A broadly applicable protein-polymer adjuvant system for antiviral vaccines

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  Figure EV1. Schematic illustration of the PPCDQ nanovaccine induce a humoral and cellular immune response against virus infection. PPCDQ nanovaccine was synthesized by PLA-Porphyrin-Co2+, DSPE-PEG2000, QS-21, and histidine-tagged antigen. After intramuscular injection, the PPCDQ nanovaccine drained into the lymph nodes, efficiently activated DCs and then activated T cells. Furthermore, the activated CD4+ helper T cells (TH cells) activated B cells to differentiate into plasma cells (PCs) to produce antibodies. Neutralizing antibodies and cytotoxic T lymphocytes (CTLs) against virus infection together. This figure was created with BioRender (https://biorender.com/).

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  Figure 1. Construction, characterization, and immunization of HA@PPCDQ. (A) Schematic illustration of the preparation process of PPCDQ nanovaccine (The positions of all materials in the diagram are relative positions drawn to display the antigens). (B) The procedure of HA@PPCDQ production. PPCD nanocarrier, QS-21, and three kinds of His-tagged HA protein were mixed and incubated in PBS buffer to self-assemble into HA@PPCDQ nanovaccines (His-HA-NPs, C-His-Fc-HA-NPs, and N-His-Fc-HA-NPs). (C) TEM images of PPCDQ (NPs), His-HA-NPs, C-His-Fc-HA-NPs, and N-His-Fc-HA-NPs. The rose-red circles in the picture are part of the protein bound to the surface of the micelle. Scale bars: 50 nm. (D) Hydrodynamic diameter distribution of each nanoparticle (n = 3 biological replicates per group). (E) Schematic of C57/BL6 vaccination. Mice from each group were prime/boost-vaccinated (I.M.) with different vaccines at week 0 and week 2. Serum was collected every week. All mice were euthanized at week 6. (F–H) Hemagglutination inhibition (HI) titers (F), microneutralization (MN) titers (G), and HA-specific total IgG (H) at 3 weeks after immunization (n = 10 animals per group). Data information: Data in (F–H)…

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  Figure EV2. Efficiency of PPCD anchoring His-tagged proteins. (A) Construction strategy of three expression plasmids. CMV was used as a promoter, an IgG1 signal peptide (SP) was fused to the N-terminal and an 8 × His tag was fused to the N-terminal or C-terminal. Fc tag was fused to the C-terminal of HA. (B) SDS-PAGE gel of C-His-HA (1), C-His-Fc-HA (2), and N-His-Fc-HA (3). (C, D) Native PAGE gel (D) and anchoring efficiency (C) of PPCD micelles with different proportions of His-HA proteins (n = 3 biological replicates per group). (E, F) Native PAGE gel (E) and statistical graph of binding efficiency (F) of PPCD micelles to three kinds of HA proteins (n = 3 biological replicates per group). (G, H) Practical size (G) and zeta potential (H) of PPCDQ combined with or without protein (n = 3 biological replicates per group). Data information: Data in (C, F, G, H) are mean ± SD, statistical analysis in (H) was performed by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05.

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  Figure EV3. The synergistic effect of PPCD and QS-21 is critical for antibody production. (A–C) HI titers (A), MN titers (B), and HA-specific total IgG (C) of immunized C57/BL6 mice at each time point (n = 10 animals per group). Arrows represent the time points of vaccination. (D, E) C57/BL6 mice were immunized with different dose (5, 10, and 20 μg) of His-HA-NPs (D) or C-His-Fc-HA-NPs (E). HI titers were detected at indicated time point (n = 5 animals per group). (F) C57/BL6 mice were intramuscularly or subcutaneously immunized with 5 μg His-HA-NPs and HI titers of each time point were tested (n = 10 animals per group). (G) C57/BL6 mice were immunized with different formulations and HI titers were detected at 3 weeks post vaccination (n = 10 animals per group). (H) C57/BL6 mice were immunized intramuscularly with the mixture of different adjuvants (Alum, AddaVax, AddaS03, and QS-21) and His-HA or His-HA@PPCD, respectively. HI titers were detected at 3 weeks post vaccination (n = 10 animals per group). (I) C57/BL6 mice were immunized with His-HA@PPCDQ containing different doses of QS-21(5, 10, and 20 μg). HI titers were detected at 3 weeks post vaccination (n = 5 animals per group). (J)…

