Abstract

   Using nanoparticles (NPs) as a platform for multivalent antigen display
   is an effective strategy to increase the immunogenicity of subunit
   vaccines, which can induce high levels of humoral and cellular
   immunity. In addition, antigens that target antigen-presenting cells
   (APCs) can further increase their immunogenicity. To date, there are no
   commercially available ASFV vaccines available worldwide. The present
   study developed a dendritic cell (DC)-targeting ASFV biomimetic
   nanovaccine. First, a high-affinity and specific nanobody (Nb)
   targeting DCs was screened and expressed in tandem with B and T-cell
   epitopes of highly immunogenic p30, p54, p72, pB602L, and CD2V proteins
   of ASFV (Nb-rAg). The Nb-rAg complexes were then loaded onto
   azabisphosphonate-terminated phosphorus dendrimers (PPHs) to construct
   PPH-Nb-rAg NPs, which were subsequently coated with ASFV-infected
   activated porcine alveolar macrophage (PAM) membranes to prepare the
   PPH-Nb-rAg@PM biomimetic nanovaccine. Finally, the immune efficacy of
   the nanovaccine was evaluated in mice. Notably, compared with the PBS,
   rAg, Nb-rAg, and PPH-Nb-rAg immunization groups, the PPH-Nb-rAg@PM
   immunization group exhibited stronger ASFV antigen-specific humoral and
   cellular immune responses. Single-cell RNA sequencing (scRNA-seq)
   revealed that immunization with PPH-Nb-rAg@PM increased the proportions
   of B cells, T cells, NK cells, plasma cells, and macrophages in the
   mouse spleen. Further analysis revealed that PPH-Nb-rAg@PM immunization
   increased the numbers of memory B cells and plasma cells in the mouse
   spleen, and the numbers of CD4 + T cells, CD8 + T cells and NK cells
   also increased compared with those in the control group. These results
   suggest that PPH-Nb-rAg@PM is a promising and effective candidate
   vaccine against ASFV.

Graphical abstract

   [40]graphic file with name 12951_2025_3593_Figa_HTML.jpg

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12951-025-03593-7.

   Keywords: African swine fever, Multiepitope nanovaccine, B-cell
   response, T-cell response

Introduction

   African swine fever (ASF) is caused by the ASF virus (ASFV), and its
   clinical syndrome is characterized by rapid onset and symptoms such as
   high fever, dyspnea, and extensive hemorrhaging of the skin and various
   internal organs in pigs [[41]1, [42]2]. Since August 2018, ASF has
   spread throughout China and other adjacent Asian countries, causing
   substantial losses to the pig industry in these regions [[43]3, [44]4].

   ASFV is an enveloped double-stranded DNA virus belonging to the
   Asfarviridae family, and its genome is approximately 170 ~ 193 kb and
   encodes 150 ~ 167 proteins [[45]5]. This virus is able to evade
   elimination by the host immune system effectively, and viral protein
   functions are mostly unknown, leading to considerable obstacles in the
   development of ASF vaccines. Most reported vaccines still have various
   problems and cannot or can only partially exert protective effects. For
   example, some natural or artificial attenuated vaccines or attenuated
   gene knockout vaccines may have obvious side effects or provide
   protection only against specific ASFV strains, whereas inactivated
   vaccines cannot effectively protect experimental pigs from virulent
   strain infection [[46]6–[47]9]. In addition, subunit vaccines and DNA
   vaccines also cannot effectively protect pigs from infection
   [[48]10–[49]12]. There is no commercially available ASF vaccine thus
   far.

   Owing to the complexity of the structure and function of the ASFV
   protein, screening protective antigens suitable for application in
   subunit vaccine development is difficult. Nevertheless, some viral
   proteins can induce neutralizing antibodies and have certain viral
   neutralizing activities, providing the possibility for the development
   of new types of vaccines. The ASFV p54 and p30 proteins can induce
   neutralizing antibodies in pigs, but immunized pigs are not fully
   protected against lethal infections [[50]13]. In addition to inducing
   neutralizing antibodies, p72 and p30 can also activate cytotoxic T
   lymphocyte responses [[51]11]. Other ASFV envelope or membrane
   proteins, such as CD2v, p12, and D117L, may also induce neutralizing
   antibodies and provide serotype-specific cross-protective immunity
   [[52]14, [53]15]. Oura et al. confirmed that the clearance of CD8 + T
   cells led to complete loss of resistance in pigs to infection with the
   virulent ASFV OUR/T88/1 strain [[54]16], indicating that relying solely
   on humoral immunity cannot provide effective immune protection against
   ASFV infection and that cellular immunity also plays an important role
   in immune protection, emphasizing that T-cell epitopes are necessary
   for the development of ASF vaccines.

   Dendritic cells (DCs) serve as a bridge between innate and adaptive
   immunity and are critical in initiating immune responses and immune
   regulation [[55]17]. Immature DCs continuously capture pathogens such
   as viruses and bacteria to activate themselves into mature DCs and
   migrate to lymph nodes, where they act as antigen-presenting cells
   (APCs) to present antigens and nonantigen-specific costimulatory
   signals from pathogens to T cells to activate helper T cells, killing T
   cells and B cells [[56]18]. Therefore, DCs are usually utilized as
   antigen targets for the induction of efficient and strong immune
   responses.

   For the design of DC-targeting vaccines, the practical application of
   traditional DC receptor-specific monoclonal antibodies (McAbs)
   [[57]19–[58]23], ligands [[59]24], and single-chain variable fragments
   [[60]25] is greatly restricted because of their enormous molecular
   weight, complex structure and high cost. As an alternative, the heavy
   chain variable region, also known as the nanobody (Nb), is isolated
   from camelids, providing another complete, new and much smaller
   DC-targeting vehicle. Compared with traditional antibodies, Nbs have
   many unique advantages, such as a smaller molecular weight (~ 15 vs.
   150 kDa), smaller size (~ 4 vs. 14.2 nm), high affinity, ease of
   modification and engineering, and low immunogenicity [[61]26, [62]27],
   making them ideal antigen delivery vehicles for DC targeting. XC motif
   chemokine receptor 1 (XCR1) is selectively expressed on CD8 + DCs with
   cross-antigen presentation ability [[63]28], enhances specific
   cytotoxic T cells [[64]29], and is highly conserved across different
   species, making XCR1 + DC targeting a new strategy for vaccine
   development. The present study aimed to screen porcine XCR1
   (pXCR1)-specific Nbs, and a novel ASFV antigen delivery system based on
   the screened Nbs was constructed to evaluate their immune potential.

   Dendritic macromolecules have precise molecular structures, perfect
   spatial geometric symmetry, many surface functional groups and internal
   cavities, and controllable nanoscale and highly branched
   three-dimensional structures. These characteristics make dendritic
   macromolecules important carriers for delivering drugs, genes, and
   diagnostic reagents. The major advantages of these nonviral delivery
   systems over traditional viral vectors can be highlighted in their
   simplicity of synthesis, strong stability, tunable modifications, and
   biosafety profile [[65]30]. Azabis phosphonate-terminated phosphorus
   dendrimers (PPHs) with iminodi (methylphosphate) terminals can serve as
   nanocarriers to load drugs and have anti-inflammatory activity, which
   can activate the immune system and induce natural killer (NK) cell
   proliferation; the latter plays an important role in immune regulation
   in anticancer and anti-infection activities [[66]31]. In addition, PPHs
   with iminodi (methylphosphate) termini have been proven to be a
   universal protein delivery system since they possess terminal phosphate
   anionic groups and a benzene ring-rich branched scaffold structure and
   can complex proteins through electrostatic interactions, hydrogen
   bonding, cation-Π interactions, and hydrophobic interactions [[67]32].

   As an appealing emerging cell-mimicking platform, cell membrane-coated
   biomimetic nanovaccines may have better performance in the face of ASFV
   challenge. Cell membrane-coated biomimetic nanoparticles (NPs) can
   perform multiple functions, such as targeted recognition and
   immunomodulation, which are attributed to their inherent expression of
   diverse pathogen-associated receptors (PRRs) on derived cell membranes
   [[68]33, [69]34]. Additionally, nanomedicines can be enhanced after
   they are camouflaged with preactivated macrophage membranes (PMs)
   because of their immune escape ability and prolonged circulation, which
   results in less clearance by the mononuclear phagocyte system [[70]35,
   [71]36]. For example, a PM-camouflaged nanoplatform consisting of
   branched polyethylenimine, short hairpin RNA targeting Ptpn2 and
   doxorubicin can reduce phagocytosis by macrophages and significantly
   prolong the circulation time in vivo, thus enhancing the antitumor
   activity of NPs [[72]37]. However, no biomimetic NPs have been applied
   for the development of ASF vaccines.

   Porcine alveolar macrophages (PAMs), the main target cells after ASFV
   infection in pigs in vivo, and one of the pivotal members, as the first
   line defence against invaded viruses, may abundantly express specific
   receptor(s) that target ASFV when infected by ASFV. ASFV infection can
   also activate many types of PRRs (such as RIG-I, MDA5 and LGP2) of the
   Toll-like receptor (TLR) pathway [[73]38], which can recognize specific
   pathogen-associated molecular patterns (PAMPs), activate innate
   immunity and exert anti-viral effects [[74]39]. Therefore, we
   hypothesized that ASFV-infected PAM membranes may likewise express high
   levels of ASFV-relevant TLRs on their membrane surface and that
   biomimetic NPs coated with these PMs may have longer retention times
   than traditional vaccines do.

   This study developed a DC-targeting ASFV biomimetic nanovaccine. First,
   ASFV B-cell epitopes that can induce neutralizing antibodies and T-cell
   epitopes that can induce a cellular immune response were screened and
   combined with a recombinant antigen (rAg); phage display technology was
   used to screen specific Nbs against the DC surface ligand XCR1, and the
   Nbs and rAg genes were tandemly expressed to prepare Nb-rAg; and Nb-rAg
   was coupled with PPH phosphorus dendrimers to prepare PPH-Nb-rAg NPs,
   which were then coated with ASFV-infected PAM membranes to prepare the
   PPH-Nb-rAg@PM biomimetic nanovaccine. Finally, the immune efficacy of
   the nanovaccine was evaluated in mice. The advantages of this new
   vaccine are as follows: it utilizes phosphate terminal dendritic
   molecules coupled with Nb-rAg targeted to DCs and disguises them with
   preactivated membranes to construct NPs. Simple ingredients and longer
   circulation times in vivo. Compared with ordinary vaccines, the
   different components of NPs can activate NK cell proliferation,
   inducing more durable, stronger cellular and humoral immune responses.

