Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18.

More than 170 types of human papilloma viruses (HPV) exist with many causing proliferative diseases linked to malignancy in indications such as cervical cancer and head and neck squamous cell carcinoma. Characterization of antibody levels toward HPV serology is challenging due to complex biology of oncoproteins, pre-existing titers to multiple HPV types, cross-reactivity, and low affinity, polyclonal responses. Using multiplex technology from MSD, we have developed an assay that simultaneously characterizes antibodies against E6 and E7 oncoproteins of HPV16 and 18, the primary drivers of HPV-associated oncogenesis. We fusion tagged our E6 and E7 proteins with MBP via two-step purification, spot-printed an optimized concentration of protein into wells of MSD 96-well plates, and assayed various cynomolgus monkey, human and HPV+ cervical cancer patient serum to validate the assay. The dynamic range of the assay covered 4-orders of magnitude and antibodies were detected in serum at a dilution up to 100,000-fold. The assay was very precise (n = 5 assay runs) with median CV of human serum samples ~ 5.3% and inter-run variability of 11.4%. The multiplex serology method has strong cross-reactivity between E6 oncoproteins from human serum samples as HPV18 E6 antigens neutralized 5 of 6 serum samples as strongly as HPV16 E6. Moderate concordance (Spearman's Rank = 0.775) was found between antibody responses against HPV16 E7 in the multiplex assay compared to standard ELISA serology methods. These results demonstrate the development of a high-throughput, multi-plex assay that requires lower sample quantity input with greater dynamic range to detect type-specific anti-HPV concentrations to E6 and E7 oncoproteins of HPV16 and 18.

Development and Standardization of a High-Throughput Multiplex Immunoassay for the Simultaneous Quantification of Specific Antibodies to Five Respiratory Syncytial Virus Proteins.

Human respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in (premature) newborns and causes respiratory illness in the elderly. Different monoclonal antibody (MAb) and vaccine candidates are in development worldwide and will hopefully become available within the near future. To implement such RSV vaccines, adequate decisions about immunization schedules and the different target group(s) need to be made, for which the assessment of antibody levels against RSV is essential. To survey RSV antigen-specific antibody levels, we developed a serological multiplex immunoassay (MIA) that determines and distinguishes antibodies against the five RSV glycoproteins postfusion F, prefusion F, Ga, Gb, and N simultaneously. The standardized RSV pentaplex MIA is sensitive, highly reproducible, and specific for the five RSV proteins. The preservation of the conformational structure of the immunodominant site Ø of prefusion F after conjugation to the beads has been confirmed. Importantly, good correlation is obtained between the microneutralization test and the MIA for all five proteins, resulting in an arbitrarily chosen cutoff value of prefusion F antibody levels for seropositivity in the microneutralization assay. The wide dynamic range requiring only two serum sample dilutions makes the RSV-MIA a high-throughput assay very suitable for (large-scale) serosurveillance and vaccine clinical studies.IMPORTANCE In view of vaccine and monoclonal development to reduce hospitalization and death due to lower respiratory tract infection caused by RSV, assessment of antibody levels against RSV is essential. This newly developed multiplex immunoassay is able to measure antibody levels against five RSV proteins simultaneously. This can provide valuable insight into the dynamics of (maternal) antibody levels and RSV infection in infants and toddlers during the first few years of life, when primary RSV infection occurs.

Vascular Regeneration in Ischemic Hindlimb by Adeno-Associated Virus Expressing Conditionally Silenced Vascular Endothelial Growth Factor.