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  Figure 2. HA@PPCDQ facilitates antigen uptake and delays antigen release. (A) Frequencies of Cy5 positive DCs (B220-CD11c+MHC-II+, top panel) and macrophages (B220-CD11b+F4/80+, bottom panel) in iLNs at 4 h post injection. (B) Antigen uptake experiment of DCs and macrophages in vitro. (B) represented the distribution of HA (red) and lysosome (green) in RAW264.7 and DC2.4 cells after incubation of equivalent iFlourTM 594-tagged His-HA or His-HA-NPs for 12 h. The blue staining indicated Hoechst-stained nuclei. Scalar bar: 20 μm (left), 5 μm (right). (C) Statistical graphs of Cy5 positive DCs (n = 5 animals per group). (D) Statistical graphs of Cy5 positive macrophages (n = 5 animals per group). (E) Statistical graphs of the HA mean fluorescence intensity (MFI) analyzed by ImageJ software (n = 3 biological replicates per group). (F) The distribution of iFlourTM 594-tagged His-HA or His-HA-NPs in inguinal lymph nodes at day 0 and day 7 post injection. (G, H) Light sheet microscopy visualizing iFlourTM 488-tagged His-HA-NPs in inguinal lymph node (G) and axillary lymph node (H) at 4 h post injection. (a–e) represented the different angles of lymph nodes. 360° views are available in Movie EV1…

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  Figure EV4. His-HA-NPs rapidly accumulate in inguinal lymph nodes and flow to remote lymph nodes. (A) Representative flow cytometric plots of Cy5 positive DCs (B220-CD11c+MHC-II+, top panel) in iLNs at 4 h post injection. (B) Statistical graphs of Cy5 positive DCs (n = 5 animals per group). (C) Representative flow cytometric plots of Cy5 positive macrophages (B220-CD11b+F4/80+). (D) Statistical graphs of Cy5 positive macrophages (n = 5 animals per group). (E) ILNs were harvested at 4 h post injection for the living image by IVIS Spectrum system. (F) Statistical graphs of the total radiant efficiency analyzed by Living Image Vision 4.4 (n = 5 animals per group). (G) Transgenic mice CD11c-EYFP were immunized with equal mass of iFlourTM 594-tagged His-HA or His-HA-NPs. Inguinal, popliteal, mesenteric, axillary, and cervical lymph nodes were obtained at 4 h post injection. Antigen distribution of these lymph nodes was shown by the cryosections. Scalar bar: 1000 μm (left), 200 μm (right). Data information: Data in (B, D, F) are mean ± SD, statistical analysis was determined by one-way ANOVA with Tukey’s multiple comparisons test.

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  Figure 3. HA@PPCDQ promotes DC activation and depletes CD169+ macrophages. (A) FCM analysis of DC activation markers MHC-II, CD80, and CD86. (B) Represented the statistical graphs of the normalized MFI of MHC-II, CD80, and CD86 (n = 5 biological replicates per group). (C) FCM analysis to determine the percentages of CD11c+MHC-II+DCs, CD11c+CD80+ DCs, and CD11c+CD86+DCs in iLN at 2 days post boost immunization (n = 6 animals per group). (D) Statistical results of subcapsular sinus macrophage (SSM, B220-CD11b+F4/80-CD169+) and medullary sinus macrophage (MSM, B220-CD11b+F4/80+CD169+) in iLN at 2 days post boost immunization (n = 5 animals per group). (E) Spleens at 2 days post boost immunization were processed into single cells for FCM analysis, B220- non-B cells were identified into three distinct populations: tissue-resident macrophage (MФ, B220-F4/80hiCD11bint), monocyte-derived MФ (B220-F4/80intCD11bhi) and neutrophil (B220-F4/80-CD11bhi). (F) CD169 expression of tissue-resident MФ, monocyte-derived MФ, and neutrophils. (G) Statistical results of tissue-resident MФ, monocyte-derived MФ, and neutrophils in the spleen (n = 5 animals per group). (H) Statistical results of the normalized MFI…

