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bioneedle(biodegradable mini implant) delivery of vaccine

Best seminar from First semester student of niper ahmedabad : seminar given by Vishal Goyani, Department of Pharm.Analysis,NIPER-Ahmedabad

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  • To address all the limitations of needle injections, research is beingcarried out on alternative delivery methods and alternative routes
  • implants frombiological material incorporating the vaccine. Examples are siliconeimplants , sugar-glass needle and the Bioneedle.
  • Bioneedles™:Bioneedle Technologies Group BV, Eindhoven, The NetherlandsUsing a material that is quickly biodegradable, capable of holding any type of vaccine, and able to withstand high-speed injection Bioneedle creators have addressed the major problems of needle-based injections.
  • compressed air drivenfor use time and againContinue to monitor progress made on the technologies by Glide-Pharma and Bioneedle,Alza Corporation. .penetrate through the skin causing less Bioneedles were safe and showed minimal adverselocal and systemic effectsLess external skin damage than with conventional needles.The Bioneedle is inserted into a patient using an air compressor. Once beneath the skin, the needle dissolves and releases a vaccine
  • expensive bulky transport, storage and distribution to vaccination centers - many children out of reach: difficult circumstances, remoteness - in many African countries: < 50% vaccination coverage - failing cold chain: too hot (sun) too cold (freezer)
  • injection takes 5 seconds - 60 injections per hour problems - dedicated clean workplace not always feasible (dangerous situations) - professional medical staff - line moves slowly along stationary table >moving line
  • Injections, although safe in the developedcountries and relatively painless, may cause serious stress, fear andconcern in children as well as parents .This may lead to reducedparticipation in vaccination programs.increase in compliance No needles are perceivedwhich is expected to lead to less stress and fear in children aswell as in parents.This is advantageous to specially western country,
  • thanks to ultra-portability, thermo-stability, and dry delivery… Bioneedles solve all problems related to the cold chain and to the use of syringes, needles, and vials.
  • A course of three (3) vaccine injections are given with the second injection at least one month after the first dose and the third injection given six months after the first dose.Afterward an immune system antibody to HBsAg is established in the bloodstream. The antibody is known as anti-HBsAg. This antibody and immune system memory then provide immunity to hepatitis B infection.[3] The first vaccine became available in 1981
  • don’t forgate to tell aim
  • When developing a new delivery system for vaccines, the mostimportant requirement is that the delivery via the new system is asleast as effective as the conventional delivery system. Besides theadvantages of needle free delivery systems, as described above,aspects such as improved immunogenicity by using adjuvants, dosereduction and higher stability of the formulation are all advantagesthat make the alternative even more attractive.In this paper hepatitis B surface antigen has been formulated inBioneedles and compared in vitro and in vivo with conventionalinjections. LpxL1 has been compared to aluminum hydroxide as anadjuvant and the heat stability and dose reduction has been evaluatedfor liquid and Bioneedle formulations.
  • In the text thethree sources are referred to as manufacturer A, manufacturer B andmanufacturer C.