Materials and methods

Screening, identification and recombination of immune-dominant B and T-cell
epitopes

   To optimize the immune characteristics of the vaccine as much as
   possible and improve the antibody level and activity after
   immunization, neutralizing active epitopes in ASFV proteins were
   carefully screened according to previous reports. These epitopes were
   distributed mainly in the p30, p54, p72, pB602L, and CD2V proteins of
   ASFV. These preliminarily screened epitopes were then artificially
   synthesized and further identified for their reactivity with
   inactivated ASFV-antibody-positive pig serum to determine B-cell
   epitopes via dot-ELISA. Briefly, the synthesized epitope peptide was
   dissolved in phosphate-buffered saline (PBS) and coated onto an ELISA
   plate (2 µg/well) at 4°C overnight. Then, the peptide coating solution
   was discarded, and the samples were blocked with 200 µl/well 2.5% dry
   milk for 2 h at 37°C. After washing with PBST 3 times, each peptide was
   incubated with 10 portions of inactivated pig anti-ASFV
   antibody-positive serum (1:10 dilution) for 1 h at 37°C. The plates
   were washed 3 times with PBST and then incubated with horseradish
   peroxidase (HRP)-conjugated goat anti-swine IgG (H&L) (1:5,000). After
   washing with PBST 3 times, 100 µl/well 3,3’,5,5’-tetramethylbenzidine
   (TMB) was added, and the mixture was incubated for 15 min in the dark.
   Finally, 3 M H[2]SO[4] (50 µl/well) was added to stop the colorimetric
   reaction, and the optical density (OD) at 450 nm was read via an
   automated ELISA plate reader (BioTek; Agilent Technologies, Inc., USA).

   In accordance with previous reports, T-cell epitope peptides were
   synthesized to evaluate their ability to activate splenocyte activation
   and subsequent IFN-γ secretion. Briefly, 6-week-old mice were immunized
   with ASFV p30, p72, or CD2v recombinant protein. At 7 d after
   immunization, the spleens were dissected, and the splenocytes were
   isolated, counted, and seeded into a 96-well plate at a density of
   1 × 10^6/ml per well. Then, 10 µg of epitope peptide dissolved in PBS
   was added to each well, and the plate was cultured at 37 °C in a 5%
   CO[2] incubator for 60 h. Cell proliferation was measured via the Cell
   Counting Kit-8 (CCK-8) assay. Then, the cells were plated and treated
   as described above, and an ELISpot assay was performed to analyze the
   proportion of IFN-γ-positive splenocytes. Non-immunized healthy mouse
   splenocytes were used as the negative control.

Preparation of pXCR1-specific Nb and Nb-rAg recombinant proteins

   The pXCR1 gene was synthesized, cloned, and inserted into the pET-30a
   expression vector. After that, the pET-30a-pXCR1-His plasmid was
   transformed into E. coli BL21 (DE3) competent cells, which were induced
   with 1.0 mM IPTG to express the pXCR1 protein. After purification with
   a nickel column, the pXCR1 protein was used to immunize adult male
   bactrian camel at a dose of 5.0 mg each time at 2-week intervals.

   After the 5 immunizations, the pXCR1 protein antibody titer in the
   camel serum was tested via indirect ELISA (iELISA). Then, whole blood
   was collected, peripheral blood lymphocytes (PBMCs) were isolated, and
   total RNA from PBMCs was extracted and reverse transcribed into cDNA,
   followed by amplification of the VHH gene by 2 rounds of nested PCR and
   cloning of the VHH gene into the phagemid display vector pCANTAB-5E
   through the Pst I and Not I restriction sites (NEB, Ipswich, MA, USA)
   and electronical transformation into fresh E. coli TG1 competent cells
   to construct the VHH phage display library. The M13K07 helper phage was
   utilized to rescue the VHH phage library, and anti-pXCR1-specific Nb(s)
   were screened by 4 rounds of biopanning via phage display technology.
   The specific phage particles were significantly enriched after 4 rounds
   of biopanning. Seventy-two individual colonies were randomly selected
   for sequencing and classification according to sequence diversity in
   the third complementarity-determining region (CDR3). The specificity
   and affinity of the Nb(s) screened were subsequently evaluated via
   anti-M13/HRP-conjugated phage ELISA.

   The gene corresponding to the Nb with the highest affinity for the
   pXCR1 protein was coupled with the optimized ASFV rAg gene by a
   flexible linker (hereafter abbreviated as Nb-rAg), and the Nb-rAg gene
   fragment was cloned and inserted into the pET-30a plasmid and
   transformed into E. coli BL 21(DE3) competent cells to induce the
   expression of the Nb-rAg protein. The Nb-rAg protein was purified with
   a nickel column for subsequent experiments.

Preparation of iminodi(methylphosphate)-capped phosphorus dendrimers (PPHs)
and PPH-Nb-rAg

   In accordance with previous reports, third-generation iminodi
   (methylphosphate) terminal dendrimer molecules were synthesized
   [[75]40]. The PPHs and Nb-rAg proteins were resolved in sterile water
   separately, and then the PPH was added dropwise into the Nb-rAg
   solution at mass ratios of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, and 8:1 to
   determine the optimal mass ratio of the two. The solutions were stirred
   at room temperature (RT) for 24 h until they turned light white. The
   mixtures were subsequently centrifuged at 15,000 × g at RT for 20 min
   to separate the solid and liquid phases, and the supernatant and
   precipitate were harvested separately. The concentration of residual
   Nb-rAg in the supernatant was assayed via a bicinchoninic acid (BCA)
   assay kit to calculate the encapsulation efficiency (EE) and loading
   capacity (LC) of the PPH-Nb-rAg NPs. The precipitate was subsequently
   resuspended in sterile water and stored at 4 °C until further use.

   The EE of Nb-rAg by the PPH-Nb-rAg NPs was calculated via the following
   formula: EE (%) = (initial Nb-rAg amount - unencapsulated free
   Nb-rAg)/initial Nb-rAg amount. The LC of Nb-rAg by the PPH-Nb-rAg NPs
   was calculated via the following formula: LC (%) = (initial Nb-rAg
   amount - unencapsulated free Nb-rAg)/total amount of PPH-Nb-rAg NPs.

Preparation of the PPH-Nb-rAg@PM NPs

   The ASFV (0.01 MOI) and PAMs were cocultured at 37 °C for 12 h. After
   washing 3 times with PBS, 2 ml of ice-cold Tris-MgCl[2] buffer (pH 7.4,
   0.01 M Tris, 0.001 M MgCl[2]) supplemented with 1% PMSF (v/v, to
   prevent protein degradation) was added, and the cells were collected
   via a cell scraper. An ultrasonic disruptor was used for ultrasonic
   lysis to obtain a cell homogenate. Then, 1 M sucrose solution was added
   to the cell homogenate to achieve a final concentration of 0.25 M,
   followed by centrifugation at 2,000 × g for 10 min at 4 °C. The
   supernatant was collected and centrifuged at 14,000 × g for 30 min at
   4 °C, and the milky precipitates were collected. The final precipitates
   were PMs, which were stored at 4 °C for further use.

   The PPH-Nb-rAg NPs were mixed with PMs at a mass ratio of 1:1 and then
   coextruded through a 200 nm porous polycarbonate membrane for 20 cycles
   to form biomimetic PPH-Nb-rAg@PM NPs. PPH-Nb-rAg@M NPs were prepared
   simultaneously, where M or PM denoted the ordinary or preactivated
   membrane.

Characterization of the membrane-encapsulated biomimetic PPH-Nb-rAg@PM NPs

   The size distributions and surface potentials of the PPH-Nb-rAg@M and
   PPH-Nb-rAg@PM NPs were analyzed via dynamic light scattering (DLS) with
   Malvern Zetasizer Nano ZS ZEN3600 equipment (Malvern, Worcestershire,
   U.K.).

   The morphology of the prepared PPH-Nb-rAg NPs was observed via
   field-emission scanning electron microscopy (FE-SEM, SU8100, Hitachi,
   Tokyo, Japan) at a voltage of 3.0 kV and transmission electron
   microscopy (TEM, Hitachi H7800, Tokyo, Japan) at an accelerating
   voltage of 120.0 kV. The morphology of the prepared PPH-Nb-rAg@PM NPs
   was also observed via TEM. For TEM imaging, the PPH-Nb-rAg,
   PPH-Nb-rAg@M or PPH-Nb-rAg@PM NPs in an aqueous solution (2 mg/ml,
   5 µl) were deposited onto a carbon-coated copper grid and air-dried
   before measurement. The particle size distribution was analyzed with
   ImageJ software ([76]http://rsb.info.nih.gov/ij/download.html). For
   size distribution analysis, at least 200 particles were randomly
   selected from different SEM images to obtain the size distribution
   histogram. For the stability analysis, the PPH-Nb-rAg@PM NPs were
   resuspended in 10% FBS + DMEM or saline at a final concentration of
   1.0 mg/ml. The particle size was measured every 3 d for 15 d, and each
   measurement was repeated three times.

   The protein components of the PPH, Nb-rAg, PPH-Nb-rAg, PPH-Nb-rAg@M
   NPs, PPH-Nb-rAg@PM NPs and preactivated macrophage lysates were
   determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
   (SDS-PAGE) analysis. Different samples with equal amounts of protein
   (30 µg/sample) were assayed via a BCA assay kit (except for the PPH
   that did not carry proteins), mixed with 5 × SDS loading buffer and
   boiled for 5 min at 95 °C. The samples were subsequently analyzed via
   SDS-PAGE. The expression of TLR2, TLR4, and TLR6 in the PPH,
   PPH-Nb-rAg@M, and PPH-Nb-rAg@PM NP groups was detected via western
   blotting.

In vitro cytotoxicity and cellular uptake assay

   To evaluate the cytotoxicity of the PPH-Nb-rAg@PM NPs, mouse monocyte
   macrophage leukemia cells (RAW264.7) and mouse dendritic cells (DC2.4)
   were plated in 96-well plates at a density of 1 × 10^4 cells/well.
   Approximately 24 h later, the cells were incubated with 100 µl of DMEM
   containing 0, 30, 60, 120, or 240 µg/ml rAg, PPH, Nb-rAg, PPH-Nb-rAg,
   or PPH-Nb-rAg@PM NPs for 24 h. Then, cell viability was determined via
   a CCK-8 kit (Beyotime Biotechnology, Shanghai, China) following the
   manufacturer’s instructions. The cell viability was calculated on the
   basis of the OD values at 450 nm. For each sample, 5 parallel wells
   were treated and analyzed to calculate the mean ± SD.

   To evaluate the cellular uptake efficiency of the PPH-Nb-rAg@PM NPs by
   the main APCs, RAW264.7 and DC2.4 cells were seeded into 24-well plates
   at a density of 5 × 10^4 cells/well. Approximately 24 h later, the
   cells were incubated with 50 µg of rAg, Nb-rAg, PPH-Nb-rAg, or
   PPH-Nb-rAg@PM NPs for another 24 h. Then, the old culture medium was
   discarded, and the cells were fixed at -20 °C in precooled 70% alcohol
   at 4°C for 30 min. After washing with PBS 3 times, the cells were
   incubated with inactivated ASFV-antibody-positive pig serum at a
   dilution of 1:400 for 1 h at RT, followed by staining with Alexa Fluor
   488-conjugated goat anti-swine IgG (H&L) at a dilution of 1:500 for 1 h
   at RT in the dark. The cell nuclei were stained with DAPI (Beyotime
   Biotechnology) for 5 min. Images were acquired via fluorescence
   microscopy (Olympus Corporation, Tokyo, Japan).

