Rational design of a ‘two-in-one’ immunogen DAM drives potent immune response against mpox virus

Abstract

The global outbreak of the mpox virus (MPXV) in 2022 highlights the urgent need for safer and more accessible new-generation vaccines. Here, we used a structure-guided multi-antigen fusion strategy to design a ‘two-in-one’ immunogen based on the single-chain dimeric MPXV extracellular enveloped virus antigen A35 bivalently fused with the intracellular mature virus antigen M1, called DAM. DAM preserved the natural epitope configuration of both components and showed stronger A35-specific and M1-specific antibody responses and in vivo protective efficacy against vaccinia virus (VACV) compared to co-immunization strategies. The MPXV-specific neutralizing antibodies elicited by DAM were 28 times higher than those induced by live VACV vaccine. Aluminum-adjuvanted DAM vaccines protected mice from a lethal VACV challenge with a safety profile, and pilot-scale production confirmed the high yield and purity of DAM. Thus, our study provides innovative insights and an immunogen candidate for the development of alternative vaccines against MPXV and other orthopoxviruses.

Main

Since the first case of mpox was confirmed in the United Kingdom in May 2022, the mpox outbreak has resulted in more than 91,000 infections around the world (https://www.who.int/). Mpox is a viral zoonotic disease caused by MPXV, an Orthopoxvirus belonging to the family Poxviridae (https://ictv.global/taxonomy). Three other orthopoxviruses are pathogenic to humans: variola virus (VARV), the causative agent of smallpox, cowpox virus (CPXV) and VACV¹. Based on similarity, the less virulent VACV was used as a live virus vaccine administered by scarification during the successful eradication of smallpox in the first half of the 20th century². Two smallpox vaccines are approved for use in the current mpox outbreak: JYNNEOS (also known as IMVANEX or IMVAMUNE) and ACAM2000 (refs. ^3,4), both VACV-based, live virus vaccines.

The poxviral genome consists of a large linear double-stranded DNA and has the capacity to encode up to 200 viral proteins⁵. Nearly half of the viral proteome is dedicated to impeding innate immunity in host cells^6,7,8, whereas our knowledge of the poxvirus antigens stimulating protective host immunity is currently incomplete. Therefore, the large number of viral proteins introduced by live virus vaccination, many of which cannot induce an effective antiviral immune response and may impair the host immune response^6,7,8, create uncertainty and safety risks, especially for the immunocompromised populations.

Poxviruses can produce two antigenically distinct forms of infectious virions known as intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs), which are thought to mediate transmission between hosts and within hosts, respectively^9,10. Alternative vaccines against poxviruses using DNA vaccine or subunit vaccine strategies with a safer profile than live virus vaccines have been attempted. Co-immunization of several IMV antigens (mainly D8, L1, H3 and A27) together with EEV antigens (B5 and A33) provides effective protection against lethal challenge with VACV in mice^{11,12,13,14,15,16,17,18,19} or MPXV challenge in nonhuman primate models^20,21,22,23. However, multi-antigen co-immunization, both for nucleic acid-based and protein-based vaccines, faces some important challenges. Because the bioavailability of each component, as well as the expression efficiency, in the case of nucleic acid vaccines, cannot be effectively controlled, a sustained balanced immune response between different immunogens can hardly be achieved, posing a formidable challenge for the stability and maximization of vaccine protection. In addition, the cocktail immunization approach will result in high production costs, difficult transition to industrial production and product approval.

In this study, we describe a strategy for MPXV vaccine development based on structure-guided immunogen design and multi-antigen fusion. Two MPXV antigens, M1 and A35, were used to develop a ‘two-in-one’ version of the chimeric immunogen, which we named DAM (in tandem fusion of double A35s and M1s). DAM elicited antibody responses that cross-recognized other pathogenic poxviruses, but were most potent against MPXV antigens. DAM was superior compared to co-immunization with M1 and A35 in terms of specific antibody responses and protection against in vivo VACV challenge in mice. The MPXV-specific neutralizing antibody titer induced by DAM vaccination was markedly higher than that elicited by live VACV scarification. The safety profile provided by alum-adjuvanted DAM, as well as the high yield and purity after purification from the industry-standard stable Chinese hamster ovary (CHO) cell line, indicated a potential for vaccine development.

Results

Single-chain dimerization maintains A35 immunogenicity

MPXV A35 is a type II transmembrane protein comprising 181 amino acids (Fig. 1a) and shares high sequence identity with orthologs of other species (Extended Data Fig. 1). VACV A33, the homolog of MPXV A35, forms a homodimer on the EEV membrane, due to interprotomer disulfide bonds located in the membrane-proximal stalk region and non-covalent interactions between the A33 ectodomains (Fig. 1a)^24,25. To assess the immunogenicity of MPXV A35, a full-length form of the A35 ectodomain (A35^Ecto, R58–T181), which contains the conserved interprotomer disulfide bond-related cysteines (C62 of A35), was expressed in Expi293F cells. Analytical gel filtration profile and analytical ultracentrifugation assay indicated the purified A35^Ecto protein was dimeric (~29.6 kDa) in solution (Fig. 1c and Extended Data Fig. 2a). Gel electrophoresis under both non-reducing and reducing conditions indicated that the single peak of dimeric A35^Ecto after exclusion chromatography was heterogeneous in nature, due to the presence of different A35^Ecto dimers formed by the disulfide bond and/or non-covalent interactions (Fig. 1c). A35^Ecto protein formulated with AddaVax (an MF-59-like squalene adjuvant) as adjuvant was administered intramuscularly (i.m.) to BALB/c mice with three doses of 10 μg each at 3-week intervals. The A35^Ecto protein elicited a potent specific antibody response with an endpoint titer of nearly 10⁶ after the third dose (Fig. 1d). Because the intermolecular disulfide bond triggered heterogeneity of the A35^Ecto dimer and could pose challenges in both subsequent design of the chimeric MPXV immunogen and the production of subunit vaccines, a truncated form of the A35 extracellular domain protein (A35^ΔDB, Ser90–Thr181), which contains the full set of known neutralizing epitope-related residues, based on its VACV A33 counterpart, but lacks the stalk region containing the disulfide bond, was expressed in Expi293F cells. The analytical gel filtration and analytical ultracentrifugation assay showed that A35^ΔDB was expressed as a single peak with a size of ~27 kDa, the molecular weight of a A35^ΔDB dimer (Fig. 1c and Extended Data Fig. 2b). A surface plasmon resonance (SPR) assay confirmed the interaction between A35^ΔDB and A27D7 (Fig. 1e), a cross-neutralizing antibody that recognizes the dimeric epitope of VACV A33 and its orthopoxviruses ortholog²⁶, suggesting that A35^ΔDB was a dimer in solution, despite the absence of the intermolecular covalent bond. However, i.m. administration of two doses of 10 μg A35^ΔDB to BALB/c mice at 3-week intervals did not elicit A35-specific IgG responses (Fig. 1d), while low titers of A35-specific IgG were detected after the third dose (Fig. 1d) compared to A35^Ecto vaccination. Mass spectrometry analysis of the two bands detected by SDS–PAGE in reducing conditions for A35^Ecto and A35^ΔDB (Fig. 1c) indicated they differed in the number of glycosylation sites and the size of the glycan moieties attached (Extended Data Figs. 3 and 4). To investigate the influence of the stalk region in maintaining the immunogenicity of MPXV A35, two A35^Ecto mutants that lacked the interprotomer disulfide connection, A35^Ecto-C62A, which contained a Cys62Ala substitution, and A35^Ecto-ΔC62, which contained a Cys62 deletion (Ser64–Thr181) were expressed and purified. Both mutants behaved as homogenous homodimers (Extended Data Fig. 2d–g) and, when administered to BALB/c mice with two doses of 10 μg each at 3-week intervals, elicited a robust A35-specific response similar to that induced by A35^Ecto (Extended Data Fig. 2h). These results suggest that the stalk region is dispensable for the dimerization of A35^Ecto in vitro, but is required for its immunogenicity.