  • when the formulations were freeze dried in the presence of trehalose the immunogenicity is preserved. Due to freeze drying, the aluminum salt forms aggregates and the antigen entrapped in these aggregates cannot be released. Apparently, trehalose has a good stabilising effect probably by adsorbing onto the aluminum particles thereby minimizing the formation of aggregates
  • Bioneedleswere dissolved by incubation in 1 ml water, adding 5 μl ofa 120 kilonovo units/ml solution of 1,4-α-D-glucanglucanohydrolase(Novozymes, Denmark) and 5 μl of a 330 units/ml solution of pullulanase(Novozymes, Denmark)during 1 h at room temperatureAkilonovo unit is the amount of enzyme which degrades 4870 mg starchdrymatter, Merck soluble amylum, per hour under standard conditionsMethod:In short, the sample was pre-incubated in microwells (3 or 4 dilutions)pre-coated with a mixture of mouse monoclonals specific for differentepitopes on the ‘a’ determinant of HBsAg. Affinity purified goatantibody to HBsAg conjugated to horse radish peroxidasewas thenadded. After washing, to remove sample and unbound conjugate,peroxidase activity is detected by addition of 3,3′,5,5′-tetramethylbenzidine(TMB) substrate and hydrogen peroxide. The enzymereaction was terminated with sulfuric acid. Substrate conversion wasdetermined spectrophotometrically at 450 nm.Each ELISA plate contained a reference HBsAg sample (liquidformulation stored at 4 °C) for calculation of the antigen concentrationin the samples.An effective pullulanase enzyme that hydrolyzes alpha-1,6 glycosidic bonds present in starch side chains and amylopectin. α-Amylase is an enzyme that hydrolyses alpha-bonds of large alpha-linked polysaccharides such as starch and glycogen
  • Freeze dried formulationswere reconstituted with water prior to the measurementsNo need to readBAKI:Far UVspectra were taken at 25 °C in the region of 200–260 nm. A proteinconcentration of 40 μg/ml was used in 1 mm Quartz cuvettes. The scanvelocity was 0.1 nm/swith step lengths of 0.5 nm. The monochromatorbandwidth was 1 nm. The CD spectra were corrected for buffer andsmoothedwithasmoothingfactor of5.Chirascan™CDNNsoftwarewasused to provide an estimate of the secondary structure. The software isbased on algorithms designed for fitting CD spectra of proteins.circular dichroism (CD) refers to the differential absorption of left and right circularly polarizedlight. UV CD is used to investigate the secondary structure of proteins. this phenomenon will be exhibited in absorption bands of any optically active molecule. As a consequence, circular dichroism is exhibited by biological molecules, because of their dextrorotary and levorotary components. Even more important is that a secondary structure will also impart a distinct CD to its respective molecules. Therefore, the alpha helix of proteins and the double helix of nucleic acids have CD spectral signatures representative of their structures.
  • All in vivo experiments were approved by the animal ethicscommittee of the NVI.Formulations used for the heat stability study were incubated for3 weeks at either 4 °C, 37 °C 50 °C or 60 °C prior to immunization
  • (paper2008:Although Bioneedles will be applied in humans using compressed air, a preliminary trial with a prototype delivery system in mice cadavers (data not published) showed that the pressure was too high for animals as small as mice. Therefore, Bioneedles have been subcutaneously implanted in mice by using a trocart and mandarin, identical to the implantation of identification chips in animals. Although the trocart is a large needle, it did not cause inconvenience to none of the mice after recovery. Once inserted, the Bioneedle could still be localized by palpation for 30 s–1 min. Thereafter, the Bioneedle could not be palpated any longer. After implantation of the Bioneedles no oedema, redness or pain as signs of local inflammation were observed. The animals did not show any signs of discomfort and showed normal behavior regarding feeding and drinking during the time of the trial.)Bioneedle is applied subcutaneously in the neck between theears. In animals as small as mice, Bioneedles cannot be deliveredintramuscularly.
  • The steps of "indirect" ELISA follows the mechanism below:-A buffered solution of the protein antigen to be tested for is added to each well of a microtiter plate, where it is given time to adhere to the plastic through charge interactions.A solution of non-reacting protein, such as bovine serum albumin, or casein is added to block any plastic surface in the well that remains uncoated by the protein antigen.Next the primary antibody, generally in the form of serum is added, which contains a mixture of the serum donor's antibodies, of unknown concentration, some of which may bind specifically to the test antigen that is coating the well.Afterwards, a secondary antibody is added, which will bind any antibody produced by a member of the donor's species (for example, an antibody produced in a mouse that will bind any rabbit antibody). This secondary antibody often has an enzyme attached to it, which has a negligible effect on the binding properties of the antibody.A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme. The color change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen. This can be helpful in a clinical setting, and in R&D.The higher the concentration of the primary antibody that was present in the serum, the stronger the color change. Often a spectrometer is used to give quantitative values for color strength.