In vivo biodistribution of Cy5.5-labeled PPH-Nb-rAg@PM NPs

   rAg, Nb-rAg, PPH-Nb-rAg, and PPH-Nb-rAg@PM NPs were labeled with the
   fluorescent probe Cy5.5. Six-week-old female BALB/c mice were randomly
   divided into 4 groups, with 3 mice in each group. Two hundred
   microliters of PBS containing Cy5.5-rAg, Cy5.5-Nb-rAg,
   PPH-Cy5.5-Nb-rAg, or PPH-Cy5.5-Nb-rAg@PM was injected through the tail
   vein (injection dosage: Cy5.5-rAg = 1.5 mg/kg). In vivo fluorescence
   imaging of the mice was performed at 4, 8, 12, 24 and 48 h
   postinjection via a Live Imaging System IVIS Spectrum CT (PerkinElmer,
   Waltham, USA) with an excitation filter at 625 nm and an emission
   filter at 680 nm.

   The mice were sacrificed at 48 h postinjection, the liver, spleen,
   lung, kidney and lymph nodes of each group were isolated separately,
   and the fluorescence distribution in each organ was detected via an in
   vivo imaging system.

Mouse immunization

   Six-week-old female BALB/c mice were randomly divided into 5 groups
   (n = 8 for each group) and immunized subcutaneously with 200 µl of PBS
   containing rAg, Nb-rAg, PPH-Nb-rAg or PPH-Nb-rAg@PM NPs on d 0 and 14
   (rAg = 30 µg/mouse), and aluminum hydroxide and CPG-1826 were used as
   adjuvants. The control group received 200 µl of PBS. Three d prior to
   immunization, whole blood samples were collected to separate the serum
   as the negative control. Then, whole blood was collected from the tail
   vein at 28, 35, and 49 d after immunization, and the serum was isolated
   and stored at -80 °C for further analysis. At 28 d after immunization,
   some of the mice were euthanized with carbon dioxide in an appropriate
   euthanasia chamber. Spleens from mice in different immunization groups
   were collected under sterile conditions, and splenocytes were isolated
   for further experiments.

Serum IgG and its subtype or isotype antibody titer detection

   iELISA was employed to detect serum-specific IgG and its subtype or
   isotype antibody titers against ASFV rAg. Briefly, 400 ng/well ASFV rAg
   dissolved in PBS was coated in 96-well ELISA plates at 4 °C overnight.
   The plates were subsequently blocked with 2.5% dry milk in PBS for 2 h
   at RT. After being washed with PBST 3 times, the samples were incubated
   with serially diluted immunized mouse serum (100 µl/well) for 1 h at
   37 °C, followed by washing with PBST 3 times. Then, the samples were
   incubated with goat anti-mouse IgG (H&L), IgG1 (H&L), IgG2a (H&L),
   IgG2b (H&L), IgG2c (H&L), IgG3 (H&L), IgA or IgM antibodies
   (Proteintech, Wuhan, China) for 1 h at 37 °C and washed with PBST 3
   times. The following operations were performed as described previously
   for Dot-ELISA.

Flow cytometry (FCM) assay

   At 28 d postimmunization, the spleens of the mice in each group were
   isolated, and splenocyte suspensions were prepared. Before the
   experiments, the cells were incubated with 15 g/L EDTANa[2] solution
   for 5 min at 37 °C to prevent red blood cell contamination. The cells
   were stained with a fluorescence-labeled antibody against surface
   marker proteins and then subjected to FCM. For the NK cell
   proliferation assay, the splenocytes were stained with anti-CD3-APC and
   anti-CD49-FITC antibodies in the dark. For T-cell immune response
   analysis, the cells were stained with anti-CD3-APC and anti-CD4-FITC or
   anti-CD8-FITC antibodies. For DC maturation detection, the cells were
   stained with anti-CD11c-FITC, anti-CD80-APC, anti-CD86-APC, or
   anti-MHCII-APC antibodies. For analysis of the GC-B-cell population,
   the cells were stained with anti-CD19-FITC, anti-CD95-APC, and
   anti-GL7-Pacific blue antibodies. For analysis of plasma cells,
   anti-B220-FITC and anti-CD138-APC antibodies were used. For analysis of
   T follicular helper (Tfh) cells, anti-CD4-FITC, anti-PD-1-APC, and
   anti-CXCR5-PE antibodies were used. For analysis of effector memory T
   cells (T[EM]) and central memory T cells (T[CM]), anti-CD4-FITC,
   anti-CD44-PE, and anti-CD62L-APC antibodies were used. For analysis of
   memory B cells, anti-B220-PE, anti-CD38-FITC, anti-IgD-PE/Cy7,
   anti-GL7-Pacific Blue, and anti-IgG-APC antibodies were used. The
   single-cell suspensions were detected via CytExpert (Beckman Coulter,
   USA), and the data were analyzed via CytExpert software version 2.6.

ELISpot assay

   Antigen-specific splenocytes from immunized BALB/c mice were analyzed
   via mouse IFN-γ and IL-4 ELIspot kits (DAKEWE, Beijing, China).
   Briefly, splenocytes were isolated from each immunized group at 28 d
   postimmunization, and single-cell suspensions were prepared. Then, the
   cells were plated into 96-well commercial ELISpot plates at a density
   of 5 × 10^5 cells/well, and the plates were precoated with anti-IFN-γ
   or -IL-4 antibodies. Approximately 24 h later, the cells were
   restimulated with rAg at a final concentration of 10 µg/ml for 36 h,
   with 3 replicates for each sample. The plates were then cultured at
   37 °C in 5% CO[2] for 48 h. After incubation, the plates were incubated
   with a biotinylated IFN-γ detection antibody and a streptavidin-HRP
   conjugate. 3-Amino-9-ethylcarbazole (AEC) substrate solutions were
   added and incubated for 20 min, followed by rinsing with ddH[2]O and
   drying. The number of IFN-γ- or IL-4-positive T cells was calculated
   via a CTL-ImmunoSpot^®S6 cell reader/ImmunoSpot 7.0.15.1 software
   (Cellular Technology Limited, Shaker Heights, OH, USA) and normalized
   to the number of spots/10^6 splenocytes.

Lymphocyte proliferation assay

   At 28 d after immunization, spleen lymphocytes from the mice in each
   immunized group were collected and plated in 96-well plates at a
   density of 1 × 10^6 cells/ml (100 µl/well). After 24 h, the cells were
   restimulated with ASFV rAg at a final concentration of 10 µg/ml for
   72 h at 37 °C in 5% CO[2]. Then, 10 µl of CCK-8 was added to each well,
   and the mixture was cultured at 37 °C in 5% CO[2] for 3 h. The
   proliferation of lymphocytes was evaluated via a CCK-8 assay. The cell
   proliferation rate was represented as the stimulation index (SI). SI=
   (the mean of OD 450 nm values of the immunized lymphocyte wells)/(the
   mean of OD 450 nm values of unimmunized cells).

Statistical analysis

   To ensure that the results were reproducible, all the experiments were
   performed independently at least three times. Statistical significance
   was determined by Student’s t-test when two groups were compared or by
   one-way analysis of variance (ANOVA) when more than two groups were
   compared. A P-value of < 0.05 was considered statistically significant.

Results

Screening and identification of Nbs against pXCR1

   To develop an ASF nanovaccine targeting DCs, pXCR1-specific Nb(s) were
   screened. The present study first expressed and purified the pXCR1
   protein in prokaryotic cells. After the protein concentration was
   measured and immunized bactrian camel, whole blood was collected, and
   PBMCs were isolated. Total RNA was extracted and reverse transcribed
   into cDNA. Then, pXCR1-specific Nb(s) were screened via phage display
   technology (Fig. [77]1A). SDS-PAGE analysis revealed that the purified
   recombinant His-tagged pXCR1 protein was successfully obtained with the
   expected size of 38 kDa (Fig. [78]1B). The western blotting results
   indicated that the recombinant pXCR1 protein could specifically react
   with the anti-His McAb (Fig. [79]1C). The purified pXCR1 protein was
   used to immunize bactrian camel and as a coating antigen to screen
   specific Nbs.

Fig. 1.

   [80]Fig. 1
   [81]Open in a new tab

   Screening and identification of nanobodies against pXCR1 protein. (A)
   Schematic diagram of antigen-specific nanobody screening. (B) SDS-PAGE
   analysis of the purification of pXCR1 protein. (C) Western blotting
   identification of His-pXCR1 protein using mouse anti-His antibody as
   the primary antibody. (D) After 5 immunization, the serum antibody
   titers against pXCR1 protein. (E) Enrichment of the specific VHH phage
   particles against pXCR1 protein after 4 rounds of bio-panning. (F and
   G) ELISA analysis of periplasmic extracts reacted with pXCR1 protein
   from 72 clones. (H) Amino acid sequence alignment of isolated Nbs. CDR3
   variable domain was used to distinguish different Nbs. (I) ELISA was
   performed to detect the affinity of Nb6, Nb21 and Nb50 to pXCR1
   protein. (J) ELISA analysis of the reaction specificity of Nb6, Nb21
   and Nb50 with pXCR1. (K) Co-ip identification of Nb21 interacted with
   pXCR1 protein, Nb35 was used as a negative control. (L) Confocal
   analysis of screened Nb21 reactivity with pXCR1 protein. Images were
   obtained using a Zeiss LSM510 laser scanning inverted confocal
   microscope with a Zeiss 63X/1.4 NA oil immersion objective. Scale bar:
   10 μm

   The anti-serum titer of bactrian camel against the pXCR1 protein was
   1:256,000 after 5 immunizations according to the iELISA results
   (Fig. [82]1D), suggesting a good immune response of bactrian camel to
   the pXCR1 protein. Then, PBMCs were isolated from whole blood to
   extract total RNA, which was reverse transcribed into cDNA. After
   nested PCR amplification, double digestion, ligation, and
   transformation, a phage display VHH library was successfully
   constructed against the pXCR1 protein, consisting of approximately
   4.2 × 10^8 individual colonies with good diversity (data not shown).

   After 4 rounds of biopanning, the specific VHH phage particles against
   the pXCR1 protein were enriched (Fig. [83]1E). Then, 72 individual
   colonies were randomly selected from the products of the fourth round
   of biopanning, and periplasmic extracts were extracted for further
   iELISA. Among the 72 individual colonies, 41 specifically bind with the
   pXCR1 protein (Fig. [84]1F and G) and were subsequently sequenced.
   Finally, 3 different pXCR1-specific Nbs were successfully screened
   according to the amino acid sequence of the CDR3 hypervariable region
   and named pXCR1-Nb6, -Nb21, and -Nb50 (Fig. [85]1H). pXCR1-Nb21 showed
   the highest affinity (Fig. [86]1I) and good specificity (Fig. [87]1J)
   for the pXCR1 protein. Coimmunoprecipitation (co-IP) was performed to
   detect whether the prepared Nb21 could react with pXCR1. Co-IP revealed
   that Nb21, but not the control Nb35 against the ASFV p54 protein
   screened previously, pulled down pXCR1 (Fig. [88]1K). Confocal
   microscopy revealed that Nb21 effectively reacted with XCR1 in DC2.4
   cells (Fig. [89]1L). Therefore, Nb21 was selected as the delivery
   vehicle for ASFV antigens in the following experiments.