Fig. 1: Single-chain dimeric design maintains the immunogenicity of MPXV A35.

a, Schematic representation of MPXV A35 (top) and crystal structure of VACV A33 ectodomain (PBD 4M1G)²⁶ (bottom). Cysteine (C62)-mediated interprotomer disulfide bond, the N terminus and C terminus of the protein are labeled. TM, transmembrane domain. b, Schematic representation of A35^scDimer (top) and the cartoon representation of the A35^scDimer design (bottom). The N terminus, C terminus and the amino acids at the fusion site are shown. c, Analytical gel filtration profile of A35^Ecto (top), A35^ΔDB (middle) or A35^scDimer (bottom) protein with HiLoad 16/600 Superdex 200 pg column. Reducing and non-reducing SDS–PAGE analyses of the peak fraction and the mass in kilodaltons (kDa) of the selected molecular weight marker (M) species are shown. The blue arrowhead indicates the difference of A35^Ecto in reduced and non-reduced SDS–PAGE. The SDS–PAGE analyses are representative of three independent experiments. d, ELISA of IgG titers specific to A35^ΔDB, A35^Ecto or A35^scDimerafter dose 1 (day 19), dose 2 (day 40) and dose 3 (day 54) immunization with each immunogen (n = 5). a,b, Structures generated using Pymol (https://pymol.org/2/). Data indicate the mean ± s.d. P values were determined by one-way analysis of variance (ANOVA) with multiple-comparison tests. NS, not significant; P > 0.05. e,f, SPR assay showing the binding of A35^ΔDB (e) and A35^scDimer (f) to the A27D7 antibody. The saturation curve is shown by the blue line, and the BIAcore diagrams are displayed in red. mAU, milli-absorbance unit.

To eliminate the heterogeneity caused by disulfide bonds in the expression and purification procedures, we designed a single-chain dimeric form of A35 (A35^scDimer). We modeled the monomeric structure of A35 using the AlphaFold program²⁷ and obtained the structure of dimeric A35 based on the VACV A33 dimer structure. In the dimeric A35, the N and C termini of the two protomers were positioned as the same side of the molecule and adjacent to each other (Fig. 1b). Considering the role of the stalk region in the immunogenicity of A35, residues Ser64–Ser89 were used as an intermolecular linker to combine the two protomer (Fig. 1b). The A35^scDimer expressed in Expi293F cells was purified as a single peak of ~28 kDa (Fig. 1c and Extended Data Fig. 2c) and correctly folded to possess an exposed dimeric neutralizing antibody epitope, as demonstrated by binding to A27D7 in SPR assays (Fig. 1f). When injected i.m. in BALB/c mice in three doses of 10 μg each at 3-week intervals, A35^scDimer elicited A35-specific IgG at titers of nearly 10⁴ after dose 2, and reached 10⁵ after dose 3 (Fig. 1d), which was markedly higher than that of A35^ΔDB after any of the doses and similar to A35^Ecto immunization after dose 3 (Fig. 1j). Taken together, the single-chain dimeric design of A35^scDimer effectively eliminated the natural heterogeneity of A35^Ecto while maintaining its immunogenicity.

Structure guides the design of DAM chimeric immunogen

To produce a chimeric immunogen that would provide protection against both EEVs and IMVs, we sought to combine the IMV antigen M1 with the A35^scDimer. Because L1 and M1 have only two or four amino acid differences (varying between different strains; Extended Data Fig. 5), the structure of VACV L1, which suggests a possible dimeric morphology form of L1 (refs. ^28,29,30), was used as reference in the design of the chimeric immunogen. The immunogen, called DAM, was expressed as a continuous polypeptide chain with one M1 inserted between the two A35 domains of the A35^scDimer and one M1 inserted at the C terminus, to maintain the potent M1 dimeric morphology (Fig. 2a). No exogenous sequences were introduced. DAM was expressed at high levels in Expi293F cells and was purified from the supernatant with high purity (Fig. 2b). Analytical gel filtration indicated DAM was expressed as a single peak with a size of more than 70 kDa (Fig. 2b). Size-exclusion chromatography coupled to multi-angle light scattering (SEC–MALS) further confirmed the molecular weight at 75.5 kDa (Fig. 2c).

Fig. 2: The chimeric immunogen DAM shows full antigenicity and exposed epitopes of two components.

a, Schematic representation of the DAM design. SP, signal peptide. Numbers indicate the amino acids contained in A35 (red) and M1 (blue) domains of DAM. b, Analytical gel filtration profile of DAM protein with HiLoad 16/600 Superdex 200 pg. Reducing SDS–PAGE analysis of the peak fraction and the mass in kDa of the selected molecular weight marker species are shown. Protein purification and SDS–PAGE analysis are representative of more than three experiments. c, Mass measurement and elution profile of DAM determined by SEC–MALS. Molar mass and refractive index are plotted. d,f, SPR assay of binding characteristics of DAM (d) or M1 (f) to antibody 7D11. The raw and fitted curves are displayed as red and black lines, respectively. e, SPR assay of DAM binding to antibody A27D7. The saturation curve is displayed in blue, and the BIAcore diagrams are displayed in red. g, BLI assay of biotin-labeled DAM linked to streptavidin (SA) biosensors saturated with the DAM neutralizing antibodies A27D7 (top) or 7D11 (bottom) and further incubated for additional binding to 7D11 (top) or A27D7 (bottom) to assess the competition for the DAM M1 and A35 epitopes. h, Cryo-EM density map and fitted model of DAM in complex with A27D7-Fab and 7D11-scFv. A35 dimer, red; M1, blue; A27D7-Fab, orange; and 7D11-scFv, cyan.