  • Higher titer means higher concentration
  • Read and understand
  • Fig. 1. IgG titers in mice (n=8); L: Liquid formulations, F: freeze dried formulations,HepB: hepatitis B surface antigen of manufacturer C, Al: aluminum hydroxide, S.C:subcutaneous, I.M intramuscular. Engerix-B: commercial available vaccine from GSK.DISCUSSION:IM>SC , FREEZE DRYING HAS NO ADVERSE EFFECT ON FREEZE DRYINGThe commercial hepatitis B vaccine is a liquid vaccine of HBsAgadjuvated to Al(OH)3 and delivered intramuscularly (L[HepB+Al]_IM).Since Bioneedles are freeze dried formulations that can only bedelivered subcutaneously in mice, the effect of freeze drying and theroute of immunization on the IgG titer were evaluated with liquidvaccine formulations. The commercial available Engerix-B vaccine, analum adjuvated vaccine, was used to evaluate the difference betweensubcutaneous and intramuscular injections. Intramuscular injectionsperformed significant better (p=0.003) than subcutaneous injections.To evaluate the effects of freeze drying, HBsAg was freeze dried,reconstituted in water and injected subcutaneously. The reconstitutedfreeze dried formulations induced comparable IgG titers as the liquidformulations injected subcutaneously but with liquid HepB formulationsone non responder was found within the group. Liquid HepBformulations were tested several times and always showed one ortwo non responders after subcutaneous injection
  • Fig. 2. IgG titers after one (14 days) and two immunizations (42 days). L: liquidformulation; B: Bioneedle formulation, HepB: HBsAg from manufacturer B, Al: Al(OH)3,S.C: subcutaneous, I.M: intramuscular. Asterisks (*) indicate titers that are significantdifferent (*pb0.005) from the liquid i.m. group for the titers after 14 days. Asterisks (**)indicate titers that are significant different (*pb0.005) from the liquid i.m. group for thetiters after 42 days. Titers are average titers of the responders.Thelowernumber of animalsafter 14 days is due to insufficient serum from some animals to perform the ELISA test.In a pilot experiment, Bioneedle formulations of HepBadjuvatedwith alum performed not as good (several non responders) as theconventional liquid vaccine delivered via the intramuscular route. Toimprove the Bioneedle formulations another adjuvant, the LPSderivative lpxL1, was evaluated as well as no adjuvant at all (Fig. 2).Mice were immunized with formulations containing no adjuvant,with aluminum hydroxide or with lpxL1 as adjuvant.With Bioneedle formulations containing alum comparable IgGtiters were induced as with liquid formulations containing alum, (L[HepB+Al]_sc), but with non responders within the group. WhenlpxL1 was used as an adjuvant or when no adjuvant was used at all, allmice responded in the group.When lpxL1 is present in either liquid or Bioneedle formulations,comparable IgG titers were induced as with the intramuscularconventional vaccine, L[HepB+Al]_im. After one vaccination(14 days) Bioneedle formulations with lpxL1 showed the best immunogenicityresults. Allmice responded and the IgGtiterwas significantly(pb0.001) higher as compared to the intramuscular L[HepB+Al]_imgroup. After 2 immunizations (42 days) the IgG responses werecomparable to the responses in the L[HepB+Al]_im group.