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  Figure EV5. HA@PPCDQ promotes DC activation and depletes CD169+ subcapsular sinus macrophages. (A) Representative flow cytometric plots of CD11c+MHC-II+DCs, CD11c+CD80+ DCs, and CD11c+CD86+DCs in iLNs at 2 days post boost immunization. (B) Representative flow cytometric plots of subcapsular sinus macrophage (SSM, B220-CD11b+F4/80-CD169+) and medullary sinus macrophage (MSM, B220-CD11b+F4/80+CD169+) in iLN. (C–E) C57/BL6 mice were immunized with different formulations, and iLNs were collected for FCM. (C) Representative flow cytometric plots of SSM and MSM. Statistical graphs of SSM (D) and MSM (E) (n = 5 animals per group). (F) Cryosections of inguinal lymph nodes were incubated with FITC anti-mouse CD169 (Siglec-1) and Brilliant Violet 421™ anti-mouse/human CD45R/B220. Images were taken on a Nikon super-resolution spinning-disk confocal microscopy. Scalar bar: 100 μm (left) and 50 μm (right). Data information: Data in (D), and (E) are mean ± SD, statistical analysis was determined by one-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01, ****P < 0.0001.

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  Figure 4. T cell immune responses in HA@PPCDQ-vaccinated mice. (A) Representative flow cytometric plots of CD4+ and CD8+ effector memory T cells (TEM, CD62L-CD44+) at 2 weeks post boost vaccination. (B) Statistical results of TEM, TCM (n = 6 animals per group), and CD69+ cells (n = 5 animals per group) in CD4+ T cells. (C, D) Representative flow cytometric plots of central memory T cells (TCM, CD62L+CD44+) in CD4+ T cells (C) and CD8+ T cells (D). (E) Statistical results of TEM, TCM (n = 6 animals per group), and CD69+ cells (n = 5 animals per group) in CD8+ T cells. (F) ELISpot assays were conducted for IFN-γ (F, top) and IL-4 (F, bottom) secretion in splenocytes. (G) Statistical results of IFN-γ and IL-4 secreting cells (n = 6 animals per group). (H, I) Splenocytes were stimulated with homologous His-tagged HA for 18 h, Golgi Stop and Golgi Plug were added 6 h before the end of the stimulation. The percentages of IFN-γ+, IL-4+, and TNF-α+ in CD4+ T cells (H) and IFN-γ+, IL-2+, and TNF-α+ in CD8+ T cells (I) were determined by ICCS (n = 6 animals per group). Data information: Data in (B, E, G, H, I) are presented as mean ± SD. Statistical significance was calculated by one-way ANOVA with…

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  Figure 5. HA@PPCDQ induces germinal center responses. (A) Representative flow cytometric plots of HA-specific GC B cells (CD95+GL7+HA+). (B, C) Statistical results of GC B cells (CD95+GL7+) and HA-specific GC B cells (n = 5 animals per group). (D) The percentages of HA-specific GC Tfh cells (CD25+OX40+) were determined by AIM assay. (E, F) Statistical results of GC Tfh cells (CXCR5+PD1+) and HA-specific GC Tfh cells (n = 5 animals per group). (G) Representative flow cytometric plots of HA-specific switched memory B cells (MBC, IgD-CD38+HA+). (H, I) Statistical results of switched MBCs (B220+IgD-CD38+) and HA-specific switched MBC (n = 5 animals per group). (J) Cryosections of inguinal lymph nodes were incubated with a germinal center staining cocktail containing the following antibodies: Brilliant Violet 421™ anti-mouse/human CD45R/B220, Alexa Fluor® 594 Goat anti-mouse IgG and Alexa Fluor® 488 anti-mouse/human GL7. Scalar bar:1000 μm. (K) HA-specific ASCs were analyzed by ELISpot. (L) The number of GCs calculated according to the quantity of GL7 positive cell clusters (n = 6 animals per group). (M, N) The number of ASCs in lymph node (M) and bone marrow (N) (n = 6 animals per group). (O)…