Prefusion-stabilized Hantaan virus glycoprotein nucleic acid vaccine elicits potent neutralizing antibody responses via germinal center activation

Domain: biology
Published: 2026-03-14
Editorial outcome: minor_revision

Abstract
Old World orthohantaviruses, including Hantaan virus (HTNV), cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia. Available inactivated vaccines often induce low neutralizing antibodies and short-term protection. We evaluated nucleic acid vaccines expressing a prefusion-stabilized HTNV glycoprotein in female BALB/c mice. Both DNA and mRNA-LNP versions elicited robust neutralizing antibodies by strongly activating germinal centers, which protected mice against high-dose HTNV challenge. We further tested heterologous prime-boost regimens, where mice primed with inactivated vaccine received different boosters. All boosters increased neutralizing titers, but only the prefusion-stabilized glycoprotein mRNA-LNP vaccine raised titers to the level achieved by its own full primary vaccination course. This demonstrates the immunogen’s superiority in developing next-generation vaccines and its unique ability to potently recall memory B cells induced by suboptimal inactivated vaccines. Thus, prefusion-stabilized glycoprotein-based nucleic acid vaccines are promising candidates for advanced orthohantavirus vaccine development.

Introduction
Rodent-borne Orthohantavirus (hereafter referred to as hantaviruses) causes two severe, often fatal human diseases; their distribution aligns with that of their natural rodent reservoirs 1, 2, 3. In Eurasia, “Old World” hantaviruses (OWHs) such as Hantaan virus (HTNV), Dobrava-Belgrade virus, and Puumala virus (PUUV) cause hemorrhagic fever with renal syndrome (HFRS), which has a mortality rate of up to 15% 3, 4, 5. The Seoul virus (SEOV) is distributed worldwide along with its natural host Rattus norvegicus 3, 4, 5. In the Americas, “New World” hantaviruses (NWHs) such as Sin Nombre virus and Andes virus (ANDV) cause hantavirus pulmonary syndrome (also called hantavirus cardiopulmonary syndrome), with mortality rates reaching up to 45% 2, 3. Climate change and urbanization are altering rodent host distributions, leading to unexpected hantavirus outbreaks 6, 7. In East Asia, an inactivated HTNV vaccine derived from mouse brain (Hantavax™) is licensed in South Korea, and several bivalent inactivated vaccines against HTNV and SEOV are used in China 8. Field investigations have shown that while these inactivated vaccines can induce a binding antibody response, the resulting neutralizing antibody (NAb) titers are often low and transient 8. Therefore, designing immunogens capable of eliciting high-titer, durable NAb responses remains a critical need for developing effective prophylactic vaccines against hantaviruses.

Hantaviruses, belonging to the family Hantaviridae and genus Orthohantavirus, are enveloped viruses with a tri-segmented negative-sense RNA genome 1, 3, 9. The genome encodes an RNA polymerase, a nucleocapsid protein (NP), and a glycoprotein (GP) precursor that is proteolytically cleaved into N-terminal Gn and C-terminal Gc subunits 1, 3, 9. On the virion surface, Gn and Gc form a metastable heterotetrameric spike 9, 10, 11, 12, 13, 14. Gn mediates cellular receptor attachment and shields cryptic epitopes on Gc, which is a class II fusion protein that drives membrane fusion during endocytosis 9, 10, 11, 12, 13, 14. Both Gn and Gc are targets of NAbs, driving vaccine development efforts focused on these proteins individually or in combination 15, 16, 17, 18, 19, 20. Recent advances in structural biology have resolved the architecture of the hantavirus GP, including the Gn head (GnH) and the Gc ectodomain 11, 12, 21, 22. These structures provide a mechanistic basis for neutralization by both previously known and newly isolated NAbs 23, 24, 25, 26, 27, 28.

Despite these efforts, the rational design of vaccines development for hantaviruses has lagged compared to that for other viruses, such as coronaviruses and respiratory syncytial virus (RSV) 29, 30. Building on recent structural insights, we engineered an HTNV GP immunogen by introducing three cysteine mutations to stabilize its prefusion conformation and prevent structural rearrangements. We then evaluated this engineered GP delivered via both a DNA vaccine platform and a lipid nanoparticle (LNP)-encapsulated mRNA vaccine. This work provides proof-of-concept for a rational, structure-based antigen design strategy in developing an efficacious hantavirus vaccine. These findings support the further investigation and development of this strategy.

Results
Prefusion-stabilized HTNV GP DNA vaccination elicits a significant NAb response
The HTNV GP contains multiple endogenous disulfide bonds 31. As the HTNV Gc is a class II membrane fusion protein, we hypothesized that engineering additional disulfide bonds would increase the energy barrier for the conformational change required for membrane fusion.

First, we introduced an inter-protomer disulfide bond within Gc by replacing glycine 835 with cysteine (G835C; numbering corresponds to the GP precursor) to increase Gn/Gc spike stability, designating this mutant as GP-C1 (Fig. 1a, Supplemental Fig. 1a-b) 13. Because the GnH is loosely attached to Gc, we next introduced an inter-subunit disulfide bond by engineering serine 291 and threonine 731 to cysteine (S291C/T731C). This design was based on structural modeling of the HTNV GP using the prefusion glycoprotein complex of ANDV (PDB: 6Y5F) 12 ; the resulting mutant was designated GP-C2 (Fig. 1a, 1b, Supplemental Fig. 1a). Finally, we constructed a triple mutant combining all three mutations (S291CT/T731C/G835C), designated GP-C3 (Fig. 1a, Supplemental Fig. 1a).

To determine whether these mutations stabilized the prefusion state, we expressed C-terminal Myc-tagged proteins and analyzed their migration under reducing and non-reducing SDS-PAGE conditions, alongside a WAASR mutant, which produces an uncleaved GP precursor (Fig. 1c) 9.

Under reducing conditions, wild-type (WT) GP migrated primarily as a ~50 kDa monomer (representing Gc), while the WAASR mutant appeared as an uncleaved ~120 kDa precursor (Fig. 1c). In contrast, GP-C2 and GP-C3 showed a prominent high-molecular-weight band (>480 kDa) (Fig. 1c), indicating the preservation of Gn/Gc heterotetramers despite β-mercaptoethanol (β-ME) treatment. Under non-reducing conditions, while all constructs showed some tetrameric form, the monomeric Gc band was nearly absent in GP-C3 and substantially reduced in GP-C2 compared to WT (Fig. 1c). When cloned into the pVAX1 DNA vaccine vector, GP-C1, GP-C2, and GP-C3 maintained their stabilized conformation, with GP-C3 showing the strongest effect (Supplemental Fig. 1c).

We next performed a syncytium formation assay to evaluate functional stabilization. In 293T cells (chosen for their high transfection efficiency at 24 hours; Supplemental Fig. 1d), the GP-C2 and GP-C3 mutants produced fewer syncytia than WT, with GP-C3 yielding the fewest GFP-positive syncytia (SFig. 1d, Supplemental Fig. 1e). These data confirm that the engineered cysteine mutations stabilize the prefusion conformation of HTNV GP.

