A vaccine targeting antigen-presenting cells through CD40 induces protective immunity against Nipah disease

Abstract

Nipah virus (NiV) has been recently ranked by the World Health Organization as being among the top eight emerging pathogens likely to cause major epidemics, whereas no therapeutics or vaccines have yet been approved. We report a method to deliver immunogenic epitopes from NiV through the targeting of the CD40 receptor of antigen-presenting cells by fusing a selected humanized anti-CD40 monoclonal antibody to the Nipah glycoprotein with conserved NiV fusion and nucleocapsid peptides. In the African green monkey model, CD40.NiV induces specific immunoglobulin A (IgA) and IgG as well as cross-neutralizing responses against circulating NiV strains and Hendra virus and T cell responses. Challenge experiments using a NiV-B strain demonstrate the high protective efficacy of the vaccine, with all vaccinated animals surviving and showing no significant clinical signs or virus replication, suggesting that the CD40.NiV vaccine conferred sterilizing immunity. Overall, results obtained with the CD40.NiV vaccine are highly promising in terms of the breadth and efficacy against NiV.

Keywords: CD40-targeting vaccine; Henipavirus; Nipah virus; cross-neutralizing responses; dendritic cell-based vaccine; epitope mapping; germinal center reaction; neutralizing antibodies; prophylactic vaccine; sterilizing immunity.

Graphical abstract

Figure 1

Epitope mapping of NiV antigens associated with the CD40 mAb (A) Open reading frames encoding the G, F, and N proteins in the NiV (Bangladesh strain) genome are highlighted in blue, orange, and red, respectively (center). Full-length amino acid sequences were screened for predicted HLA-I (NetMHC 4.0) HLA-II (NetMHCII 2.3), and linear B cell epitopes (BepiPred 2.0). For HLA-I and -II, the color intensity is representative of the density of predicted epitopes, with coverage of at least 30% of the worldwide HLA. (Top) Ectodomain (ECD) of the G protein and (bottom) F and N down-selected domains associated with the CD40.NiV vaccine. (B) Amino acid conservation of vaccine antigens.

Figure 2

CD40.NiV induces NiV neutralizing IgG and polyepitopic cellular T cell responses in hCD40Tg mice (A) Schematic representation of the study design, comparing responses to CD40.NiV to the non-targeted NiV G ECD (with poly-ICLC). Blood samplings are represented by diamonds. (B) IgG titers specific to NiV G ECD were determined by Luminex technology on days 0, 21, and 28 post-immunization with either CD40.NiV (red) or non-targeted NiV G ECD protein (blue), with poly-ICLC. The mean fluorescence intensity (MFI) (±SEM) is represented. Non-parametric Kruskal-Wallis tests with Dunn’s multiple comparison post hoc test, ∗p < 0.05, ∗∗p < 0.01, ns non-significant. (C) Avidity of NiV G-specific IgG measured by Luminex on day 21 (3 weeks post-prime) and 28 (1 week post-boost), comparing mice immunized with non-targeted NiV G versus CD40.NiV, with poly-ICLC. The color legends and statistical analyses are the same as in (B). (D) Neutralization activity of sera from mice immunized with NiV G ECD versus CD40.NiV (with poly-ICLC) measured first by a Luminex-based inhibition assay. The color legends and statistical analysis are the same as in (B). (E) In vitro neutralization assay performed on NiV-B-infected cells using serial-diluted sera collected on day 28 post-immunization. The IC₅₀ values were calculated and are reported as titers (log(1/IC₅₀)). The dotted line indicates the limit of quantification (LOQ) for the neutralization titer. Non-parametric Mann-Whitney unpaired t tests; ∗p < 0.05. (F) IFN-γ T cell responses to the NiV G, F, and N overlapping peptide pools assessed from spleens by ELISpot 1 week post-boost. The total number of spots are reported per million splenocytes (background subtracted). NiV G (blue), N (green), and F (orange). Data are representative of at least two experiments.

Figure 3

Cellular and humoral responses of AGMs vaccinated with CD40.NiV (A) Schematic representation of the study design. Diamonds are representative of sampling. (B) Blood NiV G-specific IgG (left) and IgA (right) titers, in poly-ICLC- (black) and CD40.NiV (+poly-ICLC)-vaccinated AGMs (red) on days 0, 10, 21, 35, and 56 post-prime. Individual Ig titers (log(1/EC₅₀)) are shown. The whiskers indicate the medians (min-max) for each group and at each time point. The prime and boost are indicated by arrows. The dotted line indicates the LOQ for the Ig titer. Non-parametric Kruskal-Wallis tests with Dunn's multiple comparison post hoc test; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, non-significant. (C) In vitro NiV-B neutralization activity of AGM sera measured post-immunization and post-challenge (80 days post-infection [dpi]): black line, poly-ICLC group (n = 3); red line, CD40.NiV with poly-ICLC (n = 9); gray line, modeling of the decrease in neutralizing Abs after the peak of the response (day 35). (D) Cross-neutralization was assessed with serial dilutions of five AGM sera collected 21 and 35 dpi added to cells infected with VSVΔG-NiVF/G harboring the NiV G and F proteins, either from the Bangladesh (plain circles), Malaysia (open circles), or Cambodia (open diamonds) strain or from the Hendra virus (plain triangles). Results are reported as the log of the IC₅₀. Dotted line: LOQ given by sera from the poly-ICLC group of AGMs. GenBank references of the NiV G and F proteins expressed by rVSV and homology of the amino acid sequence are detailed in Table S1. (E) In vitro HeV neutralization activity of AGM sera measured 2 weeks post-boost (35 dpi): black dots, poly-ICLC group (n = 3); red dots, CD40.NiV with poly-ICLC (n = 9). Mann-Whitney unpaired t test, ∗∗p < 0.01. (F) Total specific IFN-γ responses to the NiV G, F, and N antigens assessed in PBMCs on days 21 and 35 post-immunization, as in Figure 2 for the spleen. The number of spots is reported per million PBMCs (background subtracted). The dotted line represents the threshold of positivity, defined by the mean response (±3 SD) of the non-vaccinated AGM group. Non-parametric Kruskal-Wallis tests with Dunn’s multiple comparison post hoc test; ∗p < 0.05; ns non-significant.