In SPR assays, DAM was recognized by the VACV L1-specific neutralizing antibody 7D11 and the VACV A33-specific neutralizing antibody A27D7 (Fig. 2d,e), indicating individual epitopes were exposed. Compared to the interaction between M1 and 7D11, 7D11 bound to DAM with a comparable on-rate constant (DAM k_on, 8.57 × 10⁵ M⁻¹s⁻¹; M1 k_on 1.44 × 6 M⁻¹s⁻¹) but a much slower off-rate constant (DAM k_off, 9.08 × 10⁻⁴ s⁻¹; M1 k_off 4.76 × 10⁻³ s⁻¹; Fig. 2d,f), which could be due to the divalent M1 epitopes in the DAM and the resulting avidity effects. The steady state affinity of DAM for A27D7 was 1.48 μM (Fig. 2e), nearly four times higher than that of A35^scDimer. Bio-layer interferometry (BLI) indicated there was no competition between 7D11 and A27D7 when binding DAM concurrently (Fig. 2g), indicating the M1 and A35 epitopes remained accessible and were not overlapping.

We could not determine the intact cryo-electron microscopy (cryo-EM) structure of DAM bound to the antigen-binding fragment (Fab) of A27D7 and the single-chain fragment variable (scFv) of 7D11 (DAM–A27D7-Fab–7D11-scFv) with high resolution, due to the preferred orientation of the particles and the flexible connection between domains. Using different sample preparation methods, the structure of the complex was determined in two ways, referred to as CS1 and CS2 (Extended Data Table 1). CS1 provides a density map of the intact DAM with the A35 dimer and the two 7D11-scFv-bound M1 domains visible, whereas only half of A27D7-Fab was present (Extended Data Fig. 6). CS2 determined a density map of an intact A27D7-Fab, but only the A35 half of DAM (Extended Data Fig. 7). Combining CS1 and CS2 displayed the complete map of the DAM–A27D7-Fab–7D11-scFv complex (Fig. 2h). A model fitted on published atomic models of 7D11–VACV L1 (PDB 2I9L)²⁸ and A27D7–VACV A33 (PDB 4M1G)²⁶ complexes showed that DAM adopts a ‘chest and arm’ configuration, with the A35 dimer acting as a chest-shaped core and M1s prominently located in its flanks (Fig. 2h). The combined cryo-EM density maps present a quaternary complex, in which the density of either two 7D11-scFvs or A27D7-Fab can be confirmed (Fig. 2h). These data confirm that the M1 and A35 epitopes are exposed and that DAM has full antigenicity.

DAM elicits a high level of cross-reactive antibodies

To assess the immunogenicity of DAM, BALB/c mice were administered i.m. with three 10 μg doses of DAM, M1, A35^scDimer, 5 μg M1 combined with 5 μg A35^scDimer or PBS, all adjuvanted with AddaVax, at 3-week intervals. In addition, a group of BALB/c mice were immunized with 10⁷ plaque forming units (PFUs) of live VACV strain Tian Tan (VACV-VTT) by tail scarification once at day 0 (Fig. 3a). ELISA of immunogen-specific IgG after each dose at day 19 (after dose 1), day 40 (after dose 2) and day 54 (after dose 3) indicated DAM elicited a robust DAM-specific antibody response of 10⁵ after dose 1 (Fig. 3b). The antibody response increased steadily after the second and third doses, reaching a titer of almost 10⁷ after dose 3 (Fig. 3b). The A35-specific IgG titers were slightly lower than those for M1 after dose 3 (Fig. 3c), but the antibody responses against both epitopes were much stronger compared to either immunization with M1 or A35^scDimer alone or co-immunization with M1 plus A35^scDimer, especially after dose 1 and after dose 2 (Fig. 3c). Compared to the post-dose 2 timepoint, DAM-induced A35-specific IgG increased slightly, while M1-specific IgG remained at the same level after dose 3 (Fig. 3c).

Fig. 3: DAM is a superior immunogen that elicits cross-reactive antibodies.

a, Time-course measurements of immunization, serum collection, viral challenge and lung tissue collection in 6-week-old female BALB/c mice vaccinated i.m. with three 10 μg doses of DAM, M1, A35^scDimer, 5 μg M1 combined with 5 μg A35^scDimer or PBS, all adjuvanted with AddaVax, at 3-week intervals (n = 5 for each group), or immunized by scarification once at day 0 with 10⁷ PFUs per milliliter of live VACV (n = 5), followed by i.n. challenge with 7 LD₅₀ of VACV-WR. Serum samples analyzed after dose 1 (day 19), dose 2 (day 40) and dose 3 (day 54); lung tissue analyzed at days 1–5, 7 and 9 after challenge (n = 3 or 4 for each timepoint). b, ELISA of DAM-specific, A35^scDimer-specific or M1-specific IgG titers at day 19 (after dose 1), day 40 (after dose 2) and day 54 (after dose 3) in the serum of BALB/c mice as in a, and of VACV-specific IgG titers that used the VACV-infected cell lysates as antigen in the serum of VACV-scarified BALB/c mice at day 19, day 40 and day 54 after live VACV scarification. c, ELISA of A35-specific (left) and M1-specific (right) IgG titers after dose 1 (day 19), after dose 2 (day 40) and after dose 3 (day 54) in BALB/c mice as in a. d, ELISA of cross-reactive antibodies to VACV A33, VARV A36 and MPXV A35 induced by DAM or live VACV vaccination after dose 3 immunization (day 54, n = 5). e, PRNT assay of the VACV-WR (IMV form)-specific neutralizing activity of serum from BALB/c mice immunized with M1 (n = 5), M1 + A35^scDimer (n = 5) or DAM (n = 5) at day 40 (after dose 2) or day 54 (after dose 3), or live VACV (n = 5) at day 54 after scarification. f, PRNT assay of the MPXV-specific neutralizing activity in the serum of BALB/c mice immunized with DAM (n = 5) at day 54 (after dose 3) or scarified with live VACV (n = 5) at day 54 after scarification. The values shown in b–f indicate the mean ± s.d. P values in b–f were determined by one-way ANOVA with multiple-comparison tests. NS, P > 0.05.

The VACV A33-specific neutralizing antibody A2C7 does not bind to ECTV or CPXV²⁶, indicating a serological difference between the A35 homologs, despite having high sequence identity. DAM-elicited antisera showed cross-reactivity to VACV A33 and VARV A36 (Fig. 3d), but the binding was lower compared to MPXV A35. Antisera from mice with VACV scarification showed stronger recognition of VACV A33 and VARV A36 compared to that of MPXV A35 (Fig. 3d). In a plaque reduction neutralization test (PRNT) assay, serum collected after dose 2 and dose 3 of DAM vaccination showed potent neutralization against the IMV forms of VACV strain Western Reserve (VACV-WR), with a mean 50% PRNT titer (PRNT₅₀) of 5,623 (10^3.75) after dose 3, compared to co-immunization with M1 + A35^scDimer (10^3.23) or VACV scarification (10^2.79; Fig. 3e). In addition, serum collected after dose 3 of DAM vaccination elicited a robust neutralizing antibody response to MPXV, with the PRNT₅₀ titer of up to 30,903 (10^4.49), 28-fold higher than that induced by VACV scarification (Fig. 3f). These data suggest that DAM is a strong immunogen, with significant immunogenicity and neutralizing antibody elicitation against MPXV and VACV.