  • Fig. 3. IgG2a/IgG1 response after 42 days. L: liquid formulation; B: Bioneedleformulation, HepB: HBsAg from manufacturer B, Al: Al(OH)3, S.C: subcutaneous, I.M.:intramuscular. Asterisks (*) indicate titers that are significant different from the liquidi.m. group. Titers are average titers of the responders.The sera after 42 dayswere also analyzed for IgG1 andIgG2a antibodyresponses. Fig. 3 shows the IgG2a/IgG1 ratio. Formulations containinglpxL1 showed significant higher IgG2a/IgG1 ratios as compared to theintramuscular group with alum. This was true for both Bioneedleformulations and liquid formulations. With Bioneedle formulationscontaining aluminum hydroxide no IgG2a could be detected
  • Fig. 4. Dose response. Antibody titers after 42 days (2 immunizations). Mice (n=10)have been immunized with conventional formulations containing alum via i.minjections and with Bioneedle formulations (inserted subcutaneously) containinglpxL1. The dose of adjuvant is kept constant whereas the dose of antigen is variable(2, 1, 0.5 and 0.1 μg hepatitis B/dose). HepB: HBsAg frommanufacturer A, Al: Al(OH)3,S.C: subcutaneous, I.M: intramuscular. Titers are averages of the responders. Asterisks(*) indicate titers within the L[HepB+Al] groups that are significant different fromthe L[2 μgHepB+Al]_IM group (*pb0.005). Asterisks (**) indicate titers within theB[HepB+lpxL1] groups that are significant different from the B[2 μg HepB+lpxL1]group.The dose response for conventional liquid formulations L[HepB+Al]_im was compared to the dose response of Bioneedle lpxL1-HBsAgformulations delivered subcutaneously. Fig. 4 shows that both theliquid formulations and Bioneedle formulations showed a plateauvalue for the IgG titer with 2, 1 and 0.5 μgHepB. For both formulationsa significant decrease (pb0.005) was only observed when formulationscontained 0.1 μg HepB.
  • TO BE DELETEDFig. 5. IgG titers and IgG2a/IgG1 ratio. N=10. The effect of delivery route(intramuscular and subcutaneous), of the adjuvant (Alum or lpxL1) and of theformulation (liquid or Bioneedle) are compared. Titers are averages of the responderswithin a group. Asterisks (*) indicate titers that are significant different from the L[2 μgHepB+Al]_IM group. P-values between other groups are indicated in the graph. HepB:HBsAg from manufacturer A, Al: Al(OH)3, S.C: subcutaneous, I.M: intramuscular.In order to discriminate between the subcutaneous and intramuscularroutes, liquid formulations L[HepB+lpxL1] were injected bothintramuscularly and subcutaneously (Fig. 5). Just like alum containingformulations (see Fig. 1), lpxL1 formulations induced significanthigher IgG titers after 42 days (p=0.012) via intramuscular injectionsas compared to subcutaneous injections. Via the intramuscular route,lpxL1 formulations (L[HepB+lpxL1]_IM) showed comparable immunogenicityto aluminum hydroxide formulations(L[HepB+Al]_IM).The L[HepB+lpxL1]_sc and B[HepB+lpxL1]_sc formulations showedin this experiment significant (p-values were respectively 0.0012 and0.0011) lower IgG titers as compared to the L[HepB+Al]_IMformulations whereas in Fig. 2 comparable titers were induced withthese formulations.The IgG2a/IgG1 ratio for the liquid formulations containingaluminum hydroxide, L[HepB+Al]_IM, was significant lower thanfor all formulations containing lpxL1. All formulations containinglpxL1 showed similar IgG2a/IgG1 ratio's irrespective of delivery routeor of formulation (liquid or Bioneedle).