BACKGROUND: Critical limb ischemia (CLI) is the extreme manifestation of peripheral artery disease, a major unmet clinical need for which lower limb amputation is the only option for many patients. After 2 decades in development, therapeutic angiogenesis has been tested clinically via intramuscular delivery of proangiogenic proteins, genes, and stem cells. Efficacy has been modest to absent, and the largest phase 3 trial of gene therapy for CLI reported a worsening trend of plasmid fibroblast growth factor. In all clinical trials to date, gene therapy has used unregulated vectors with limited duration of expression. Only unregulated extended expression vectors such as adeno-associated virus (AAV) and lentivirus have been tested in preclinical models. METHODS AND RESULTS: We present preclinical results of ischemia (hypoxia)-regulated conditionally silenced (CS) AAV-human vascular endothelial growth factor (hVEGF) gene delivery that shows efficacy and safety in a setting where other strategies fail. In a BALB/c mouse model of CLI, we show that gene therapy with AAV-CS-hVEGF, but not unregulated AAV or plasmid, vectors conferred limb salvage, protection from necrosis, and vascular regeneration when delivered via intramuscular or intra-arterial routes. All vector treatments conferred increased capillary density, but organized longitudinal arteries were selectively generated by AAV-CS-hVEGF. AAV-CS-hVEGF therapy reversibly activated angiogenic and vasculogenic genes, including Notch, SDF1, Angiopoietin, and Ephrin-B2. Reoxygenation extinguished VEGF expression and inactivated the program with no apparent adverse side effects. CONCLUSIONS: Restriction of angiogenic growth factor expression to regions of ischemia supports the safe and stable reperfusion of hindlimbs in a clinically relevant murine model of CLI.

Multivalent Conjugates of Sonic Hedgehog Accelerate Diabetic Wound Healing.

Despite their preclinical promise, few recombinant growth factors have been fully developed into effective therapies, in part, due to the short interval of therapeutic activity after administration. To address this problem, we developed nanoscale polymer conjugates for multivalent presentation of therapeutic proteins that enhance the activation of targeted cellular responses. As an example of this technology, we conjugated multiple Sonic hedgehog (Shh) proteins onto individual hyaluronic acid biopolymers to generate multivalent protein clusters at defined ratios (i.e., valencies) that yield enhanced Shh pathway activation at equivalent concentrations relative to unconjugated Shh. In this study, we investigated whether these multivalent conjugates (mvShh) could be used to improve the therapeutic function of Shh. We found that a single treatment with mvShh significantly accelerated the closure of full-thickness wounds in diabetic (db/db) mice compared to either an equivalent dose of unconjugated Shh or the vehicle control. Furthermore, we identified specific indicators of wound healing in fibroblasts and endothelial cells (i.e., transcriptional activation and cell migration) that were activated by mvShh in vitro and at concentrations approximately an order of magnitude lower than the unconjugated Shh. Taken together, our findings suggest that mvShh conjugates exhibit greater potency to activate the Shh pathway, and this multivalency advantage improves its therapeutic effect to accelerate wound closure in a diabetic animal model. Our strategy of multivalent protein presentation using nanoscale polymer conjugates has the potential to make a significant impact on the development of protein-based therapies by improving their in vivo performance.

Sustained endothelial expression of HoxA5 in vivo impairs pathological angiogenesis and tumor progression.

HoxA5 is expressed in quiescent endothelial cells (EC), but absent in activated angiogenic EC. To examine the efficacy of targeting HoxA5 therapeutically to quell pathologic or tumor angiogenesis, we generated an inducible, transgenic mouse model of sustained HoxA5 expression in ECs. During pathologic angiogenesis, sustained HoxA5 regulates expression several angiogenic effector molecules, notably increased expression of TSP-2 and reduced expression of VEGF, thus leading to inhibition of pathological angiogenesis in tissues. To evaluate if this impressive reduction of vascularization could also impact tumor angiogenesis, HoxA5 mice were bred with a mouse model of de novo squamous carcinogenesis, e.g., K14-HPV16 mice. Activation of EC-HoxA5 significantly reduced infiltration by mast cells into neoplastic skin, an early hallmark of progression to dysplasia, reduced angiogenic vasculature, and blunted characteristics of tumor progression. To evaluate HoxA5 as a therapeutic, topical application of a HoxA5 transgene onto early neoplastic skin of K14-HPV16 mice similarly resulted in a significant impairment of angiogenic vasculature and progression to dysplasia to a similar extent as observed with genetic delivery of HoxA5. Together these data indicate that HoxA5 represents a novel molecule for restricting pathological and tumorigenic angiogenesis.

