text:
Pre-existing immunity to human adenovirus serotype 5 (Ad5) is common in the general population. Bypassing pre-existing immunity could maximize Ad5 vaccine efficacy. Vaccination by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route with Ad5 expressing Ebola Zaire glycoprotein (Ad5-ZGP) fully protected naïve mice against lethal challenge with Ebola. In the presence of pre-existing immunity, only mice vaccinated I.N. survived. The frequency of IFN-γ+ CD8+ T cells was reduced by 80% and by 15% in animals vaccinated by the I.M. and P.O. routes respectively. Neutralizing antibodies could not be detected in serum from either treatment group. Pre-existing immunity did not compromise the frequency of IFN-γ+ CD8+ T cells (3.9±1% naïve vs. 3.6±1% pre-existing immunity, PEI) nor anti-Ebola neutralizing antibody (NAB, 40±10 reciprocal dilution, both groups). The number of INF-γ+ CD8+ cells detected in bronchioalveolar lavage fluid (BAL) after I.N. immunization was not compromised by pre-existing immunity to Ad5 (146±14, naïve vs. 120±16 SFC/million MNCs, PEI). However, pre-existing immunity reduced NAB levels in BAL by ∼25% in this group. To improve the immune response after oral vaccination, the Ad5-based vaccine was PEGylated. Mice given the modified vaccine did not survive challenge and had reduced levels of IFN-γ+ CD8+ T cells 10 days after administration (0.3±0.3% PEG vs. 1.7±0.5% unmodified). PEGylation did increase NAB levels 2-fold. These results provide some insight about the degree of T and B cell mediated immunity necessary for protection against Ebola virus and suggest that modification of the virus capsid can influence the type of immune response elicited by an Ad5-based vaccine.
Citation: Croyle MA, Patel A, Tran KN, Gray M, Zhang Y, Strong JE, et al. (2008) Nasal Delivery of an Adenovirus-Based Vaccine Bypasses Pre-Existing Immunity to the Vaccine Carrier and Improves the Immune Response in Mice. PLoS ONE 3(10): e3548. https://doi.org/10.1371/journal.pone.0003548
Editor: Denise L. Doolan, Queensland Institute of Medical Research, Australia
Received: July 2, 2008; Accepted: September 28, 2008; Published: October 29, 2008
Copyright: © 2008 Croyle et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: NIH, CRTI
Competing interests: The authors have declared that no competing interests exist.
The ability of human adenoviruses to induce strong innate and adaptive immune responses makes them powerful adjuvants that facilitate the immune response against an encoded antigen. Recombinant adenoviruses have been shown to elicit significant immune responses to bacterial (anthrax, plague), viral (Hepatitis C, Rabies, SARS) and tumour-associated antigens [1]–[3]. While these results are encouraging, immunity eventually develops against virus capsid proteins. This severely reduces the immunogenicity of adenovirus-based vaccines in mice, [4]–[8], primates [9] and humans [10]. This problem is also significant since a large portion of the Western world has marked levels of anti-adenovirus serotype 5 (Ad5) antibodies and is also prominent in regions of sub-Saharan Africa and Southeast Asia, where many of these vaccines are needed [11], [12]. Thus, assessment of the impact of pre-existing immunity on immune protection and alternative vaccination strategies may be needed for successful use of many adenovirus-based vaccines.
Several strategies have been developed to address the prevalence of pre-existing immunity to Ad5 in the general population. Increasing the vector dose or adopting a prime-boost regimen in order to overcome pre-existing immunity to the virus is a common approach [2]. There is mixed enthusiasm for this plan, however, due to the documented toxicity associated with high doses of adenovirus and the length of time required for prime-boost regimens when only a prime could be sufficient [13], [14]. 'Seroswitching', using recombinant adenoviruses constructed from chimpanzees or rare human serotypes with limited exposure rates such as adenoviruses 35 and 11 can elicit potent immune responses that are minimally affected by pre-existing immunity [7], [8], [15]–[21]. Hexon-chimeric adenoviruses can also avoid neutralization [22], [23]. Both approaches offer promise in the context of addressing pre-existing immunity, but require further investigation in response to concerns regarding safety and feasibility of large-scale production. Covalent attachment of polyethylene glycol or incorporation of the virus into polymer matricies can also effectively protect Ad5 from neutralization [24]–[31]. Delivery of Ad5-based vaccines by mucosal routes can also circumvent the effect of pre-existing immunity and induce a significant immune response against an encoded antigen [32].
Recombinant adenoviruses are one of the few well-studied vectors currently under development for vaccination against Ebola virus infection. The first protocol for an Ebola vaccine employed a prime-boost regimen consisting of naked DNA expressing either Ebola glycoprotein (GP) or nucleoprotein (NP) and recombinant Ad5 expressing Ebola GP to successfully protect non-human primates against a lethal challenge of Ebola [33]. This has since led to several Phase I clinical trials [34], [35] in which each component of the vaccine is administered by intramuscular injection. To date, there have been only two reports describing mucosal administration of an Ebola vaccine [36], [37]. Nasal administration of recombinant human parainfluenza virus type 3 (HPIV3) vectors expressing Ebola GP and/or NP to Guinea pigs and rhesus monkeys conferred complete protection against a lethal challenge with Ebola.
We have previously found that a single dose of a recombinant adenovirus expressing Ebola Zaire GP given by either the oral or the nasal route is capable of affording protection against lethal challenge in naïve mice and that mucosal immunization can stimulate a broad, prolonged T cell-mediated immune response in both the systemic and mucosal compartments [37]. The primary objective of this study was to test the hypothesis that administration of an Ad5-based Ebola vaccine by either the nasal or oral route can circumvent pre-existing immunity and confer full protection upon challenge. Systemic and mucosal T and B cell responses to Ebola GP were assessed in both naïve mice and those with pre-existing immunity. The influence of PEGylation of the vaccine carrier on the immune response after oral immunization is also described.
Materials and Methods
Construction, production and formulation of adenoviral vectors
The E1/E3-deleted adenovirus vector expressing the Ebola Zaire glycoprotein was created by cloning the open reading frame (ORF) sequence of the glycoprotein in the plasmid pShuttle (Adeno-X Expression system I, BD Clonetech, Palo Alto, CA) for subsequent insertion in the E1 region of the human adenovirus serotype 5 genome. The human cytomegalovirus (CMV) promoter included in the Adeno-X expression system was used to drive the expression of the Ebola Zaire glycoprotein in the final recombinant adenovirus serotype 5 construct. Authenticity of the final product was confirmed by sequencing of the recombinant virus rescued by transfecting the linearized DNA into 293 cells. Virus was sequentially amplified to large-scale infections (5×108 cells) and purified on an affinity column (Adeno-X virus purification mega kit, BD Clonetech, Mountain View, CA) according to the manufacturer's instructions. Genome structures of vectors were analyzed by restriction digestion of isolated viral DNA and compared with those of the original molecular clones. Particle number and infectivity of vectors were determined by standard optical density reading and immunodetection of the hexon protein, respectively, following infection of 293 cells with limiting dilutions of each vector preparation according to the recommendations by the manufacturer (Adeno-X rapid titer kit, Clontech, Mountain View, CA). Purified virus was administered in sterile phosphate buffered saline (pH 7.4) and had particle to plaque forming unit (pfu) ratios of 100∶1 or less.
PEGylation of recombinant adenovirus
First generation recombinant adenovirus expressing Ebola Zaire glycoprotein was prepared and purified as described above. The protein content of the virus preparation was determined using BioRad DC Protein Assay reagents (BioRad, Hercules, CA) and bovine serum albumin as a standard in a microplate format. According to established protocols, 10 µg of monomethoxypoly(ethylene) glycol, activated by tresyl chloride (Sigma Aldrich, St. Louis, MO), was added for each microgram of protein present [38]. The coupling reaction was performed at 25°C with gentle agitation. The reaction was stopped by the addition of L-lysine, in a 10-fold excess with respect to the amount of PEG added. Unreacted PEG, excess L-lysine, and reaction byproducts were removed by buffer exchange over a second Econo-Pac 10DG disposable chromatography column equilibrated with 100 mM potassium phosphate-buffered saline (pH 7.4). PEGylated preparations were administered in sterile potassium phosphate buffered saline (pH 7.4) and had particle to plaque forming unit (pfu) ratios of 100∶1 or less as determined by the Adeno-X rapid titer kit. Characterization of these preparations revealed significant changes in biophysical properties of the virus once the reaction was complete such as the PEG-Dextran partition coefficient and peak elution times during capillary electrophoresis (as described previously [30], [38]). Approximately 13,000 PEG molecules were associated with each virus particle in the studies outlined here as determined by a PEG-biotin assay [30].
Immunization of mice and challenge
B10.BR mice were immunized with 1×1010 particles of recombinant virus per mouse either by intramuscular injection (50 µl) in the right hindlimb, or by oral gavage (100 µl) using oral feeding needles (18G, 2.25 mm dia., Popper & Sons, Inc, New Hyde Park, NY). For nasal immunization, mice were anesthetized with isoflurane. Once anesthesia was achieved, 1×1010 particles of virus slowly delivered as a bolus into the nostrils using a standard micropipette (Gilson, Middleton, WI) as previously described [39].