  • Fig. 6. Stability of Bioneedle formulations of HepB after 1 week and 3 weeks incubationat 4 °C, 37 °C, 50 °C and 60 °C as measured with an antigen ELISA. L: liquid formulation,B: Bioneedle formulation, HepB: HBsAg, Al: Aluminum hydroxide. Asterisks (*)represents recoveries of L[HepB+Al] formulations that are significant lowerthan L[HepB+Al] _4 °C (Pb0.001 as determined by the Student t test). Asterisks(**) represents recoveries of B[HepB] formulations that are significant lower thanB[HepB] _4 °C (Pb0.001 as determined by the Student t test). Asterisks (***) representsrecoveries of B[HepB+lpxL1] formulations that are significant lower than B[HepB+lpxL1]) _4 °C (Pb0.001 as determined by the Student t test).Bioneedle formulations containing plain HepB or HepB+lpxL1 andconventional liquid formulations of HepBadjuvated to alum wereincubated at different temperatures. The antigenic recoveries areshown in Fig. 6.The recoveries shown in Fig. 6 are average values of 3 experiments. Ineach experiment each formulation was incubated in duplicate or intriplicate at the different temperatures. After 1 and 3 weeks each samplewas measured in an antigen ELISA. The conventional liquid vaccineformulation L[HepB+Al] showed a significant decrease in antigenicityafter 3 weeks at 37 °C. After 1 week at 60 °C a recovery of 40%was foundand the recovery decreased to 20% after 3 weeks at 60 °C.Bioneedle formulations containing only HBsAg retained allantigenicity after 3 weeks at 37 °C. A significant decrease (pb0.001)was found after 3 weeks at 50 °C. After 1 week at 60 °C a recovery inantigenicity was found of 60%. Unlike the conventional formulation nofurther decrease in antigenicity was observed after incubation of 2more weeks at 60 °C. Bioneedle formulations containing lpxL1 showedexcellent stability. After 3 weeks at 50 °C this formulation lost 10% ofits antigenicity. Although a significant decrease in antigenicity wasonly found after 3 weeks at 60 °C a recovery in antigenicity of 60% wasstill found with the B(HepB+lpxL1) formulations whereas for liquidformulations (L[HepB+Al]) only 20% recovery was found.
  • CONCLUSION:When lpxL1 was included in theformulations, no significant change in α-helix content was observedafter 3 weeks storage at temperatures up to 60 °C.Fig. 7. Circular dichroism: α-helix content of formulations incubated for 3 weeks atdifferent temperatures. L: liquid formulation, F: freeze dried formulations. Presentedvalues are the average of 3 independent samples. Asterisks (*) represent liquidformulations with a significant lower α-helix content as compared to the liquid HepBformulation at 4 °C. Asterisks (**) represent freeze dried formulations with a significantlower α-helix content as compared to the freeze dried HepB formulation at 4 °C.Liquid formulations and freeze dried formulations of hepatitis Bsurface antigen in the presence and absence of lpxL1 were stored for3 weeks at 4 °C, 37 °C, 50 °C and 60 °C and the tertiary structure of theantigen was determined with circular dichroism.As shown in Fig. 7, both liquid and freeze dried formulations ofonly HBsAg showed a decrease in α-helix content at highertemperatures. For liquid samples, L[HepB], a significant decrease(pb0.05) in α-helix content was observed at 50 °C as compared to4 °C, whereas for freeze dried samples, F[HepB], this significantdecrease was only observed at 60 °C. When lpxL1 was included in theformulations, no significant change in α-helix content was observedafter 3 weeks storage at temperatures up to 60 °C. This was true forboth liquid and freeze dried formulations. When liquid HBsAg wasincubated overnight at 100 °C the protein denatured, resulting in adecrease of the α-helix content to only 25%.