Screening and identification of ASFV dominant B- and T-cell epitopes

   41 B-cell epitopes and 11 T-cell epitopes of the p30, p54, p72, CD2v,
   and pB602L proteins were identified on the basis of previous studies in
   the NCBI database (Tables [90]S1 and [91]S2). To further screen the
   dominant B-cell epitopes used for the development of the ASFV candidate
   vaccine, these epitope peptides were synthesized and used as coated
   antigens, and inactivated positive sera from ASFV-recovered pigs were
   used as primary antibodies to perform Dot-ELISAs to detect their
   reactivity. The results revealed that the p30-B1, -B6, p54-B1, -B6,
   p72-B10, -B13, CD2v-B1, -B2, -B6, and pB602L-B3, -B7 peptides reacted
   well with the positive serum of ASFV-recovered pigs (OD450[nm] > 1.5)
   (Additional file 1: Figure [92]S1), and they were selected as the
   dominant B-cell epitopes for nanovaccine design.

   For T-cell epitopes, the identified T-cell epitope peptides were
   synthesized, and their ability to promote splenocyte proliferation was
   first tested. When the threshold was set to a cell proliferation rate
   greater than 30%, p30-T2, p72-T3, and CD2V-T2 cells met the
   requirements (Additional file 2: Figure [93]S2A). ELISpot analysis of
   T-cell epitopes stimulating IFN-γ secretion also revealed that p30-T2,
   p72-T3, and CD2V-T2 exhibited stronger stimulating effects (Additional
   file 2: Figure [94]S2B). Thus, these epitopes were selected as the
   dominant T-cell epitopes for ASFV candidate vaccine design. These
   selected epitopes are highly conserved between different type II ASFV
   strains (Additional file 3: Figure [95]S3) and are currently widely
   prevalent in China and Southeast Asian countries. In addition, the
   molecular simulation results revealed that these epitopes are
   distributed on the surface of the corresponding protein structure
   (Additional file 4: Figure [96]S4).

Transcriptome analysis of ASFV-infected PAM membranes

   To obtain ASFV-infected PAM membranes, ASFV was cocultured with PAMs
   for 12 h. Then, the changes in the gene transcriptome of ASFV-infected
   PAMs were analyzed via high-throughput transcriptome sequencing
   technology. The heatmap revealed that the host genes whose expression
   was upregulated after ASFV infection were strongly associated with
   chemokines, proinflammatory cytokines, and the antiviral immune
   response (Additional file 5: Figure [97]S5A). Gene Ontology (GO)
   analysis revealed that ASFV stimulation activated the immune response,
   pattern recognition receptors (PRRs), inflammatory response, and
   inflammation-related pathways (Additional file 5: Figure [98]S5B).
   Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
   analysis revealed that ASFV stimulation led to enrichment of the TLR
   signaling pathway, inflammation-related pathways and PRRs (Additional
   file 5: Figure [99]S5C).

   As the main PRRs expressed on the surface of macrophages and other
   immune cells, TLRs can recognize different PAMPs derived from exogenous
   bacteria, viruses, parasites, etc [[100]41]. Thus, TLR expression in
   PAMs infected with ASFV was detected via qPCR. qPCR revealed that the
   expression of TLR2, TLR4, and TLR6 mRNAs gradually increased with
   increasing stimulation time (Additional file 5: Figure [101]S5D-F).
   Western blotting revealed that the expression of TLR2, TLR4 and TLR6
   significantly increased (Additional file 5: Figure [102]S5G). An
   indirect fluorescence assay (IFA) also revealed that the expression of
   TLR2, TLR4 and TLR6 on both the surface and cytoplasm of PAMs also
   increased (Additional file 5: Figure [103]S5H).

Preparation and characterization of biomimetic PPH-Nb-rAg@PM NPs

   The third generation of azabisphosphonate-terminated sodium phosphite
   terminal dendrimers (PPHs) was synthesized via a divergent iterative
   method with hexachlorocyclotriphosphazene as the core on the basis of
   previous reports (Fig. [104]2A, Additional file 6: Figure [105]S6)
   [[106]40]. Then, prokaryotic expression, purification, and
   identification of the Nb-rAg protein were conducted. As shown in
   Fig. [107]2B, the purified Nb-rAg protein (~ 77 kDa) was successfully
   obtained, and it reacted well with inactivated ASFV-antibody-positive
   pig serum (Fig. [108]2C and D).

Fig. 2.

   [109]Fig. 2
   [110]Open in a new tab

   Characterization of PPH-Nb-rAg@PM NPs. (A) Chemical structure of the
   synthesized phosphorus dendrimers. (B) SDS-PAGE analysis of purified
   Nb-rAg protein. (C) ELISA analysis of the reactivity of rAg protein
   with 10 inactivated ASFV antibody-positive pig serums. (D) Western
   blotting analysis of rAg reactivity with inactivated ASFV
   antibody-positive pig serum. (E) Hydrodynamic size PPH, Nb-rAg,
   PPH-Nb-rAg and PPH-Nb-rAg@PM NPs (n = 3). (F) Zeta potential of PPH,
   Nb-rAg, PM, PPH-Nb-rAg, PPH-Nb-rAg@M and PPH-Nb-rAg@PM NPs (n = 3). (G)
   Representvie SEM image of PPH-Nb-rAg. Scale bar: 500 nM. (H) Size
   distribution of PPH-Nb-rAg NPs. (I) Representvie TEM image of
   PPH-Nb-rAg NPs. Scale bar: 200 nM. (J) Representvie TEM image of
   PPH-Nb-rAg@M NPs. Scale bar: 200 nM. (K) Representvie TEM image of
   PPH-Nb-rAg@M NPs. Scale bar: 200 nM. (L) SDS-PAGE analysis of the
   protein composition of the prepared PPH-Nb-rAg@M, PPH-Nb-rAg@PM NPs and
   pre-activated macrophage cell lysates. (M) Western blotting analysis of
   TLR2, TLR4, TLR6 expression on PPH-Nb-rAg@M, PPH-Nb-rAg@PM NPs. (N) DLS
   detection of size changes of PPH-Nb-rAg@PM NPs in PBS, 10% FBS+DMEM and
   0.9% saline (n = 3)

   In preliminary experiments, the optimal protein encapsulation
   efficiency (EE) and loading content (LC) of PPH were determined by
   using different mass ratios of PPH and Nb-rAg. When the mass ratio of
   PPH to Nb-rAg was 3:1 in sterile water for 24 h, the optimal EE
   (76.79%) and LC (34.28%) were obtained, as detected with a BCA assay
   kit (Table [111]S3). The hydrodynamic size of the PPH-Nb-rAg NPs was
   169.0 ± 5.6 mm, which was larger than that of the PPH and Nb-rAg alone,
   indicating the successful formation of the PPH-Nb-rAg NPs
   (Fig. [112]2E). Finally, the PMs were extracted and coated on the
   surface of PPH-Nb-rAg via coextrusion to prepare PPH-Nb-rAg@PM NPs
   [[113]42]. Moreover, PPH-Nb-rAg coated with ordinary macrophage
   membranes (PPH-Nb-rAg@M) was also prepared and used as a control.

   Dynamic light scattering (DLS) analysis revealed that the hydrodynamic
   diameter ranges of PPH, Nb-rAg, PPH-Nb-rAg and PPH-Nb-rAg@PM were
   160.8 ± 3.1, 10.8 ± 1.3, 169.0 ± 5.6, and 182.6 ± 3.7 nm, respectively
   (Fig. [114]2E). Compared with that of PPH-Nb-rAg, the hydrodynamic
   diameter of PPH-Nb-rAg@PM increased by approximately 13.0 nm,
   indicating the successful decoration of the surface of the PPH-Nb-rAg
   NPs with macrophage membranes (~ 13 nm in thickness) (Fig. [115]2E).
   After Ms or PMs decoration, the zeta potential changed from − 26.2
   (PPH-Nb-rAg) to -11.6 mV (PPH-Nb-rAg@M) and − 12.02 mV (PPH-Nb-rAg@PM)
   (Fig. [116]2F), which was similar to the surface potential of the PMs.
   Scanning electron microscopy (SEM) revealed that the PPH-Nb-rAg NPs had
   a uniform spherical shape with a mean particle diameter of 169.0 nm
   (Fig. [117]2G and H). The TEM images revealed a typical sphere-like
   core-shell structure with a thin uniform membrane on the surface of
   both the PPH-Nb-rAg@M and PPH-Nb-rAg@PM NPs compared with the naked
   spherical PPH-Nb-rAg core (Fig. [118]2I-K). These results suggest that
   PMs were successfully coated on the surface of the PPH-Nb-rAg NPs.

   Next, SDS-PAGE combined with western blotting was performed to confirm
   the success of Nb-rAg loading and PMs decoration. SDS-PAGE analysis
   revealed that the protein compositions of PPH-Nb-rAg@M and
   PPH-Nb-rAg@PM were different from those of normal macrophage lysates
   (Fig. [119]2L), and a marked target band of approximately 77 kDa could
   be observed in the PPH-Nb-rAg@M and PPH-Nb-rAg@PM lanes. Western
   blotting confirmed that both the PPH-Nb-rAg@M and PPH-Nb-rAg@PM NPs
   carried membrane surface marker proteins, such as TLR2, TLR4, and TLR6,
   and their expression levels were greater in the PPH-Nb-rAg@PM than in
   the PPH-Nb-rAg@M NPs (Fig. [120]2M), confirming the successful loading
   of Nb-rAg, Ms and PMs, respectively. Next, the stability of the
   PPH-Nb-rAg@PM NPs at RT was determined by suspending them in 0.9%
   saline, PBS or 10% FBS + DMEM for 15 d. As shown in Fig. [121]2N, the
   hydrodynamic size of PPH-Nb-rAg@PM remained almost unchanged during
   incubation, indicating that the prepared PPH-Nb-rAg@PM NPs are stable.

Cytotoxicity, cellular uptake and biodistribution assays

   A CCK-8 assay was used to evaluate the viability of RAW264.7 and DC2.4
   cells after incubation with rAg, PPH, Nb-rAg, PPH-Nb-rAg or
   PPH-Nb-rAg@PM NPs for 24 h in vitro. The results indicated that even
   when the highest concentration reached 240 µg/ml, these NPs did not
   exhibit notable cytotoxicity to either RAW264.7 or DC2.4 cells
   (Fig. [122]3A and B), suggesting that the PPH-Nb-rAg@PM NPs have good
   cytocompatibility.

Fig. 3.