We then evaluated the immunogenicity of these GP mutants in BALB/c mice, using the inactivated vaccine as a positive control (Fig. 1e). Mice received three vaccinations at 4-week intervals, with serum collected 2 weeks after each dose. Throughout the study, all vaccinated mice remained active and healthy, with no clinical signs of distress or systemic toxicity observed, indicating the favorable safety profile of the vaccine candidates. All GP immunogens induced robust humoral responses, with GP-C3 performing best (Fig. 1f, g). While GP-specific IgG levels at week 2 were similar between GP-C3 and WT, the GP-C3 group already exhibited significantly higher NAb titers against HTNV than the WT and inactivated vaccine groups (Fig. 1g). Antibody titers peaked at day 42 in all groups, with GP-C3 again inducing the highest levels of both GP-specific IgG and NAbs. Notably, by day 70, the NAb titer in the inactivated vaccine group had declined by half, whereas both NAb and GP-specific antibody titers in the GP-C3 group remained at their peak levels, suggesting robust antibody affinity maturation (Fig. 1f, g).

We also assessed cross-neutralizing activity against SEOV, a clade II hantavirus with considerable sequence similarity to HTNV 25. At both 14 and 70 days post-immunization, GP-C3 induced the highest SEOV NAb titers among all groups (Fig. 1h, Supplemental Fig.1f). Although GP-C2 elicited strong HTNV-specific responses at day 14, its SEOV NAb titer was lower than those induced by GP-C1 and WT (the difference was not significant), but increased by day 70 (Fig. 1h, Supplemental Fig.1f ). Collectively, these results indicate that the disulfide-bond-stabilized GP-C3 is an ideal immunogen, inducing potent homologous and cross-neutralizing antibodies.

Finally, we investigated the T-cell responses induced by the DNA vaccines. Splenocytes were harvested 70 days post-immunization (Fig. 1e). Upon in vitro stimulation with HTNV-GP peptides, GP-C1, GP-C2, and GSP-C3 induced stronger IFN-γ and IL-2 recall responses than the inactivated vaccine, with GP-C3 eliciting the highest spot-forming cells (SFCs) levels (Fig. 1i, j). Correspondingly, flow cytometry analysis showed that the percentages of IFN-γ-secreting CD4+ and CD8+ T cells were significantly higher in the GP-C3 group than in all other groups (Fig. 1k, l, Supplemental Fig. 2a, b). A similar pattern was observed for TNF-α-secreting T cells (Supplemental Fig. 2c, d). In contrast, Th2-associated cytokine responses (IL-4 and IL-10 SFCs) were similar across all DNA vaccine groups and were not significantly improved over the inactivated vaccine (Fig. 1m, n). Flow cytometry analysis of IL-4-secreting CD4+ and CD8+ T cells yielded consistent results (Fig. 1o, p, Supplemental Fig. 2e, f). Together, these data demonstrate that the prefusion-stabilized HTNV GP DNA vaccine, particularly the GP-C3 construct, elicits a potent Th1-skewed cellular immune response.

Prefusion-stabilized HTNV GP DNA vaccine confers potent protection against viral challenge and tissue injury
To determine whether prefusion-stabilized HTNV GP DNA vaccination confers protective immunity, mice were challenged with 5 × 105 focus-forming units (FFUs) of HTNV strain 76-118 via intramuscular (i.m.) injection two weeks after the final immunization. All animals were euthanized three days post-infection, and viral RNA loads in the lung, liver, kidney, and spleen were quantified by qRT-PCR.

The viral RNA level was markedly reduced in all immunogen-vaccinated groups compared to the pVAX1 vector control group (Fig. 2a, b, Supplemental Fig. 3a, b). In the lung (Fig. 2a), GP-C3 immunization reduced viral RNA to 9.24% of the vector control level, corresponding to a 10.8-fold reduction. This protective efficacy surpassed that of the inactivated vaccine (1.7-fold reduction) and the wild-type (WT) GP vaccine (1.6-fold reduction). The GP-C1 and GP-C2 constructs also conferred strong protection, with 6.5-fold and 5.4-fold reductions in lung viral RNA, respectively. A similar trend was observed in other tissues. In the liver (Fig. 2b), GP-C3 immunization led to a 7.2-fold reduction in viral RNA. Notably, although GP-C1-induced antibody titers were not significantly higher than those elicited by the inactivated vaccine or WT GP, it still provided substantial protection, reducing viral RNA in the lung, liver, and spleen by 6.5-, 5.2-, and 6.1-fold, respectively, suggesting protective mechanisms beyond peak antibody titers.

Consistent with the viral load data, immunofluorescence staining for HTNV nucleocapsid protein (NP) revealed substantially less antigen in the lung, kidney, and spleen tissues of GP-C3-immunized mice compared to the pVAX1, inactivated vaccine, and WT GP groups (Fig. 2c, Supplemental Fig. 3c-g).

Hematological and serological analyses further demonstrated the protective efficacy of the vaccines. Total white blood cell (WBC) and lymphocyte (LYM) counts in all immunogen-vaccinated groups were maintained close to the normal range for adult BALB/c mice and were significantly lower than the elevated levels in the pVAX1 control group (Fig. 2d, e). Neutrophil (NEU) counts were comparable across all vaccinated groups (Supplemental Fig. 3h). Assessment of liver and kidney function showed that the GP-C3 group had the lowest levels of aspartate aminotransferase (AST; Fig. 2f) and creatinine (CREA; Fig. 2g). Alanine aminotransferase (ALT), albumin (ALB), and blood urea nitrogen (BUN) levels in all immunogen groups were also within the normal range (Supplemental Fig. 3i, j, k), indicating effective protection against HTNV-induced hepatic and renal injury.

Histopathological analysis of tissue sections from challenged mice provided direct evidence of protection (Fig. 2h, i, Supplemental Fig.3l, m). Lungs of mice immunized with the pVAX1 (Vector) exhibited discernible pathology, including thickened alveolar septa, whereas lung morphology in the GP-C3 group remained largely normal (Fig. 2h). The pVAX1 group also displayed kidney injury, manifested as renal tubulointerstitial hemorrhage and erythrocyte accumulation within glomerular capillary lumina with luminal obstruction; as well as liver damage characterized by hepatocellular edema, and splenic inflammation. In contrast, these pathological alterations were markedly attenuated in the GP-C3 group, which showed no discernible pathological alterations in any of these tissues.

Collectively, these results demonstrate that the prefusion-stabilized HTNV GP DNA vaccine, particularly the GP-C3 construct, provides robust and comprehensive protection against HTNV challenge, effectively suppressing viral replication and preventing virus-induced tissue injury.

Prefusion-stabilized HTNV GP DNA vaccine elicits prolonged and durable NAb response
Given the protective efficacy of the prefusion-stabilized vaccine, we next evaluated the longevity of the humoral immune response it elicits. Mice received three immunizations at 4-week intervals, and serum and splenocytes were analyzed at day 180 for long-term immune evaluation (Fig. 3a).

