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taylor bröst whitney

taylor bröst whitney

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taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

taylor bröst whitney

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Oncoimmunology , 05 Jan , 7 3 : e DOI: Monoclonal antibody therapy is effective in treating HER2-driven mammary carcinomas, but its utility is limited by high costs, side effects and development of resistance. Active vaccination may represent a safer, more effective and cheaper alternative, although the induction of strong and durable autoantibody responses is hampered by immune-tolerogenic mechanisms. Using a novel virus-like particle VLP based vaccine platform we show that directional, high-density display of human HER2 on the surface of VLPs, allows induction of therapeutically potent anti-HER2 autoantibody responses.

The VLP platform may represent an effective tool for development of vaccines against other non-communicable diseases. Immunotherapy represents a major breakthrough in the treatment of cancer.

Specifically, the serum half-life 2—4 weeks of the mAbs 8 , 9 requires that new doses are administered continuously every third week. This may lead to hypersensitivity reactions requiring premedication with cortisol or anti-histamine 11 and treatment failure. These limitations have prompted investigation into strategies for development of anti-HER2 vaccines capable of triggering the patient's own immune system to produce anti-tumor Abs.

Monomeric proteins are generally weak immunogens 23 and simple subunit vaccines based on a soluble protein antigen in an adjuvant formulation have almost exclusively failed in clinical trials due to insufficient efficacy. In the context of anti-cancer vaccines multiple mechanisms moreover prevent induction of immune responses against self. Their repetitive surface structures facilitate complement fixation and B cell receptor clustering, which activate the innate immune system and leads to greater B cell activation.

We have recently developed a highly versatile antigen display platform that unlike existing technologies, effectively facilitates directional covalent attachment of large vaccine antigens at high density on the surface of VLPs. Interaction between SpyTag and SpyCatcher results in spontaneous formation of an isopeptide bond, which occurs at high efficiency in a wide variety of protein contexts and buffer conditions. We demonstrate that the vaccine effectively overcomes B-cell tolerance and induce high-titer therapeutically potent anti-HER2 IgG, which i prevent tumor growth in wild-type FVB mice grafted with mammary carcinoma cells expressing human HER2 and ii prevent spontaneous development of human HER2-positive mammary carcinomas in tolerant transgenic mice.

The results also provide strong proof-of-concept for the use of the VLP platform to develop self-antigen based vaccines against non-communicable diseases. Lane 1 contains uncoupled Spytagged VLP. Specifically, the lane contains both unconjugated VLP subunit ~ Subsequent dialysis of the reaction mixtures reduced the amount of unconjugated SpyCatcher-HER2 antigen present in the vaccine formulation Fig.

We evaluated the immunogenicity and the therapeutic potential of the vaccine in wild type mice grafted with mammary carcinoma cells expressing HER2. It is noteworthy that vaccinations completely prevented tumor growth in 4 of 5 mice inoculated with tumor fragments Fig. These encouraging results obtained in mice not expressing human HER2 led us to test the vaccine in transgenic mice, which are immunologically tolerant to human HER2. HER2-VLP vaccinations were administered in the tibialis anterior muscle every second week starting either 5 weeks after cell injection or 2 weeks after fragment implantation.

A, D tumor growth curves, each line represents one mouse. All vaccinated mice remained tumor-free until one year of age, whereas all untreated mice developed carcinomas, which started at two months of age Fig.

B, E Mean number of mammary carcinomas per mouse tumor multiplicity. Each point represents the mean ± SEM of 3—13 individual sera of different mice.

Moreover, the mice that developed carcinomas had significantly reduced tumor multiplicity compared to both untreated and pHURT-vaccinated mice Fig. As immunoprevention of mammary carcinogenesis was shown to be mediated by antibodies and T cell cytokines, 39 we studied the pattern of cytokines elicited by vaccination in F1 mice.

We found that HER2-VLP vaccination induced specific increases in the levels of various cytokines involved in chemotaxis, antigen presentation and type 1 helper T cell response Supplementary Fig.