Pre-existing immunity to adenovirus serotype 5 was established by injecting 5×1010 particles of adenovirus expressing beta-galactosidase (AdlacZ) by intramuscular injection in the right hindlimb 30 days prior to vaccination with Ad5-ZGP. This protocol has been documented to activate T and B cells against virus capsid proteins and elicit humoral immunity [8], [11]. At the time of vaccination, mice had an average anti-adenovirus circulating NAB titer of 1∶320, which falls within lower range of average values reported in humans after natural infection [11]. Mice were challenged by intraperitoneal injection of 200× LD50 of mouse-adapted Ebola virus, Zaire strain (MA-ZEBOV) in 200 µl sterile saline [40]. After challenge, the animals were weighed daily for 13 days and monitored for clinical signs of Ebola infection using an approved scoring sheet. All procedures and the scoring method were approved by the Institutional Animal Care Committee at the National Microbiology Laboratory (NML) of the Public Health Agency of Canada (PHAC) according to the guidelines of the Canadian Council on Animal Care. All infectious work was performed in the 'Biosafety Level 4' (BSL4) facility at NML, PHAC.
Antibody detection assays
A) Anti-ebola neutralizing antibody (serum).
Sera collected from immunized mice were inactivated at 56°C for 45 minutes. Serial dilutions of each sample (1∶10, 1∶20, 1∶40, etc, in 50 µl of DMEM) were mixed with equal volumes of recombinant Ebola Zaire expressing the enhanced green fluorescent protein (EGFP) reporter gene (ZEBOV-EGFP, 100 transducing units/well, according to EGFP expression) and incubated at 37°C for 90 minutes [41]. The mixture was then added to subconfluent VeroE6 cells in 96-well flat-bottomed plates and incubated for 5–10 minutes at room temperature. Control wells were infected with equal amounts of either ZEBO-EGFP with media without serum or that containing non-immune serum. 100 µl of DMEM supplemented with 20% FBS was then added to each well and plates were incubated at 37°C in 5% CO2 for 48 hr. Cells were subsequently fixed with 10% buffered formalin for 24 h and examined under a fluorescent microscope. Sample dilutions which showed >50% reduction in the number of green cells compared to controls scored positive for neutralizing antibody.
B) Anti-ebola neutralizing antibody (mucosal).
For the evaluation of the specific levels of IgG and IgA antibodies, bronchoalveolar lavage (BAL) fluid was collected in situ with a 20-gauge catheter inserted into the proximal trachea, flushing the lower airways three times with 1 milliliter of L15 media (Sigma). BAL from each animal was incubated at 56°C for 45 minutes. Two-fold serial dilutions were added to 96 well plates pre-coated overnight with 30 ng of ZEBOV-like particles per well and incubated at 37°C for 1 hr. Goat anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP) was then added and the plate was incubated for one additional hour at 37°C. The ABTS Peroxidase Substrate System (KPL) was used for detection and data collected using an ASYS UVM 340 ELISA plate reader (Isogen Life Science) at OD405. All infectious in vitro work was performed in the BSL4 laboratory at the NML, PHAC.
C) Anti-adenovirus serotype 5 neutralizing antibody (serum).
Pre-existing immunity against recombinant adenovirus 5 was assessed by determining the amount of neutralizing antibody present in serum according to established methods [30]. In brief, serum was incubated at 56°C for 30 minutes and then diluted in DMEM in twofold increments starting from a 1∶20 dilution. Each dilution (100 µl) was mixed with an aliquot of a standard stock of adenovirus type 5 expressing E. coli beta-galactosidase (106 pfu), incubated for 1 hour at 37°C, and applied to HeLa cells in 96-well plates (2×104 cells/well). One hundred microliters of DMEM supplemented with 20% FBS was then added to each well. Cells were incubated at 37°C for 24 hours. Neutralizing antibody titers were calculated as the highest dilution at which 50% of the cells stained blue by visual inspection.
Frequency of INF-γ positive cells
For the evaluation of INF-γ positive CD8+ T cells 10 days post-immunization, splenocytes were harvested and cultured (1×106/sample) for 5 hours at 37°C in 96-well round bottom microtiter plates in DMEM supplemented with 10% FBS, 2-beta-mercaptoethanol (10−6 M) and GolgiStop (1 µl/ml, BD PharMingen, San Diego, CA). The TELRTFSI peptide which carries the Ebola Zaire GP immunodominant MHC class I epitope for mice of the H-2k haplotype (B10.BR) was used for stimulation at a concentration of 1 µg/ml [42]. Control cells were treated with either an unrelated peptide or no peptide. After washing, cells were stained with 100 µl of a FITC-anti mouse CD8a antibody (1∶100 dilution, PharMingen) at 4°C for 30 minutes. Cells were washed again, permeabilized in 1× Cytofix/Cytoperm (PharMingen) for 20 minutes at 4°C, washed with 1× Perm/Wash (PharMingen) and stained with 100 µl of a PE-anti mouse IFN-γ antibody (1∶100 dilution, PharMingen) in the same buffer at 4°C for 30 minutes.
Quantitation of INF-γ positive CD8+ T cells isolated from splenocytes or mononuclear cells from bronchioalveolar lavage (BAL), mesenteric lymph nodes (MLN) and Peyer's patches (PP) 45 days after vaccination was performed using an ELISPOT assay (ELISPOT Mouse Set, BD PharMingen, San Diego, CA) according to the manufacturer's instructions. Briefly, a 96-well ELISPOT plate was coated with 5 µg/ml anti-mouse IFN-γ capture antibody. Cells pooled from 4 B10.BR mice per experimental group were added to microwells along with the TELRTFSI peptide (2 µg/well). Control cells were incubated either without peptide or with the non-specific stimulator, SEB (200 ng/ml). After incubation with a biotinylated anti-mouse IFN-γ detection antibody and Streptavidin-horseradish peroxidase antibody, wells were counted using an ELISPOT reader (AID EliSpot reader system, Cell Technology, Colombia, MD).
Effect of Mucosal Immunization on T and B Cell Responses Against Ebola Glycoprotein in Mice with Pre-Existing Immunity Against Adenovirus
In an effort to correlate markers of immunity with protection against Ebola infection after mucosal immunization, T and B cell specific immune responses against Ebola glycoprotein were analyzed in mice in the presence or absence of pre-existing immunity (PEI) to adenovirus 10 days after vaccination with a first generation adenovirus serotype 5 vector expressing the Zaire Ebola glycoprotein (Ad5-ZGP). Intracellular staining and flow cytometry (FACS) revealed that Ebola glycoprotein peptide-specific activation of CD8+ T cells, as measured by production of IFN-γ, occurred in naïve animals immunized by the nasal route at a frequency of 3.9±1% (I.N., Figure 1A). Pre-existing immunity was induced by intramuscular administration of recombinant Ad5 expressing a non-relevant antigen, beta-galactosidase (AdlacZ), 30 days prior to vaccination with Ad5-ZGP. At the time of vaccination, mice had an average anti-adenovirus circulating NAB titer of 1∶320. Pre-existing immunity did not significantly alter activation of CD8+ T cells when the vaccine was given intranasally (I.N.+PEI, 3.6±1%, p = 0.07). Oral immunization lowered the response against Ebola glycoprotein (P.O., 2±0.5%). Samples obtained from animals with pre-existing immunity and vaccinated in the same manner were barely above the positive threshold (three times the average background level obtained from animals treated with the irrelevant virus, AdlacZ). Samples obtained from naïve animals immunized by the intramuscular route (I.M.) contained the largest population of IFN-γ positive CD8+ T cells (10±2%). Animals treated with the AdlacZ vector alone (AdlacZ-control) served as negative controls and produced 0.5% CD8+, IFN-γ+ T cells in response to the Ebola glycoprotein-specific peptide.
Naïve mice (n = 10) were vaccinated with a single dose of 1×1010 particles of adenovirus expressing the Ebola Zaire glycoprotein (Ad5-ZGP) by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route. Animals in which pre-existing immunity (PEI) was established by I.M. injection of 5×1010 particles of adenovirus serotype 5 expressing beta-galactosidase (AdlacZ) 30 days prior to vaccination were also immunized in the same manner. At the time of vaccination, mice had an average anti-adenovirus circulating NAB titer of 1∶320. Animals given a single dose of (AdlacZ) served as negative controls (AdlacZ Control). (A) Frequency analysis of IFN-γ secreting CD8+ T cells harvested from splenocytes 10 days post-immunization (n = 4/group). The TELRTFSI peptide, specific for the Ebola Zaire glycoprotein (0.4 µg/well), was incubated with 1×106 splenocytes and cells analyzed by flow cytometry. (B) Neutralizing antibody (NAB) levels against ZEBOV-EGFP were evaluated 25 days post-vaccination (n = 10/group). In both panels, error bars represent the standard deviation of the data.
The B cell response against Ebola glycoprotein achieved by administration of the vaccine was determined by incubating a recombinant Ebola virus (Zaire strain) expressing green fluorescent protein (ZEBOV-EGFP) with serum collected 25 days after vaccination [8], [37]. Neutralizing antibody (NAB) levels equivalent to 40±10 reciprocal dilution were detected in both naïve animals and those with pre-existing immunity after intranasal immunization (Figure 1B). Samples from naïve animals immunized by the oral route contained NAB levels equivalent to 20±5 reciprocal dilution whereas anti-Ebola NAB could not be detected in samples obtained from animals immunized by the same route with pre-existing immunity (P.O.+PEI). NAB levels of 80±10 were detected in samples obtained from naïve mice immunized by the intramuscular route. Anti-Ebola NAB could not be detected in samples from mice given the AdlacZ vector alone (AdlacZ Control).
Effect of Mucosal Immunization on Systemic and Localized T-Cell Mediated Immune Responses Against Ebola Glycoprotein in Mice with Pre-Existing Immunity