HTNV GP-specific IgG antibody titers in the GP-C3 group remained significantly higher than those in the WT and inactivated vaccine groups at this late time point (Fig. 3b). Notably, NAb titers in the GP-C3 group showed no evident decline, maintaining high levels at day 180 (144.91 ± 40.06) compared to day 70 (172.81 ± 33.99; see also Fig. 1h), indicating a remarkably durable neutralizing antibody response (Fig. 3c).

A well-coordinated immune response often involves an initial pro-inflammatory Th1 phase followed by a shift toward a Th2 profile, which supports antibody affinity maturation and memory B cell (MBC) formation 32. To assess T-cell immunity over time, we performed ELISpot assays for IFN-γ and IL-4 recall responses at day 180. Only the inactivated vaccine and the GP-C3 construct maintained substantial levels of IFN-γ-secreting T cells, whereas responses in the other DNA vaccine groups diminished considerably by 6 months (Fig. 3d). Flow cytometry analysis corroborated these findings, showing no significant difference in the frequencies of IFN-γ- and TNF-α-secreting CD4+ and CD8+ T cells between the GP-C3 and inactivated vaccine groups (Fig. 3e, f, Supplemental Fig. 4a-d).

In contrast, a clear Th2-skewed response emerged in the long term. ELISpot analysis revealed that GP-C1, GP-C2, and GP-C3 all elicited high levels of IL-4-secreting cells at day 180 (Fig. 3g). Specifically, the GP-C3 group exhibited the highest frequencies of IL-4-secreting CD4+ and CD8+ T cells (Fig. 3h, i, Supplemental Fig. 4e, f), indicating a robust and sustained Th2-type response.

Together, these results demonstrate that the GP-C3 immunogen induces high and durable NAb titers, which are maintained for at least 6 months. This sustained humoral immunity is associated with a coordinated immune shift, where an early Th1 response transitions into a dominant and persistent Th2-skewed profile over time.

Prefusion-stabilized HTNV GP DNA vaccination elicits robust primary germinal center responses
Germinal center (GC) formation, driven by T follicular helper (Tfh) cells, is essential for B cell affinity maturation and the generation of high-affinity antibodies 33, 34, 35. Given that the prefusion-stabilized GP-C3 immunogen induced potent and durable NAb responses, we hypothesized that it would efficiently promote Tfh cell differentiation and GC formation.

To test this, BALB/c mice received a single i.m. dose of the different vaccines. Draining lymph nodes (LNs) were analyzed 7 days post-immunization—a well-established peak for germinal center formation—by flow cytometry seven days post-immunization. The frequency of Tfh cells (CD4+ B220− PD1+ CXCR5+) in the LNs of GP-C3-immunized mice was significantly higher than in mice immunized with the empty vector, inactivated vaccine, or WT GP (Fig. 4a, b, c, Supplemental Fig. 5a).

Consistently, GP-C3 vaccination also induced a significantly higher frequency of GC B cells (CD19+ CD38− CD95+) compared to the control groups (Fig. 4d, e, Supplemental Fig. 5b).

We next performed histological immunofluorescence staining to visualize GC structures directly. GCs were identified as GL7+ B cell clusters within B220+ follicles and distinct from adjacent CD3+ T cell zones. The number of detectable GCs was significantly greater in the GP-C3 group than in all other groups (Fig. 4f, g).

These data demonstrate that a single dose of the prefusion-stabilized GP-C3 immunogen potently initiates a germinal center reaction, characterized by robust Tfh cell expansion and GC B cell accumulation, providing a cellular basis for its superior antibody responses.

mRNA-LNP delivery of prefusion-stabilized HTNV GP elicits potent humoral and cellular immunity
Having established GP-C3 as the lead immunogen in the DNA vaccine platform, we next evaluated its performance in a clinically relevant mRNA-LNP platform. The mRNA was synthesized with a canonical Cap1 structure and incorporated N1-methylpseudourine (m1Ψ) nucleoside modifications to enhance stability and translational efficiency while reducing innate immunogenicity (Fig. 4a, Supplemental Fig. 6a).

Both GP-WT and GP-C3 mRNAs were successfully encapsulated into LNPs with properties suitable for immunization (Fig. 5a, b, Supplemental Fig. 6b, c). In vitro, these mRNA-LNPs mediated efficient expression of HTNV GP in Huh7 cells, as detected by Gc protein, while empty LNPs showed no signal (Fig. 5c). To evaluate immunogenicity, mice received two i.m. doses (2 or 10 μg) of GP-WT or GP-C3 mRNA-LNP at a 3-week interval, with empty LNPs and inactivated vaccine serving as controls (Fig. 5d). As observed in DNA vaccine regime, no clinical signs of distress or systemic toxicity were observed, indicating a similarly favorable safety profile orf the mRNA-LNP platform.

The mRNA-LNP vaccines, particularly at the 10 μg dose, elicited potent antibody responses. Serum analysis revealed that the 10 μg GP-C3 group produced significantly higher HTNV GP-specific IgG titers than all other groups (Fig. 5e). Crucially, this group also achieved the highest neutralizing antibody (NAb) titers against HTNV (Fig. 5f).

Interestingly, despite the self-adjuvanting properties of mRNA-LNPs, the NAb levels induced by GP-WT and 2 μg GP-C3 mRNA-LNP were comparable to, rather than substantially exceeding, those of the inactivated vaccine. This underscores the intrinsic potency of the prefusion-stabilized GP-C3 immunogen across delivery platforms.

Furthermore, the GP-C3 mRNA-LNP vaccine elicited strong cross-neutralizing activity against SEOV, with titers reaching a level comparable to the homologous HTNV response (Fig. 5g). A direct comparison between vaccine platforms, accounting for their respective bleeding endpoints, confirmed that the mRNA-LNP formulation achieved higher peak NAb titers than the DNA vaccine (HTNV: 247.16 vs. 170.12; SEOV: 242.64 vs. 94.18), as summarized in Supplemental Table 1.

We next evaluated cellular immunity. Splenocytes harvested four weeks post-immunization were stimulated with HTNV GP peptide pools. Consistent with the DNA vaccine results, the 10 μg GP-C3 mRNA-LNP group mounted the strongest IFN-γ and IL-2 recall responses among all groups (Fig. 5h, i). Flow cytometry confirmed significantly higher frequency of IFN-γ-secreting CD4⁺ and CD8⁺ T cells in this group (Fig. 5j, k, Supplemental Fig 6d, e). In contrast, IL-4 responses across mRNA-vaccinated groups were similar to those observed with the DNA vaccine platform, with GP-C3 showing no marked enhancement (Fig. 5l, m, n, Supplemental Fig. 6f, g). Additionally, IL-10 levels in the GP-WT groups were lower than in the inactivated vaccine group (Fig. 5o).