A side-by-side comparison of the results obtained in Delta16 mice Fig. Even more so when one considers that Delta16 mice received only three vaccinations, vis-à-vis the lifetime vaccination schedule of F1 mice.

It should be kept in mind that F1 mice harbor two versions of the transgene, a full-length HER-2 and a Delta 16 isoform, which together could hamper the breakage of immune tolerance and increase tumor resistance to immune attacks.

The resistance of F1 mice to immunopreventive vaccines was shown also by the limited effects obtained after pHuRT vaccination, which in different hosts elicited significant protective responses. To further assess the ability of the HER2-VLP vaccine to induce high-affinity anti-HER2 IgG, kinetic binding experiments were performed using an Attana A Quartz Crystal Microbalance Biosensor, which allows for direct and label free analysis of the kinetic properties of the association between different molecules.

Analysis of vaccine-induced anti-HER2 IgG showed the characteristics of multiple binding factors with different binding kinetics, consistent with a polyclonal IgG population Fig.

Approximately seconds after sample injection, the Hz response curve showed a constant dissociation rate, which at this point was in a similar range to that measured for trastuzumab Fig. A IgG subtypes elicited by the indicated vaccinations, determined by flow cytometry of SK-OV-3 cells after indirect immunofluorescence with subtype-specific secondary Abs.

Each bar represents the mean and SEM of 2—5 individual sera of different mice obtained two weeks after the third vaccination. Each point represents the mean±SEM of 3—5 sera. Binding is shown as change in frequency over time ΔHz. The black curve represents the real-time trace, while the red curve shows the fit of the dissociation rate measured for  seconds — seconds after injection.

Dissociation rate constants K d were obtained by applying a dissociation rate model using the TraceDrawer software. As expected, when BT C5 cells were cultured in the same conditions, trastuzumab had no effect. A Inhibition of agar colony number, counted 18 days after seeding. Each bar represents the mean and SEM inhibition of colony number in two independent plates. Inhibition by trastuzumab or anti-HER2 sera was calculated in reference to wells containing medium alone and normal mouse serum, respectively.

B Dark-field micrographs of colonies in agar. White bar corresponds to  μm. HER2 is an important target antigen for vaccine development, and a wide variety of technologies have been deployed over the years. Besides, recombinant expression of HER2 in Drosophila S2 insect cells has the potential advantage of improved antigen uptake by APCs due to the addition of insect glycans.

Specifically, the paucimannose insect glycan structure terminates in mannans, which has been reported to induce improved uptake by professional APCs and dendritic cells in particular, due to membrane bound mannose receptor expression on these cells. Besides levels of LPS were not measured in the final vaccine preparation. We have previously demonstrated that prevention of tumor onset in HER2 vaccinated transgenic mice was mediated by anti-HER2 Abs, 48 and that protection was dependent on induction of a rapid and strong Ab response to keep at bay the unrelenting mammary carcinogenesis of transgenic mice.

To appraise the potency of the HER2-VLP vaccine we compared it head-to-head with a highly optimized DNA vaccine 50 in a mouse model of aggressive mammary carcinogenesis. BT to a similar extent as trastuzumab, and could even inhibit 3D growth of the trastuzumab-resistant clone BT C5. In human clinical trials, treatment with multiple anti-HER2 mAb i.

Importantly, mechanisms of resistance to HER2-targeted therapy are not limited to alterations in HER2, but may also involve e. The strong preclinical efficacy data shown in this study underline the potential benefits of an active vaccination approach directed against HER2 and support further clinical evaluation of the HER2-VLP vaccine. To this end, dose-escalation and formulation studies e. Moreover, preclinical and early clinical studies suggest that the integration of an effective anti-HER2 vaccine, like HER2-VLP, in current therapeutic regimes could build on the immunogenic tumor cell death induced by therapeutic mAbs and chemotherapy, leading to a powerful and prolonged anti-HER2 immune response.