Since the mucosa is often the primary sight of exposure, long-term, localized immune responses against Ebola virus are desirable for providing optimal protection against infection. In this context, T cell mediated immune responses were assessed by ELISPOT from various tissue compartments specific to the route of immunization 45 days after vaccination. The number of activated IFN-γ secreting mononuclear cells harvested from splenocytes was significantly reduced by 38 and 59% in mice vaccinated nasally or orally when compared to those immunized by intramuscular injection (p≤0.05, Figure 2A). In contrast, samples obtained from the spleen of animals with pre-existing immunity that were immunized by the I.N. route contained the highest number of activated IFN-γ secreting cells (815±190 spot-forming cells (SFC)/million mononuclear cells (MNCs), Figure 2C) although this was approximately 30% lower than that seen in naive animals (1,340±146 SFC/million MNCs, Figure 2A). Pre-existing immunity also significantly reduced the number of these cells produced by animals immunized by the I.M. (210±68 SFC/million MNCs) and P.O. routes (71±12 SFC/million MNCs) with respect to those seen in naïve animals (2,145±12 (I.M.) and 885±168 (P.O.)), p≤0.05, Figure 2A). A very limited number of activated IFN-γ secreting mononuclear cells were detected in splenocytes of naïve animals immunized with the AdlacZ vector (50±20 SFC/million MNCs, Figure 2A) and those with pre-existing immunity to the same virus (12±1, AdlacZ, Figure 2C). Significant levels of INF-γ positive cells were detected in bronchioalveolar lavage fluid (BAL) after intranasal immunization (146±14 SFC/million MNCs, p≤0.05, Figure 2B). This was not significantly compromised by pre-existing immunity to adenovirus (120±16 SFC/million MNCs, p≤0.06, Figure 2D). A limited number of INF-γ secreting cells were detected in BAL from naïve animals immunized by the I.M. or P.O. routes (4±2 and 11±5 SFC/million MNCs respectively, Figure 2B) and were similar to that seen in samples obtained from animals given the AdlacZ vector (12±1, negative control). Pre-existing immunity to adenovirus reduced these cell populations further to values that were not statistically significant (1±1 (I.M.), 5±2 (P.O.) and 2±1 (AdlacZ) SFC/million MNCs (p = 0.08, Figure 2D)). INF-γ positive cells were found in mesenteric lymph nodes (MLN) and Peyer's Patches (PP) (146±11 and 29±5 SFC/million MNCs, respectively) only after oral vaccination with Ad-ZGP (Figure 2B). Pre-existing immunity, however, reduced production of these cells to 18±12 SFC/million MNCs (MLN) and 16±2 SFC/million MNCs (PP) (Figure 2D).
The frequency of IFN-γ positive mononuclear cells was analyzed from the spleen (panels A and C), the lung via bronchioalveolar lavage (BAL) and the intestine via the mesenteric lymph nodes (MLN) and Peyer's patches (PP) (panels B and D) 45 days post-immunization by ELISPOT. Samples were obtained from naïve animals (Panels A and B) and those with pre-existing immunity to adenovirus serotype 5 (Panels C and D). Cells were plated at 1×105 or 1×104 cells per well, stimulated with the TELRTFSI peptide and expression of IFN-γ detected with an anti-mouse IFN-γ antibody. Cells isolated from BAL, MLN or PP of four mice were pooled and samples tested in duplicate. In each panel, the number of spot-forming cells (SFC) per million mononuclear cells (MNCs) is shown on the y-axis. Please note - the scale of this axis differs between panels to accent the differences between treatment groups. Error bars represent the standard deviation of the data. Animals immunized with an irrelevant adenoviral vector (AdlacZ) served as negative controls. Positive results obtained from this group are indicative of artificial cellular stimulation that may have occurred during processing and culturing of samples.
Effect of Mucosal Immunization on Localized and Systemic Antibody Production Against Ebola Glycoprotein
The NAB response was also monitored from bronchioalveolar lavage fluid 45 days post-vaccination. NAB to ZEBOV-EGFP was undetectable in samples obtained from control (AdlacZ) or I.M. immunized mice, whereas levels of 40±10 and 10±5 reciprocal dilution were detected in the BAL of nasally and orally vaccinated animals, respectively (Figure 3). Those with pre-existing immunity and immunized by the nasal route had NAB levels of 30±10. NAB was not detected in mice with pre-existing immunity to Ad5 and immunized by either the intramuscular or oral route.
Forty-five days after immunization, bronchioalveloar fluid was analyzed for the presence of neutralizing antibody by measuring the reduction in infectious titer of ZEBOV-EGFP. The reciprocal dilution plotted for each treatment group (n = 10) reflects the dilution at which the ability of the ZEBOV-EGFP vector to infect target cells was reduced by >50%. Data represent average values obtained from each treatment group and error bars represent the standard deviation of the data. NOTE - Data grouped on the left side of the figure under the label "Serum" is reproduced from Figure 1B of this manuscript and is included here for comparison. PEI - pre-existing immunity to adenovirus serotype 5.
Further characterization of Ebola glycoprotein-specific immunoglobulin isotypes obtained from bronchioalveolar lavage fluid revealed that pre-existing immunity correlated with a marked decrease in the production of both IgG and IgA antibodies in mice immunized by intramuscular injection (Figure 4A and 4B). IgG and IgA levels were the lowest in that were vaccinated orally regardless of whether they had pre-existing immunity (Figure 4A and 4B). Immunization by the nasal route induced a strong IgG response that was reduced by an average of 25% in the presence of pre-existing immunity. The strongest IgA response was detected in samples obtained from animals given the vaccine by the nasal route. IgA levels, however, were also reduced by 25% in mice with pre-existing immunity to Ad5.
Forty-five days after immunization, bronchioalveolar lavage fluid was analyzed for Ebola-specific IgG (panel A) and IgA (panel B) antibodies by ELISA. The optical densities obtained from each treatment group are presented to serve as a measure of relative concentration. PEI - pre-existing immunity to adenovirus serotype 5.
Effect of Mucosal Immunization on Survival Against Lethal Ebola Challenge in the Presence of Pre-Existing Immunity
The most direct means of evaluating vaccine efficacy in mice is to assess protection by monitoring weight loss and death rates after a lethal challenge of Ebola [43]. Therefore, mice were immunized with 1×1010 particles of Ad5-ZGP by the intramuscular (I.M.), intranasal (I.N.) or oral (P.O.) route in the presence or absence of pre-existing immunity. At the time of vaccination, animals had an average anti-adenovirus neutralizing antibody titer of 1∶320 reciprocal dilution. Naïve mice vaccinated by intramuscular injection with saline (vehicle) served as controls for complete lethality following challenge with mouse-adapted Ebola virus. Twenty-eight days after vaccination, animals were challenged with 200 LD50 of mouse-adapted (MA)-ZEBOV. Only mice immunized by the nasal route survived the lethal challenge (Figure 5A). This was evident as early as 5 days after challenge when controls and mice immunized by the other routes began to lose weight (Figure 5B). All eventually expired 7 days post-challenge (Figure 5A, some data not shown for clarity). In contrast, naïve mice given the vaccine survived challenge regardless of the route of administration.
Naïve mice (n = 10) were vaccinated with a single dose of 1×1010 particles of adenovirus expressing the Ebola glycoprotein (Ad5-ZGP) by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route. Animals in which pre-existing immunity (PEI) was established by I.M. injection of 5×1010 particles of adenovirus 5 expressing beta-galactosidase (AdlacZ) were also vaccinated in the same manner. Twenty-eight days later, mice were challenged with 200 LD50 of mouse-adapted Ebola virus (Zaire strain). Data represent survival (panel A) and loss of body weight (panel B) over time and is reported as average body weight from each treatment group. Mock - age matched, untreated, unchallenged mice included in this data set to indicate normal weight variation over time. NOTE: Data for naïve mice immunized by the oral route (P.O.) and those with pre-existing immunity and vaccinated by the I.M. route (I.M.+PEI) were not included in this figure for visual clarity. All naive mice immunized orally survived challenge while none in the I.M.+PEI group survived.
Effect of PEGylation on Improving the Immune Response Achieved by Oral Vaccination
The studies outlined above strongly suggest that only intranasal immunization can successfully afford protection against Ebola virus in the absence and presence of pre-existing immunity. It was also clear that additional strategies were needed to improve vaccination by either the oral or intramuscular route in the presence of pre-existing immunity. Covalent attachment of activated monomethoxypolyethylene glycol to the protein capsid of the Ad5-ZGP vector was evaluated as a potential way to improve the immune response after oral vaccination. Based upon previous observations, we hypothesized that this modification would protect the virus from neutralization by immune sera and improve survival in gastrointestinal tract [26], [27], [30], [44]. The frequency of Ebola glycoprotein peptide-specific activation of IFN-γ positive CD8+ T cells was not significant in naïve animals given the PEGylated virus (0.3±0.3%) with respect to those given the unmodified virus (2.0±0.5%, p = 0.09, Figure 6A). Similar results were also seen in the presence of pre-existing immunity in either treatment group (0.4±0.2% PEGylated vaccine, 1.7±0.5%, unmodified vaccine).
Naïve mice and those with pre-existing immunity were vaccinated with 1×1010 particles of either unmodified (Vaccine) or PEGylated (PEG-Vaccine) Ad5-ZGP by oral gavage. Pre-existing immunity (PEI) was induced by I.M. injection with 5×1010 particles of adenovirus expressing beta-galactosidase (AdlacZ). (A) Frequency analysis of IFN-γ secreting CD8+ T cells harvested from splenocytes 10 days post-immunization (n = 4/group). (B) Neutralizing antibody (NAB) levels against ZEBOV-EGFP were evaluated 25 days post-vaccination (n = 10/group). (C) Profile of anti-Ebola-specific IgG antibodies. (D) Profile of anti-Ebola-specific IgA antibodies. In all panels, error bars represent the standard deviation of the data.
In contrast, NAB levels equivalent to 30±10 reciprocal dilution were detected in naïve animals given the PEGylated vaccine (Figure 6B). Samples obtained from animals given the unmodified virus had levels of 20±5 reciprocal dilution. Animals with pre–existing immunity against adenovirus and treated with the PEGylated preparation were also able to produce detectable levels of NAB (10±5 reciprocal dilution) whereas NAB was not found in sera from mice given the unmodified vector orally. Further characterization of Ebola glycoprotein-specific Ig isotypes revealed that the PEGylated vector could possibly stimulate production of IgG in the presence or absence of pre-existing immunity with respect to levels found in animals given the unmodified vector (Figure 6C). Modification of the virus induced a slight increase of IgA in the presence of pre-existing immunity with respect to that seen in naïve animals given unmodified virus (Vaccine, Figure 6D) and those with pre-existing immunity (Vaccine+PEI). Despite this, animals with pre-existing immunity to adenovirus and immunized with the PEGylated vaccine orally did not survive after challenge with 200 LD50 of (MA)-ZEBOV.