In summary, the prefusion-stabilized GP-C3 immunogen delivered via mRNA-LNP elicits robust and balanced immune responses, characterized by high-titer, cross-reactive NAbs and a Th1-skewed cellular profile, confirming its efficacy across nucleic acid vaccine platforms.

mRNA-LNP delivery of prefusion-stabilized HTNV GP provides optimal protection against HTNV infection
To evaluate protective efficacy, vaccinated mice were challenged with 5 × 105 FFUs of HTNV at four weeks post immunization and analyzed three days later (Fig. 5d). qRT-PCR analysis showed markedly reduced HTNV RNA levels in the lungs, liver, and kidneys of all immunogen-vaccinated groups compared to the LNP placebo group (Fig. 6a, b, Supplemental Fig. 7a, b). Notably, the GP-C3 mRNA-LNP group exhibited the lowest viral burden, with lung RNA levels significantly lower than those in the GP-WT group and approaching sterile protection (Fig. 6a). Consistent with this, immunofluorescence staining for HTNV NP antigen revealed minimal viral protein in the lung, kidney, and spleen tissues of GP-C3-vaccinated mice (Fig. 6c, Supplemental Fig. 7c-g). These results indicate that GP-C3 mRNA-LNP immunization effectively restricts viral replication and dissemination.

Analysis of hematological and serological parameters further demonstrated protection from virus-induced pathology. White blood cell (WBC), lymphocyte (LYM), and neutrophil (NEU) counts in the GP-C3 group were significantly lower than in the LNP placebo group but remained within the normal range for healthy mice (Fig. 6d, e, f). Levels of the liver injury markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were also lower in all immunogen-vaccinated groups compared to the LNP placebo (Fig. 6g, Supplemental Fig.7h), suggesting protection against virus-induced liver injury. Furthermore, the GP-C3 group maintained near-normal levels of renal function markers, including albumin (ALB), blood urea nitrogen (BUN), and creatinine (CREA) (Fig. 6h, Supplemental Fig.7i, j), suggesting effective protection against HTNV-induced kidney damage.

Histopathological examination provided morphological evidence of protection. Lung sections from the LNP placebo group showed discernible pathology, including thickened alveolar septa (Fig. 6i). In contrast, all immunogen-vaccinated groups displayed attenuated lung injury to varying degrees, with the GP-C3 group exhibiting the least severe alveolar structural disruption (Fig. 6i). Similarly, examination of liver, kidney, and spleen sections revealed that GP-C3-immunized mice developed only mild pathological alterations, with no typical signs of inflammatory infiltration or notable edema observed (Fig. 6j, Supplemental Fig. 7k, l).

In summary, the prefusion-stabilized GP-C3 immunogen delivered via mRNA-LNP confers optimal protection against high-dose HTNV challenge, effectively suppressing viral replication, preserving critical organ function, and preventing acute tissue injury.

mRNA-LNP delivery of prefusion-stabilized HTNV GP elicits robust germinal center responses
Since the GP-C3 mRNA-LNP vaccine induced robust NAb responses and protection, we hypothesized that it could also efficiently trigger primary GC reactions. To test this, inguinal LNs were collected from mice 7 days post-vaccination for analysis. Flow cytometry revealed that Tfh cells constituted nearly one-third of the CD4+ T cell population in the GP-C3 group, significantly higher than in the GP-WT and inactivated vaccine groups (Fig. 7a, b), indicating a potent GC response. GC B cells stimulated by Tfh and follicular dendritic cells rapidly proliferate and undergo antibody diversification, differentiating into MBCs and long-lived plasma cells (LLPCs) 33, 34, 35. Consistently, the frequency of CD19+ CD38− GL7+ CD95+ GC B cells was also significantly elevated in the GP-C3 group compared to these controls (Fig. 7c, d). Histological immunofluorescence analysis of LNs confirmed these findings, showing that both the number and area of GCs were greatest in the GP-C3 group (Fig. 7e, f). These results demonstrate that the prefusion-stabilized GP-C3 mRNA-LNP vaccine is highly effective at inducing a strong primary germinal center response.

mRNA-LNP delivery of prefusion-stabilized HTNV GP effectively boosts immunity primed by inactivated vaccine
Despite decades of field usage in endemic areas, epidemiological investigations have revealed comparably low and transient levels of HFRS- inactivated vaccine-induced NAbs 36, 37. We therefore investigated whether a heterologous boost with GP-C3 nucleic acid vaccines could recall and amplify this pre-existing immunity. Mice were primed with two doses of inactivated vaccine and, 180 days later, received a booster immunization with different vaccine regimens, with phosphate-buffered saline (PBS) serving as a naïve placebo for all three doses (Fig. 8a). Two weeks after the final booster dose, the mice were sacrificed and blood samples were collected for splenocyte immunoassays.

As expected 20, 38, a homologous booster with the inactivated vaccine increased titers of both HTNV GP-specific IgG and NAbs (Fig. 8b, c). Notably, heterologous boosting with all nucleic acid vaccines further enhanced these responses. The GP-C3 mRNA-LNP booster elicited the highest levels of both GP-specific antibodies and NAbs among all groups (Fig. 8b, c). Interestingly, the delivery platform significantly impacted the outcome, as boosting with the GP-C3-based DNA vaccine resulted in lower NAb titers than with the GP-WT mRNA vaccine, likely reflecting the adjuvant effect of the LNP system. Critically, a single GP-C3 mRNA-LNP booster elevated NAb titers to a level comparable to that achieved by a full primary course of GP-C3 mRNA vaccination (Fig. 8c).

We further evaluated the cellular immune responses. The GP-C3 mRNA-LNP booster elicited higher frequencies of IFN-γ-secreting CD4+ and CD8+ T cells compared to the inactivated vaccine booster, although the difference compared to the GP-WT mRNA or GP-C3 DNA vaccines was not statistically significant (Fig. 8d, e, Supplemental Fig. 8a, b). A similar pattern was observed for IL-4-secreting CD4+ and CD8+ T cells, with the 10 μg GP-C3 mRNA dose showing the most pronounced effect (Fig. 8f, g, Supplemental Fig. 8c, d).

Collectively, a single booster dose of the prefusion-stabilized HTNV GP mRNA-LNP vaccine potently recalls and expands immunity primed by suboptimal inactivated vaccines, achieving neutralizing antibody levels equivalent to a full primary mRNA vaccination course (Fig. 9).

Methods
Ethics statement
The experiments were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. The protocols were approved by the Animal Ethics Committee of the Air Force Medical University (Fourth Military Medical University; approval no. FMMU-20200403).