  • Fig. 8. Immune response in mice (n=10) after 2 vaccinations with heat treatedformulations. L: liquid formulation; B: Bioneedle formulation; HepB: HBsAg (manufacturerA); Al: (Al(OH)3. Liquid formulations were incubated for 4 h at 100 °C, 3 weeksat 4 °C, 37 °C or 50 °C prior to intramuscular injection. Bioneedle formulations wereincubated for 3 weeks 4 °C, 37 °C or 50 °C prior to subcutaneous insertion. Titers areaverage titers of all 10 mice (responders and non responders). Antigenicity recovery asdetermined with an ELISA is indicated above. (^) antigenicity recovery that aresignificant different from the same formulation stored 3 weeks at 4 °C. (*) Significantdifferences in IgG (*), IgG1 (**) and IgG2a (***) within the liquid formulations ascompared to L[HepB+Al]_4 °C. (#) Significant differences in IgG (#), IgG1(##) andIgG2a (###) within the Bioneedle formulations as compared to B[HepB]_4 °C.Differences are significant when pb0.05 as determined with a Student t test.The immunogenicity and antigenicity of heat treated formulations isshown in Fig. 8. As shown in Fig. 6, a significant decrease in antigenicitywas found for liquid formulations (54% recovery) and Bioneedleformulations of HepB (67% recovery) incubated for 3 weeks at 50 °C.Both liquid formulations and Bioneedle formulations inducedcomparable IgG titers after heat treatment, regardless of antigenicrecoveries. In the negative control group (overnight at 100 °C) whichlost all antigenicity, immunogenicity also disappears. For both liquidformulations and for Bioneedle formulations a decrease in IgG2awas observed as compared to the conventional L[HepB+Al]_imgroup, although for the Bioneedle formulation this decrease was lessevident (p=0.007 for group L[HepB+Al]_50 °C and p=0.029 forgroup B[HepB]_50 °C).
  • pain-free no infection through contamination

bioneedle bioneedle(biodegradable mini implant) delivery of vaccine bioneedle bioneedle(biodegradable mini implant) delivery of vaccine Presentation Transcript

  • Bioneedles as alternative delivery system for hepatitis B vaccine
    VISHAL N. GOYANI
    PHARMACEUTICAL ANALYSIS
  • Presentation outline
    Introduction
    Research paper
    Materials and methods
    Results & Discussion
    Conclusion
  • INRODUCTION
  • Vaccinations
    Vaccination is the administration of antigenic material to produce immunity  to a disease
    About 1 billion vaccinations every year
    Cost-effective life-saver for children
    Smallpox eradicated
    The conventional way for vaccine delivery is SC or IM route by using syringe & needle
  • Vaccine delivery technologies
    Auto-disable syringes
    Prefilled syringes or droppers
    Needle-free injections (jet injector)
    Biodegradable implants (Bioneedles™)
    Microneedles
    Transdermal delivery
    Buccal /sublingual delivery
    Inhalation delivery
    Reconstitution devices
  • Bioneedles™
    Mini implant
    Biodegradable polymer
    Vaccine incorporated
    Thermostable
    16 mm long & 1.2 mm wide
    Volume of 4.0 to 4.5 μl
  • Bioneedles delivery
    IM or SC
    Bioneedle Applicator
    < 1 millisecond
    Pain-free application
    Polymer dissolves
    Vaccine released
  • Why Bioneedle™ ?
    Ultra portability
    No degradation
    No need for cold chain
    Advantage to developing
    countries (African countries) reaching even the most remote children
  • Advantage at vaccination center
    No reconstitution
    No need for clean workplace
    Dry delivery
    No risk of contamination
    No vaccine wastage
    1000 vaccinations/hour
    20x faster campaign
    Less medical staff
  • Oh dear, No fear, bioneedle is hear
    No needle fear because pain-free
    No infection through needle-stick injury
    No infection through contamination
    Prevents 25 million infections of HepB, HepC, HIV per year from reuse
  • Waste elimination
    Contaminated
    needles, syringes, and vials
    require proper waste disposal
    Bioneedle™
    no waste
  • Hepatitis B Vaccine
    For the prevention of hepatitis B virus infection
    Vaccine contains one of the viral envelope proteins, hepatitis B surface antigen
    DOSE: A course of three vaccine injections
    MOA: Antibody to HBsAg is established in the bloodstream which then provide immunity to hepatitis B infection
  • RESEARCH PAPER
  • INTRODUCTION
    Bioneedles as alternative delivery system for hepatitis B vaccine
    New system should at least as effective as the conventional delivery system
    LPS-derived LpxL1 compared to Al(OH)3 as an adjuvant and the heat stability and dose reduction has been evaluated for liquid and Bioneedle formulations
  • Materials and methods
  • Materials
    Yeast derived, commercial GMP grade, hepatitis B surface antigen (HBsAg)
    Control vaccine: Engerix-B 20 μg/mL from GSK
    lpxL1 LPS from ImsaVac, Netherlands
    Al(OH)3, Alhydrogel 2% from BrenntagBiosector, Denmark
    Bioneedles: thermoplastic starch by injection moulding
    Bioneedle Technologies Group's proprietary procedures
  • Formulations
    Different formulation:
    1) HBsAg
    2) HBsAg + Al(OH)3 3) HBsAg +lpxL1
    All formulations contained 2 μg HBsAg
    Adjuvants either 50 μg Al(OH)3 or 0.5 μg lpxL1
    Liquid formulations (L)
    1) Subcutaneous injections :500μl
    2) Intramuscular injections :100μl
  • Cont..
