A two-component modular approach for enhancing T-cell activation utilizing a unique anti-FcγRI-streptavidin construct and microspheres coated with biotinylated-antigen

Walsh, Mary F.; Banas, Jeffrey A.; Mudzinski, Stanley P.; Preissler, Mark T.; Graziano, Robert F.; Gosselin, Edmund J.

doi:10.1016/s1389-0344(02)00089-8

Cited by 20 publications

(15 citation statements)

References 46 publications

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“…Ampicillin-resistant transformants were screened for insertion by PCR, followed by restriction endonuclease analysis and sequencing of miniprep DNA to confirm the in-frame fusion construct. Plasmid DNA was used for liposome-mediated transfection of 1 ϫ 10 4 NSO cells (ATCC), a non-Ig-synthesizing murine myeloma cell line (68). Supernatants from transfected hybridomas were then screened by flow cytometry for the presence of the fusion protein.…”

Section: Methodsmentioning

confidence: 99%

“…Finally, antirabbit-alkaline phosphatase (AP) or anti-mouse-AP was added for 1 h, respectively; the wells were washed again, and AP substrate was added. The anti-hFc␥RI-PspA fusion protein was purified using a nickel affinity column or protein L, also as previously described (51,68). Purified antihFc␥RI-PspA fusion protein was run on an 8% polyacrylamide SDS-PAGE gel (Pierce, Rockford, IL).…”

Section: Methodsmentioning

confidence: 99%

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Mucosal Immunization with an Unadjuvanted Vaccine That Targets Streptococcus pneumoniae PspA to Human Fcγ Receptor Type I Protects against Pneumococcal Infection through Complement- and Lactoferrin-Mediated Bactericidal Activity

Bitsaktsis

Iglesias

et al. 2012

Infect Immun

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Targeting an antigen to Fc receptors (FcR) can enhance the immune response to the antigen in the absence of adjuvant. Furthermore, we recently demonstrated that intranasal immunization with an Fc␥R-targeted antigen enhances protection against a category A intracellular mucosal pathogen, Francisella tularensis. To determine if a similar strategy could be applied to the important pathogen Streptococcus pneumoniae, we used an improved mucosal FcR-targeting strategy that specifically targets human Fc␥R type I (hFc␥RI). A humanized single-chain antibody component in which the variable domain binds to hFc␥RI [antihFc␥RI (H22)] was linked in a fusion protein with the pneumococcal surface protein A (PspA). PspA is known to elicit protection against pneumococcal sepsis, carriage, and pneumonia in mouse models when administered with adjuvants. Anti-hFc␥RI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFc␥RI transgenic (Tg) mice in the absence of adjuvant. The hFc␥RI Tg mice receiving anti-hFc␥RI-PspA exhibited elevated S. pneumoniae-specific IgA, IgG2c, and IgG1 antibodies in serum and bronchoalveolar lavage fluid. Neither immunogen was effective in protecting WT mice in the absence of adjuvant, but when PspA was targeted to hFc␥RI as the anti-hFc␥RI-PspA fusion, enhanced protection against lethal S. pneumoniae challenge was observed in the hFc␥RI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFc␥RI-PspA-immunized Tg mice showed enhanced complement C3 deposition on bacterial surfaces, and protection was dependent upon an active complement system. Immune serum also showed an enhanced bactericidal activity directed against S. pneumoniae that appears to be lactoferrin mediated.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mucosal Immunization with an Unadjuvanted Vaccine That Targets Streptococcus pneumoniae PspA to Human Fcγ Receptor Type I Protects against Pneumococcal Infection through Complement- and Lactoferrin-Mediated Bactericidal Activity

Bitsaktsis

Iglesias

et al. 2012

Infect Immun

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“…Fc receptors (FcR), 3 including Fc␥Rs, on APCs can enhance humoral and cellular immunity (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Fc␥Rs are classified based on their molecular weight, IgG-Fc-binding affinities, IgG subclass-binding specificity, and cellular distribution.…”

Section: N Umerous Studies Have Demonstrated That Targeting Ag Tomentioning

confidence: 99%

Utilization of Fc Receptors as a Mucosal Vaccine Strategy against an Intracellular Bacterium, Francisella tularensis