Cells, plasmids, viruses
Vero E6 (ATCC , CRL-1586), Huh7 (Procell, Wuhan, China; CL-0120), and HEK-293T (Clontech, now Takara Bio, 632180) cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO , USA) supplemented with l-glutamine, sodium pyruvate, and 10% fetal bovine serum (Sigma-Aldrich). The GP gene of HTNV strain 76-118 (GenBank: NC_005219.1) was codon-optimized and synthesized by GenScript (Nanjing, China). The gene was cloned into the pCAGGS mammalian expression vector using using the NovoRec® plus One Step PCR Cloning Kit (Novoprotein, Suzhou, China) to generate a construct with a C-terminal Myc tag, following the manufacturer’s instructions 69. Internal signal peptide mutant WAASR was generated through site-directed mutagenesis using primer pairs WAASR-F and WAASR-R and amplifying the full-length pCAGGS-GP plasmid. After digesting with Dpn I, the prefusion-stabilized HTNV GP was obtained via cysteine mutations to generate additional disulfide bonds. Full-length cysteine-mutated GP was obtained through overlapping PCR by amplifying each mutated cysteine upstream and downstream segment. Double and triple cysteine mutations were generated based on a sequencing-verified nonmutation template. The resulting multiple pCAGGS-GP plasmids were Sanger-sequencing verified and subcloned into the pVAX1 vector. Thus, the pCAGGS vector was utilized for in vitro assays requiring high transient expression, while the pVAX1 vector, a backbone optimized for DNA vaccines, was employed for all animal immunization studies. HTNV (strain 76-118) and SEOV (strain SR-11) stocks were propagated in Vero E6 cells. The HFRS bivalent-inactivated vaccine (HANPUWEITM) used in this study was produced by the Changchun Research Institute of Biological Products Co., Ltd. (Changchun, China). The inactivated vaccine was produced from inactivated and purified HTNV PS-6 and SEOV L-99 strains propagated in hamster kidney cells. Mice were vaccinated with 10 μg of HTNV inactivated vaccine (the manufacturer's instructions were referred to for the immunization dose), which was diluted in phosphate-buffered saline (PBS).

mRNA synthesis, LNP encapsulation, and evaluation
mRNA was synthesized in vitro using T7 RNA polymerase-mediated transcription from a linearized DNA template derived from the plasmid pUC-simple (Sangon Biotech, Shanghai, China). The template encodes codon-optimized HTNV GP with or without a triple cysteine mutation, incorporating the 5'- and 3'-untranslated regions and a poly-A tail. Lipid nanoparticle (LNP) formulations were prepared following a standard procedure previously described for siRNAs 70. Briefly, ethanol-dissolved ionizable lipid SM102, 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and DMG-PEG 2000 were combined in a molar ratio of 50:10:38.5:1.5. The lipid mixture was combined with mRNA at a ratio of 1:2 in 20 mM citrate buffer (pH 4.0) using a T-mixer. Formulations were dialyzed with PBS (pH 7.4), concentrated, passed through a 0.22-μm sterile filter, and stored at –80 °C until use. All formulations were tested for particle size, distribution, and encapsulation. The resultant mRNA-LNPs were deposited on carbon-coated Formvar grids, stained with 1% phosphotungstic acid, and examined using transmission electron microscopy. Huh7 cells were seeded in 24-well plates containing coverslips (5 × 105 cells/well). After 18 h, the cells were treated with mRNA-LNPs or empty LNPs. Six hours later, the spent medium was replaced with Opti-MEMTM I reduced-serum medium (Thermo Fisher Scientific, Waltham, MA, USA). Twenty-four hours later, the cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100, blocked with 3% bovine serum albumin (BSA), and incubated with the HTNV Gc-specific monoclonal antibody 3G1, and then with Cy3-conjugated goat anti-mouse IgG. Finally, the coverslips were observed using a BX60 fluorescence microscope (Olympus).

Native-PAGE, SDS-PAGE, and Western blotting
Plasmids bearing each HTNV GP construct were transfected into HEK-293T cells using the Hieff Trans Liposomal Transfection Reagent (Yeasen Biotech, Shanghai, China) according to the manufacturer's instructions. After 24 h, the cells were harvested and lysed on ice with a radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China) for 30 min. The lysate was clarified through centrifugation at 12,000 × g for 5 min. The protein levels in the supernatant were quantified using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and equal amounts of protein in each group were incubated with anti-c-Myc magnetic beads (MedChemExpress, Monmouth Junction, NJ, USA) overnight. After washing thrice with IP lysis buffer, each sample was separated, and one copy of each sample was directed loaded onto a 3–20% gradient polyacrylamide gel (GenScript) and subjected to electrophoresis under non-denaturing, non-reducing conditions (Native-PAGE). The other copy of each sample added with 5% (v/v) β-Mercaptoethanol (β-ME) and boiled was separated using SDS-PAGE. Both proteins were transferred to polyvinylidene fluoride membranes and blocked in tris-buffered saline containing 5% nonfat powdered milk. Membranes were blotted with rabbit antibodies against Myc-tag (Sangon Biotech, Shanghai, China) at 1:5,000 dilution and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sangon Biotech) at 1:10,000 dilution. After washing with PBS twice, the membranes were incubated with high-sensitivity luminol (Abbkine Biotechnology, Atlanta, GA, USA) and imaged using an electrochemiluminescence system (Tanon, Shanghai, China).

Syncytia formation assay
WT and prefusion constructs of HTNV GP were subcloned into the high expression pCAGGS vector by truncating the C-terminal six amino acids (ΔC6), and each plasmid was co-transfected with pCAGGS-ITGB3 and pCAGGS-GFSP at a ratio of 1:1:1 into HEK-293T cells, with a total of 1 μg per well in a 24-well plate. After 24 h, the culture medium was replaced with a citric acid solution (pH 5.0) and incubated at 37 °C for 30 min. Subsequently, the number of syncytia formed through cell fusion was visualized using an inverted fluorescence microscope (Olympus IX71, Tokyo, Japan). The degree of membrane fusion for the observation field in each group was calculated by averaging the number of multinucleated cells in three independent biological replicates of that group.

DNA vaccine immunization regime
All vaccine immunogenicity-related experiments were performed using 6–8-week-old female BALB/c mice purchased from the Animal Centre of Air Force Medical University. Which represent a standard and well-characterized model for the initial proof-of-concept evaluation of vaccine-induced immune responses. The mice were maintained in individually ventilated cages under regular ambient room temperature (21 °C; RT), 60% humidity, and a 12 h/12 h light/dark cycle, and were randomly divided into seven groups (10–12 mice per group): six DNA-immunized groups (Vector, WT, WAASR, GP-C1, GP-C2, and GP-C3) and an HFRS-inactivated vaccine control group. Mice were immunized with 50 μg (25 μg per leg) of the DNA vaccine through i.m. injection on days 0, 28, and 56. Serum was collected 2 weeks after each immunization and stored at −80 °C until specific antibody IgG and NAb assays were performed to assess the level of vaccine-induced humoral immune response. Two weeks after the third immunization, five mice from each group were randomly selected, and splenic lymphocytes were isolated for ELISpot and intracellular cytokine staining to evaluate the level of vaccine-induced T cell immune response. The remaining five mice in each group were subjected to HTNV strain 76-118 challenge with 5 × 105 FFUs via i.m. injection. Three days post-infection—corresponding to the peak of viral replication in murine model—tissues from various organs were collected and examined for pathological damage and viral load. For the long-term immunization regime, mice were immunization as above, while the serum and splenocytes were collected at day 180.