Enhanced angiogenic efficacy through controlled and sustained delivery of FGF-2 and G-CSF from fibrin hydrogels containing ionic-albumin microspheres.

Neo-vessel formation in ischemic tissues relies on numerous growth factors and cell fractions for the formation of mature, stable, functional vasculature. However, the efforts to regenerate tissues typically rely on the administration of a single growth factor or cells alone. Conversely, polymeric matrices have been investigated extensively to deliver multiple growth factors at pre-determined rates to form stable blood vessels in ischemic tissues. We report on a novel sequential delivery system of a fibrin hydrogel containing ionic-albumin microspheres that allows for the controlled release of two growth factors. The use of this system was investigated in the context of therapeutic angiogenesis. Material properties were determined based on degree of swelling measurements and degradation characteristics. Release kinetics of model angiogenic polypeptides FGF-2 and G-CSF were determined using ELISA and the bioactivity of released protein was evaluated in human endothelial cell cultures. The release of growth factors from ionic-albumin microspheres was significantly delayed compared to the growth factor released from fibrin matrices in the absence of spheres. The scaffolds were implanted in a murine critical limb ischemia model at two concentrations, 40 ng (low) and 400 ng (high), restoring 92% of the blood flow in a normally perfused limb using a fibrin hydrogel releasing FGF-2 containing albumin-PLL microspheres releasing G-CSF (measured by LDPI at the high concentration), a 3.2-fold increase compared to untreated limbs. The extent of neo-vessel formation was delineated by immunohistochemical staining for capillary density (CD-31+) and mature vessel formation (α-SMA+). In conclusion, our study demonstrated that the release kinetics from our scaffold have distinct kinetics previously unpublished and the delivery of these factors resulted in hindlimb reperfusion, and robust capillary and mature vessel formation after 8 weeks compared to either growth factor alone or bolus administration of growth factor.

Whole-mount imaging of the mouse hindlimb vasculature using the lipophilic carbocyanine dye DiI.

The availability of transgenic disease backgrounds and the accessibility of molecular research reagents have contributed to make the mouse ischemic hindlimb the model of choice for many studies of angiogenesis, and to investigate new treatments for peripheral artery disease. A limitation of these models involves our inability to easily visualize the regenerated vascular architecture. Approaches such as micro-computed tomography and micro-angiography are expensive, technically demanding and not available to many laboratories. Here we describe a rapid and inexpensive adaptation of a vascular staining procedure for precise imaging of the mouse hindlimb vasculature. We introduced two technical modifications and an analytical extension to the original method including (i) pre-skinning of the muscle prior to fixation that preserves tissue integrity, (ii) mild pressure-desiccation subsequent to fixing that enhances resolution and image penetration, and (iii) reconstruction of confocal data into 3D images. The procedure provides resolution that is equivalent or superior to other approaches at a fraction of the cost, time and technology required.

Synergistic angiogenic effect of codelivering fibroblast growth factor 2 and granulocyte-colony stimulating factor from fibrin scaffolds and bone marrow transplantation in critical limb ischemia.

Increasing evidence suggests that therapeutic angiogenesis strategies utilizing cytokines and stem cells are necessary to treat traumatic vascular events such as critical limb ischemia and peripheral artery disease. In this study, basic fibroblast growth factor 2 (FGF-2) and granulocyte-colony stimulating factor (G-CSF) were immobilized in fibrin matrices and codelivered in combination with unfractionated bone marrow cells. Hindlimb ischemia was induced on young (6-7 weeks) Balb/C mice, and fibrin gels containing 100 ng/mL of FGF-2 and G-CSF were implanted adjacent to the ligation points. In addition, 1×10(6) bone marrow (BM) cells were injected into five locations in the ischemic muscle immediately after ligation and artery excision. Hindlimb reperfusion was determined by Laser Doppler Perfusion Imaging and immunohistochemistry for CD31+ and smooth muscle actin-positive cells at 2, 4, and 8 weeks postsurgery to identify capillary formation and maturation. A fluorescent vessel painting technique was also utilized to determine the extent of angiogenesis and arteriogenesis in the hindlimb at 8 weeks postsurgery. The codelivery of FGF-2 and G-CSF in combination with BM cells led to enhanced therapeutic recovery in critical limb ischemia Balb/C mice after 8 weeks of treatment with 87.2% blood flow recovery and a significant increase (p<0.05) in capillary formation in comparison to growth factor delivery or BM cell administration alone.