   [123]Fig. 3
   [124]Open in a new tab

   Cytotoxicity and in vivo distribution of prepared PPH-Nb-rAg@PM NPs.
   Cell viability of RAW264.7 (A) and DC2.4 (B) cells after incubation
   with 0, 30, 60, 120, 240 μg/ml rAg, Nb-rAg, PPH, PPH-Nb-rAg and
   PPH-Nb-rAg@PM NPs for 24 h. Then cell viability of each group was
   detected using a CCK-8 kit. (C) Uptake of rAg, Nb-rAg, PPH-Nb-rAg, and
   PPH-Nb-rAg@PM NPs by RAW264.7 cells. (D) FCM analysis of fluorescence
   intensity of (C), the fluorescence intensity of the rAg treatment group
   was normalized as 100%. (E) Uptake of rAg, Nb-rAg, PPH-Nb-rAg, and
   PPH-Nb-rAg@PM NPs by DC2.4 cells. (F) FCM analysis of fluorescence
   intensity of (E), the fluorescence intensity of the rAg treatment group
   was normalized as 100%. (G) Detection and biodistribution of Cy5.5-rAg,
   -Nb-rAg, -PPH-Nb-rAg and -PPH-Nb-rAg@PM NPs. (H) Biodistribution of
   fluorescent antigens in the liver, spleen, lung, kidney, and lymph
   nodes were analyzed

   APCs are responsible for the uptake and processing of various antigens
   and play a critical role in inducing immune response. Thus, the
   cellular uptake of rAg, Nb-rAg, PPH-Nb-rAg and PPH-Nb-rAg@PM NPs by
   RAW264.7 and DC2.4 cells was investigated via IFA. The results revealed
   that the fluorescence intensity of RAW264.7 cells treated with Nb-rAg
   was 2.47 times stronger than that of those treated with rAg, and that
   of the PPH-Nb-rAg group was approximately 1.77 times stronger than that
   of the Nb-rAg group (Fig. [125]3C and D), indicating that the
   conjugation of Nb-rAg with PPH promoted the cellular uptake of Nb-rAg.
   The fluorescence intensity of the PPH-Nb-rAg treatment group was 4.8
   times stronger than that of the PPH-Nb-rAg@PM group (Fig. [126]3C and
   D), implying that PPH-Nb-rAg NPs decorated with PMs effectively
   hindered their uptake by macrophages and prolonged their half-life. For
   DC2.4 cells, the fluorescence intensity of the PPH-Nb-rAg@PM treatment
   group was 1.17 times that of the PPH-Nb-rAg group (Fig. [127]3E and F),
   indicating that llike PPH-Nb-rAg, PPH-Nb-rAg@PM NPs are also able to be
   efficiently uptaken by DC2.4 cells.

   To investigate the distribution of the PPH-Nb-rAg@PM NPs in vivo,
   BALB/c mice were intravenously injected with Cy5.5-rAg, Cy5.5-Nb-rAg,
   PPH-Cy5.5-Nb-rAg, or PPH-Cy5.5-Nb-rAg@PM NPs, and fluorescence imaging
   was performed at the indicated time points via a live imaging system.
   As shown in Fig. [128]3G, the fluorescence intensity of the Cy5.5-rAg
   group was the weakest and reached the highest value at 12 h
   post-injection, after which it gradually decreased. The fluorescence
   intensities of the Cy5.5-Nb-rAg and PPH-Cy5.5-Nb-rAg groups were both
   greater than those of the Cy5.5-rAg group and decreased beginning at
   24 h post-injection. Notably, the fluorescence intensity of the
   PPH-Cy5.5-Nb-rAg@PM group was greater than that of the other three
   groups, and strong fluorescence continued to be observed even at 48 h
   post-injection (Fig. [129]3G). To further investigate the in vivo
   distribution of these NPs, the mice were sacrificed at 48 h, and their
   major organs were collected and imaged. As shown in Fig. [130]3H, the
   PPH-Cy5.5-Nb-rAg@PM group presented the strongest Cy5.5 fluorescence
   intensity in the lungs, lymph nodes, and spleens among all the groups,
   which was consistent with the in vivo fluorescence results, indicating
   that avoiding engulfment by macrophages effectively prolonged the
   half-life of the PPH-Nb-rAg@PM NPs in vivo.

PPH-Nb-rAg@PM NPs promote NK cell proliferation

   Studies have shown that the PPH can induce NK cell proliferation
   [[131]31]. NK cells act as regulatory cells of the immune system and
   are able to release perforin and IFN-γ to combat virus-infected cells
   and to regulate innate and adaptive immune reactions. Therefore,
   whether PPH-Nb-rAg@PM NPs can regulate the proliferation of NK cells in
   mice was investigated. Mouse PBMCs were isolated and stimulated with
   PBS, IL-2, IL-2 + PPH, IL-2 + PPH-Nb-rAg, or IL-2 + PPH-Nb-rAg@PM,
   after which an FCM assay was carried out to detect the population of
   CD3-CD49 + phenotype NK cells. IL-2 was included as the positive
   control. The results revealed that the proportion of CD3-CD49 + NK
   cells in the IL-2 + PPH-stimulated group was 21.2%, which was 1.19
   times greater than that in the IL-2 treatment group (Fig. [132]4A and
   B), and the proportions of CD3-CD49 + NK cells in the IL-2 + PPH-Nb-rAg
   and IL-2 + PPH-Nb-rAg@PM groups were 24.31% and 26.41%, respectively,
   which were 1.15 and 1.25 times greater than those in the IL-2 treatment
   group (Fig. [133]4A and B). However, the number of CD3-CD49 + NK cells
   in the IL-2 + PPH-Nb-rAg group and the IL-2 + PPH-Nb-rAg@PM group did
   not significantly differ (P > 0.05, Fig. [134]4B), indicating that the
   proliferation of NK cells is largely dependent on the PPH, which can
   inherently stimulate NK cell activation.

Fig. 4.

   [135]Fig. 4
   [136]Open in a new tab

   PPH-Nb-rAg@PM NPs promotes NK cells proliferation in vitro and in vivo.
   Representative flow cytometry plots (A) and quantification (B) of
   CD3-CD49 + phenotype NK cells proportion after mice PBMCs were
   stimulated with PBS, IL-2, IL-2 + PPH, IL-2 + PPH-Nb-rAg and
   IL-2 + PPH-Nb-rAg@PM NPs (n = 3). ELISA analysis of the cell culture
   supernatants IFN-γ (C) or perforin (D) secretion after mice PBMCs were
   stimulated with the above mentioned NPs (n = 3). (E and F) Represent
   flow cytometry plots and quantification of CD3-CD49 + NK cells in
   spleen of each group at 28 d after immunization (n = 3). (G) ELISA
   analysis of mice serum IFN-γ contents at 28 d post immunization
   (n = 3). (H) ELISA analysis of mice serum perforin contents at 28 d
   post immunization (n = 3)

   The expression levels of IFN-γ and perforin, two important
   pathogenic-killing cytokines in PBMC culture supernatants, were
   detected via ELISA. The ELISA results revealed that, compared with the
   IL-2 treatment, the IL-2 + PPH treatment dramatically stimulated the
   secretion of IFN-γ into the supernatant, which was increased by
   1.2-fold (Fig. [137]4C), and the IL-2 + PPH-Nb-rAg and
   IL-2 + PPH-Nb-rAg@PM treatment groups were increased by 1.39- and
   1.45-fold, respectively, compared with the IL-2 treatment group
   (Fig. [138]4C). There was no marked difference between the
   IL-2 + PPH-Nb-rAg and the IL-2 + PPH-Nb-rAg@PM groups (Fig. [139]4C).
   In terms of perforin, the levels in the IL-2 + PPH, IL-2 + PPH-Nb-rAg
   and IL-2 + PPH-Nb-rAg@PM groups were 2.04, 2.75, and 2.82 times greater
   than those in the IL-2 treatment groups (21.71, 29.24, 30.04 and 10.64
   pg/ml, respectively), and no marked difference was observed between the
   IL-2 + PPH-Nb-rAg and IL-2 + PPH-Nb-rAg@PM groups (Fig. [140]4D).

   Next, the proliferation of NK cells in vivo was further investigated.
   Splenocytes were isolated from different immunized mice at 28 d after
   immunization and then subjected to FCM. Compared with the PBS group,
   immunization with rAg, Nb-rAg, or PPH-Nb-rAg or PPH-Nb-rAg@PM NPs
   increased the number of NK cells with the CD3-CD49 + phenotype by 1.07,
   1.26, 1.37 and 1.48 times, respectively, and the PPH-Nb-rAg@PM NPs
   induced the highest percentage of CD3-CD49 + phenotype NK cells among
   all the groups (Fig. [141]4E and F). Splenocyte culture supernatant
   IFN-γ analysis via ELISA revealed that the PPH-Nb-rAg@PM immunization
   group presented the highest level of supernatant IFN-γ (Fig. [142]4G),
   and perforin analysis revealed similar results (Fig. [143]4H). Taken
   together, these results suggest that PPH decoration endows ASFV
   antigens with an inherent immunomodulatory ability to promote NK cell
   proliferation in vitro or in vivo.

PPH-Nb-rAg@PM NP immunization induced humoral immune response

   At 28, 35, and 49 d after immunization, the levels of mouse serum
   antibodies against ASFV rAg were detected via ELISA with rAg as the
   coating antigen (Fig. [144]5A). At 28 d post-immunization, the antibody
   titer in the Nb-rAg immunization group was 5.35 times greater than that
   in the rAg group (Fig. [145]5B). Compared with the Nb-rAg group, the
   PPH-Nb-rAg group presented 13.4 times greater antibody titers
   (Fig. [146]5B). The greatest specific antibody titers were induced via
   immunization with PPH-Nb-rAg@PM NPs, which was 55.2 times greater than
   that of the PPH-Nb-rAg group (Fig. [147]5B). ELISA analysis of the
   serum antibody titers of the different groups at 35 and 49 d after
   immunization also revealed that the PPH-Nb-rAg@PM NP immunization group
   presented the highest serum titers of IgG (Fig. [148]5B).

Fig. 5.