Rawool

Bitsaktsis

et al. 2008

The Journal of Immunology

179

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Numerous studies have demonstrated that targeting Ag to Fc receptors (FcR) on APCs can enhance humoral and cellular immunity. However, studies are lacking that examine both the use of FcR-targeting in generating immune protection against infectious agents and the use of FcRs in the induction of mucosal immunity. Francisella tularensis is a category A intracellular mucosal pathogen. Thus, intense efforts are underway to develop a vaccine against this organism. We hypothesized that protection against mucosal infection with F. tularensis would be significantly enhanced by targeting inactivated F. tularensis live vaccine strain (iFt) to FcRs at mucosal sites, via intranasal immunization with mAb-iFt complexes. These studies demonstrate for the first time that: 1) FcR-targeted immunogen enhances immunogen-specific IgA production and protection against subsequent infection in an IgA-dependent manner, 2) FcγR and neonatal FcR are crucial to this protection, and 3) inactivated F. tularensis, when targeted to FcRs, enhances protection against the highly virulent SchuS4 strain of F. tularensis, a category A biothreat agent. In summary, these studies show for the first time the use of FcRs as a highly effective vaccination strategy against a highly virulent mucosal intracellular pathogen.

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“…This is important since rbhIL-2 has the potential for application in vaccines and immunotherapeutics (8,10,11,13). For example, our laboratory has recently developed a targeting strategy which has the ability to target biotinylated proteins to APC (23). We are currently exploring its use in similarly targeting cytokines to APC in order to influence the degree and direction of the immune response.…”

Section: Discussionmentioning

confidence: 99%

“…Studies also show that targeting IL-2 to tumor cells facilitates tumor elimination, presumably via enhanced T-cell activation (8,10,11,14,26). Therefore, the availability of a recombinant biotinylated human IL-2 (rbhIL-2) could not only provide a tool for monitoring T-cell activation but also facilitate the development of a therapeutic tool which utilizes single-chain variable-region antibody fragment-IL-2 fusion proteins to target IL-2 to tumors (8,10,11,14) as well as antigen-presenting cells (APC) (23). In addition, large-scale production and purification of a uniformly biotinylated IL-2 product, required for diagnostic and clinical applications, would be facilitated.…”

mentioning

confidence: 99%

Production of Genetically Engineered Biotinylated Interleukin-2 and Its Application in a Rapid Nonradioactive Assay for T-Cell Activation

Jordan

Preissler

Banas

et al. 2003

Clin Vaccine Immunol

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The development of reliable assay systems that can measure lymphocyte activation in vitro has been a major goal of immunodiagnostics. Traditionally, tritiated thymidine incorporation has been used to monitor T-cell activation. Other methods include enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay, and colorimetric assays. We have established a lymphocyte activation assay that utilizes fluorescein isothiocyanate (FITC)-streptavidin bound to recombinant biotinylated human interleukin-2 (IL-2). Utilizing recombinant DNA technology, a unique monobiotinylated human IL-2 has been created and isolated using the Promega PinPoint vector system. ELISA has been used to demonstrate streptavidin binding and recognition by a human IL-2-specific antibody. IL-2 function has been demonstrated using a murine IL-2-dependent T-cell line, CTLL-2, responsive to human IL-2. Recombinant biotinylated human IL-2 conjugated to streptavidin-FITC or streptavidin-horseradish peroxidase has been used to monitor T-cell activation in the presence of antigen as well as mitogen. The sensitivity and convenience of this method make this lymphocyte activation assay an attractive alternative to tritiated thymidine incorporation as a method for monitoring T-cell activation. In addition, the availability of a recombinant biotinylated human IL-2 will permit the production of a uniform product suitable for diagnostic and clinical application.

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A two-component modular approach for enhancing T-cell activation utilizing a unique anti-FcγRI-streptavidin construct and microspheres coated with biotinylated-antigen

Cited by 20 publications

References 46 publications

Mucosal Immunization with an Unadjuvanted Vaccine That Targets Streptococcus pneumoniae PspA to Human Fcγ Receptor Type I Protects against Pneumococcal Infection through Complement- and Lactoferrin-Mediated Bactericidal Activity

Mucosal Immunization with an Unadjuvanted Vaccine That Targets Streptococcus pneumoniae PspA to Human Fcγ Receptor Type I Protects against Pneumococcal Infection through Complement- and Lactoferrin-Mediated Bactericidal Activity

Utilization of Fc Receptors as a Mucosal Vaccine Strategy against an Intracellular Bacterium, Francisella tularensis

Production of Genetically Engineered Biotinylated Interleukin-2 and Its Application in a Rapid Nonradioactive Assay for T-Cell Activation

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