DAM protects against lethal VACV challenges in mice

To explore the protective efficacy of DAM in vivo, BALB/c mice immunized with DAM, M1, A35^scDimer, M1 + A35^scDimer, VACV-VTT or PBS as above were infected intranasally (i.n.) with 7 LD₅₀ (2 × 10⁵ PFUs) of VACV-WR at day 56 (after dose 3), and weighed daily for 2 weeks before reaching the euthanasia point (weight loss of 25% of the starting weight; Fig. 3a). Compared to mice that received PBS, which died within 6 d, DAM vaccination provided complete protection against body weight loss and mortality, at a level comparable to, if not higher, than that provided by scarification with VACV or M1 + A35^scDimer co-immunization (Fig. 4a,b). Mice immunized with M1 or A35^scDimer alone had an average weight loss of more than 20% of their starting weights and 40% of them died at day 5 and day 6 after infection (Fig. 4a,b) indicating partial protection. Time-course measurements of viral titers in the lung tissue of mice immunized and challenged as above at day 1–5, day 7 and day 9 after VACV-WR challenge indicated that mice with live VACV vaccine scarification showed rapid clearance of virus in the lungs, with only one of three mice showing detectable virus in the lung at day 2 (Fig. 4c). In the DAM-immunized mice, the virus could be detected in two of three mice and two of four mice at day 2 and day 3 after challenge, respectively, while the virus titer was nearly 1,000-fold lower than that of PBS-immunized mice at these timepoints (Fig. 4c). Virus titers were below the detection level in the VACV scarification or DAM-immunized mice after day 3 or day 4, respectively, but persisted in 50% of the mice on day 5 and reached complete clearance on day 7 in the M1 + A35^scDimer co-immunized mice (Fig. 4c), indicating co-immunization was less effective in virus clearance. In the PBS-immunized mice, virus titers were up to 10⁸ on day 2, rising to 10⁹ on day 5, and all mice died at day 5 (Fig. 4c). These results indicate that the DAM can provide complete protection to mice against the lethal VACV challenge, with improved clearance of virus in lung compared to M1 + A35^scDimer co-immunization, although the viral clearance was not as efficient as that induced by live VACV scarification.

Fig. 4: DAM provides full protection against lethal VACV challenge in mice.

a,b, Body weight (a) and survival (b) in mice as in Fig. 3a after i.n. challenge with 7 LD₅₀ of VACV-WR. c, Virus titers in the lung of BALB/c mice vaccinated i.m. with three 10 μg doses of DAM (n = 3 or 4), PBS (n = 3 or 4) or 5 μg M1 combined with 5 μg A35^scDimer (n = 3 or 4), all adjuvanted with AddaVax, at 3-week intervals, or immunized once at day 0 with 10⁷ PFUs per milliliter of live VACV (n = 3 or 4) at days 1–5, day 7 and day 9 after i.n. challenge with 7 LD₅₀ of VACV-WR. Data are presented as PFUs per gram of lung tissue. The values indicate the mean ± s.e.m.

Alum-adjuvanted DAM shows safety profile and efficacy in mice

To further evaluate the efficacy of DAM, BALB/c mice were vaccinated with three doses of 2 μg or 10 μg AddaVax-adjuvanted DAM or PBS at 3-week intervals. Either 2 μg or 10 μg DAM induced comparable DAM-specific IgG titers (Fig. 5a) and VACV-specific neutralizing antibody titer (Fig. 5b) after dose 2 (day 40) or after dose 3 (day 54). A 2 μg dose of DAM provided 100% protection against lethal challenge with VACV-WR (Fig. 5c), although VACV-WR-infected mice showed weight loss after day 2 after infection, with the lowest point (−7.7%) occurring at day 4 after infection (Fig. 5d).

Fig. 5: Alum-adjuvanted DAM vaccine provides protection against lethal VACV challenge in mice.

a, ELISA of DAM-specific IgG titers in BALB/c mice vaccinated with 2 μg (n = 5) or 10 μg (n = 5) AddaVax-adjuvanted DAM three times at 3-week intervals at day 19 (after dose 1), day 40 (after dose 2) and day 54 (after dose 3) after first immunization. The values indicated the mean ± s.d. b, PRNT assay showing the neutralizing antibody titer against the IMV form of VACV-WR at day 40 (after dose 2) and day 54 (after dose 3) in BALB/c mice immunized as in a. The values indicate the mean ± s.d. c,d, Survival curve (c) and body weight change (d) in BALB/c mice immunized with 2 μg (n = 5) or 10 μg (n = 5) DAM as in a at days 0–14 after i.n. challenge with 7 LD₅₀ of VACV-WR. The values are the mean ± s.e.m. e, ELISA showing A35-specific (left) or M1-specific (right) IgG titers in BALB/c mice at day 19 (after dose 1), day 40 (after dose 2) and day 54 (after dose 3) after immunization with 2 μg (n = 5), 10 μg (n = 5) or 30 μg (n = 5) alum-based DAM three times at 3-week intervals. The values indicate the mean ± s.d. f, PRNT assay showing the neutralizing antibody titer against the IMV form VACV-WR in BALB/c mice at day 40 (after dose 2) and day 54 (after dose 3) after immunization with 2 μg (n = 5), 10 μg (n = 5) or 30 μg (n = 5) alum-based DAM as in e after dose 2 and dose 3 (day 40 and 54 after initial immunization; n = 5). The values indicate the mean ± s.d. g,h, Survival curve (g) and body weight change (h) in BALB/c mice immunized with 2 μg, 10 μg or 30 μg alum-based DAM (as in e) during 14 d after i.n. challenge with 7 LD₅₀ of VACV-WR (n = 5). Values are the mean ± s.e.m. P values in a and b were determined by two-sided t-test; NS, P > 0.05. P values in e, f and h were determined by one-way ANOVA with multiple-comparison tests; NS, P > 0.05. For h, P values represent DAM/Alu/2 μg compared to VACV-VTT (in orange), DAM/Alu/10 μg compared to VACV-VTT (in red), DAM/Alu/30 μg compared to VACV-VTT (in blue) and DAM/Alu/2 μg compared to DAM/Alu/30 μg (in black), respectively.

Aluminum salts are adjuvants used worldwide with a high safety profile. To evaluate the protective efficacy of DAM when formulated with an alum-based adjuvant, BALB/c mice were injected i.m. with three doses of 2 μg, 10 μg or 30 μg each of alum-adjuvanted DAM at 3-week intervals. A35-specific and M1-specific IgG antibodies were robustly and equally induced with 10 μg or 30 μg DAM after dose 3 (day 54) compared to 2 μg DAM vaccination (Fig. 5e). PRNT assays indicated that mice vaccinated with 30 μg DAM generated VACV-specific neutralizing antibody exhibiting a mean PRNT₅₀ titer of 1,334 (10^3.13) after dose 3 (Fig. 5f). No statistical difference in neutralizing antibody titer was observed between 2 μg, 10 μg and 30 μg DAM after the second and third doses (Fig. 5f).

