Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of ‘epitope-resurfaced’ mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.
Taiwan 22 23 §These authors contributed equally to this work 24 25Word counts: 287 words (Abstract), 7595 words (main text) 26 Abstract 46 Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading 47 cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing 48 flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, 49 the structure of dengue VLP is poorly understood. Herein we show for the first time that 50 mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-51 protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing 52 epitopes through cryo-electron microscopy reconstruction. Mice vaccinated with highly matured 53 virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher cross-54 reactive (CR) neutralization antibodies (NtAbs) and were protected against all 4 serotypes of 55 DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. Our 56 results revealed that a "epitope-resurfaced" mature-form dengue VLP has the potential to induce 57 quaternary structure-recognizing broad CR NtAbs. 58 59 60 101 102 Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated 103to be a potential vaccine candidate, since their ordered E structures are similar to those on the 104 6 virion surface and also undergo low-pH-induced rearrangements and membrane fusion similar to 105 viral particles (Allison et al., 1995). Also, VLP vaccines present several advantages since they are 106 highly immunogenic, non-infectious, and accessible to quality control as well as increased 107 production capacity. However, we theorized that the process of VLP maturation might be similar 108 to that of dengue viral particles, with heterogeneous pr-containing structures. In this study, we 109 engineered and characterized the structure of mature DENV-2 VLPs through cryo-EM. The 110 immunological properties of mature VLPs were further compared with its immature counterparts. 111 112 113 114 7 Results 115As we theorized, the process of VLP maturation are similar to that of dengue viral 116 particles so that the wild-type particles (wtD2VLP) produced from the cells are partially 117 immature containing both prM and M proteins ( Figure S1 in Supplementary Material). To 118 overcome this problem, we engineered the DENV-2 VLPs as completely mature particles by 119 manipulating the furin cleavage site at the junction of pr and M in a DENV-2 VLP-expressing 120 plasmid (Chang et al., 2003). For comparison, we also generated immature VLP (imD2VLPs) by 121 mutating the minimal furin cleavage motif REKR to REST(Li et al., 2008), which resulted in 122 completely uncleaved prM as detected by western blot and ELISA (Figure 1). Generation of 123 mature DENV-2 VLP (mD2VLP) was much more challenging. First, we performed multiple 124 sequence alignments of the pr/M junction from several flaviviruses, including th...
Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. The vaccine candidates under development require a tetravalent immunogen to induce a balanced immunity against all four serotypes of dengue virus. Herein we show that mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher and broader neutralization antibodies (NtAbs) against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. The cryo-electron microscopy reconstruction showed that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Most importantly, maternally transferred antibodies derived from mD2VLP-vaccinated female mice protected suckling mice from lethal challenge by all four serotypes of DENV. Our results support the fact that a universal dengue vaccine that protects against all four serotypes of dengue viruses can be achieved by using an immunogen such as mD2VLP.
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