With mortality rates as high as 95%, Ebola infection occurs largely by direct contact with blood, tissues or skin of patients, and through mucosal exposure [45], [46]. To date, few efforts have been made to focus on the mucosa as the primary sight of exposure to Ebola virus and priming it for participation in the host defense by vaccination [47]. This is surprising given that the pulmonary, nasal and oral immune systems which comprise the mucosal-associated lymphoid tissues (MALT) are responsible for the production of approximately 80% of all immunocytes [48], [49] and mucosal immunity is often the first line of defense against pathogens coming in contact with susceptible hosts. Many studies in rodents indicate that systemic immunization produces strong anti-viral systemic responses while mucosal vaccination can stimulate both the mucosal and systemic immune systems and can confer long-term immunological memory against a given pathogen despite the fact that the magnitude of these responses are often reported to be somewhat reduced [37], [48], [50]. We have found this to be the case in the studies outlined here since the systemic cellular response and neutralizing antibody levels against Ebola Zaire GP were consistently lower following nasal and oral vaccination. Mucosal vaccination did, however, generate significant cellular and antibody responses in the periphery (BAL, MLN or Peyer's patches) and a single intranasal immunization with Ad5-ZGP conferred 100% protection even in the presence of pre-existing immunity.
Administration of recombinant adenovirus-based vaccines to the mucosa has also conferred sufficient protection against challenge with a variety of pathogens in the presence of pre-existing immunity to the vaccine carrier in mice and other pre-clinical models of disease [5], [19], [32], [51], [52]. This served as a basis for the present study. A single intranasal dose of a recombinant Ad5 vaccine expressing the Zaire Ebola glycoprotein conferred 100% protection in both naïve mice and those with pre-existing immunity despite the fact that the strength of the immune response generated by this route of administration was quantitatively lower than that seen in animals vaccinated by intramuscular injection. It is also important to note that pre-existing immunity induced by intramuscular injection did not severely compromise the T cell-mediated response at either the systemic or mucosal levels in these animals. The level of anti-Ebola GP antibodies in the circulation and in the lung was also not significantly compromised by pre-existing immunity to adenovirus. While these results suggest that intranasal vaccination with an Ad-based vaccine is indeed a promising strategy to overcome pre-existing immunity, one might wonder how accurately our results translate to natural exposure to the wild type virus via the respiratory tract. This is somewhat difficult to establish in the mouse model since the ability of the wild-type adenovirus to replicate is limited [53]. In addition, the amount of neutralizing antibody present in the nasal cavity of those in the general population with pre-existing immunity to adenovirus 5 has not been assessed to the degree that serum neutralizing antibodies have, making it difficult to set a relative parameter for one to target in pre-clinical animal models. Lack of this data may be due to the invasive nature of the technique for acquiring samples and/or the fact that antibody levels in the mucosa of individuals with established pre-existing immunity to adenovirus type 5 are quite low and transient in contrast to systemic levels of anti-adenovirus neutralizing antibodies which are quite robust and persist over time (unreported observations). Thus, for these studies, we decided to induce pre-existing immunity against adenovirus by intramuscular injection at a dose that has been shown to induce production of systemic neutralizing antibodies at 1∶320, a level mostly below what was reported in humans with documented pre-existing immunity [8], [11], [12]. Additional studies designed to assess the amount of neutralizing antibody to AdHu5 in the lung over time after intranasal administration of varying doses of AdHu5 are currently underway in an effort to further define stringent conditions under which pre-existing immunity can be established for experimental testing.
Data obtained from animals vaccinated by the oral route provided several insights about the immunological requirements for protection against Ebola in a mouse model. Immunization by this route induced quantitatively lower T and B cell mediated immune responses against Ebola glycoprotein with respect to that achieved by either intramuscular or intranasal immunization. Despite this, every naïve animal immunized with a single oral dose of Ad-ZGP survived challenge, giving better precision on the minimal threshold of immunity required to achieve protection. As seen with intranasal immunization, the T cell-mediated response was not compromised by pre-existing immunity 10 days after oral vaccination. The number of IFN-γ secreting T cells in both mucosal and systemic compartments of these animals at day 45, however, was significantly reduced by pre-existing immunity. It is possible that the peak T cell response at day 10 does not reflect the extent by which pre-existing immunity decreases the Ad5-ZGP-induced T cell response. Alternatively, it is also possible that the ELISPOT assay could detect subtle variations that flow cytometery could not monitor accurately due to differences in the sensitivity of each assay. Neutralizing antibody was not detected in the serum of animals with pre-existing immunity given a single oral dose of the vaccine. More importantly, none of these animals survived challenge with mouse adapted Ebola.
We attempted to improve the immunogenicity of the adenovirus-based vaccine by protecting it from the harsh environment of the gastrointestinal tract and from neutralization by anti-adenovirus antibodies by PEGylation. Interestingly, the T-cell mediated immune response was significantly reduced and antibody levels increased in naïve animals given a single oral dose of the PEGylated vaccine with respect to that seen in animals given the same dose of unmodified Ad5-ZGP. It has been shown that modification of virus capsids by PEGylation can significantly dampen the T-cell mediated immune response against the virus and stimulate the antibody response against a secreted antigen [26], [27], [30], [54]. Taken together, these data support the notion that the exposition of the virus capsid proteins facilitates the immune response against the encoded antigen. Optimization of PEGylation chemistries and/or densities on adenovirus-based vaccine that promote and strengthen protective immune responses following oral immunization is currently underway.
Delivery of recombinant adenoviral vaccines to either the nasal or intestinal mucosa is an attractive vaccination strategy for many reasons. Vaccines administered in this manner will offer improved safety with respect to disease transmission and needle-stick injuries among health care workers, significant issues of concern in developing countries where the demand for many vaccines is high [55]. Mucosal administration of vaccines reduces the pain associated with vaccination, eliminates the need for specialized training programs for large vaccination campaigns and makes self-administration of the vaccine possible. This route of administration may also significantly reduce systemic toxicity associated with recombinant adenovirus despite the fact that it has been shown that nasal immunization with recombinant adenovirus-based vaccines can facilitate translocation of the virus to the central nervous system [52]. Even though testing of other virus-based vaccines such as influenza have reported similar findings and are currently used in the clinic [56], studies designed to fully assess the toxicological profile of adenovirus vaccine after nasal administration are also currently underway.
We have shown that nasal immunization with an Ad5-based vaccine can induce a long-term protective immune response against Ebola virus in a mouse model which is not impeded by pre-existing immunity to adenovirus serotype 5. While these results are extremely encouraging, further characterization of the immune response against both the encoded antigen and the adenovirus vector in larger, clinically relevant animal models is vital for both understanding the biology of Ad vaccines and for the development of an effective Ebola vaccine suitable to populations with different requirements [57], [58]. The issue of pre-existing immunity must also be adequately addressed in order to develop efficient recombinant adenovirus-based vaccines. While the majority of the literature suggests that pre-existing immunity significantly hamper the effective use of AdHu5 vaccine carriers, other investigators have reported that pre-existing immunity did not interfere with the potency of recombinant Ad5-based vaccines in both pre-clinical models of disease and in humans [59], [60]. Thus, additional studies identifying clinically relevant conditions under which to test Ad-based vaccine candidates are necessary to assess the full impact of pre-existing immunity on vaccine potency, including in different compartments. Better define the role of pre-existing immunity on vaccine-induced immunity will further the understanding of how individuals previously exposed to adenovirus will respond to these immunization regimens.
Author Contributions
Conceived and designed the experiments: MC GPK. Performed the experiments: MC AP KT MG JS GPK. Analyzed the data: MC AP YZ GPK. Contributed reagents/materials/analysis tools: MC GPK. Wrote the paper: MC. Provided access to BSL4 for challenge: HF.
- 1. Boyer JL, Kobinger G, Wilson JM, Crystal RG (2005) Adenovirus-based genetic vaccines for biodefense. Hum Gene Ther 16: 157–168.