To test the in vivo protection of the alum-adjuvanted DAM vaccine, BALB/c mice immunized i.m. with 2 μg, 10 μg or 30 μg alum-adjuvanted DAM or alum-adjuvanted PBS three times at 3-week intervals, or scarified with live VACV once were challenged i.n. with 7 LD₅₀ of VACV-WR after dose 3 (day 56), and weighed daily for 2 weeks before reaching the euthanasia point (Fig. 3a). All DAM doses induced protection against lethal VACV infection (Fig. 5g), while the alum-based 30 μg DAM vaccine showed the highest average body weight, even when compared to live VACV scarification (Fig. 5h). Mice immunized with 2 μg or 10 μg alum-adjuvanted DAM had a reduction in body weight of up to 10.49% at day 5 or 7.81% at day 6, respectively, and recovered to near their initial body weight by week 2 after viral challenge (Fig. 5h).

Next, we evaluated the persistence of DAM-induced antibody responses. Mice that received three 10 μg doses of alum-adjuvanted DAM presented no significant reduction in DAM-specific IgG titer and neutralizing antibody levels at 6 months after dose 3 (Extended Data Fig. 8a,b), indicating long-lasting effects. The safety profile was further evaluated in mice that received a 4^th dose of 10 μg alum-adjuvanted DAM 6 months after dose 3. No significant changes in body weight were detected before (day 0) and after immunization with the 4^th dose (day 1 to day 8 after dose 4; Extended Data Fig. 8c). There were no statistically significant alterations in the functional assessment of the heart, liver and kidney based on blood biochemical parameters between mice vaccinated with dose 4 of 10 μg alum-adjuvanted DAM and mice vaccinated with dose 4 of alum-adjuvanted PBS or mice 6 months after VACV scarification, and all the blood biochemical parameters were within normal limits (Extended Data Fig. 8d–h). Histopathological examination found no obvious gross or microscopic changes in liver, heart, spleen, lung, kidney and intestine in these groups (Extended Data Fig. 8i), indicating the DAM vaccine had a favorable safety profile in mice.

To assess the MPXV-specific T cell response induced by DAM vaccination, peripheral blood mononuclear cells (PBMCs) were collected at day 7 after dose 4 and stimulated with A35 and M1 peptide pools. The cellular immune response was assessed by measuring the level of interleukin (IL)-2, IL-4 and interferon-γ cytokines in an enzyme-linked ImmunoSpot (ELISpot) assay. Moderate production of IL-2 (as a measure of the T_H1 subset of helper T cell responses) and IL-4 (the T_H2 subset of helper T cell responses) was detected in PBMCs from DAM-vaccinated mice stimulated with M1 peptide pools, while only IL-4 was differentially induced by A35 peptide pools in DAM-vaccinated mice compared to PBS-vaccinated mice (Extended Data Fig. 8j).

To develop an industry-standard stable CHO cell line for further Good Manufacturing Practice-grade production of DAM in preclinical studies, DAM was constructed without a purification tag, transduced in clinical-grade CHO cell lines and screened for antigen expression. DAM could be produced at the high yield of 1.18 g purified antigen per liter and reached 93.8% purity after one-step purification of ion-exchange chromatography as verified by size-exclusion chromatography high-performance liquid chromatography (SEC-HPLC) and gel electrophoresis analyses (Extended Data Fig. 9), suggesting the highly scalable production of DAM. The constant efficacy of alum-adjuvanted DAM, along with its favorable safety profile and high yield in industrial cell lines indicated that DAM has considerable potential for industrial development.

Discussion

In this study, we explored the possibility of using a single-immunogen vaccine as an alternative to the live VACV vaccine against MPXV. By structure-guided design, a two-component chimeric immunogen DAM was constructed and evaluated as a subunit vaccine. DAM provided superior specific antibody responses and virus clearance compared to co-immunization with both antigens and elicited a 28-fold higher MPXV-specific neutralizing response than live VACV vaccine scarification, with a safe and scalable profile.

Next-generation vaccines against poxviruses with a better safety profile are of high priority, and mpox vaccines using the mRNA platform have been proposed^{31,32,33,34,35,36,37}. However, almost all approaches have adopted a co-immunization strategy with multiple genes, which increases the difficulty and cost of industrialization. Even when five antigens were linked in tandem by 2A peptides to create a single mRNA vaccine, there were still co-immunization issues with unbalanced bioavailability and inconsistent immunogenicity, as shown by the partial immunogenicity of this vaccine against two of the five targets³¹. The chimeric DAM showed distinct advantages compared to co-immunization, by inducing higher levels of specific and neutralizing antibodies and more efficient virus clearance in vivo.

We found that the stalk region had an important role in the superior immunogenicity of MPXV A35 by increasing the molecular weight of A35, which is known to be crucial in determining the immunogenicity of proteins, especially those with smaller molecular weights, and possibly by stabilizing the dimer configuration of the extracellular domain of A35, particularly in vivo. This suggests that although a set of antigens might have good immunogenicity and protective potential for the development of poxvirus vaccines, their immunogenicity might be limited by specific conditions. Structure-guided design of immunogens, as exemplified by DAM, allows the preservation of immunogenicity of each antigen based on a thorough understanding of the structure and protective mechanism of each antigen.

Although poxviruses induce cross-reactive antibodies between species³⁸, epitope variations of several poxvirus antigens have been reported. A study of seven VACV A33-specific monoclonal antibodies²⁶ found that, of the five antibodies that neutralized VACV in the presence of complement, only A27D7, which recognizes a dimeric epitope, was a potent cross-neutralizing antibody. Despite only four amino acid differences between VACV L1 and its homolog in ECTV, EVM072, a D35N substitution in EVM072 was sufficient to cause the loss of cross-neutralizing ability of VACV neutralizing antibody M12B9 against ECTV, the causative agent of mousepox²⁹. Here we found that while DAM was able to generate cross-reactive antibodies against VARV A36 and VACV A33, it produced a more robust immune response specific to MPXV A35 compared to the other two homologs. Conversely, VACV scarification elicited an antibody response to VACV A33 and VARV A36 that was ~10-fold higher than that to MPXV A35. Although we only investigated cross-recognition against MPXV A35, we found that DAM elicited a markedly stronger neutralizing antibody response, with a PRNT₅₀ titer 28 times higher than that induced by live VACV vaccine, against MPXV. Our observations suggested that antibodies induced by MPXV antigens conferred a higher level of MPXV-specific neutralization and therefore possibly a greater protection against homologous challenge compared to those induced by vaccinia antigens. These results should be further investigated in MPXV-infected nonhuman primates. In addition, the A35 and M1 epitopes in DAM can be replaced by their orthologs from VARV or CPXV to produce a chimeric vaccine, the administration of which might elicit broader neutralization and better protection against different poxviruses. Furthermore, DAM can be applied to other vaccine platforms, such as mRNA, DNA and viral vectors, to meet a wide range of application requirements and demonstrating the wide-ranging applications of this approach.

Methods

Ethics statement

Animal studies were approved by the Committee on the Ethics of Animal Experiments of the Institute of Microbiology, Chinese Academy of Sciences (IMCAS), and conducted in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the IMCAS Ethics Committee. All challenge studies with VACV-WR were approved by the Committee on the Ethics of Animal Experiments of the IMCAS.