mRNA-LNP vaccine immunization regime
For the short-term immunization regime, groups of 6-to-8-week-old female BALB/c mice were i.m. immunized with WT or GP-C3 mRNA-LNP (2 μg or 10 μg, n = 10), or LNP placebo (10 μg, n = 10) in 100 μL PBS using an insulin syringe (BD Biosciences, San Jose, CA, USA). An HFRS- inactivated vaccine was used as a positive control. Each mouse received a booster of an equal amount and type of inactivated vaccine or mRNA-LNP on day 21 post-initial immunization. Serum samples were collected on days 14 and 28 post-immunization to detect HTNV GP-specific IgG and NAb responses. At day 28 post-initial immunization, the spleen tissues were collected to evaluate cellular immune responses via ELISpot and flow cytometry. HTNV challenge and tissues, as well as sera collection as does in the DNA vaccine regime. For the prime-boost immunization regime, mice were i.m. immunized with two doses of the HFRS-inactivated vaccine at a three-week interval and boosted with an equal dose of inactivated vaccine, 50 μg of GP-WT or GP-C3 DNA vaccine, or 10 μg of GP-WT or GP-C3 mRNA-LNP on day 180 post-initial immunization. At day 194 post-initial immunization, serum and spleen tissues were collected to evaluate HTNV GP-specific IgG and NAb responses as well as cellular immune responses.

Enzyme-linked immunosorbent assay (ELISA) based HTNV GP-specific antibody titer evaluation
Lab-defined ELISA was used to determine HTNV GP-specific antibody titers. A previously rescued recombinant vesicular stomatitis virus lacking VSVS-G but expressing HTNV GP (rVSV-HTNV GP) was used as the coating antigen 71. Briefly, 96-well ELISA plates were coated with 2.0 µg of iodixanol-purified rVSV-HTNV-GP in 100 μL ELISA Coating Buffer per well overnight at 4 °C. The plates were then washed with PBS and blocked with 3% bovine serum albumin in PBS for 1 h at RT. Mouse serum was 2-fold serially diluted starting at a 1:50 dilution in blocking buffer, added to the washed ELISA plates (100 μL/well), and incubated for 2 h at 37 °C. Following three washes with PBS, the plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000; Sangon) for 60 min at 37°C. After washing, the plates were incubated with 3,3',5,5'-tetramethylbenzidine substrate solution (TIANGEN, Beijing, China) for 15 min at 37 °C after five PBS washes, followed by quenching with 2 M sulfuric acid. The absorbance was read at 450 nm using a microplate reader (Bio-Tek, Winooski, VT, USA). Results with a positive/negative ratio >2.1 were considered positive 72. Antibody titers were defined as the reciprocal of the maximum serum dilution with a positive response.

Focus reduction neutralization test (FRNT)
The focus reduction neutralization test (FRNT) was performed based on a previously described protocol 73, with the following modifications: Vero E6 cells were seeded in 96-well plates (1 × 104 cells/well) and incubated for approximately 18 h until reaching over 90% confluence. Mouse serum was serially diluted 2-fold in PBS, starting at a ratio of 1:10. The diluted sera were then mixed with 100 FFUs of HTNV (strain 76-118) or SEOV (strain SR-11) at a 1:1 (v/v) ratio in 100 μL Dulbecco’s modified Eagle’s medium (DMEM) containing 2% fetal bovine serum (FBS), and incubated at 37 °C for 1 h. After removing the mixture, the cells in the plates were overlaid with DMEM containing 2% FBS and 1.6% (w/v) carboxymethylcellulose sodium salt. After further incubation at 37 °C for 7 days, the cells were fixed with 4% PFA, permeabilized with 0.5% Triton X-100, and incubated with an HRP-conjugated HTNV NP-specific 1A8 monoclonal antibody for approximately 18 h at 4 °C. Foci representing Virus-infected cells were quantified using an AID-ELISPOT reader (Straßberg, Germany) after visualization with the precipitated TMB substrate. The neutralizing antibody titer was defined as the maximum serum dilution that conferred a 50% reduction in the formed foci (FRNT50) and calculated using the Spearman–Karber method 74.

Enzyme-linked immunospot (ELISpot) assay
Cellular immune responses, characterized by the number of IFN-γ-, IL-2-, IL-4-, and IL-10-secreting splenocytes in vaccinated mice, were assessed using pre-coated ELISPOT kits (MabTech, NackaStrand, Sweden) following the manufacturer’s protocols. Briefly, splenocytes were collected from the immunized mice, and red blood cells were removed. Freshly isolated splenocytes were resuspended in 10% RPMI-1640 medium and transferred to the coated ELISpot plates at a density of 1 × 106 cells/well. The cells were then stimulated with a 10 μg/mL HTNV Gn/Gc peptide pool (Supplemental Table 2 and 3) for 24 h at 37 °C. Positive control stimulation was performed using 1 μg/mL concanavalin A (ConA, Sigma), while negative control stimulation was conducted using RPMI-1640 medium alone. After 24 h of incubation at 37 °C, 5% CO2, biotinylated anti-mouse IFN-γ, IL-2, IL-4, or IL-10 antibodies were added to each well and incubated for 2 h at RT.

Streptavidin-horseradish peroxidase was then added and incubated for 1 h. An AEC substrate solution was subsequently added, and the reaction was stopped by rinsing the plates with deionized water. Air-dried plates were analyzed using an AID-ELISPOT reader, and the number of spot-forming cells (SFCs) per 1 × 106 cells was calculated.

T-cell intracellular cytokine staining
To assess T lymphocyte proliferation in immunized mice, 1 × 106 splenocytes were transferred into each well of a 48-well plate and stimulated with a 10 μg/mL HTNV Gn/Gc peptide pool (Tables S1 and S2) for 2 h at 37 °C. Subsequently, 10 μg/mL brefeldin A was added to each well and incubated for 4 h. Negative and positive controls were included, using dimethyl sulfoxide and a cell activation cocktail (with brefeldin A), respectively. Following two washes with PBS, splenocytes were stained with Zombie AquaTM Fixable Viability (BioLegend, San Diego, CA, USA) and fluorescent-conjugated antibodies against the surface markers CD3 (PE/Cyanine7; BioLegend), CD4 (FITC; BioLegend), and CD8 (APC/Fire 750TM; BioLegend). The splenocytes were then fixed with a permeabilization buffer (Thermo Fisher) and stained for IFN-γ (APC; eBioscience, Santiago, CA, USA), TNFα (PE, eBioscience) or IL-4 (PerCP-eFluor 710; eBioscience). All labeled lymphocytes were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Pasadena, CA, USA) and CytoExpert software (Beckman Coulter).

Measurement of viral burden in challenged mouse tissues
Mouse tissues in the RNA preservation solution were weighed and transferred to a new tube, to which TRIzol Universal RNA extraction solution (TIANGEN) was added. The tissues were then homogenized with sterile stainless-steel beads using Tissue Lyser II (QIAGEN, Dusseldorf, Germany), and RNA was isolated according to the manufacturer's instructions. One microgram of total RNA was reverse-transcribed into cDNA and amplified using SYBR Green Master Mix (Yeasen Biotech). Viral RNA levels were determined through qRT-PCR using HTNV-S-specific primer pairs (forward: 5'-TCTAGTTGTATCCCCATCGACTG-3'; reverse: 5'-ACATGCGGAATACAATTATGGC-3') and normalized to tissue weight and a standard curve.

