocks did not lubricate independently, and lubrication could be competitively inhibited with an excess of binding domain. This diblock copolymer is an example of a synthetic polymer with lubrication properties equal to lubricin under boundary mode conditions, suggesting its potential utility as a therapy for joint pathologies like osteoarthritis.

Treatment Modalities for Pathologic Fractures of the Proximal Femur Pertrochanteric Region: A Systematic Review and Meta-Analysis of Reoperation Rates.

BACKGROUND: The proximal femur represents the most common site of metastatic bone disease in the appendicular skeleton, and associated pathologic pertrochanteric femur fractures contribute to cancer-related morbidity and mortality. Controversy exists as to whether these injuries are best managed with intramedullary nailing (IMN) or with arthroplasty. METHODS: A systematic review of the literature was performed using a PubMed search following PRISMA guidelines to identify studies performed within the last 20 years regarding treatment of proximal femur metastatic lesions with either nailing or arthroplasty with a reported reoperation rate. Sixteen studies were selected for inclusion containing 1414 patients. Pooled estimates and 95% confidence intervals (CIs) for reoperation rates associated with IMN and endoprosthetic reconstruction (EPR) were separately calculated. RESULTS: The pooled estimate for reoperation for IMN was a median of 9% (95% CI, 5%-14%) and the pooled estimate for reoperation for EPR was a median of 7% (95% CI, 5%-11%). Significant heterogeneity was present in studies reporting on both treatment modalities: for IMN, I2 = 55%, and for EPR, I2 = 51%. CONCLUSION: This systematic literature review identified 16 eligible, nonrandomized, retrospective studies that reported on the results of surgical treatment for proximal femur metastatic disease. The pooled estimate of reoperation was similar between patients treated with IMN and EPR. Inconsistencies among follow-up and the study designs used limited evidence-based conclusions. As the oncologic care of patients with metastatic disease continues to evolve and improve, patient-specific needs must be carefully considered when selecting an optimal treatment strategy. LEVEL OF EVIDENCE: Level III.

Beneficial Effects of Exercise on Subendothelial Matrix Stiffness are Short-Lived.

Aerobic exercise helps to maintain cardiovascular health in part by mitigating age-induced arterial stiffening. However, the long-term effects of exercise regimens on aortic stiffness remain unknown, especially in the intimal extracellular matrix layer known as the subendothelial matrix. To examine how the stiffness of the subendothelial matrix changes following exercise cessation, mice were exposed to an 8 week swimming regimen followed by an 8 week sedentary rest period. Whole vessel and subendothelial matrix stiffness were measured after both the exercise and rest periods. After swimming, whole vessel and subendothelial matrix stiffness decreased, and after 8 weeks of rest, these values returned to baseline. Within the same time frame, the collagen content in the intima layer and the presence of advanced glycation end products (AGEs) in the whole vessel were also affected by the exercise and the rest periods. Overall, our data indicate that consistent exercise is necessary for maintaining compliance in the subendothelial matrix.

Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies.

Many microbial pathogens produce a ß-(1→6)-linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule, including bacterial, fungal, and protozoan cells. Broadly protective immune responses to this single conserved polysaccharide antigen in animals are possible but only when a deacetylated poly-N-acetyl-d-glucosamine (dPNAG; <30% acetate) glycoform is administered as a conjugate to a carrier protein. Unfortunately, conventional methods for natural extraction or chemical synthesis of dPNAG and its subsequent conjugation to protein carriers can be technically demanding and expensive. Here, we describe an alternative strategy for creating broadly protective vaccine candidates that involved coordinating recombinant poly-N-acetyl-d-glucosamine (rPNAG) biosynthesis with outer membrane vesicle (OMV) formation in laboratory strains of Escherichia coli The glycosylated outer membrane vesicles (glycOMVs) released by these engineered bacteria were decorated with the PNAG glycopolymer and induced high titers of PNAG-specific IgG antibodies after immunization in mice. When a Staphylococcus aureus enzyme responsible for PNAG deacetylation was additionally expressed in these cells, glycOMVs were generated that elicited antibodies to both highly acetylated PNAG (∼95-100% acetate) and a chemically deacetylated dPNAG derivative (∼15% acetate). These antibodies mediated efficient in vitro killing of two distinct PNAG-positive bacterial species, namely S. aureus and Francisella tularensis subsp. holarctica, and mice immunized with PNAG-containing glycOMVs developed protective immunity against these unrelated pathogens. Collectively, our results reveal the potential of glycOMVs for targeting this conserved polysaccharide antigen and engendering protective immunity against the broad range of pathogens that produce surface PNAG.

A single dose and long lasting vaccine against pandemic influenza through the controlled release of a heterospecies tandem M2 sequence embedded within detoxified bacterial outer membrane vesicles.

The influenza A virus undergoes genetic drift and shift, leaving the general population susceptible to emerging pandemic strains, despite seasonal flu vaccination. Here we describe a single dose influenza vaccine derived from recombinant outer membrane vesicles (rOMVs) that display an antigen-mapped heterospecies tandem sequence of the M2 protein from the influenza A virus, released over 30days from poly(lactic-co-glycolide) (PLGA) microparticles. Four weeks post vaccination, BALB/c mice developed high anti-M2e IgG titers that were equivalent to those generated at 8weeks in a typical prime/boost vaccine regimen. Challenge of mice with a lethal dose of mouse adapted influenza virus PR8 (H1N1) 10weeks post vaccination resulted in 100% survival for both rOMV single-dose microparticle and prime/boost vaccinated mice. Anti-M2e IgG1 and IgG2a antibody titers were weighted toward IgG1, but splenocytes isolated from rOMV single-dose microparticle vaccinated mice produced high levels of IFNγ relative to IL-4 in response to stimulation with M2e peptides, supporting a more Th1 biased immune response. The protective immune response was long lasting, eliciting sustained antibody titers and 100% survival of mice challenged with a lethal dose of PR8 six months post initial vaccination. Together, these data support the potential of controlled release rOMVs as an effective single dose, long lasting and rapidly effective vaccine to protect against influenza.