Cells and viruses

African green monkey kidney epithelial cells (Vero cells; American Type Culture Collection, CCL81), were maintained in DMEM (Invitrogen) supplemented with 10% FBS at 37 °C under 5% CO₂. Expi293F cells (Thermo Fisher Scientific) were cultured in medium at 37 °C under 5% CO₂. All cell lines tested negative for mycoplasma contamination.

VACV-WR and VACV-VTT were kindly provided by M. Fang (IMCAS) and W. Tan (National Institute for Viral Disease Control and Prevention, China CDC), respectively, and were propagated in Vero cells in culture media (DMEM containing 2% FBS plus 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin) as described before³⁹. VACV was manipulated under BSL-2 conditions. FBS used in all experiments was inactivated by heat at 56 °C for 30 min before use. Purified VACV stock was stored at −80 °C.

Mice

Specific-pathogen-free 6- to 8-week-old female BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology (licensed by Charles River) and were housed with five companions at most per cage at random. All mice used in this study were in good health and were not involved in other experimental procedures. Data collection and analysis were not performed blind to the conditions of the experiments. Mice were allowed free access to water and standard chow diet as well as provided with a 12-h light and dark cycle (temperature of 20–25 °C, humidity of 40–70%). Mice were housed under specific-pathogen-free conditions in the laboratory animal facilities at IMCAS. The challenge experiments and scarification operation using live VACV were conducted in the animal biosafety level 2 facility of IMCAS. All these experiments received ethical approval from the research ethics committee of the IMCAS. The number of mice used in each experiment is shown in the figure legends.

Protein expression and purification

The MPXV vaccine proteins (A35^scDimer and DAM), the proteins used in ELISA, were expressed with the Expi293F expression system. To express them, the coding sequences for A35^Ecto, A35^Ecto-ΔC62, A35^Ecto-C62A, A35^scDimer and DAM (the constructs were described before, GenBank: ON563414.3), as well as the A35^ΔDB (residues Ser90–Thr181, GenBank: ON563414.3), VACV A33 (residues Ser90–Asn185, GenBank: AY243312.1) and VARV A36 (residues Ser89–Asn184, GenBank: NC_001611.1) were codon-optimized and synthesized by GENEWIZ, China. For each construct, sequences encoding the signal peptide of Igκ and hexa-His tag were added to the 5′ and 3′ termini, respectively. The plasmid was transiently transfected into Expi293F cells. The supernatant was collected 5 d after transfection, and the soluble protein within the supernatant was purified by metal affinity chromatography using HisTrap HP 5 ml columns (GE Healthcare). The samples were further purified via gel filtration chromatography with HiLoad 16/600 Superdex 200 pg (GE Healthcare) in the buffer composed of 20 mM Tris-HCl (pH 7.4) and 150 mM NaCl or PBS. The eluted peaks were analyzed by SDS–PAGE for protein size and purity.

The 7D11 and A27D7 antibodies were also expressed in the Expi293F expression system. The coding sequence of the variable region of each antibody was synthesized according to the amino acid sequences submitted to the Protein Data Bank. The heavy chains were fused with the constant region of mouse IgG1 (7D11) or IgG2a (A27D7) and the light chains were fused with Igκ. Both sequences were then cloned into the pCAGGS vector. For each antibody, the heavy and light chain expressing plasmids were transiently co-transfected into Expi293F cells at a ratio of 1:1.5 using polyethylenimine. The supernatant was collected 5 d after transfection and A27D7 was purified using HiTrap Protein A HP (5 ml) columns (GE Healthcare) and 7D11 was purified using HiTrap Protein G HP (5 ml) columns (GE Healthcare) according to the manufacturer’s instructions for primary purification. The samples were further purified via gel filtration chromatography with HiLoad 16/600 Superdex 200 pg (GE Healthcare) in PBS. The eluted peaks were analyzed by SDS–PAGE for protein size and purity. The A27D7-Fab was generated by papain digestion and further purified using a Protein A column followed by gel filtration using a Superdex 200 10/300 GL column.

The proteins of MPXV-M1 and 7D11-scFv were expressed in Escherichia coli cells as inclusion bodies, and the soluble proteins were subsequently obtained by in vitro refolding via a previously described dialysis method⁴⁰. To express the MPXV-M1 protein, the cDNAs encoding residues 1–181 of MPXV-M1 (GenBank: ON563414.3) were individually cloned into the NdeI and XhoI sites of the pET-28a vector (Invitrogen). 7D11-scFv was constructed as VL-(GGGGS)₄-VH as previously described⁴¹ and cloned into the pET-21a expression vector (Invitrogen). After preparation, these soluble proteins were then purified by gel filtration on a Superdex 200 Increase 10/300 GL column (GE Healthcare) with a running buffer of PBS (pH 7.4). The purity and size of the proteins were analyzed by SDS–PAGE.

Mouse experiments

For immunization of mice, each antigen was diluted with PBS and emulsified in an equal volume of AddaVax adjuvant (InvivoGen) or alum adjuvant (InvivoGen) by a rotary mixer for 2 h at 4 °C. BALB/c mice were vaccinated via the i.m. injection. Serum samples were collected after vaccination as indicated in figure legends. For live virus immunization, 10 μl of the VACV-VTT stock (10⁹ PFUs per ml) was deposited at the base of the mouse tail, and the skin at the site of the droplet was scarified 25 to 30 times with a needle of a 0.3-ml insulin syringe. After 3 to 4 d, pustules or scabs were observed at the scarification site, indicating a successful VACV immunization.

For VACV-WR challenge experiments, mice were i.n. transduced with 50 μl of VACV-WR to a final titer of 2 × 10⁵ PFUs after the last immunization as indicated in the figure legends. Mice were weighed each day and were euthanized when they lost 25% of their initial body weight.

For lung tissue collection, the mice (n = 3 or 4) were euthanized and necropsied at 1, 2, 3, 4, 5, 7 and 9 d after challenge and lung tissues were split for virus titer determination. The tissues were weighed, homogenized and centrifugated at 212g for 10 min. Around 100 μl supernatant serial dilution from 1:10¹ to 1:10⁶ was performed on Vero cells in 12-well plates, and mixed with 300 μl culture media. After 1 h incubation at 37 °C, 5% CO₂ atmosphere, the medium was removed and the well was washed once by PBS, then overlaid with culture media containing 1% carboxymethylcellulose, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. The plates were incubated for 2 d and then plaques were visualized by crystal violet staining. The numbers of spots were measured by ELISpot reader and ImmunoSpot image analysis software (Immuno Capture 6.5.0).

ELISA

ELISA plates (Corning) were coated overnight with 2 μg ml⁻¹ of A35^ΔDB, A35^Ecto, M1, A35^scDimer, DAM or infected cell lysates in 0.05 M carbonate-bicarbonate buffer, pH 9.6, and blocked in 10% skim milk in PBS. Serum samples were diluted and added to each well. Plates were incubated with goat anti-mouse IgG-horseradish peroxidase antibody in a 1:4,000 dilution (EASYBIO) and developed with 3,3′,5,5′-tetramethylbenzidine substrate. Reactions were stopped with 2 M hydrochloric acid, and the absorbance was measured at 450 nm using a microplate reader (PerkinElmer). The endpoint titer was defined as the highest reciprocal dilution of serum to give an absorbance greater than 2.5-fold of the background values. Antibody titer below the limit of detection was determined as half the limit of detection.

