Protein Nanoparticle-Mediated Delivery of Recombinant Influenza Hemagglutinin Enhances Immunogenicity and Breadth of the Antibody Response

Badten, Alexander J; Ramírez, A. Gonzaga; Hernandez-Davies, Jenny E.; Albin, Tyler J.; Jain, Aarti; Nakajima, Rie; Felgner, Jiin; Davies, D. Huw; Wang, Szu-Wen

doi:10.1021/acsinfecdis.2c00362

Cited by 2 publications

(8 citation statements)

References 110 publications

(194 reference statements)

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“…To conjugate CBU1910 onto the surface of the E2 protein NP, we used an affinity strategy that we had developed for conjugating green fluorescent protein (GFP) and influenza hemagglutinin (HA). 56 An E2 NP displaying 60 cysteines on its surface (E279C) 28 allows for conjugation of a synthesized maleimide-tris-NTA (mal-tNTA) linker, 56 which enables a His-tagged protein to couple to the NP. This protocol was performed for CBU1910 as shown in Figure 2A.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…28 We have reported the generation of tNTA-E2 nanoparticles previously. 56 Purified E2 (E279C) in 20 mM HEPES and 100 mM NaCl (pH 7.3) was incubated with an 8.5× molar excess of TCEP (Thermo Fisher Scientific; dissolved in Milli-Q water). A 10× molar excess of maleimido cyclic tris-NTA (mal-tNTA) (diluted to 4 mg/mL in DMF) was added to the E2 and incubated at room temperature for 2 h and then at 4 °C overnight.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Attachment of CBU1910-(His) 6 to tNTA-E2 nanoparticles followed similar procedures established using other protein-(His) n antigens. 56 Briefly, a 10× molar excess of NiCl 2 was incubated with tNTA-E2 for 2 h at room temperature and subsequently purified from unchelated Ni using Zeba spin desalting columns into 20 mM HEPES + 360 mM NaCl buffer pH 7.3. To test optimal conjugation, varying molar ratios of (His) 6 -tagged CBU1910 (previously synthesized 47 ) were added to the Ni-tNTA-E2 and incubated at room temperature for 2 h. The Ni-tNTA-E2 + CBU1910-(His) 6 reaction required optimization to yield unaggregated/precipitated constructs, the details of which are described in the Supporting Information.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Attachment of CBU1910-(His) 6 to tNTA-E2 nanoparticles followed similar procedures established using other protein-(His) n antigens . Briefly, a 10× molar excess of NiCl 2 was incubated with tNTA-E2 for 2 h at room temperature and subsequently purified from unchelated Ni using Zeba spin desalting columns into 20 mM HEPES + 360 mM NaCl buffer pH 7.3.…”

Section: Methodsmentioning

confidence: 99%

“…Direct genetic fusion of protein antigens onto virus-like particles has shown some success with particular platforms and therefore was explored with the E2 protein nanoparticle; however, expression and correct folding into a soluble protein assembly needs to be empirically tested. 18,50−52 The introduction of polyhistidine tags on recombinant proteins to bind to Ni-NTA-based matrices is a well-established protein purification methodology, 53−55 and we previously applied this complexation-based approach in nanoparticle-mediated delivery of influenza hemagglutinin antigen; 56 here, we used it as the basis for loading polyhistidine-tagged (His-tag) CBU1910 antigen onto E2 NPs. To attach protein antigens, covalently and modularly, onto the surface of E2 NPs, the versatile protein− protein conjugation method, SpyTag/SpyCatcher, was implemented.…”

Section: ■ Introductionmentioning

confidence: 99%

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Engineering Protein Nanoparticles Functionalized with an Immunodominant Coxiella burnetii Antigen to Generate a Q Fever Vaccine

Ramirez,

Felgner,

Jain

et al. 2023

Bioconjugate Chem.

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Coxiella burnetii is the causative agent of Q fever, for which there is yet to be an FDA-approved vaccine. This bacterial pathogen has both extra- and intracellular stages in its life cycle, and therefore both a cell-mediated (i.e., T lymphocyte) and humoral (i.e., antibody) immune response are necessary for effective eradication of this pathogen. However, most proposed vaccines elicit strong responses to only one mechanism of adaptive immunity, and some can either cause reactogenicity or lack sufficient immunogenicity. In this work, we aim to apply a nanoparticle-based platform toward producing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugation of the immunodominant outer membrane protein antigen (CBU1910) to the E2 nanoparticle to obtain a consistent antigen orientation: direct genetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-tagged CBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we found that the ST/SC approach yielded nanoparticles loaded with the highest number of antigens while maintaining stability, enabling formulations that could simultaneously co-deliver the protein antigen (CBU1910) and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Using protein microarray analyses, we found that after immunization, antigen-bound nanoparticle formulations elicited significantly higher antigen-specific IgG responses than soluble CBU1910 alone and produced more balanced IgG1/IgG2c ratios. Although T cell recall assays from these protein antigen formulations did not show significant increases in antigen-specific IFN-γ production compared to soluble CBU1910 alone, nanoparticles conjugated with a CD4 peptide epitope from CBU1910 generated elevated T cell responses in mice to both the CBU1910 peptide epitope and whole CBU1910 protein. These investigations highlight the feasibility of conjugating antigens to nanoparticles for tuning and improving both humoral- and cell-mediated adaptive immunity against C. burnetii.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%