Co-delivery of FGF-2 and G-CSF from gelatin-based hydrogels as angiogenic therapy in a murine critical limb ischemic model.

Peripheral artery disease and critical limb ischemia have become prevalent health risks in the United States due to an increasing elderly population and the prevalence of obesity and diabetes mellitus. Although highly invasive endarterectomy is the most popular method for treatment, angiogenic therapies based on growth factor administration are quickly becoming a popular alternative. Enzymatic degradation of these factors in vivo may be avoided by their incorporation in a delivery vehicle where the growth factor's release rate can be controlled by altering the vehicle's properties (i.e. cross-linking density, material selection, biodegradation, etc.). Herein, we report on the immobilization and controlled release of human recombinant basic fibroblast growth factor (FGF-2) and human recombinant granulocyte colony-stimulating factor (G-CSF) from ionic, gelatin-based hydrogel scaffolds to re-establish perfusion and induce capillary outgrowth in a murine hindlimb ischemic model. In vitro studies showed that endothelial cell proliferation was highly depended on FGF-2, whereas G-CSF stimulated migration and formation of a tubular network. When FGF-2 and G-CSF were used in combination there was an 82% increase in endothelial branch point formation compared to control groups. Leg reperfusion was assessed with laser Doppler perfusion imaging, while capillary outgrowth in the ischemic leg was evaluated using CD31(+) and alpha-SMA immunostaining. The co-delivery of G-CSF (1000 ngml(-1)) and FGF-2 (1000 ng ml(-1)) from the gelatin hydrogels resulted in a 3-fold increase in the perfusion levels and a 2-fold increase in capillary density and positive alpha-SMA vessels compared to the empty vehicle group. In conclusion, the co-delivery of FGF-2 and G-CSF was superior to bolus administration or the delivery of either factor alone in promoting reperfusion and mature vessel formation.

The effect of the controlled release of basic fibroblast growth factor from ionic gelatin-based hydrogels on angiogenesis in a murine critical limb ischemic model.

The localized delivery of exogenous, angiogenic growth factors has become a promising alternative treatment of peripheral artery disease (PAD) and critical limb ischemia. In the present study, we describe the development of a novel controlled release vehicle to promote angiogenesis in a murine critical limb ischemic model. Ionic, gelatin-based hydrogels were prepared by the carbodiimide-mediated amidation reaction between the carboxyl groups of gelatin or poly-L-glutamic acid molecules and the amine groups of poly-L-lysine or gelatin molecules, respectively. The degree of swelling of the synthesized hydrogels was assessed as a function of EDC/NHS ratios and the pH of the equilibrating medium, while the release kinetic profile of basic fibroblast growth factor (FGF-2) was evaluated in human fibroblast cultures. The degree of swelling (DS) decreased from 26.5+/-1.7 to 18.5+/-2.4 as the EDC concentration varied from 0.75 to 2.5 mg/ml. Eighty percent of the FGF-2 was released at controlled rates from gelatin-polylysine (gelatin-PLL) and gelatin-polyglutamic acid (gelatin-PLG) hydrogel scaffolds over a period of 28 days. Cell adhesion studies revealed that the negatively charged surface of the gelatin-PLG hydrogels exhibited superior adhesion capabilities in comparison to gelatin-PLL and control gelatin surfaces. Laser Doppler perfusion imaging as well as CD31(+) capillary immunostaining demonstrated that the controlled release of FGF-2 from ionic gelatin-based hydrogels is superior in promoting angiogenesis in comparison to the bolus administration of the growth factor. Over 4 weeks, FGF-2 releasing gelatin-PLG hydrogels exhibited marked reperfusion with a Doppler ratio of 0.889 (+/-0.04) which was 69.3% higher than in the control groups.