A systematic mutational analysis identifies a 5‐residue proline tag that enhances the <i>in vivo</i> immunogenicity of a non‐immunogenic model protein

Rahman, Nafsoon; Islam, Mohammad Monirul; Kibria, Golam; Unzai, Satoru; Kuroda, Yutaka

doi:10.1002/2211-5463.12941

Cited by 9 publications

(6 citation statements)

References 41 publications

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“…5A). This is in line with our previous reports [20] and corroborates that small proteins are not or are poorly immunogenic [27]. The C5I tagged D3ED3 showed an increased serum anti-D3ED3 IgG titre than monomeric D3ED3, whereas Ms showed the strongest IgG titre (Fig.…”

Section: Effect Of D3ed3 Oligomerization Type On Immune Responsesupporting

confidence: 92%

Biophysical and biochemical nature of amorphous protein oligomers determines the strength of immune response and the generation of T‐cell memory

Kibria,

Shiwaku,

Brindha

et al. 2023

The FEBS Journal

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Here, we used domain 3 of dengue virus serotype 3 envelope protein (D3ED3), a natively folded globular low‐immunogenicity protein, to ask whether the biophysical nature of amorphous oligomers can affect immunogenicity. We prepared nearly identical 30 ~ 50 nm‐sized amorphous oligomers in five distinct ways and looked at any correlation between their biophysical properties and immunogenicity. One oligomer type was produced using our SCP tag (solubility controlling peptide) made of 5 isoleucines (C5I). The others were prepared by miss‐shuffling the SS bonds (Ms), heating (Ht), stirring (St) and freeze–thaw (FT). Dynamic light scattering showed that all five formulations contained oligomers of approximately identical sizes with hydrodynamic radii (Rh) between 30 and 55 nm. Circular dichroism (cd) indicated that the secondary structure content of oligomers formed by stirring and freeze–thaw was essentially identical to that of the native monomeric D3ED3. The secondary structure content of the Ms showed moderate changes, whereas the C5I and heat‐induced (Ht) oligomers exhibited a significant change. The Ms contained D3ED3 with intermolecular SS bonds as assessed by nonreducing size exclusion chromatography (SEC). Immunization in JcL:ICR mice showed that both C5I and Ms significantly increased the anti‐D3ED3 IgG titre. Ht, St and FT were only mildly immunogenic, similar to the monomeric D3ED3. Cell surface CD marker analysis by flow cytometry confirmed that immunization with Ms generated a strong central and effector T‐cell memory. Our observations indeed suggest that controlled oligomerization can provide a new, adjuvant‐free method for increasing a protein's immunogenicity, yielding a potentially powerful platform for protein‐based (subunit) vaccines.

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Section: Effect Of D3ed3 Oligomerization Type On Immune Responsesupporting

confidence: 92%

Biophysical and biochemical nature of amorphous protein oligomers determines the strength of immune response and the generation of T‐cell memory

Kibria,

Shiwaku,

Brindha

et al. 2023

The FEBS Journal

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“…Plates were then incubated at 37 °C for 1 h. After washing the plates thrice with PBS-0.05% Tween-20, each well was filled with 100 μL of anti-mouse IgG HRP conjugate (Thermo Fisher Scientific, Waltham, MA, USA) at a 1:3000 dilution (or its subclasses-IgG1, 1:25,000 dilution/IgG2a, 1:3000 dilution) in 0.1% BSA-PBS-Tween-20 and incubated at 37 °C for 1 h. As a substrate, OPD (o-phenylenediamine dihydrochloride) was added. Color intensities were measured at 492 nm using a microplate reader (SH9000 Lab, Hitachi High-Tech Science Co., Tokyo, Japan) immediately after stopping the reaction with 1 N sulfuric acid (50 µL/well) [ 29 , 30 , 31 ]. Antibody titers were calculated from the power fitting of OD 492 nm versus the reciprocal of the antisera dilution, using a cutoff of OD 492 nm = 0.1 above the background value.…”

Section: Methodsmentioning

confidence: 99%

Antisera Produced Using an E. coli-Expressed SARS-CoV-2 RBD and Complemented with a Minimal Dose of Mammalian-Cell-Expressed S1 Subunit of the Spike Protein Exhibits Improved Neutralization

Yoshizue

Brindha

Wongnak

et al. 2023

IJMS

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E. coli-expressed proteins could provide a rapid, cost-effective, and safe antigen for subunit vaccines, provided we can produce them in a properly folded form inducing neutralizing antibodies. Here, we use an E. coli-expressed SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as a model to examine whether it yields neutralizing antisera with effects comparable to those generated by the S1 subunit of the spike protein (S1 or S1 subunit, thereafter) expressed in mammalian cells. We immunized 5-week-old Jcl-ICR female mice by injecting RBD (30 µg) and S1 subunit (5 µg) according to four schemes: two injections 8 weeks apart with RBD (RBD/RBD), two injections with S1 (S1/S1), one injection with RBD, and the second one with S1 (RBD/S1), and vice versa (S1/RBD). Ten weeks after the first injection (two weeks after the second injection), all combinations induced a strong immune response with IgG titer > 105 (S1/RBD < S1/S1 < RBD/S1 < RBD/RBD). In addition, the neutralization effect of the antisera ranked as S1/RBD~RBD/S1 (80%) > S1/S1 (56%) > RBD/RBD (42%). These results indicate that two injections with E. coli-expressed RBD, or mammalian-cell-produced spike S1 subunit alone, can provide some protection against SARS-CoV-2, but a mixed injection scheme yields significantly higher protection.

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“…4-week-old JCl mice were injected subcutaneously with 30 µg of BPTI four times. Particle radii were measured with DLS just before injection by taking an aliquot from the injected sample; experimental details are described in Rahman et al ( 2020a , b ) …”

Section: Aggregation and Immunogenicitymentioning

confidence: 99%

Biophysical studies of amorphous protein aggregation and in vivo immunogenicity

Kuroda

2022

Biophys Rev

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Amorphous protein aggregates are oligomers that lack specific, high-order structures. Soluble amorphous aggregates are smaller than ~1 µm. Despite their lack of high-order structure, amorphous protein aggregates exhibit specific biophysical properties such as reversibility of formation, density, conformation, and biochemical stability. Our mutational analysis using a Solubility Controlling Peptide (SCP) tag strongly suggests that amorphous aggregation of small globular proteins can significantly increase in vivo immune response and that the magnitude of enhanced immune responses depends on the aggregates’ biophysical and biochemical properties. We propose that SCP tags might help develop subunit (protein) adjuvant-free (immunostimulant-free) vaccines by controlling the aggregation propensity of target proteins.

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A systematic mutational analysis identifies a 5‐residue proline tag that enhances the in vivo immunogenicity of a non‐immunogenic model protein

Cited by 9 publications

References 41 publications

Biophysical and biochemical nature of amorphous protein oligomers determines the strength of immune response and the generation of T‐cell memory

Biophysical and biochemical nature of amorphous protein oligomers determines the strength of immune response and the generation of T‐cell memory

Antisera Produced Using an E. coli-Expressed SARS-CoV-2 RBD and Complemented with a Minimal Dose of Mammalian-Cell-Expressed S1 Subunit of the Spike Protein Exhibits Improved Neutralization

Biophysical studies of amorphous protein aggregation and in vivo immunogenicity

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