The infected cell lysate for use in antibody binding assays was prepared by inoculating two T-150 flasks of Vero cell monolayers with VACV-WR. After 3 d, infected cells were harvested and collected by centrifugation at 212g for 5 min. The cell pellets were resuspended in 1 ml PBS in a grinding tube, followed by grinder homogenization. The homogenates were centrifuged at 212g for 5 min to pellet nuclei, and the supernatants were centrifuged at 112,845g for 30 min. The pellets were resuspended in PBS, and aliquots were stored at −80 °C.

VACV-WR neutralization assays

IMV of VACV-WR plaque reduction neutralization assays were carried out in duplicate by mixing ∼150 PFUs of IMV form VACV-WR with 2-fold dilutions of heat-inactivated sera from individual mouse at a concentration from 1:40 to 1:81,920 in a 400 μl final volume of culture media. After 2 h of incubation at 37 °C, a portion of the incubated mixture was then added to one well of a 12-well plate containing a confluent monolayer of Vero cells (American Type Culture Collection). After 1 h at 37 °C in a 5% CO₂ atmosphere, the medium was removed and the well was washed once by PBS, overlaid with culture media containing 1% carboxymethylcellulose, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. The plates were incubated for 2 d and then plaques were fixed with 4% neutral buffered formaldehyde and visualized by crystal violet staining. The numbers of spots were measured by ELISpot reader and ImmunoSpot image analysis software (Immuno Capture 6.5.0) and the PRNT₅₀ was calculated through the equation ‘log(inhibitor) versus normalized response -- variable slope’ by GraphPad.

MPXV neutralization assays

MPXV plaque reduction neutralization assays were conducted in the animal biosafety level 3 facility in Wuhan Institute of Biological Products. Briefly, the experiments were carried out in duplicate by mixing ∼150 PFUs of MPXV-IMV (strain WIBP-MPXV-001)⁴² with heat-inactivated sera of 4-fold dilutions from individual mice at a concentration from 1:40 to 1:40,960 or 1:40 to 1:655,360 in a 400 μl final volume of culture media. Neutralization and infection steps were the same as in the VACV-WR neutralization assays. The plates were incubated for 3–4 d and then plaques were fixed with 4% neutral buffered formaldehyde and visualized by crystal violet staining. PRNT₅₀ was calculated in the same way as in VACV-WR neutralization assays.

SPR assay

SPR binding experiments were carried out using a BIAcore 8000 device (GE Healthcare) at 25 °C in the single-cycle mode. All proteins for SPR assays were exchanged to PBST buffer (10 mM Na₂HPO₄; 2 mM KH₂PO₄, pH 7.4; 137 mM NaCl; 2.7 mM KCl; 0.05% Tween-20). The monoclonal antibody A27D7 or 7D11 expressed in vitro was captured by mouse IgG antibodies immobilized on the CM5 chip, respectively. Gradient concentrations of A35^ΔDB (from 1,000 to 16,000 nM for A27D7), A35^scDimer (from 1,000 to 16,000 nM for A27D7), M1 (from 1 to 16 nM for 7D11) and DAM (from 250 to 4,000 nM for A27D7, and from 1 to 16 nM for 7D11) were then used to flow over the chip surface at 30 μl min⁻¹ and the real-time response was recorded. After each cycle, the sensor surface was regenerated using 10 mM glycine-HCl (pH 1.5). The binding kinetics were analyzed using a 1:1 binding model with the software BIAevaluation version 4.1 (GE Healthcare). K_D values for SPR assays were calculated using a 1:1 (Langmuir) binding fit model with the BIAcore 8K evaluation software. The SPR binding assays were conducted three times, and the proteins from different batches were used in the replication experiment.

Binding competition assay

The biotin-labeled DAM was immobilized to an SA biosensor at 30 °C for 180 s. The associations of A27D7 monoclonal antibody and 7D11 monoclonal antibody were measured on the Octet RED96 (ForteBio) for 120 s at 30 °C by exposing the sensors to 460 nM monoclonal antibodies in PBST; then the degree of additional binding was assessed by exposing the sensors to a second round of monoclonal antibodies (460 nM in PBST) in the presence of the first Fab (460 nM) for 120 s at 30 °C. The data were collected by Octet Data Acquisition software 9.0. These assays were conducted three times, and the proteins from different batches were used in the replication experiment.

Cryo-EM data collection and three-dimensional reconstruction

We prepared two kinds of cryo-samples for the complex of DAM bound to A27D7-Fab and 7D11-scFv (DAM–A27D7-Fab–7D11-scFv) by using the same bath of complex protein samples. For the preparation of the cryo-sample 1, the 4 μl of complex protein sample of DAM–A27D7-Fab–7D11-scFv in 1.0 mg ml⁻¹ was applied to glow-discharged Quantifoil R 1.2/1.3 holey carbon grids with 2 nm continuous carbon on top and blotted for 0.5 s with a humidity of 100% before being plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher). For the cryo-sample 2, 4 μl of the same batch of the DAM–A27D7-Fab–7D11-scFv (1.0 mg ml⁻¹) was applied to a Cu Quantifoil R 1.2/1.3 holey carbon grid with glow discharged for 60 s and then blotted for 3 s with a humidity of 100% before being plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher).

The data collection processes for the two cryo-samples are the same. Briefly, the frozen grides were loaded onto a Titan Krios cryo-transmission electron microscope (Thermo Fisher) that is equipped with a BioQuantum energy filter (Gatan), operated at 300 kV for data collection. Automatic data collection was performed using EPU software (Thermo Fisher). Movies were recorded with a K3 direct electron in a super-resolution counting mode at pixel size of 0.69 Å. The exposure was performed with a dose rate of 26 e⁻ pixel s⁻¹ and an accumulative dose of 60 e⁻/Ų for each movie, which was fractionated into 32 sub-frames. The final defocus range of the datasets was approximately 1.0–2.0 μm.

The detailed data processing workflow is summarized in Extended Data Figs. 5 and 6. The drift correction of all stacks was performed with MotionCor2 (ref. ⁴³) to generate 2× binned images. Initial contrast transfer function values for each micrograph were calculated with CTFFIND4.1 (ref. ⁴⁴). The subsequent image processing and reconstruction were performed using cryoSPARC⁴⁵.

For the cryo-sample, 1,160,786 particles were picked from 25,181 micrographs. Then the picked particles were extracted and subjected to two rounds of reference-free two-dimensional (2D) classification in cryoSPARC. A clean dataset with 126,552 particles from good 2D classes was selected and the initial model was generated by cryoSPARC ab-initio reconstruction. After the second round of heterogeneous refinement, the predominant class contained a subset of 62,949 good particles. These particles were subjected to nonuniform refinement, which yielded a reconstruction at ~7.54 Å resolution as determined by the Fourier shell correlation cutoff value of 0.143. For the cryo-sample 2, a total of 3,416,737 initial particles from 11,622 micrographs were picked and extracted with the box size of 320 pixels. After three rounds of iterative 2D classification, a clean set of 1,036,929 particles was selected to generate the initial models and heterogeneous refinement and resulted in four distinct volumes. Then, one volume containing ~28.1% of total particles was subjected to two rounds of nonuniform refinement, which yielded a final density map at 3.88 Å resolution estimated by the gold-standard Fourier shell correlation cutoff value of 0.143.