    The freeze dried formulations(F) :500 μl
    freeze dried using a Leybold GT 4/6 freeze dryer
    Bioneedles (B) were filled, by using a specially designed filling apparatus, with liquid formulations containing 5% w/w of D-trehalosedi-hydrate then freeze dried
  • Hepatitis B antigen ELISA
    For HBsAg quantification
    Bioneedles were dissolved
    by incubation in 1 ml water
    by using α-Amylase&
    pullulanase
    Commercial kit of Abbott,
    Murex HBsAg version 3
    was used
  • UV Circular dichroism
    Used to investigate the secondary structure of antigen
    Chirascan™ (Applied Photophysics, UK)
    Liquid and freeze dried formulations were stored for 3 weeks at 4 °C, 37 °C, 50 °C and 60°C prior to the measurements
    concentration of antigen 40 μg/ml at 25 °C
    Scan region of 200–260 nm
    Scan velocity was 0.1 nm/s
    Chirascan™CDNN software was used to provide an estimate of the secondary structure
  • In vivo studies
    BalB/C mice of 10–14 g
    Prior to immunization, all animals were anesthetized with a mixture of 1-isoflurane, N2 and O2
    For the dose response study, the hepatitis B doses used were 2 μg, 1 μg, 0.5 μg and 0.1 μg
    Liquid formulations were either injected I.M in the rear leg, S.C. in the groin
  • Cont..
    Freeze dried formulations were reconstituted In 500 μl water prior to subcutaneous injection
    Bioneedles were implanted in mice by using a sterilized trocar with mandrin S.C not I.M
    Two weeks after primary immunization, blood was collected
    Four weeks after the booster vaccination, all mice were sacrificed by bleeding
  • IgG, IgG1 and IgG2a ELISA
    Polystyrene microtiter plates were coated HBsAg diluted in phosphate buffered saline (PBS)
    The plates were incubated overnight at room temperature. The plates were washed with tap water containing Tween 80.
    Serum was added in series of three fold dilutions (in PBS)
    After 2 h of incubation at 37 °C the plates were washed and goat anti mouse immunoglobulin conjugated to horse radish peroxidase was added
  • Cont..
    The conjugate was incubated for 1.5 h at 37 °C.
    After washing the plates, 100 μl TMB substrate solution was added to each well.
    The reaction was stopped after 10 min with 2 M H2SO4 and the absorbance was measured at 450 nm.
    The titre was determined as the dilution factor at which the absorbance was 50% of the maximum absorbance
  • Statistics
    Antibody titers are expressed as the mean log10 titer of eight or ten independent observations plus the standard errors of the means.
    Antigenic recoveries are expressed as the mean of three independent observations, plus the mean standard errors of the means.
    Statistical evaluations are done with a Student t test (one way ANOVA) by using a two tailed distribution and a two sample unequal variance.