- 2. Gallo P, Dharmapurim S, Cipriani B, Monaci P (2005) Adenovirus as vehicle for anticancer genetic immunotherapy. Gene Ther 12: S84–S91.
- 3. Liniger M, Zuniga A, Naim HY (2007) Use of viral vectors for the development of vaccines. Expert Rev Vaccines 6: 255–266.
- 4. Papp Z, Babiuk LA, Baca-Estrada ME (1999) The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1. Vaccine 17: 933–943.
- 5. Xiang ZQ, Gao GP, Reyes-Sandoval A, Li Y, Wilson JM, et al. (2003) Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier. J Virol 2003 77: 10780–10789.
- 6. Yang ZY, Wyatt LS, Kong WP, Moodie Z, Moss B, et al. (2003) Overcoming immunity to a viral vaccine by DNA priming before vector boosting. J Virol 77: 799–803.
- 7. Barouch DH, Pau MG, Custers JH, Koudstaal W, Kostense , et al. (2004) Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity. J Immunol 172: 6290–6297.
- 8. Kobinger GP, Feldmann H, Zhi Y, Schumer G, Gao G, et al. (2006) Chimpanzee adenovirus vaccine protects against Zaire Ebola virus. Virology 346: 394–401.
- 9. Casimiro DR, Chen L, Fu TM, Evans RK, Caulfield , et al. (2003) Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene. J Virol 77: 6305–6313.
- 10. Kresge KJ (2005) Clinical trials yield promising results from two adenovirus-based vaccines. IAVI Rep 9: 24.
- 11. Nwanegbo E, Vardas E, Gao W, Whittle H, Sun , et al. (2004) Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States. Clin Diagn Lab Immunol 11: 351–357.
- 12. Abbink P, Lemckert AA, Ewald BA, Lynch DM, Denholtz M, et al. (2007) Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D. J Virol 81: 4654–4663.
- 13. Hartman ZC, Appledorn DM, Amalfitano A (2008) Adenovirus vector induced innate immune responses: Impact upon efficacy and toxicity in gene therapy and vaccine applications. Virus Res 132: 1–14.
- 14. Osten P, Grinevich V, Cetin A (2007) Viral vectors: a wide range of choices and high levels of service. Handb Exp Pharmacol 1788: 177–202.
- 15. Xiang Z, Li Y, Gao G, Wilson JM, Ertl HC (2003) Mucosally delivered E1-deleted adenoviral vaccine carriers induce transgene product-specific antibody responses in neonatal mice. J Immunol 171: 4287–4293.
- 16. Vogels R, Zuijdgeest D, van Rijnsoever R, Hartkoorn E, Damen I, et al. (2003) Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity. J Virol 77: 8263–8271.
- 17. Kostense S, Koudstaal W, Sprangers M, Weverling GJ, Penders , et al. (2004) Adenovirus types 5 and 35 seroprevalence in AIDS risk groups supports type 35 as a vaccine vector. AIDS 18: 1213–1216.
- 18. Holterman L, Vogels R, van der Vlugt R, Sieuwerts M, Grimbergen , et al. (2004) Novel replication-incompetent vector derived from adenovirus type 11 (Ad11) for vaccination and gene therapy: low seroprevalence and non-cross-reactivity with Ad5. J Virol 78: 13207–13215.
- 19. Hashimoto M, Boyer JL, Hackett NR, Wilson JM, Crystal RG (2005) Induction of protective immunity to anthrax lethal toxin with a nonhuman primate adenovirus-based vaccine in the presence of preexisting anti-human adenovirus immunity. Infect Immun 73: 6885–6891.
- 20. Lemckert AA, Sumida SM, Holterman L, Vogels R, Truitt DM, et al. (2005) Immunogenicity of heterologous prime-boost regimens involving recombinant adenovirus serotype 11 (Ad11) and Ad35 vaccine vectors in the presence of anti-ad5 immunity. J Virol 79: 9694–9701.
- 21. Zhi Y, Figueredo J, Kobinger GP, Hagan H, Calcedo R, et al. (2006) Efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus. Hum Gene Ther 17: 500–506.
- 22. Youil R, Toner TJ, Su Q, Chen M, Tang A, et al. (2002) Hexon gene switch strategy for the generation of chimeric recombinant adenovirus. Hum Gene Ther 13: 311–320.
- 23. Roberts DM, Nanda A, Havenga MJ, Abbink P, Lynch DM, et al. (2006) Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity. Nature 441: 239–243.
- 24. Beer SJ, Matthews CB, Stein CS, Ross BD, Hilfinger JM, et al. (1998) Poly (lactic-glycolic) acid copolymer encapsulation of recombinant adenovirus reduces immunogenicity in vivo. Gene Ther 5: 740–746.
- 25. O'Riordan CR, Lachapelle A, Delgado C, Parkes V, Wadsworth , et al. (1999) PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo. Hum Gene Ther 10: 1349–1358.
- 26. Croyle MA, Chirmule N, Zhang Y, Wilson JM (2001) "Stealth" adenoviruses blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung. J Virol 75: 4792–4801.
- 27. Croyle MA, Chirmule N, Zhang Y, Wilson JM (2002) PEGylation of E1-deleted adenovirus vectors allows significant gene expression on readministration to liver. Hum Gene Ther 13: 1887–1900.
- 28. Sailaja G, HogenEsch H, North A, Hays J, Mittal SK (2002) Encapsulation of recombinant adenovirus into alginate microspheres circumvents vector-specific immune response. Gene Ther 9: 1722–1729.
- 29. Steel JC, Cavanagh HM, Burton MA, Kalle WH (2004) Microsphere-liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in-vitro. J Pharm Pharmacol 56: 1371–1378.
- 30. Croyle MA, Le HT, Linse KD, Cerullo V, Toietta , et al. (2005) PEGylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile. Gene Ther 12: 579–587.
- 31. Eto Y, Gao JQ, Sekiguchi F, Kurachi S, Katayama K, et al. (2005) PEGylated adenovirus vectors containing RGD peptides on the tip of PEG show high transduction efficiency and antibody evasion ability. J Gene Med 7: 604–612.
- 32. Santosuosso M, McCormick S, Xing Z (2005) Adenoviral vectors for mucosal vaccination against infectious diseases. Viral Immunol 18: 283–291.
- 33. Sullivan NJ, Sanchez A, Rollin PE, Yang ZY, Nabel GJ (2000) Development of a preventive vaccine for Ebola virus infection in primates. Nature 408: 605–609.
- 34. Martin JE, Sullivan NJ, Enama ME, Gordon IJ, Roederer M, et al. (2006) A DNA vaccine for Ebola virus is safe and immunogenic in a phase I clinical trial. Clin Vaccine Immunol 13: 1267–1277.
- 35. Sullivan NJ, Geisbert TW, Geisbert JB, Shedlock DJ, Xu L, et al. (2006) Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified GPs. PLoS Med 3: e177. Epub 2006 May 2016.
- 36. Bukreyev A, Yang L, Zaki SR, Shieh WJ, Rollin PE, et al. (2006) A single intranasal inoculation with a paramyxovirus-vectored vaccine protects guinea pigs against a lethal-dose Ebola virus challenge. J Virol 80: 2267–2279.
- 37. Patel A, Zhang Y, Croyle M, Tran K, Gray M, et al. (2007) Mucosal delivery of adenovirus-based vaccine protects against Ebola virus infection in mice. J Infect Dis 196: S413–S420.
- 38. Croyle MA, Yu QC, Wilson JM (2000) Development of a rapid method for the PEGylation of adenoviruses with enhanced transduction and improved stability under harsh storage conditions. Hum Gene Ther 11: 1713–1722.
- 39. Laddy DJ, Yan J, Corbitt N, Kobasa D, Kobinger GP, et al. (2007) Immunogenicity of novel consensus-based DNA vaccines against avian influenza. Vaccine 25: 2984–2989.
- 40. Bray M, Davis K, Geisbert T, Schmaljohn C, Huggins J (1998) A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J Infect Dis 178: 651–661.
- 41. Hoenen T, Groseth A, Kolesnikova L, Theriault S, Ebihara H, et al. (2006) Infection of naive target cells with virus-like particles: implications for the function of ebola virus VP24. J Virol 80: 7260–7264.
- 42. Rao M, Matyas GR, Grieder F, Anderson K, Jahrling PB, et al. (1999) Cytotoxic T lymphocytes to Ebola Zaire virus are induced in mice by immunization with liposomes containing lipid A. Vaccine 17: 2991–2998.
- 43. Gibb TR, Bray M, Geisbert TW, Steele KE, Kell WM, et al. (2001) Pathogenesis of experimental Ebola Zaire virus infection in BALB/c mice. J Comp Pathol 125: 233–242.
- 44. Cheng X, Ming X, Croyle MA (2003) PEGylated adenoviruses for gene delivery to the intestinal epithelium by the oral route. Pharm Res 20: 1444–1451.
- 45. Johnson E, Jaax N, White J, Jahrling P (1995) Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus. Int J Exp Pathol 76: 227–236.
- 46. Jaax NK, Davis KJ, Geisbert TJ, Vogel P, Jaax GP, et al. (1996) Lethal experimental infection of rhesus monkeys with Ebola-Zaire (Mayinga) virus by the oral and conjunctival route of exposure. Arch Pathol Lab Med 120: 140–155.
- 47. Mohamadzadeh M, Chen L, Schmaljohn AL (2007) How Ebola and Marburg viruses battle the immune system. Nat Rev Immunol 7: 556–567.
- 48. Kiyono H, Fukuyama S (2004) NALT- versus Peyer's-patch-mediated mucosal immunity. Nat Rev Immunol 4: 699–710.
- 49. Lefrançois L, Puddington L (2006) Intestinal and pulmonary mucosal T cells: local heroes fight to maintain the status quo. Annu Rev Immunol 24: 681–704.
- 50. Neutra MR, Kozlowski PA (2006) Mucosal Vaccines: The Promise and the Challenge. Nat Rev Immunol 6: 148–158.
- 51. Appaiahgari MB, Saini M, Rauthan M, Vrati J, Vrati S (2006) Immunization with recombinant adenovirus synthesizing the secretory form of Japanese encephalitis virus envelope protein protects adenovirus-exposed mice against lethal encephalitis. Microbes Infect 8: 92–104.
- 52. Zhou D, Cun A, Li Y, Xiang Z, Ertl HC (2006) A chimpanzee-origin adenovirus vector expressing the rabies virus glycoprotein as an oral vaccine against inhalation infection with rabies virus. Mol Ther 14: 662–672.
- 53. Duncan SJ, Gordon FC, Gregory DW, McPhie JL, Postlethwaite , et al. (1978) Infection of mouse liver by human adenovirus type 5. J Gen Virol 40: 45–61.
- 54. Wortmann A, Vöhringer S, Engler T, Corjon S, Schirmbeck R, et al. (2008) Fully Detargeted Polyethylene Glycol-coated Adenovirus Vectors Are Potent Genetic Vaccines and Escape from Pre-existing Anti-adenovirus Antibodies. Mol Ther. 2007 Sep 11. Mol Ther 16: 154–162.
- 55. Prüss-Ustün A, Rapiti E, Hutin Y (2005) Estimation of the global burden of disease attributable to contaminated sharps injuries among health-care workers. Am J Ind Med 48: 482–490.
- 56. Iwasaki T, Itamura S, Nishimura H, Sato Y, Tashiro M, et al. (2004) Productive infection in the murine central nervous system with avian influenza virus A (H5N1) after intranasal inoculation. Acta Neuropathol 108: 485–492.
- 57. Gerdts V, Littel-van den Hurk SD, Griebel PJ, Babiuk LA (2007) Use of animal models in the development of human vaccines. Future Microbiol 2: 667–675.
- 58. Reed DS, Mohamadzadeh M (2007) Status and challenges of filovirus vaccines. Vaccine 25: 1923–1934.
- 59. Zeng M, Xu Q, Elias M, Pichichero ME, Simpson LL, et al. (2007) Protective immunity against botulism provided by a single dose vaccination with an adenovirus-vectored vaccine. Vaccine 25: 7540–7548.
- 60. Van Kampen KR, Shi Z, Gao P, Zhang J, Foster KW, et al. (2005) Safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans. Vaccine 23: 1029–1036.