   [149]Fig. 5
   [150]Open in a new tab

   PPH-Nb-rAg@PM NPs immunization elicits humoral immune response. (A)
   Schedule of mouse immunization and sampling. Mice were immunized with
   PBS, rAg, Nb-rAg, PPH-Nb-rAg and PPH-Nb-rAg@PM NPs respectively twice
   with a 14-days intervals by the subcutaneous injection route. (B) Serum
   rAg-specific IgG titers at 28, 35, 49 d after immunization of each
   group was detected by ELISA. (C) Serum rAg-specific IgG2b, IgG2c, IgG3,
   IgA and IgM titers at 28 d after immunization of each group was
   determined by ELISA. (D and E) Serum rAg-specific IgG2a and IgG1 titers
   at 28, 35, 49 d after immunization of each group was detected by ELISA.
   (F) The IgG1/IgG2a ratio at 28, 35, 49 d after immunization of each
   group was calculated. (G) Standard virus-neutralizing assay. (H) IFA
   analysis of the effect of different mice immunized serum on ASFV p30
   protein expression. (I) Fluorescence density analysis of (H)

   To assess the polarization of the immune response comprehensively,
   mouse serum IgG2b, IgG2c, IgG3, IgA and IgM titers against rAg at 28 d
   after immunization were determined via ELISA. Compared with those in
   the other groups, the titers of IgG2b, IgG2c, IgG3, IgA, and IgM in the
   groups immunized with the PPH-rAg-Nb@PM NPs were greater
   (Fig. [151]5C). In addition, compared with the other groups, the
   PPH-rAg-Nb@PM group presented higher IgG1 and IgG2a titers
   (Fig. [152]5D and E). Th1 cells and Th2 cells are two different
   subtypes of T cells, both of which play important roles in host defence
   and immune regulation and are associated with the production of
   different types of antibodies. Th1 cells are involved mainly in
   cell-mediated immune responses, such as the activation of macrophages
   and cytotoxic T cells. Th2 cells are involved mainly in humoral
   immunity, assisting in the activation of B cells and the production of
   antibodies. In mice, Th1 cells mainly produce IgG2a-type antibodies,
   whereas Th2 cells mainly produce IgG1-type antibodies. Subtype analysis
   by ELISA revealed that immunization with rAg, Nb-rAg, PPH-Nb-rAg and
   PPH-Nb-rAg@PM resulted in higher IgG1 than IgG2a levels (Fig. [153]5D
   and E), and the ratio of IgG1 to IgG2a was greater than 1.0, the
   PPH-rAg-Nb@PM NP immunization group presented the greatest
   IgG1-to-IgG2a ratio (Fig. [154]5F), implying a Th2-biased immune
   response.

PPH-Nb-rAg@PM NPs induces ASFV-neutralizing antibodies

   After incubation of the NPs-immunized serially diluted mice serum (1:2,
   1:4, 1:8, 1:16, 1:32) with 100 TCID[50] of ASFV suspension for 2 h at
   37^oC, then the titer of each viral sample was assayed using TCID[50].
   The results showed that for the rAg- or Nb-rAg-immunized group, when
   the dilution ratio of mice serum was 1:2, it could partially neutralize
   the activity of ASFV (Fig. [155]5G), the ratio was 1:4 for the
   PPH-Nb-rAg-immunized group, and 1:8 for the PPH-Nb-rAg@PM group
   (Fig. [156]5G).

   By detecting the expression of the ASFV p30 protein, it was observed
   that for the rAg-immunized group, a 1:2 dilution of mice serum
   partially suppressed ASFV p30 protein expression, and the
   Nb-rAg-immunized group showed the same results (Fig. [157]5H and I).
   For the PPH-Nb-rAg-immunized group, 1:4 but not 1:8 dilution of mice
   serum partially suppressed ASFV p30 protein expression, while the
   effective dilution increased to 1:8 in the PPH-Nb-rAg@PM-immunized
   group (Fig. [158]5H and I). These results indicate that PPH-Nb-rAg@PM
   induces higher neutralization antibodies against ASFV than other
   groups.

PPH-Nb-rAg@PM NP immunization induced systemic cellular immune responses

   CD4 + and CD8 + T cells are key immune cells involved in the
   ASFV-specific adaptive immune response. CD4 + T cells play a critical
   role in promoting B-cell differentiation and sustaining the response of
   CD8 + cytotoxic T cells. Therefore, the T-cell responses in the spleens
   of immunized mice elicited by rAg, Nb-rAg, PPH-Nb-rAg, and
   PPH-Nb-rAg@PM NPs were further evaluated. At 28 d post vaccination, the
   proportion of CD3 + CD4 + T lymphocyte differentiation induced by
   Nb-rAg was 1.05 times greater than that induced by rAg, which induced
   by PPH-Nb-rAg was 1.12 times greater than that induced by Nb-rAg
   (Fig. [159]6A and B), and that induced by PPH-Nb-rAg@PM NPs was 1.52
   times greater than that induced by PPH-Nb-rAg (Fig. [160]6A and B),
   which was the highest among all the groups. The proportion of
   CD3 + CD8 + T cells in the Nb-rAg group was 1.08 times greater than
   that in the rAg group, that in the PPH-Nb-rAg group was 1.11 times
   greater than that in the Nb-rAg group (Fig. [161]6C and D), and that in
   the PPH-Nb-rAg@PM NP group was the highest among the different groups,
   reaching 1.94 times greater than that in the PPH-Nb-rAg group
   (Fig. [162]6C and D). These results suggest that the PPH-Nb-rAg@PM NPs
   are able to elicit more effective CD4 + and CD8 + T-cell immune
   responses.

Fig. 6.

   [163]Fig. 6
   [164]Open in a new tab

   PPH-Nb-rAg@PM NPs immunization elicits cell immune response in vivo. (A
   and B) Representative flow cytometry plots and quantification analysis
   of the percentage of CD3 + CD4 + T cells of each group at 28 d post
   immunization with PBS, rAg, Nb-rAg, PPH-Nb-rAg and PPH-Nb-rAg@PM NPs
   (n = 3). (C and D) Representative flow cytometry plots and
   quantification analysis of the percentage of CD3 + CD8 + T cells of
   each group at 28 d post immunization. (E) Shematic diagram of immunized
   mice splenocytes that were re-stimulated with rAg. (F and G) Analysis
   of IFN-γ secreting splenocytes clones re-stimulated with ASFV rAg
   protein were determined by ELISpot assay. (H and I) Analysis of IL-4
   secreting splenocytes clones re-stimulated with ASFV rAg protein were
   determined by ELISpot assay. (J and K) Splenocytes isolated from each
   immunized group were re-stimulated with rAg for 60 h, and cell culture
   supernatants IL-4 and IFN-γ concentration were detected by ELISA,
   respectively. (L) The proliferation ability of lymphocyte of each group
   was detected using CCK-8 assay, and then the proliferation index was
   calculated based on the OD450 nm value

   Cellular immune responses benefit the generation of effective immunity
   and the clearance of invasive pathogens [[165]43]. Splenocytes isolated
   from immunized mice were restimulated with the rAg protein, followed by
   ELISpot measurement of IFN-γ- and IL-4-positive cells (Fig. [166]6E).
   Although the proportions of IFN-γ- and IL-4-secreting cells increased
   to some extent in each immunized group compared with those in the PBS
   control group at 28 d after immunization, the PPH-Nb-rAg@PM NP group
   presented the greatest number of IFN-γ- and IL-4-secreting cells
   (Fig. [167]6F-I). In addition, the IFN-γ and IL-4 levels in the
   splenocyte culture supernatants of each group were also detected via
   ELISA. Compared with the other groups, the PPH-rAg-Nb@PM NP group
   secreted the highest levels of extracellular IFN-γ (Fig. [168]6J) and
   IL-4 (Fig. [169]6K).

   The proliferation of lymphocytes reflects the host immune status
   [[170]44]. Thus, splenocytes isolated from each immunized group were
   restimulated with rAg for 72 h. CCK-8 assay results revealed that the
   proliferative capacity of lymphocytes isolated from PPH-Nb-rAg@PM
   NP-immunized mice was greater than that of the other groups
   (Fig. [171]6L).

DC-targeting PPH-Nb-rAg@PM NPs promote BMDC maturation in vivo

   pXCR1-specific Nb was fused at the N-terminus of the ASFV rAg fragment,
   resulting in Nb-rAg, PPH-Nb-rAg and PPH-Nb-rAg@PM NPs capable of DC
   targeting. To further evaluate the effects of DC-targeting NPs on BMDC
   maturation in vivo, splenic DCs were isolated from immunized mice at
   36 h after immunization, and an FCM assay was subsequently conducted to
   detect the phenotypes associated with DC maturation. The percentages of
   CD11c + CD80 + DCs in the PPH-rAg-Nb@PM NP-immunized group were 1.62
   times greater than those in the PBS-immunized group, 1.41 times greater
   than those in the rAg-immunized group, 1.35 times greater than those in
   the Nb-rAg-immunized group, and 1.12 times greater than those in the
   PPH-Nb-rAg-immunized group (Fig. [172]7A). Analysis of
   CD11c + CD86 + and CD11c + MHCII + BMDCs also revealed that ASFV rAg
   conjugated with a specific Nb indeed promoted BMDC maturation more
   efficiently, especially the PPH-rAg-Nb@PM NPs, which exhibited the
   strongest ability to promote BMDC maturation (Fig. [173]7B and C).

Fig. 7.

   [174]Fig. 7
   [175]Open in a new tab

   PPH-Nb-rAg@PM NPs immunization promotes BMDCs maturation. The
   splenocytes of the PBS, rAg, Nb-rAg, PPH-Nb-rAg and PPH-Nb-rAg@PM NPs
   immunized mice were isolated at 36 h post immunization and then were
   subjected to FCM assay. Representative flow cytometry plots and
   quantification analysis of CD11c + CD80+ (A), CD11c + CD86+ (B),
   CD11c + MHCII + phenotype (C) BMDCs proportion were presented. (D-G)
   The isolated splenocytes were cultured for 48 h and supernatants were
   collected for detection of the secretion of IFN-γ (D), IL-12p70 (E),
   IL-4 (F), and IL-13 (G) using ELISA. (H and I) The isolated splenocytes
   were subjected to qPCR analysis of the mRNA expression of IRF4 and IRF8
   that associated with BMDCs maturation

   Mature BMDCs can secrete various cytokines, which play important roles
   in the immune response, inflammatory response, and antigen
   presentation. For example, IFN-γ and IL-12 are capable of promoting
   Th1-type immune responses. IL-4 and IL-13 participate in the Th2-type
   humoral immune response. At 28 d postimmunization, the expression of
   these cytokines in isolated splenocytes after restimulation with rAg
   was further analyzed via ELISA. The results revealed that
   PPH-Nb-rAg@PM-immunized mice produced the highest levels of the
   Th1-type cytokines IFN-γ and IL-12p70 (Fig. [176]7D and E), as well as
   the Th2-type cytokines IL-4 and IL-13 (Fig. [177]7F and G).

   To further confirm that the PPH-Nb-rAg@PM NPs had greater immune
   efficiency in inducing BMDC maturation, the expression of several key
   regulators that participate in regulating BMDC maturation was assayed.
   Owing to the lack of available commercial antibodies, the expression
   levels of the IR4 and IR8 mRNAs were detected. qPCR results revealed
   that immunization with rAg, Nb-rAg or PPH-Nb-rAg indeed upregulated IR4
   and IRF8 mRNA expression, especially in PPH-Nb-rAg@PM NP-immunized mice
   (Fig. [178]7H and I). These results suggest that, compared with
   ordinary ASFV rAg, PPH-Nb-rAg@PM NPs are able to promote splenic DC
   maturation more efficiently.