However, it is possible that the increased immunogenicity of the antigen obtained e. Also, it is possible that antibodies raised against the VLP backbone may cause off-target effects if they cross-react with self-antigens in the vaccine recipient. Several cancer-associated antigens have been identified as promising targets for immunotherapy and hence potential vaccine antigens. Passive immunotherapy using licensed mAbs has moreover generated valuable safety and efficacy data to guide rational vaccine design.

On the other hand, the lack of a generally effective method to facilitate induction of therapeutically potent immune responses to a self-antigen has hampered progress in the development of anti-cancer vaccines. Consequently, the modular VLP-display system represents a simple, versatile and effective tool to overcome B-cell immune tolerance, which can be used as a generic platform for the design of self-antigen based vaccines.

Harvesting of the cell culture supernatant was done by centrifugation and filtration three days after the final split. The pHuRT DNA vaccine consisted of 50 μg plasmid diluted to a final volume of 40 μl per mouse in final concentrations of 0. The DNA vaccine was injected into the tibial muscles 20 μl in each muscle then the muscle tissues were immediately subjected to electroporation. Serenella M. Delta16 mice, transgenic for an alternative splicing isoform of HER2, Delta16, were kindly gifted by Dr.

To study the inhibition of tumor growth two different experiments were performed. Starting 5 weeks after cell injection, mice were immunized every second week with the HER2-VLP vaccine for the entire lifetime of the mouse. Control group consisted of untreated mice.

Starting 2 weeks after fragment implantation, mice were vaccinated every second week with HER2-VLP vaccine for the entire lifetime of the mouse. Serum samples were collected periodically, one day before every vaccination. Female mice were monitored weekly by palpation and tumor dimensions were measured with calipers.

Masses with a mean diameter exceeding 3 mm were considered tumors. Anti-HER2 avidity assay was performed as described. Freshly made Urea 8M was added to the wells for 5 min reference plates were incubated with PBS followed by a wash. The plates were developed using o-phenylenediamine substrate. The enzymatic reaction was stopped by adding 1 M H 2 SO 4. IgG avidity index values were calculated as the mean OD value of urea-treated wells triplicates divided by the mean OD value of control wells, multiplied by Hereafter, a mixture of sera and binding buffer was allowed to flow through the columns by gravity.

Two-fold dilutions of the analyte, prepared in running buffer, were injected over the surface and the association and dissociation was monitored for 95 s and  s, respectively. The binding surface was regenerated after each injection by applying 10 mm Glycine, pH 2 for 30 s. Data was processed in the Attana Attache software Attana AB and curve fitting was performed using the TraceDrawer software Ridgeview Instruments applying a dissociation rate model.

In vitro sensitivity of BT trastuzumab-sensitive and BT C5 trastuzumab-resistant cells to HER2-VLP-induced antibodies was evaluated in three-dimensional cultures, which better represent tumor architecture than bi-dimensional cultures. This work was also supported by a grant to P-L Lollini pier.

The authors would like to thank Jens Hedelund Madsen for technical assistance. This work was supported by grant IG to P-L. Tania Balboni and Veronica Giusti are recipients of Ph. Arianna Palladini and Marianna L.

Ianzano are recipients of research fellowships Assegni di Ricerca from the University of Bologna. All in vivo experiments were approved by the institutional review board of the University of Bologna, authorized by the Italian Ministry of Health and done according to Italian and European laws and guidelines. Read article at publisher's site DOI :

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  • Arianna Palladini and Marianna L. När andningen har avtagit, använd en tå nypa för att bekräfta musen är helt bedövad. Börjar på 15 dagar efter parning, kontrollera honorna regelbundet för tecken på graviditet stor utskjutande omkrets. Cancer vaccines inducing antibody production: more pros than cons. Pihl J 2 ,.
  • Curr Opin Immunol ; 10 —5. Antibody therapy of cancer. Sprutan ska hållas så att ingen ompositionering måste ske för att injicera innehållet. Den teknik som används här för injektion var kritisk för detta experiment. The Netherlands: Elsevier; [ Google Scholar ].

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