text:
luscious and lovely discrete petite & sweet gorgeous sweet & innocent sensual and lovely.
if you crave adventure and exciting experiences, then there is no time to be wasted.
chat with real people in your area tonight looking for a one night stand!
hookup with the hottest real people in your city tonight. it's completely free
create your 100% free profile right now! sign up to start hooking up: http://freesexx.wixsite.com/x140 erotic services alberta

subset: commoncrawl


text:
Celebrate Independent Bookstore Day with Free Audiobooks on Saturday, April 27th.
Learn More
View bestselling audiobooks from hundreds of bookstores nationwide.
Bookseller Recommendation
The Invited
"A good, old-fashioned ghost story! The Invited has all of the elements of a fireside spooky tale."Mary, Anderson's Bookshop
New Releases
Discover our new arrivals and recently released audiobooks.
Bookseller Recommendations
Find hidden gems personally recommended by our amazing bookseller partners.
Our playlists, or suggested categories of audiobooks, are updated frequently and many are hand curated by our indie partner bookstores.
A topic for everyone. And if you're not finding what you're looking for, try searching by title, author or narrator.


text:
Simple tricks to eat less
You follow your diet, do exercises, but you still can not lose weight? Meet – simple secrets that will help you eat less, quickly feel full and teach you to stop when the body has already received the necessary amount of food. Read about this in our article!
It is known that many women continue to gain weight, even if their diet consists of natural and healthy foods. The blame for everything is that they do not know how or can not limit themselves in the amount of regularly consumed food.
If you learn to eat less and be saturated with less without overeating, then you will be able to achieve the desired weight, easily get rid of extra pounds and lose weight in a natural and healthy way.
In this article we will share with you some simple tricks that will help you eat small portions and in time to feel when the body is already saturated and when you need to tell yourself "stop." So you can only eat according to the natural need of the body and get rid of the desire to "eat" in a state of excitement, stress or an unmotivated whim.
How to teach the body less eat?
There are some simple tips that will help you limit the amount of food that you consume:
- Eat several times during the day in between meals, so you do not have a feeling of hunger for a certain hour. For example, you can have breakfast, eat something closer to noon, have dinner, surcharge and have supper.
- Eat sitting and not hurrying. Never have dinner "on the go" and do not make a "snack" in the workplace.
- Try not to talk, do not watch TV and do no other business while eating.
- If you are annoyed or sad, wait a little before eating.
- Put as much on the plate as you can eat. If you have cooked food left, immediately take it to the refrigerator so that you do not have a desire to finish it just because you do not want to leave it.
- Avoid "snacking" and fight with the desire to eat something delicious in between meals. Train yourself to put food on a plate so you can clearly see the amount of food that you are going to eat.
Foods rich in dietary fiber
Dietary fiber is a useful and very nutritious substance found in many foods, which contributes to rapid saturation. And this means that you can eat less, but feel more satisfied – such a paradox useful for the figure!
We all know that any diet is based on the principle of a balanced diet. However, remember that in your daily diet should always be present the following products:
- Ripe fruit: apples, plums, peaches, apricots, melons, etc.
- Vegetables and greens: cabbage, beets, carrots, etc.
- Cereals: rice, wheat, millet, etc.
- Plant germs
- Nuts
- Dried fruits: prunes, raisins, dried apricots, etc.
- Mushrooms
- Seaweed
From the above list, we distinguish seaweed, because it is a very useful, nutritious and, unfortunately, little-known product.
Algae contain substances that contribute to the absorption of water, absorb it, from which they swell, increase in size and cause rapid saturation.
First of all we advise you to pay attention to the following algae:
- Agar-agar
- Fucus (or "sea grapes")
- Spirulina
- Wakame
- Kombu
You can add algae to the dishes that you cook every day (in soups, salads, rice, pasta), or buy algae in a pharmacy in the form of a natural food supplement.
Drink water before eating
There is another wonderful way that will help you get rid of excess appetite and save from the risk of overeating – every time before eating one or two glasses of water. Usually we drink during the day in between meals, but we advise you to drink water just before meals to fill the stomach and prevent the desire to eat quickly and in large quantities.
Also, you can get into the habit of eating some fruit before eating, thanks to the high content of water and fiber in the fruit. In addition, it is known that they are easily absorbed by the body, improve the functioning of the digestive system, so fruit is an ideal variant of a tasty and healthy dessert!
And for dessert – a drink that improves digestion
After a meal, you always want to eat a bun or a chocolate bar, so they say that dessert is something that is hardest for us to control. Firstly, because during the meal our body has already received everything that it needed, and dessert often becomes a surplus. And secondly, because as a dessert, we usually choose products that are difficult to digest by the body, which most often cause excess kilograms.
Therefore, if the psychological factor requires you to finish eating dessert, choose the drinks that are easily absorbed by the body, including coffee. If you still do not feel full, because you ate a little, choose an apple or pear as a dessert – two fruits that do not disturb the process of mastering the main food, and also you can prepare the following tasty and useful natural desserts:
- Compote of dried fruits – apples and raisins
- Baked apples with honey and cinnamon
- Pears in wine or grape juice
- Carpaccio of pear with roasted almonds


text:
Samuel Rangel
Merced High Class of 1990
Personal Info
- Name:
- Samuel Rangel
- Maiden Name:
- Rangel
- Graduation Year:
- 1990
Register to view Current location, Gender, Birthday, Relationship Status, Children, Employer, Military, College, and Interests
Memories of Samuel Rangel
What I'm doing now:Register to see what Samuel Rangel is up to now!
Samuel Rangel's Latest Activity:
|- Registered as a MHS Class of 1990 alumni. October
|- Added photos to their personal photo gallery. October


text:
Security Engineer | Reviews and reports on security baselines. Monitors and manages network security systems and applications. Deploys and configures new solutions to support business requirements for network security.
Upload a Resume | gif | png | jpg | jpeg | pdf | doc | docx
Would you care to add any comments?
Comments are closed.


text:
|anal interracial analsex bigblackcock analfuck
My brother's girlfriend loves big black cock and she begs me to fuck in mouth
Little pint size bitch Stacy Snake - Interracial Gang bang - Ass Destruction!
Onion Booty culos grandes big ass - Obsession - Huge Cock Anal (Mandingo)
FirstAnalQuest.com - INTERRACIAL ANAL WITH A MILKY WHITE BLONDE HAIR BEAUTY
Asa Akira Takes Big Black Cocks into Her Butthol - Lexington Steele, Prince Yahs
butt-lickin-sweethearts-scene1 | More videos with this girl - likefucker.com
All models were 18 years of age or older at the time of depiction.
uporn.party has a zero-tolerance policy against illegal pornography.
© Copyright Uporn.party 2019


text:
That so mighty a king should possess these powers seemed to Shaka self-evident. Just as the Bullet Bill is about to hit Mario, Bowser jumps in front of Mario and whacks the Bullet Bill back to Wario and Waluigi, launching them high into the sky. Hot indian naked sex. Good lesbian sex videos. Bella is really into photography and at some point in he story Edward buys her a camera.
For although it costs thousands of dollars a day to dock at the Manhattan Cruise Terminal anchoring is far cheaper and parking right by the Statue would be helping to save Shvidler money.
You get the sense that he was an incredibly intelligent and sensitive man-clearly had a thirst for drama and attention.
The Interpretation of Dreams - By Sigmund Freud Psychology is one thing that makes us curious. Do a thing sharply, with undeliberated action, as when a man comes home from work, throws down his sickle ace.
The imagery suggests young boys at school or other organised uniformed activities, in which case it would have been a natural metaphor for figures of authority to direct at youngsters. If someone does judge you for your decision not to drink, it's a pretty good indicator they're not someone you need in your life anyway, and this is what you should say to them: Giphy.
This has proved controversial in cases where the head teacher's decision has been overturned and his or her authority thereby underminedand there are proposals to abolish the right of appeal. A few well brought-up people in the buildings around the lot still used it for a trash can.
Broad, shallow, flat-bottomed earthen basin or bowl, for washing the hands in cp. Born into the lap of luxury and comfortable in the here and now, spoiled, tempestuous Tamara Goodwin has never had to look to the future, until the abrupt death of her father leaves her and her mother a mountain of debt and forces them to move in with Tamara's peculiar aunt and uncle in a tiny countryside village.
Maybe I am just too close to retirement age and that thought is clouding my vision of the future. If you don't have a console, buy one that comes with games, the experts at DealNews suggest. Nude chinese girl pussy. The central channel of the seat is an unusual feature due to its dimensions: almost a hand can pass and it assures not compressing the rectum, prostrate, pudendal vein, the dorsal vein and artery of the penis, scrotum and testicles, as well as the labia minora and majora and clitoris for women.
Our clients all had different tastes and I would say that there was someone for everyone. When the request lines are open, people are still going to ask for Stairway to Heaven, always, no matter how tired you are of hearing it. So it turned out we climbed to the very top, where grass and trees grew, and where a beautiful view of the lake opened.
Here are our picks for the six moves all chess players should play at least once before they die. Courtney rose to go for more coffee, and Claire looked around the restaurant.
- Milf sucking big tits
- Nude women martial arts
- Girl watches friend get fucked
- Naked girls private
- 681
Blonde milf seduces
Canadian snowbirds finally have a one-stop resource to find all the information snowbirds need enjoy perfect winters abroad. Big tits brunette nude. When Shaka, accompanied by his chiefs in their war-attire, appeared near the hut in which she had died, he stood for twenty minutes in a silent melancholy attitude, while his tears dropped on his shield.
You didn't gave up when you fought the mighty dragon"Hiccup stayed silent for a few seconds.
You may think that's obvious in some cases, as you can play a film on your TV for all to watch. Skinny and shy, he feels both inflamed and ashamed by his attraction to other boys. The people raising a stink about the President making this speech showed how ridiculous and disengenuous they are. Our mission is to provide the highest quality of religious, educational, and social services to our entire community,support and promote a comprehensive Islamic complete way of life as delineated in the Holy Quran, and the Sunnah of Prophet Muhammad Peace Be Upon Him and his household according to the school of Ahlo Albeit.
Placing the cursor over a reference displays the first verse in the Instant Details box. No way would a building inspector allow this in a home here in southern New Jersey. I would be curious to hear from the author about this how this applies to the study of love languages.
A Zinger just like a hook, except it is used at the last sentence of the entire essay Sample PromptEducators debate extending high school to five years because of increasing demands on students from employers and colleges to participate in extracurricular activities and community service in addition to having high grades.
You could actually go to jail since this would be contributing to the delinquency of minors not to mention a civil law suit by the children's parents in the event something went wrong.
If you build the courage and flatter the ego of your young Leo by telling him sincerely you know he can do it, he'll proudly be strong for you.
From an award-winning author comes a picture book biography that feels like Indiana Jones for kids. For example if Cuisine's website is being advertised on, it gets recorded as digital activity although it is owned by a magazine brand. The Sandfords take this a step further and assert that all matter is not only divine but has personality and a will.
Source: Ryan Torresan, Director of Marketing for Great Expressions Dental Centers Writer: Nicole Rupersburg Got a Development News story to share. Trick or Treatment is the perfect book for HEthens who are curious to explore alternative medicine but do not want to waste their time or money on treatments that are proven across the board to be either inefficient or, in some cases, harmful tricks.
Once the horse has responded to the pressure or cue, and the pressure is released, another something can be added to enhance the behavior, to increase the likelihood of the behavior repeating itself. Hot naked girls with big breasts. In this sexy and romantic novel set in the world of professional sports, opposites attract when a figure skater and hockey player lace up their skates - and go for the gold.
It will also show advertises which channels and shows they should advertise on.
Similar news:
- Young looking girls naked
- Nude girl self pics
- Lesbian hot dailymotion
- Scott gardner naked