PPH-Nb-rAg@PM NPs stimulate the activation of Tfhs and induce germinal center
(GC) B cells and plasma cells

   DC maturation is able to induce a humoral immune response and
   facilitate the differentiation of T cells into Tfh cells. CD4 + Tfh
   cells reside in the spleen and are critical for the differentiation of
   GC-B cells into memory B cells and long-lived plasma cells [[179]45].
   Therefore, CD4 + Tfh cells in the mouse spleen at 28 d postimmunization
   were investigated. Compared with the PBS treatment group, immunization
   with rAg increased the percentage of CD4 + CXCR5 + PD-1 + Tfh cells by
   1.48-fold (9.72% vs. 6.55%), whereas the percentage of
   CD4 + CXCR5 + PD-1 + PD-1 + Tfh cells in the Nb-rAg-immunized group was
   1.67-fold greater than that in the rAg group (16.24% vs. 9.72%), the
   percentage of CD4 + CXCR5 + PD-1 + Tfh cells in the PPH-Nb-rAg group
   was 1.21-fold greater than that in the Nb-rAg group (19.65% vs.
   16.24%), the PPH-Nb-rAg@PM NPs group showed the highest percentage of
   CD4 + CXCR5 + PD-1 + Tfh cells and was 1.26-fold greater than that in
   the PPH-Nb-rAg group (24.71% vs. 19.65%) (Fig. [180]8A and B).

Fig. 8.

   [181]Fig. 8
   [182]Open in a new tab

   Detection of Tfh cells, GC-B cells and plasma cells from the immunized
   mice. (A) Gating strategy and representative flow cytometry plots of
   CD4 + CXCR5 + PD1 + Tfh cells, and quantitative analysis (B) of Tfh
   cells in the spleen (n = 3). (C) Gating strategy and representative
   flow cytometry plots of CD19 + CD95 + GL7 + GC-B cells in the spleen.
   (D) The percentage of GC-B cells (n = 3). (E) Gating strategy and
   representative flow cytometry plots of B220-CD138 + plasma cells in the
   spleen. (F) The percentage of plasma cells in the spleen (n = 3)

   The transcription regulatory factors Blimp-1, PD-1, and Bcl-6 are
   involved in regulating the production, development and differentiation
   of Tfh cells. Thus, the mRNA and protein levels of these transcription
   factors were further detected in the splenocytes of each immunized
   group. qPCR revealed that the PPH-Nb-rAg@PM NPs induced the highest
   levels of Blimp-1, PD-1, and Bcl-6 mRNA expression (Additional file 7:
   Figure [183]S7A-C). The western blotting results revealed that
   immunization with rAg, Nb-rAg or PPH-Nb-rAg promoted the expression of
   the Blimp-1, PD-1, and Bcl-6 proteins, and the PPH-Nb-rAg@PM NPs
   induced the highest expression levels of these proteins (Additional
   file 7: Figure [184]S7D).

   After antigen stimulation, GCs first form in secondary lymphoid organs
   such as the spleen, and then, antigen-specific B cells migrate to GCs
   to form GC-B cells [[185]46, [186]47]. With antigen stimulation and Tfh
   assistance, some GC-B cells differentiate into plasma cells or memory B
   cells [[187]48]. Thus, whether PPH-Nb-rAg@PM NP immunization could
   promote the production of GC-B cells was first determined at 28 d
   postimmunization. FCM revealed that the percentages of
   CD19 + CD95 + GL7 + phenotype GC-B cells were 0.30%, 0.44%, 0.65%,
   2.3%, and 5.08% in the PBS-, rAg-, Nb-rAg-, PPH-Nb-rAg-, and
   PPH-Nb-rAg@PM NP-immunized groups, respectively. The PPH-Nb-rAg@PM
   group had the highest value, which was 11.55 times greater than that of
   the rAg group (Fig. [188]8C and D), indicating that PPH-Nb-rAg@PM NP
   immunization promoted the precursor production of plasma cells.

   Next, the proportion of B220-CD138 + phenotype plasma cells in the
   splenocyte population was determined. Compared with those of the PBS
   group (the proportion of plasma cells was 1.33%), the percentage of
   plasma cells with the B220-CD138 + phenotype of the mice immunized with
   rAg was 3.06%, which was 2.30 times greater than that of the PBS group;
   the percentages of the Nb-rAg, PPH-Nb-rAg groups were 6.94%, 10.39%,
   and were 2.27, 3.40 times greater than those of the rAg group; and the
   percentage of the PPH-Nb-rAg@PM NP-immunized group was the highest
   (13.41%), which was 4.38 times greater than that of the rAg group
   (Fig. [189]8E and F).

PPH-Nb-rAg@PM NPs promoted memory B- and T-cell responses

   Memory B cells form in the GC after primary stimulation by antigens,
   and when re-exposed to the same specific antigen, they play an
   important role in producing faster and stronger antibody-mediated
   immune responses. Thus, splenic memory B cells were also analyzed
   (Fig. [190]9A). The results revealed that after rAg restimulation of
   splenocytes that were isolated at 28 d after immunization, the
   B220 + CD38 + IgG + IgD-GL7- phenotype memory B-cell proportion was
   markedly greater in the PPH-Nb-rAg@PM group than in the PBS, rAg,
   Nb-rAg and PPH-Nb-rAg groups (66.42% vs. 8.54%, 26.19%, 42.51%, and
   54.91%, respectively) and was 2.54 times greater than that in the rAg
   group (Fig. [191]9B and C).

Fig. 9.

   [192]Fig. 9
   [193]Open in a new tab

   Detection of memory B cell, CD80 + memory B cells and central memory T
   cells in mice spleen. (A) Experimental schematic diagram. (B) Gating
   strategy and representative flow cytometry plots of
   B220 + CD38 + IgG + IgD-GL7- memory B cells. (C) Quantitative analysis
   of the percentage of memory B cells (n = 3). (D) Gating strategy and
   representative flow cytometry plots of CD80 + CD19 + IgD-IgG + memory B
   cells. (E) Quantitative analysis of the percentage of memory B cells
   (n = 3). (F) Gating strategy and representative flow cytometry plots of
   CD4 + CD44 + CD62L + central memory T cells. (G) The percentage of
   central memory T cells in immunized mice spleen

   CD80 is one of the key proteins expressed on the surface of memory B
   cells and is involved in regulating B-T-cell interactions as well as
   Tfh generation [[194]49, [195]50]. Thus, the expression of CD80 on the
   surface of the IgG + memory B cells was detected, and the results
   revealed that the percentage of CD19 + IgG + IgD-CD80 + memory B cells
   in the PPH-Nb-rAg@PM-immunized group was markedly greater than that in
   the PBS, rAg, Nb-rAg and PPH-Nb-rAg groups (71.51% vs. 14.13%, 28.18%,
   31.21%, 37.15%, respectively) (Fig. [196]9D and E).

   Central memory T (T[CM]) cells circulate within the lymphatic system
   and proliferate and differentiate into effector T cells quickly to
   exert immune protection once they encounter the same antigen again.
   Boosting local and systemic memory T-cell responses is an effective
   strategy for obtaining long-term immunity, which is one of the pivotal
   features of an ideal vaccine. CD4 + CD44 + CD62L + T cells in the mouse
   spleen are considered typical TCMs with strong viability and can exist
   in the body for a long period. Thus, the splenocytes of each immunized
   group were isolated and restimulated with rAg, followed by FCM of the
   T[CM] proportion. After the boost immunization, although the
   proportions of CD4 + CD44 + CD62L + T cells were greater in the rAg,
   Nb-rAg and PPH-Nb-rAg groups than in the PBS treatment group (12.80%,
   25.24%, 27.43% vs. 7.70%, respectively), the PPH-Nb-rAg@PM group
   presented the highest number of CD4 + CD44 + CD62L + T cells (32.23%),
   which was 2.52 times greater than that of the rAg-immunized group
   (Fig. [197]9F and G). The frequency of effector memory T (T[EM]) cells
   in mouse splenocytes was further investigated. The percentage of
   CD4 + CD44 + CD62L- phenotype T[EM] cells in the PPH-Nb-rAg@PM
   NP-immunized group was markedly greater than that in the PBS, rAg,
   Nb-rAg and PPH-Nb-rAg groups (21.33% vs. 3.39%, 6.36%, 9.36%, and
   14.93%, respectively) (Additional file 8: Figure [198]S8).

Single-cell transcriptomics analysis of immune cells in the spleen before and
after immunization

   To further investigate the mechanism of the immune response induced by
   the PPH-Nb-rAg@PM NPs, scRNA-seq was performed. After initial quality
   control, 9959 and 8059 cells were obtained for single-cell
   transcriptome analysis in the PBS and PPH-Nb-rAg@PM groups,
   respectively. Uniform manifold approximation and projection (UMAP)
   identified eight cell subsets in the mouse spleen: T_NK cells, B cells,
   erythroblast cells, DCs, macrophages, neutrophils, endothelial cells,
   and plasma cells (Fig. [199]10A). Marker genes, including those in
   nonimmune and immune cell fractions, were analyzed to identify cell
   types. Among the identified cells, nonimmune cells primarily consisted
   of erythroblasts (marked by Spc24) and endothelial cells (marked by
   Cdh5). The immune cells included T_NK cells (marked by Cd3d), B cells
   (marked by Cd19), DCs (marked by Cd209a), macrophages (marked by
   Cd163), and plasma cells (marked by Jchain) (Fig. [200]10B). Analysis
   of the selected canonical markers for different cell subsets revealed
   that Cd3g, Cd3d and Nkg7, but not Ncam1, were highly expressed in T_NK
   cells; Cd19, Cd79a and Cd79b were highly expressed in B cells; Cdca5,
   Spc24 and Stmn1 were highly expressed in erythroblasts; Flt3 and Cd209a
   were highly expressed in DCs, while Itgam were highly expressed in
   neutrophils cells; Cd163, C1qa, C1qb and Csf1r were highly expressed in
   macrophages; Ly6g, Ncf1, Cd177, Sorl1, Csf3r and Retnlg were highly
   expressed in neutrophils; Pecam1 and Cdh5 were highly expressed in
   endothelial cells; and Mzb1 and Jchain were highly expressed in plasma
   cells (Fig. [201]10C), further confirming that the identified cell
   subsets were correct.

Fig. 10.

   [202]Fig. 10
   [203]Open in a new tab

   Single-cell analysis of splenocytes from mice immunized with
   PPH-Nb-rAg@PM. (A) UMAP analysis of splenocyte subsets. (B) Expression
   of cell-type-specific markers. (C) Markers for different cell subsets
   in mice splenocytes. (D) UMAP plot showed the expression profiles of
   mice spleens in PBS and PPH-Nb-rAg@PM groups, respectively. (E) Number
   of T_NK cells, B cells, erythroblast cells, DCs, macrophages,
   neutrophils, endothelial cells, plasma cells subsets of PBS and
   PPH-Nb-rAg@PM immunized mice spleens. The cell number of each
   population was calculated by multiplying the total number of isolated
   immune cells by the percentage of the population

   UMAP analysis of the quantitative changes in different cell subsets
   revealed that, compared with those in the PBS group, the numbers of
   T_NK cells, B cells, macrophages, and plasma cells in the spleens of
   the PPH-Nb-rAg@PM-immunized mice markedly increased (Fig. [204]10D and
   E), indicating that immunization with the PPH-Nb-rAg@PM NPs elicited
   extensive humoral and cellular immune responses in the mouse model.
   Compared with that in the PBS group, the population of neutrophils
   obviously decreased in the PPH-Nb-rAg@PM-immunized group (Fig. [205]10D
   and E), as neutrophils are associated with the inflammatory response.
   These results suggest that immunization with PPH-Nb-rAg@PM effectively
   inhibits the inflammatory response in vivo.