Tissue histological immunofluorescence and histopathology
Three days post HTNV challenge, whole blood samples from each mouse were collected. The livers, spleens, lungs, and kidneys of the mice were excised and weighed. Part of the tissue was preserved in a non-freezing tissue RNA preservation solution, and the remaining tissues were cut into small pieces and placed in 4% PFA for hematoxylin and eosin staining and tissue immunofluorescence. Tissues fixed in 4% PFA for 24 h were processed into paraffin-embedded microtomy sections. For fluorescence immunohistochemistry, paraffin sections were sequentially incubated with the mAb-1A8 antibody and Cy3-conjugated goat anti-mouse IgG (DNA vaccine regime) or FITC-conjugated goat anti-mouse IgG (mRNA-LNP regime), and the nuclei were stained with DAPI. Finally, the slices were sealed with an anti-fluorescence quenching sealing agent (Solarbio, Beijing, China). Images were captured using a Pannoramic MIDI instrument (Thermo Fisher) and analyzed using CaseViewer V2.4.0 software (3D HISTECH, Budapest, Hungary). For histopathological detection, paraffin-embedded microtome sections from mouse lung, liver, kidney, and spleen tissues were deparaffinized, rehydrated, and stained with hematoxylin and eosin, followed by treatment with ethanol and xylene.

Measurement of biochemical index
Blood from vaccinated mice was collected 3 days post-HTNV challenge and mixed with 1.5 mg/mL K2-ethylenediaminetetraacetic acid (EDTA). Analysis of WBC, lymphocyte, and neutrophil counts was performed using a BC-2800vet automatic blood cell analyzer (Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China). Serum samples were analyzed to determine the levels of ALT, AST, ALB, BUN, and CREA using an automated biochemical analyzer (ChemRay 800; Rayto Life and Analytical Sciences Co., Ltd., Shenzhen, China).

Detection of Tfh and GC B cells and GCs in situ
To detect Tfh and GC B cells, groups of 6-to-8-week-old female BALB/c mice were intramuscularly immunized with only one dose. Inguinal LNs were collected at 7 days post-immunization and single-cell suspensions were obtained. For Tfh analysis, lymph node cells were incubated with fluorescently conjugated antibodies against CD4 (FITC; BioLegend), B220/CD45R (APIC/Cy7; BioLegend), PD1 (PerCP-eFluor 710; eBioscience), and CXCR5 (PE/Cy7; BioLegend). GC B cells were labeled with CD19 (FITC; BioLegend) and CD95 (PerCP-Cy5.5). For the in situ detection of GCs, mouse inguinal LNs were harvested and fixed in 4% PFA for 30 min at RT. Snap-freezing was performed in a Tissue-Tek Cryomold using an optimal cutting temperature compound (Yeasen Biotech) in a dry ice-cooled bath of 2-methylbutane. Subsequently, 8-μm-thick tissue sections were blocked with PBS containing 5% goat serum and FcR blocking reagent (Biolegend). The sections were then stained with antibodies against CD3, B220, and GL7 for 18 h at 4 °C, and detected using TSA-dendron fluorophores. Nuclei were stained with DAPI. Images were acquired using a Zeiss LSM710 confocal microscope (Oberkohen, Badenwalburg, Germany) and processed and analyzed using CaseViewer V2.4.0 software.