Figure 4

Protection assay in AGMs (A) Survival curve. Nine AGMs were immunized twice with CD40.NiV and challenged with 10² PFUs of NiV-B (intratracheal route) (red line). Eight naive animals were used as controls (black dotted line). Gehan-Breslow-Wilcoxon test, ∗∗∗p < 0.001. (B) Clinical scores and temperatures of naive (black) and vaccinated AGMs (red). Gray dashed line score threshold for ethical considerations (left); median temperature at day 0 (right). (C) Hematoxylin and eosin staining of collected tissues from the spleen, lungs, and brain (frontal cortex) at necropsy. Representative staining of naive AGMs is shown in (a) and (b) (follicular degeneration and depletion in spleen) and (d) and (e) (interstitial pneumonia with alveolar edema and syncytial formation in the lungs) and of vaccinated AGMs in (c) (follicular hyperplasia in the spleen) and (f) (normal lung tissue). No obvious changes were observed in the brain (frontal cortex).

Figure 5

Viral dissemination post-challenge (A) (Left) Amount of viral NiV RNA quantified by RT-PCR (N gene) in PBLs. Values were normalized against those of the GADPH housekeeping gene. Genomic RNA was measured by RT-qPCR in nasal (middle) and pharyngeal (right) swabs. Mean values (±SEM) are presented for the non-vaccinated (black, n = 7) versus CD40.NiV-vaccinated (red, n = 9) groups. (B) Same as in (A) for fluids collected at necropsy. (C) Representative panels of immunofluorescent staining of NiV N protein (red) and DAPI (blue) in the lung, spleen, and brain tissues collected from the poly-ICLC versus CD40.NiV (+poly-ICLC) groups.

Figure 6

Myeloid and lymphoid populations post-challenge (A) Percentage of CD20⁺ B cells among live cells determined by flow cytometry for non-immunized (black, n = 5) versus vaccinated AGMs (red, n = 9). Dotted line: median percentage of all animals at day 0. For each time point, percentages are compared to the initial amount in each animal group. Friedman paired t test, ∗∗∗p < 0.001. (B and C) As in (A), percentage of CD3⁺ T cells (B) with evolution of CD4/CD8 T cell ratios (C). (D) CD14⁺ monocytes exhibiting inflammatory (CD14^low CD16⁺, solid), intermediate (CD14⁺ CD16^hi, square), and classical (CD14^hi, CD16⁻, dashed) phenotypes in non-vaccinated (n = 3, black) versus CD40.NiV AGMs (n = 9, red).

Figure 7

Characterization of gene expression profiles induced after prime-boost vaccinations (A) Principal-component analysis based on the full transcriptomic profile of vaccinated AGMs sampled at day 0 (before vaccination) (yellow); day 1 after prime vaccination (purple); 1 day before the boost, corresponding to day 21 (orange); and 1 day after the boost (day 22) (sky blue). (B) Venn diagram of differential expression of genes (DEGs) comparing VAC day 1 to 0, 22 to 21, and 22 to 0 (adjusted p value ≤ 0.05, absolute log2 fold change [FC] ≥ 0.58). (C) Volcano plot of DEGs observed between VAC day 22 and 0. The down- (190) and upregulated (583) genes are shown in blue and red, respectively. The top 35 most upregulated and down-regulated genes based on the log2FC are depicted. (D) Gene set enrichment analysis was performed on the DEGs with an adjusted p value ≤ 0.05 between VAC day 22 and 0 using the ClusterProfiler v.4.4.4 R package. The density plot shows the top 25 pathways based on the adjusted p value and normalized enrichment score. (E) The highest ranked network of DEGs (adjusted p value ≤ 0.05, absolute log2FC ≥ 0.58) between VAC days 22 and 0 was obtained from the protein-protein interaction (PPI) network using the MCODE plug-in of Cytoscape. The DEGs in the network are colored based on the log2FC values. (F) Heatmap of gene expression belonging to the MCODE network at days 0 (green), 1 (salmon), 21 (blue), and 22 (purple). Gene expression levels in the heatmap are shown as Z scores. Significantly differentially expressed genes in post-boost versus post-prime are indicated by a square.