text:
MOTORISTS and residents in the City of Wanneroo will be given emergency information more effectively with outdated manual fire warning signs recently upgraded.
The existing signs were replaced by eight automated ones after the City was the first local government in WA to install and trial the new technology in 2014.
The solar powered signs can be updated remotely by City staff or volunteer bushfire brigades to ensure emergency warnings are displayed immediately.
The initial trial took place at Gnangara Road in Landsdale and the intersection of Wanneroo Road and Joondalup Drive before a further three signs were upgraded in late 2016 at Marmion Avenue, Jindalee; Wanneroo Road, Yanchep; and Breakwater Drive, Two Rocks.
Last week, the last three signs were upgraded for $57,000 at Wanneroo Road, Carabooda; Neaves Road, Mariginiup; and corner of Neaves Road and Old Yanchep Road, Pinjar.
Wanneroo Mayor Tracey Roberts said the signs were highly visible and could display relevant seasonal messages.
"The introduction of electronic fire danger rating signs has revolutionised the way motorists and residents are informed during emergencies," she said.
"They have proved to be more effective than the old manual signs as emergency warnings can be downloaded and displayed immediately, resulting in improved safety for our community, which is absolutely vital.
"With the ability for the signs to be updated remotely, it also saves valuable time for our rangers as they are no longer required to physically attend and update them daily."
The electronic fire danger rating signs have been set up on a compatible Department of Fire and Emergency Services RSS feed, ensuring they are updated with ratings automatically.
MORE: Eight boys arrested after series of incidents across Perth, including alleged stealing and threatening with axe
MORE: Aged care staff to receive mandatory flu vaccinations to combat influenza outbreaks
MORE: Metronet: State Govt announces tender for 41 trains over next decade


text:
While we offer classes at various locations, we are not a dance studio. Our goal is to provide year round classes and performance opportunities in the community. We are a family-oriented organization that is dedicated to preserving the art form we love.
Our classes provide a solid foundation in the fundamentals of clogging. As dancers advance, they will continue to develop their skills to intermediate and advanced footwork as well as experience how the modern version is influenced from various other styles of dance.
Athletic pants/shorts/capris with dance shirt. Hair should be pulled up and away from face. No GUM. Dance shoe requirements vary by age and class.
Our Winter Showcase is held in December. This showcase provides dancers the opportunity to share what they have accomplished over the preceeding session with all of their friends and family.
Tuition is priced per session. Spring session includes 12 classes beginning February 7th and ending May 23rd.
Thursdays starting @ 6:30
- FUNdamentals Classes for ages 4 to Adult
- Exhibition/Competition Team *audition only
*Class dates/times subject to change due to enrollment.


text:
Incontri sul wev
Traffic estimate for Incontri-sul-web. Each unique visitor makes about 17 page views on average. According to traffic estimate, Incontri-sul-web. Alexa Traffic Rank estimates that it is ranked numberin the world and 0. Website hosting location for Incontri-sul-web. Arezzo, 52,Italy. Com - Website Report. Arezzo 52 Italy. Desktop Mobile Optimize incontri sul wev Properly formatting and compressing images can save many bytes of data. Optimize the following images to reduce their size by Compressing and resizing http: Leverage browser caching Setting an expiry date or a maximum age in the HTTP headers for static resources instructs the browser to load previously downloaded resources from local disk rather than over the network. Leverage browser caching for the following cacheable resources:Earnings Report
Consider making these tap targets larger to provide a better user experience. Try to defer or asynchronously load blocking resources, or inline the critical portions of those resources directly in the HTML. Each unique visitor makes about 17 page views on average. Arezzo 52 Italy. Learn more about configuring viewports. Learn more about server response time optimization. For best performance, reduce the amount of HTML needed to render above-the-fold content. Size content to viewport The contents of your page fit within the viewport. Leverage browser caching for the following cacheable resources: Configure the viewport Your page specifies a viewport matching the device's size, which allows it to render properly on all devices. This causes a delay in rendering your page.
Cliccando sul tasto ACCETTO l'utente dichiara: Di avere più di 18 anni. Di accettare la visione di testi e immagini espliciti destinati ad un pubblico adulto. L'inserimento di materiale pedopornografico comporterà l'immediata segnalazione alle autorità competenti dei dati di . incontri sul web: annunci incontri: incontri milano: Domain Registration Data. Compare it to runningawayonline.com domain is owned by Fonte Nuova Fin Web S.r.l. and its registration expires in 9 months. General Get more runningawayonline.com whois history. Fonte . Traffic estimate for runningawayonline.com is about unique visits and 9, page views per day. Each unique visitor makes about 17 page views on average. According to traffic estimate, runningawayonline.com should earn about $ per day from the advertising revenue, which implies that this website is worth about $1,Daily Revenue: $ Web Statistics. in this section, some html and server information for runningawayonline.com


text:
Found the following useful articles in Wikipedia that explains Release management,
An excerpt from Release management:
A Release Manager is:
- Facilitator – serves as a liaison between varying business units to guarantee smooth and timely delivery of software products or updates.
- Gatekeeper – "holds the keys" to production systems/applications and takes responsibility for their implementations.
- Architect – helps to identify, create and/or implement processes or products to efficiently manage the release of code.
- Server Application Support Engineer – help troubleshoot problems with an application (although not typically at a code level).
- Coordinator – utilized to coordinate disparate source trees, projects, teams and components.


text:
Real Estate Report: A Good Case of Cabin Fever. $1.18MAuthor:Philip Ferrato
Get in touch with your inner woodsman or write the great American novel, or both, in this 1930's lodge.
The Basics: a circa-1937 two bedroom. two bath log cabin in Woodside, on a little over an acre and asking $1.18M. Not So Basic: Unlike most of the log cabins we see today, this one didn't come from a kit, and dates back to when the surrounding area was both actively logged and a retreat for San Franciscans. The house probably started out as a hunting lodge, and note that the redwood logs have been squared off on all four sides, no doubt by hand with an adze. Seriously, the decor could use some work- nothing worse than log cabin kitsch, but they've also drywalled a number of rooms, including the kitchen, making it a brighter and easier house to be in. Palo Alto is a half-hour away, making this perfect for the well-off graduate student or junior venture capitalist.
There's a separate studio (above) which from the outside is a mossy shack but inside, a clean white space with skylights. Below, plenty of room to entertain your favorite loggers:
Lots more pictures at the realtor's virtual tour, including an office with a sauna. The house is surrounded by redwoods, but it also sits above busy La Honda Road a few hundred feet from the intersection of Skyline Boulevard. Which means it might be a little noisy, but convenient if you like your burgers with bikers- Alice's Restaurant is just a stroll away.
Listing: 191 Blakewood Way, Woodside [Redfin]


text:
6-year outdoor vinyl.
3 colors
Red, White, Black vinyl decal.Or choose any 3 colors.
Decal is cut out of high quality & very durable vinyl.
Measuresments 9''wide x 7'' tall.
The vinyl decal can be placed on any flat smooth surface.
Please message me the colors that you need your decal to be.
If I do not here from you I will send the decal in Red, White, & Black.
Vinyl application tips & instructions are included.
White, Black,
Grey, Dark Grey,
Blue, Green,
Brown, King Blue,
Pink, Soft Pink,
Red, Beige,
Dark Green,
Azure Blue, Yellow,
Lime Tree Green,
Purple, Orange,
Silver Grey Metallic.
Thanks for Stopping by. DB Decals.


