Recombinant proteins are an attractive choice as a safe alternative to traditional live attenuated vaccines. However, most small-size proteins are poorly immunogenic, and adjuvants, whose mode of action remain to be fully clarified, are needed for increasing their immunogenicity. Here, we report the effects of short solubility controlling peptide tags (SCP-tags) on the immunogenicity of DENV3 envelope protein domain 3 (3ED3; 103 residues, 11.46 kDa) in ICR and Swiss albino model mice. The attachment of a 4-Ile SCP-tag (C4I-tag) increased the hydrodynamic radius of 3ED3 from 2.2 ± 0.09 to 111 ± 146 nm as assessed by dynamic light scattering in phosphate buffered saline at 37 • C, indicating that the C4I-tag oligomerized 3ED3. Immunization at 30 µg/dose showed that the untagged 3ED3 was not or poorly immunogenic, whereas the C4I-tag increased its immunogenicity by up to 39-fold as assessed by the IgG level measured using ELISA. Moreover, the increased antibody level was sustained for over 6 months after immunization and a high number of effector and central memory T cells were generated. These observations provide solid and quantitative evidence for the hypothesis that subvisible aggregates with hydrodynamic radii of 100 nm can increase immunogenicity and that SCP-tag can establish a long-term, target-specific immune response in a way adequate for the development of a peptide/protein-based DENV vaccine.
Dengue virus infection, a highly prominent health concern, has caused many health complications, positive cases, and deaths in Bangladesh in previous years. However, the prevalence of this infection and fatality rates in 2022 has shattered all prior records. The dengue virus vector, mosquitoes, found a high prevalence of infection due to the weather’s favorable conditions for breeding in the months of June and July. While there is presently no particular vaccination for dengue infection, awareness of its epidemiology, pathogenesis, signs, and symptoms may aid in the development of improved diagnostic and treatment strategies. The government should also improve the infrastructure of cities to make prevent mosquito breeding and the spread of dengue infection.
25Dengue viruses are classified into four serotypes (DENV1~4), and the severe forms of dengue 26 disease, the dengue hemorrhagic fever and shock syndrome, are caused by sero-cross-reacting 27 antibodies. However, the residue determinants of the serospecificity and sero-cross-reactivity are 28 yet to be identified. Here, we report an epitope grafting mutational analysis of the serospecificity 29 and cross-serospecificity of the envelope protein domain 3 (ED3; 107 residues, ~11.6kDa), 30 which contains two major putative epitopes of DENVs. To this end, we constructed ED3 from 31 DENV3 (3ED3) and DENV4 (4ED3), and six epitope-grafted variants, where we transferred 32 epitope 1 (L 304 I, K 305 D, V 309 M, and S 310 A) and/or epitope 2 (D 383 N, K 384 S, K 387 T, and N 389 H) of 33 4ED3 onto 3ED3 and vice versa. Mice immunization using 3ED3 and 4ED3 generated serotype-34 specific antisera, as expected. Similarly, most epitope-grafted ED3s produced antisera 35 serospecific to the template ED3 with little or no cross-recognition of ED3 of the serotype from 36 which the epitopes were taken. This result indicated that a mere grafting of the epitope was not 37 sufficient to transfer serospecificity, contrary to our expectations. However, one epitope grafted 38 ED3 mutant, where epitope 1 of 3ED3 was grafted onto 4ED3 (4ED3 epi1 ), generated antisera that 39 was serospecific to both 4ED3 and 3ED3. The 4ED3 epi1 is thus a chimeric ED3 that produces 40 antisera possessing serospecificity to both 3ED3 and 4ED3. The 4ED3 epi1 provides a unique tool 41 for analyzing serospecificity and cross-reactivity in dengue, and we hope it will serve as a 42 template for trivalent and eventually tetravalent antisera. 45Dengue fever, a mosquito-borne viral disease, is caused by the dengue virus (DENV), which is a 46 flavivirus classified into four serologically distinct serotypes (DENV1~4). It is a major public 47 health issue in tropical and subtropical regions [1,2], with 390 million cases reported every year, 48 and 40% of the world's population at risk [3,4]. Primary infection by a DENV may provoke a 49 high fever for a few days, but the patient usually recovers and may gain a long-lasting immunity 50 against the infecting serotype [5]. However, a secondary heterotypic infection can lead to severe 51 syndromes such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [6,7]. 52The severity is thought to be caused by antibodies produced during the primary infection, which 53 would be cross-reacting but sub-neutralizing against the secondary infecting DENV serotype. 54This phenomenon is coined as the Antibody Dependent Enhancement (ADE) [7-9]; ADE can be 55 caused by natural dengue infection and also demonstrated in artificial immunization studies 56 [7,[10][11][12], which is a factor making the development of the dengue vaccine cumbersome [13,14]. 57The single-stranded RNA genome of DENV encodes ten gene products: The capsid (C), pre-58 membrane (prM), envelope (E), and seven nonstructural (NS) proteins [15]. The E...
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