Due to the fierce flexibility within DAM and its antibodies, we could only obtain low-resolution maps as described above. However, we fitted the structures of A27D7–VACV A33 (PDB 4M1G) and 7D11–VACV-L1 (PDB 2I9L) into those two complex density maps and combined them using CHIMERA⁴⁶, which showed a high degree of matching.

SEC–MALS

A 50 μl volume of each sample was injected onto the XBridge BEH 200 Å SEC column, 7.8 by 300 mm internal diameter; particle size, 3.5 µm; pore size, 200 Å, (Waters Corporation) on the Waters E2695 (Waters Corporation) with an isocratic elution at 0.5 ml min⁻¹ for 40 min, 50 mM PB (30 mM Na₂HPO₄·12H₂O; 20 mM NaH₂PO₄·2H₂O), 300 mM NaCl; pH 6.8 was used as the mobile phase. Ultraviolet absorbance was set at 280 nm using Empower 3 software (Waters Corporation). In tandem for detection, a MALS detector (Wyatt Technology) was set at 660 nm. BSA (Sigma-Aldrich) was used to normalize the static light scattering detector. The delay volume, band broadening parameters as well as the light scattering, and differential refractive index measurements were analyzed using Astra 8 software (Wyatt Technology).

Development of industry-standard CHO cell lines for DAM

The coding sequence for DAM protein was codon-optimized for mammalian cell expression with a signal peptide sequence added to the N terminus for protein secretion. The construct does not contain any purification tag sequence. Clinical-grade CHOZN-GS (MERCK) cell lines expressing DAM were generated and the clones with highest antigen expression and stability were selected by Shanghai JunTop Biosciences. This cell line was then used in the large-scale production of DAM. After expression, the supernatant was harvested, buffer changed to Buffer A (20 mM Tris-HCl, pH 7.5) using a 30 kDa ultrafiltration cassette (Vivaflow 200, Sartorius) and loaded onto a Capto Q (Cytiva) column. The column was then washed with two column volumes of buffer A, and the elution of purified proteins was performed by increasing the salt concentration in the buffer up to 1 M NaCl with a 20-column volume linear gradient from 0% to 100% buffer B (20 mM Tris-HCl, 1 M NaCl, pH 7.5). After one-step purification by AEX chromatography, the DAM protein was analyzed by both SDS–PAGE and SEC-HPLC.

ELISpot assay

To evaluate the cellular immune responses elicited by vaccines, PBMCs were collected at day 8 after immunization and stimulated with peptide pools (2 μg ml⁻¹ for each peptide). PMA plus ionomycin (Dakewe, 2030421) was added to positive control wells. Interferon-γ, IL-2 and IL-4 ELISpot assays were performed with ELISpot kits according to the manufacturer’s protocols (MabTech, 3311-2A, 3441-2A and 3321-2A). The numbers of spots were measured by ELISpot reader and ImmunoSpot image analysis software (Immuno Capture 6.5.0).

Analytical ultracentrifugation

Velocity sedimentation experiments were performed using a ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter), equipped with AN-60Ti rotor. The protein samples were kept in PBS (pH 7.4). Experiments were carried out at 20 °C and 205,000g, using continuous scan mode and radial spacing of 0.003 cm. Scans were collected in 3-min intervals at 280 nm. The fitting of absorbance versus cell radius data was performed using SEDFIT software and a continuous sedimentation coefficient distribution c(s) model.

Histopathology analysis

Five mice per group were necropsied on day 8 after immunization using a standard protocol. Livers, hearts, lungs, spleens, kidneys and intestines from mice were collected, fixed in 4% neutral buffered formaldehyde and embedded in paraffin. Tissue sections (5 mm) were stained with H&E and scales are shown in the figure legends.

Serum biochemical tests

Mouse serum was collected on day 8 after boost immunization for biochemical tests to analyze heart, liver and kidney function using a cobas 6000 c 501 (Roche) automated biochemical analyzer.

Mass spectrometry

Liquid chromatography separation was conducted using a Thermo Scientific EASY-nLC 1000 HPLC system. Mobile phases were composed of [A] water (0.1% formic acid) and [B] acetonitrile (0.1% formic acid). Peptides were desalted online by an in-house packed trap column (C18, 3-μm particles, 150 μm internal diameter, 2 cm length; Dr. Maisch HPLC). The trapped peptides were loaded onto an in-house packed reversed-phase C18 column, 150 μm internal diameter × 15 cm, 1.9 μm. The analytical gradient for the sample was 3–8% B in 3 min, 8–22% B over 40 min, 22–35% B over 8 min, 35–90% B over 4 min and 90% B over 5 min. Flow rate for analytical gradients was 500 nl min⁻¹. All samples were analyzed using an Orbitrap Fusion Tribrid mass spectrometer. Data was acquired on the Orbitrap Fusion MS using a resolution of 120,000 (200 m/z) for full MS scans (m/z range 350–1,550, 2.0 × 10⁵automatic gain control (AGC) target, and 50 ms maximal injection time) and using a resolution of 120,000 resolution. A data-dependent top 20 method was followed by Orbitrap HCD-MS/MS (fragmentation HCD; 33% collision energy; 1.0 × 10⁵ AGC target; maximal injection time dynamic; 30 s dynamic exclusion) and EThcD-MS/MS (fragmentation EThcD; 25% SA collision energy; 1.0 × 10⁵ AGC target; maximal injection time dynamic; 30 s dynamic exclusion) of the 20 most abundant multiply charged precursors in the MS1 spectrum. MS2 spectra were acquired at a resolution of 30,000. All MS/MS samples were confidently identified using Mascot (Matrix Science; version 2.8.0). Mascot was set up to search the databases of A35^ΔDB and A35^Ecto assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.02 Da and a parent ion tolerance of 10.0 PPM respectively. Carbamidomethyl of IAM was specified in Mascot as fixed modifications. Oxidation of DTT was specified in Mascot as variable modifications and error tolerance was specified in Mascot for the unknown modifications.

Statistics and reproducibility

No statistical method was used to predetermine sample size, but our sample size was based on existing reported research³⁸ and experience. No data points were excluded from analysis. All statistical analyses were performed using GraphPad Prism 8.0. Data are shown as the mean ± s.d. or mean ± s.e.m. An unpaired two-tailed t-test was used to determine statistical significance for comparison between two groups. P values were analyzed with t-test or one-way ANOVA with multiple-comparison tests. Differences with P < 0.05 were considered statistically significant. P > 0.05 was considered nonsignificant. Applied analytical methods and statistical significance are indicated in the corresponding legends. Data distribution was assumed to be normal, but this was not formally tested.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.