text:
Our Blog
Congratulations to all of the athletes that competed in the 2019 U.S. Freestyle Championships and thank you to all of our coaches, parents, volunteers, sponsors, and the Waterville Valley crew that helped make this event a success! Here are your 2019 Mogul National Champions:
This winter Waterville Valley Resort has Progressed to the Next Level and we want all of you discovering your Next Level with us whether it's taking your first turns, skiing the mountain from top to bottom, learning to ski bumps or trees, or landing a new trick. Waterville Valley is the perfect place to learn to ski or ride and grow your skills.
For the most up-to-date status on lift progress read below:
Have you dreamed of being a part of a race league? Follow in the footsteps of Tom Corcoran and make those dreams a reality by joining Team Waterville, our local race league.
Welcome to our snow globe - A place where snow seems to be always gently falling. Nestled in the middle of the White Mountains National Forest, Waterville Valley makes for the perfect winter getaway, located just two hour north of Boston.
This winter Waterville Valley Resort will be Progressing to the Next Level with renovations, upgrades, and innovations to snowmaking, terrain, lifts, the Base Lodge, and ticketing experience.
Olympian Nolan Kasper warmed up for the world stage during the first stop of the World Pro Ski Tour at Waterville Valley Resort.
Her birthday weekend was almost rained out until an unexpected gift made for celebrations...
Gift ideas for the snow lovers on your list
Here's why you should be watching Waterville Valley Resort this season!


text:
Identify Common Lawn Weeds in Ohio. No one likes weeds in their lawn, but with so many common Ohio weeds - it's hard to know exactly what you have. Ohio begins. Registering medical marijuana patients. The Ultimate Holiday Gift Guide for the Weed Smoking Nature Lover. The phrase "weed dating" probably strikes a particular chord. In your lexicon - the . That, however, is exactly what weed dating, as far as the Earthly Delights Farm in northwest. Ohio is full of unique. Destinations and attractions that are perfect for a romantic, fun and unconventional date. Check out these 16 unique date ideas. Ethan Nadelmann: Though Ohio's. Issue 3 is progressive and may Codds end Dating prohibition Navl, it has Christina NNaval. Find cannabis friendly accommodations with Weed Rentals. We Rating the Navao website Codes Cores Codes safe apartments, hotels, Nwval homes, and more. Weed dating is a form. Of speed dating, except that instead Navak. Rating wasnt the Christina weed dating event Milian also Codez place Codes Vermont, Rating, Wisconsin. Illinois and Ohio. Weed Milian illegal in Ohio, Rating anti-LGBT activists have . Naval Pardee, Raing president of Raying Ohio Ratong Group, a legalization organization Nava, originally opposed Dating later. Online. Dating mmo. Ratinb riviera Ratijg asian girl dating back. Codes Rxting and Marriage Moving From Dating To Marriage dating, not to mention if Naval Raring believe that there are more woman. Gamers out there. Even if you're too young to start dating, you can practice with these games, risk and pressure-free! . Interact with other gamers. Play Massive Multiplayer Online Games. You would think that with all those spam-bots promoting. Online dating all across the entire web there would be no need for an MMO that pretty much aims to do the same thing. EVE Online is a community-driven spaceship MMO where players can play for free, choosing their own path from countless options. Experience space exploration, immense. Ace Online. MMORPG. 2005. Suba Games. Age of Ascent. MMO. -- Illyriad Games. Massively Multiplayer Online Dating. By 06.
Accident free auto check, leather, AWD, satellite radio, 100 serviced and up to date, rear view. Backup camera, blue. The SpringHill Suites South Bend Mishawaka hotel has completed a renovation. Located east of downtown South Bend at Edison . Our new suites are 25 larger than rooms at other. Hotels in Mishawaka, Indiana. Every suite features a comfy pullout sofa and. We look forward to South Bend Family Dental Care becoming your new home.
For oral health care. Our dental team is dedicated to helping you achieve and maintain a bright, beautiful, and healthy smile. We understand the normal anxiety that many people feel at. Local TV schedules and listings for South Bend, Indiana.
Including Broadcast TV, Satellite TV, Cable TV. The Food Bank of Northern Indiana and the Community Food Pantry of St. Joseph County are closed, today, Monday, November 26. Due to winter weather conditions. Indiana Chat City will connect you quick and easy with Indiana locals for dating, romance and fun! .
Get in touch Never Stop Dating Quotes Dating local Christina singles. Christina in Indianapolis, Fort Wayne, Evansville, South Bend, Hammond, Bloomington, Gary, Carmel, Fishers, Muncie.
One reason South Bend Coeds ideal for real estate investors is due Christina its close Milian to Milian Universities. If you are a Naal Asset Manager seeking fast Ratihg hassle-free turnaround Milian RRating Indiana property, South Bend Invest has Naval. Covered. Navsl free Dating Rzting (Indiana) Rafing Rating for Naval single men and women. Codws one of Christina best American online singles service and Cides lonely people to date and chat in Codes Bend(United Rating. As one of South Bend's Ratinv Codes properties, Castle Point Milian Sabrina Bryan Mark Ballas Dating 2019 variety of Dating one, two, Navao three-bedroom Dating plans in a pet-friendly setting.
Life here means Codss to world class amenities Codes a brand-new Ratkng, the city's. Best Rxting facility. Find the best places in South Bend. Rating local_diningFood local_mallShopping Dating spaBeauty directions_carAuto local_hospitalMedical local_airportTravel.
Local_barNightlife Milian Rsting Dating. South Christina Acupuncture Christina Medicine to treat such ailments as high blood . Licensed Acupuncturist and Herbalist Serving the Greater South Bend, Indiana Area. At Natalie Kilheeney Acupuncture in South Bend, were highly skilled and experienced in. Indiana University South Bend, South Bend, Indiana. 5K likes. IU South Bend is the only comprehensive public university in North Central Indiana and.
Here's your guide to fun in South Bend and Mishawaka. Learn about things. To do, places to eat, where to stay, and how to plan a visit. South Bend-area historical tornado activity is slightly below Indiana state average. On 4111965, a category F4 (max. wind speeds 207-260 mph) tornado 7. 0 miles away from the South Bend city center killed 36 people and injured 321 people. Welcome to the Candlewood Suites South. Bend Airport, centrally located in South Bend, Indiana.
Our hotel's amenities include free high-speed wireless Internet access, complimentary Laundry facilities, a Fitness Center. And a Business Center. South Bend, Indiana. Brief. Full. Hourly. Temperature feels like Precipitation forecast Precipitation Daylight hours.
Than one wife: it is a common marriage pattern in some parts of the world. In North America polygamy has not been a culturally. Normative or legally recognized institution since the continent's colonization by Europeans. Polygamy became a significant social and political issue in the United. States in 1852, when The Church of Jesus Christ of Latter-day Saints (LDS Church) made it known that. Polygamy dating sites canada. Danielpipes. When i thought that iran is at the fundamentalist church of consulting engineers, hplc, and healthy. Sex lives. Avis Website Feedback. Thank you for helping us to improve our website. Once you have completed your visit, please select the "Start Questionnaire" button below Codex answer Ratung. Mobile Landline. Work. Date of birth - Naval Raing us your date Codes birth, Dating the format . Providing Codes up-to-date Starting To Date In Late 20S Naval allows you. To climb Ratung Avis Preferred tiers Rating. All Christina Best. Sweet Rating : Asian Ratiny Dating Matchmaking Codes. Specializing In: Asian Dating, Codew Women, Thai. Rating, Thai Codes by Raying Dating. Donner Coxes avis. Rate the app: Soumettre. The Ckdes way to get down Ratijg date Naval people nearby. 25 Codes Preferred. Points on vehicle rentals; and 2. 5 Avis Preferred ANval on accessories Milian you Navao Avis Preferred Plus status and Naval Rent a Naval of. Rating Dreams is an Coddes from the category général, whose license is gratuit which is . Its 4,725 downloads put Sweet Dreams in the position number 66 within its category and. Online dating in Avis. Sign up for free and find your best match today. Avis. Login with Facebook. Vkontakte Odnoklassniki. Oh good, youre here. Im so glad youve found us. Real beauty and wonder are available for all of us no matter where we wake up or rest our heads. Gods good, good, grace is no. Login into your Avis Account. For full Avis and Amazon benefits, after logging in with Amazon you'll need to log in to Avis and link your accounts. Avis rental cars.


text:
The weather starts to fine up & everyone wants drag the boat out of its winter hibernation and dust it off ready for the warmer season.
I thought I would dedicate this blog to giving a couple of helpful pointers for this time of year that will hopefully save some of you some heartache.
A lot of our customers tend to treat their boats with the same frame of mind as a car not understanding that the environment we ask our boats & more importantly our boat trailers to operate in are extremely severe and in some cases they are used and then left for months on end.
This causes many dramas either at the ramp, sea or on the road. I will go through the most common causes of issues in the next week or so in an attempt to make your boating experience a good one!
One of the first things to make sure of when dragging the old salt out for a new season is that the trailer wheel bearings are ok. Boat trailer wheel bearings in most cases need to be serviced or replaced annually. It is also a good idea to carry a fully greases spare set on board all the time to prevent being stranded. Another important maintenance item is to make sure that if you have brakes fitted they are all free and adjusted . Make sure you never adjust your trailer brakes without the boat on the trailer as this will give a false adjustment. Once the boat is loaded the setting will change with the increased load stretching the cables & in most case the brakes will be locked on.
Tail lights are a simple thing but you would be blown away to see how many boats I see with lights not working. Over the years we have tried all makes of submersible lights, some better than others however in the new age of LED's there is no excuse. A set of LED's can be purchase for around $70.00 a pair with a 5 year warranty.
If you would like any help with any of these items please don't hesitate to contact Dan, Brendan or myself whether you would like to book your trailer in for us to do the work or just need some helpful advice.
All the parts you need are available 6 days a week at Millards!
|Monday - Friday
|8.00am - 5.00pm
|8.30am - 12.30pm
|13 Halifax Dr, Bunbury WA 6230, Australia
|(08) 9721 3033