  • Results & Discussion
  • effect of freeze drying and theroute of immunization on the IgG titer
    Fig. 1. IgG titers in mice (n=8)
  • Adjuvant
  • Adjuvant:IgG2a/IgG1
  • Dose response
  • The effect of delivery route
  • Antigenicity of stressed antigen
  • Structure of stressed antigen
  • Immunogenicity of stressed antigen
  • Conclusion
  • The study shows that hepatitis B was successfully formulated with Bioneedles
    Bioneedles with lpxL1, when delivered subcutaneously, induce higher IgG titers after one vaccination than conventional vaccine
    After a booster vaccination comparable IgG titers are induced as with the conventional liquid vaccine but with higher IgG2a/IgG1 ratios
    Even better results are expected when bioneedle can be tested IM
  • Bioneedles with lpxL1 also showed much better heat stability than the conventional liquid vaccine
    Although these advantages are mainly attributed to lpxL1, the Bioneedle has the additional advantages like:
    Ultra-portability
    Thermo-stability
    Pain-free
    Contamination-free
    No waste
  • Reference
    [1] R.M. Jacobson, A. Swan, A. Adegbenro, S.L. Ludington, P.C. Wollan, G.A. Poland,
    Making vaccinesmore acceptable—methods to prevent andminimize pain and other
    common adverse events associated with vaccines, Vaccine 19 (2001) 2418–2427.
    [2] Y. Nir, A. Paz, E. Sabo, I. Potasman, Fear of injections in young adults: prevalence
    and associations, Am. J. Trop. Med. Hyg. 68 (2003) 341–344.
    [3] A. Taddio, A.L. Ilersich, M. Ipp, A. Kikuta, V. Shah, Physical interventions and
    injection techniques for reducing injection pain during routine childhood
    immunizations: systematic review of randomized controlled trials and quasirandomized
    controlled trials, Clin. Ther. 31 (Suppl 2) (2009) S48–S76.
    [4] S. Wright, M. Yelland, K. Heathcote, S.K. Ng, G. Wright, Fear of needles—nature and
    prevalence in general practice, Aust. Fam. Physician 38 (2009) 172–176.
    [5] J.U. Igietseme, F.O. Eko, Q. He, C.M. Black, Combination vaccines: design strategies
    and future trends, Expert Rev. Vaccin. 5 (2006) 739–745.
    [6] E. Mallet, B.H. Belohradsky, R. Lagos, et al., A liquid hexavalent combined vaccine
    against diphtheria, tetanus, pertussis, poliomyelitis, Haemophilusinfluenzae type B
    and hepatitis B: reviewof immunogenicity and safety, Vaccine 22 (2004) 1343–1357.
    [7] G. Kersten, H. Hirschberg, Antigen delivery systems, Expert Rev. Vaccin. 3 (2004)
    453–462.
    [8] M.A. Aziz, S. Midha, S.M. Waheed, R. Bhatnagar, Oral vaccines: new needs, new
    possibilities, Bioessays 29 (2007) 591–604.
  • Thank
    You
  • Immunogenicity & Antigenicity
    Antigenicity is the ability of a chemical structure (referred to as an Antigen) to bind specifically with certain products of adapttyive immunity: T cell receptors or Antibodies(a.k.a. B cell receptors).
    immunogenicity refers to the ability of an antigen to induce an adaptive immune response.
    Thus an antigen might bind specifically to a T or B cell receptor, but not induce an adaptive immune response
  • mandrin A stiff wire or stylet inserted into a soft catheter to give it shapeand firmness while passing  through a hollow tubular structure
    An antibody titer is a measurement of how much antibody an organism has produced that recognizes a particularepitope, expressed as the greatest dilution ratio (or its reciprocal) that still gives a positive result. ELISA is a common means of determining antibody titers.
  • Patent application title: PARENTERAL FORMULATION
    Inventors:  GijsbertusGerardusPetrus Van De WijdevenAgents:  FOLEY AND LARDNER LLP;SUITE 500Assignees:  BIONEEDLE TECHNOLOGIES GROUP B.V.Origin: WASHINGTON, DC USIPC8 Class: AA61F200FI USPC Class: 424426 Patent application number: 20100080839