PPH-Nb-rAg@PM vaccine induces protective immunity by enhancing immune cell
activation

   On the basis of single-cell sequencing data, B-cell subtypes and T-cell
   subtypes were further analyzed. For the B-cell subtype, memory B cells
   and plasma cells were identified (Fig. [206]11A). Analysis of marker
   molecules expressed on the surface of B-cell subtypes further confirmed
   that memory B cells (marker protein Ms4a1, Fig. [207]11B) and plasma
   cells (marker protein Jchain) were identified as expected
   (Fig. [208]11C). In addition, the expression patterns of surface
   proteins also revealed that Aim2 and Ms4a1 were expressed mainly on
   memory B cells, whereas Jchain and Mzb1 were expressed mainly on plasma
   cells (Fig. [209]11D). Moreover, in the spleen at 28 d
   postimmunization, significant increases in the numbers of memory B
   cells and plasma cells were observed (Fig. [210]11E and F). These
   results indicate that the PPH-Nb-rAg@PM nanovaccine enhances the
   magnitude of humoral immune responses, which is in accordance with the
   results shown in Figs. [211]8E and [212]9B.

Fig. 11.

   [213]Fig. 11
   [214]Open in a new tab

   Single-cell analysis of B and T cell subtypes from mice immunized with
   PPH-Nb-rAg@PM. (A) UMAP analysis of B cells subsets. (B) Expression of
   memory B cell-specific marker. (C) Expression of plasma cell-specific
   marker. (D) Markers for memory B cells and plasma cells in mice
   splenocytes. (E) UMAP plot showed the expression profiles of memory B
   cells and plasma cells in PBS and PPH-Nb-rAg@PM immunized mice,
   respectively. (F) Number of memory B cells and plasma cells subsets of
   PBS and PPH-Nb-rAg@PM immunized mice spleens. (G) UMAP analysis of T
   cells subsets. (H-K) Expression of CD4 + T cells (H), CD8 + T cells
   (I), NK cells (J), and NKT cells (K) cell-specific marker. (L) Markers
   for CD4 + T cells, CD8 + T cells, NK cells, and NKT cells in mice
   splenocytes. (M) UMAP plot showed the expression profiles of CD4 + T
   cells, CD8 + T cells, NK cells, and NKT cells in PBS and PPH-Nb-rAg@PM
   immunized mice, respectively. (N) Number of CD4 + T cells, CD8 + T
   cells, NK cells, and NKT cells subsets of PBS and PPH-Nb-rAg@PM
   immunized mice spleens. (O) The cross-talk between B cells, T_NK cells,
   plasma cells, neutrophils, and erythroblast

   The T_NK cell subtypes were further analyzed via scRNA-seq. UMAP
   analysis revealed that the identified T_NK cell subtypes could be
   further divided into CD4 + T cells, CD8 + T cells, NK cells and NKT
   cells (Fig. [215]11G). UMAP analysis of the expression patterns of
   surface marker proteins confirmed that CD4 + T cells (marker protein
   Cd4, Fig. [216]11H), CD8 + T cells (marker protein Cd8a,
   Fig. [217]11I), NK cells (marker protein Nkg7, Fig. [218]11J) and NKT
   cells (marker protein Gzmb, Fig. [219]11K) were correctly identified.
   Analysis of the selected canonical markers for different cell subsets
   revealed that Cd3d, Cd3e, Cd3g and Cd4 were highly expressed in CD4 + T
   cells; Cd3d, Cd3e, Cd3g and Cd8a were highly expressed in CD8 + T
   cells; Nkg7 and Gzmb were highly expressed in NK cells; and Cd3d, Cd3e,
   Cd3g and Nkg7 were highly expressed in NKT cells (Fig. [220]11L),
   confirming that the T-cell subtypes were correctly identified. UMAP
   analysis revealed that at 28 d postimmunization, the numbers of CD4 + T
   cells, CD8 + T cells and NK cells in the PPH-Nb-rAg@PM immunization
   group were markedly greater than those in the PBS control group, which
   was consistent with the FCM results shown in Figs. [221]4B and [222]8A
   and C. However, the number of NKT cells in the PPH-Nb-rAg@PM group was
   lower than that in the PBS group (Fig. [223]11M and N). Cell-cell
   communication network analysis also suggested that mouse spleen B cells
   interacted more tightly with DC clusters, T_NK cell clusters, and
   plasma cell clusters after PPH-Nb-rAg@PM immunization (Fig. [224]11O).
   Taken together, these results suggest that the PPH-Nb-rAg@PM
   nanovaccine effectively enhances the B and T-cell immune responses in
   vivo.

Discussion

   There is no vaccine or drug available for ASF globally, and the
   prevention and control of the disease relies on strict biosecurity
   measures, early diagnosis and culling of infected pigs. The subunit
   vaccine is a relatively safe vaccine composed of a variety of viral
   proteins or peptides that are immunogenic and can induce neutralizing
   antibodies in the body. However, immune-related viral proteins have not
   been fully characterized and are composed of only a limited number of
   viral proteins, such as p30, p72, p54 and CD2v, which are not enough to
   induce the body to produce strong neutralizing antibodies. The
   immunoprotective effect was low.

   DC-targeted vaccines have become a research focus in the field of
   vaccine development for animal epidemic diseases, although much of this
   research is still in its infancy. Specific nanobody-mediated ASFV
   antigen targeting to DCs is a promising strategy for the development of
   DC-targeted ASFV vaccines. The present study developed a biomimetic
   nanovaccine platform based on PM-decorated phosphorus dendrimer/Nb-rAg
   NPs for the development of a candidate vaccine for ASFV. By coupling
   specific Nbs with ASFV recombinant antigen epitopes, the antigen
   recognition ability of DCs was significantly enhanced. Compared with
   mice immunized with ordinary ASFV antigens, those immunized with Nb-rAg
   exhibited greater specific humoral or cellular immune responses.

   Among the many different delivery systems, the PPH, which is
   characterized by a highly branched three-dimensional structure, a
   functionalize surface and a uniform molecular weight, has received much
   attention [[225]51]. PPH can induce NK cell proliferation by
   specifically inhibiting CD4 + T lymphocyte proliferation, and NK cells
   play an important role in antimicrobial infection [[226]31]. Compared
   with the Nb-rAg group, the combination of the Nb-rAg complex with the
   PPH significantly promoted the proliferation of NK cells in immunized
   mice; compared with the Nb-rAg group, the PPH-Nb-rAg group presented a
   greater number of NK cell populations, and the PPH-Nb-rAg group also
   significantly promoted the proliferation of NK cells in vitro, which
   may promote the anti-ASFV infection ability of immunized pigs. In
   addition, the decoration of preactivated macrophage membranes prevents
   the NPs from being engulfed by macrophages, allowing them to have a
   longer cycle time in the body and inducing a stronger immune response.
   As demonstrated by the larger population numbers of GC B cells, memory
   B cells and plasma cells, higher levels of specific antibodies were
   induced by the PPH-Nb-rAg@PM NPs.

   In addition to their role in humoral immune responses, T cells play
   important roles in various viral infections. CD4 + T cells mediate the
   production of antibodies by B cells, coordinate the response of other
   types of immune cells, and directly initiate an immune response to
   infectious agents [[227]52]. CD4 + T cells differentiate into Th2 and
   Th1 cells that drive the adaptive immune response, and CD8 + T cells
   target infected cells and clear infection sites, mainly through
   perforin, granzyme, and the FasL pathway [[228]53]. The present results
   revealed that immunization with PPH-Nb-rAg@PM resulted in the highest
   levels of CD4 + and CD8 + T cells, and ELISpot resulted in the highest
   secretory levels of IFN-γ and IL-4 in mouse splenocytes, confirming
   that PPH-Nb-rAg@PM is superior to other types of vaccines in inducing a
   cellular immune response, as the production of IFN-γ and IL-4 by
   antigen-stimulated PBMCs is viewed as an indicator of vaccine
   immunogenicity.

   Single-cell sequencing can be used to analyze the immune cell types of
   mice directly. According to the single-cell sequencing results, after
   immunization with PPH-Nb-rAg@PM, the proportions of immune cells, such
   as T_NK cells, B_cells, DC macrophages and plasma_cells, significantly
   increased compared with those in the PBS control group, implying that
   the PPH-Nb-rAg@PM nanovaccine had a good immune effect. Further
   analysis of the B-cell subtype or T-cell subtype also suggested that
   immunization with PPH-Nb-rAg@PM elicited strong immune B and T-cell
   responses, which further confirmed the potential of PPH-Nb-rAg@PM as a
   candidate vaccine for ASFV. Owing to the high cost and biosafety
   factors, the current study did not investigate the immunoprotective
   potential of PPH-Nb-rAg@PM nanovaccines in ASFV-infected pigs, which is
   also a shortcoming. Besides, the role and mechanism of TLRs expressed
   on the surface of macrophage membrane in PPH-Nb-rAg@PM-induced humoral
   and cellular immunose is still unknown, and this also needs further
   studies in the future. However, multiepitope vaccine subunit vaccines
   have the unique advantage of high safety, which is not possessed by
   other types of vaccines. The PPH developed in this study integrates
   several reported epitopes with viral neutralizing activity. Both in
   vivo and in vitro experiments have shown that nanovaccines can induce
   higher levels of humoral and cellular immune responses than
   conventional vaccines can, and can produce higher levels of
   immunomodulators. It has great potential for development into a new
   ASFV vaccine and warrants further research. This study also provides a
   platform for the development of other viral subunit vaccines.

Conclusions

   The present study developed a nanovaccine, PPH-Nb-rAg@PM, which
   recombines multiple ASFV neutralization epitopes and then conjugates
   them with high-affinity, specific Nbs against the DC antigen receptor
   XCR1. The recombinant protein Nb-rAg, which targets DCs, was expressed
   and purified via a prokaryotic expression system, and then, Nb-rAg and
   PPH were coupled to obtain PPH-Nb-rAg. Then, the PPH-Nb-rAg NPs were
   coated with ASFV-preactivated macrophage membranes to construct the
   PPH-Nb-rAg@PM biomimetic nanovaccine. Then, the mice were immunized
   with the nanovaccine and PBS; rAg, Nb-rAg and PPH-Nb-rAg were used as
   control groups. Compared with the other control groups, the
   PPH-Nb-rAg@PM nanovaccine induced a stronger humoral immune response,
   cellular immune response, greater NK cell proliferation ability, and
   greater numbers of memory B cells, memory T cells, and plasma cells,
   indicating that the developed nanovaccine has potential as an effective
   candidate vaccine for ASFV prevention and control.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [229]Supplementary Material 1^ (4.7MB, docx)
   [230]Supplementary Material 2^ (28KB, docx)

Acknowledgements