Quantification and statistical analysis
Statistical analyses were performed using the GraphPad Prism 8.0 software. Tests, number of animals (n), median values, and statistical comparison groups are shown in the figure legends. Two-way analysis of variance (ANOVA) or One-way ANOVA followed by Tukey’s multiple comparisons test were performed. Statistical significance was set to P < 0.05 and indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "HFRS caused by Old World hantaviruses leads to severe disease in humans. The inactivated vaccine currently used in endemic areas has limited efficacy, and the development of a new vaccine platform is desired. The authors introduced one to three cysteine mutations in the GP of the Hantaan virus to stabilise it in the pre-fusion state, so that the Gn and Gc of the same molecule, and /or the Gc-Gc of two molecules, are S-S linked. Mice were immunised with the DNA plasmids and/or the mRNA-encapsulated LNPs expressing these GP mutants. Mutant with S-S binding between Gn and Gc of the same molecule and between Gc-Gc of two molecules (called GP-C3) induced the strongest neutralising antibodies in all immunisation experiments. HTNV challenge experiments were also conducted in post-immunisation mice, showing that the viral load in various organs was reduced and blood markers tested were close to normal. In addition, as there are many inactivated vaccine recipients in the actual endemic area, this vaccine candidates were administered after immunisation with inactivated vaccine, and strong neutralising antibody production was observed. Furthermore, the study also showed that the lymph nodes of the immunised mice had more germinal centre B cells than others, supporting the immunological superiority of this vaccine platform.\nThis vaccine design appears to be very useful in the prevention of HTNV infection, but there are several points that need to be addressed. It also has a number of typos, which should be checked carefully again.\nMajor comments.\n1. The histopathology is shown, but it is difficult to determine the degree of inflammation without uninfected individual controls. Uninfected histopathology should also be presented. Were the lungs inflated at necropsy? Also, please provide these pathology images at lowest magnification, as a supporting file. This is to deny that the images presented are the intended extraction of the best parts by the authors.\n2. More details on the design and structure of the mRNA vaccine are needed, including a clear description of the Cap structure and other RNA modifications.\n3. The WB image in Fig 1C shows the position of (Gn-Gc)x4, but the separation in this region is not good. Doublets are also observed. Please show (Gn-Gc)x4 using a gel with good separation in this region.\n4. Transfection efficiency is not considered in the syncytial formation experiments. The vertical axis in Fig 1d uses field as the denominator, which could varies with transfection efficiency. Therefore, I believe that for a more accurate assessment, it is necessary to calibrate with transfection efficiency.\n5. In the graph showing the number of viral copies in infected individuals, the vertical axis is linear, so there appears to be a very large difference between GP-C3 and other immuns, but what is actually a fraction of that when compared to vectors?\n6. The authors do not state whether the mice showed any weight changes after each vaccination, or whether they developed any other symptoms that might be considered side effects. This point should be included.\nMinor comments.\nLine 118. 「…, while the monomer Gc level in the GP-2 and GP-C3…」, is this a mistake for GP-C1 and GP-C2? Please verify.\n2. Line 179. It would be better for the reader if the main text describes how many days after vaccination the HTNV was challenged.\n3. Line 196. Fig 1f seems to be incorrect, is it correctly Fig 2? Please verify.\n4. Line 202. Fig 2c seems to be incorrect, is it correctly Fig S3g? Please confirm.\n5.Line 205. BUN, Fig S3i? ALB, Fig S3h?\n6. Line 606. mNA-LNP seems to be incorrect, is it correctly mRNA-LNP?\n7. The GL7 in Fig 6e is too faint and no difference can be seen in print.\n8. Normal values for blood test (cell counts, liver enzymes and renal function) are listed in Fig S3e, but it would be easier to read if each bar graph was shaded or lined to indicate normal values.", "reviewer_2": "The manuscript by Ye and coworkers reports the findings on development of a rationally designed orthohantavirus vaccine prototype. The rationale behind the study is that the vaccine currently used in the HTNV endemic regions, Hantavax, does not necessarily provide a long-lasting strong neutralizing antibody response. Hantavax, to my knowledge, is formalin-inactivated virus (the authors might perhaps include some details of the inactivation method for the vaccine?), which might lead to masking of epitopes essential for neutralization. To tackle the potential instability issues of the spike complex, the authors introduced cysteine residues to selected (based on the 3D structures of the spike complex) regions in the Gn and Gc glycoproteins. The introdution of two disulphide bridges indeed led to stabilization of the heterotetrameric HTNV spike complex based on SDS-PAGE migration, whereas introduction of a single bridge did not act as efficiently.\nThe authors then went on to test the immunogenicity of the engineered prefusion-stabilized spike in mice using both DNA and mRNA transfection. The extensive studies performed provided evidence supporting the authors' initial hypothesis that prefusion-stabilized spike complex induced more robust neutralizing antibody response than the response resulting from immunization using inactivated virus. In general, the results seem convincing.\nSome specific comments/suggestions to improve the manuscript:\n-A round of language polishing might be beneficial\n-Figure 1a, would it be possible to include similar schematic figures for all constructs tested (at least GP-C1, GP-C2, GP-C3)? This might help the reader to more easily appreciate the differences?\n-Have the authors considered of presenting the DNA and mRNA immunization data in the same graphs? This might allow the authors to shorten the manuscript and might allow comparison between the plasmid and mRNA immunizations.\nIn summary, the authors present interesting data which in my opinion are based on a solid hypothesis (approach utilized in other vaccines, as the authors point out). The conclusions and claims presented appear to be supported by the experimental data. The methods fit well for the work conducted and they have been described in adequate detail.", "reviewer_3": "Ye et al. describe the development and testing of prefusion-stabilized Hantaan virus (HTNV) vaccines. The study involved the rational design of DNA and mRNA-LNP vaccines, as well as the evaluation of their immunogenicity and protective efficacy in mice. The vaccine formulations were analyzed for their biochemical and histopathological effects, as well as their ability to induce humoral and cellular immune responses. The study also included the measurement of viral burden in challenged mouse tissues and the assessment of serological parameters. The results demonstrated that the prefusion-stabilized HTNV GP-based nucleic acid vaccines (especially the GP-C3 one) elicited robust humoral and cellular immunity, providing potential protection against HTNV-induced histopathological injury and other pathological effects. The authors did an extensive experimental work on selecting the best vaccine regarding the one they tested, however important majors and minors points need to be addressed before considering publishing this study.\nMajor points\nThe authors have already contributed significantly to the field with their previous publications on the development of an HTNV VSV-based vaccine (npj vaccines PMID: 38341504) and Hantaan virus pathogenicity (Nature Comm PMID: 38200007). Additionally, another preprint article coming from the same University and investigating glycoprotein-based nucleic acid vaccination for the effective control of HTNV epidemics is under review at npj vccines (https://doi.org/10.21203/rs.3.rs-3933421/v1). However, it's worth noting that the present study shares considerable similarity with these 2024 HTNV vaccine studies, potentially impacting the novelty of the current work.\nOne important limitation of the study, as acknowledged by the authors themselves, is the significant lack of evaluation of GP-specific B cells. The stability of GP-C3 and GP-C2 proteins allows for their characterization by SDS-PAGE +/- -mercaptoethanol. However, employing affinity chromatography coupled with fPLC purification could potentially enabling the recovery of GP-C3 and GP-C2 proteins, for further linkage to fluorophores, enabling specific B-cell sorting. Another possibility would be using Gn/Gc construct to make flow cytometry probes to sort GP-specific B cells. Evaluating affinity maturation of sorted B-cells could offer valuable information regarding the immunological response, including VH antibody classes, sequences, and mutations needed for effective neutralization of the virus and protection against HTNV infection. Regarding this study, while the authors have evaluated GC B cells numbers in LNs, information about the presence of GCs at d70 or d180 post-immunization (instead of only after 7 days) and the role of GP-C3 DNA immunogen in inducing a significant humoral memory response for long-term protection against HTNV is lacking.\nFurthermore, the paper evaluate both DNA vaccine and RNA LNP vaccine studies, which are presented in a manner that appears as two separate studies within a single article. Restructuring the presentation to focus on comparing GP-C3 as DNA or RNA vaccines would enhance the coherence of the narrative and aid in identifying the most effective HTNV GP vaccine candidate.\nMinor points:\n- Legends for Fig 1c (electrophoresis) are different from the text (line 112,113);\n- Why using two different plasmids for HTNV transfection?\n- Line 118, GP-C2 and GP-C1 (instead of GP-C3);\n- Line 121, “Fig S1b” is maybe Fig S1c?\n- Line 124, “Fig S1c” is the electrophoresis figure, not the syncytia. Maybe Fig S1b?\n- For the neutralization assay of SEOV virus, why did you choose to only take d14 sera and not d70? How the SEOV Nab levels induced by stabilized GP-C1 and GP-C2 are lesser than those induced by the attenuated Ina Vac (Fig S1d)?\n- Why scarifying mice after just 3 days of challenge? We generally wait for 5 days to do a post-challenge assess virus dissemination (PMID: 32667279). It would also be better to follow the wight loss for 14 days to see any sign of infection and/or protection.\n- In Figure 2 and figure S3, a statistical fluorescence evaluation is need as the GP-C3 seems to be significantly lowering the viral load only in the kidney, compared to attenuated Ina Vac and WT.\n- Line 196, why “Fig 1f”?\n- Statistical analysis in all graphs are confusing. To make it more powerful, it would be better to have results interpretation comparing the DNA immunogens to the attenuated Ina Vac or WT, and not to the vector.\n- A healthy cotrol (non-infected) for all tissue histology is missing in the comparison.\n- Line 234, figure “1h” is showing IFN SFC, you mean “Fig 1g”\n- Line 292, is “4d”\n- Line 295, this sentence is not true as it is now well known that one of the weakest points of mRNA vaccines, especially SARS-CoV2 mRNA vaccines is that they lack on effective long term T-cell activation.\n- One of the interesting parts of this study is the mixed vaccine protocol by priming with the inactivate Ina Vac and boosting with different GP construct. This part, in my opinion, is the most innovative and should’ve received more focus for the immune response elicited and to suggest a better way to target the immune system to a more specific and protecting HTNV immune response.\nOverall, while the study presents intriguing findings, addressing the aforementioned concerns and enhancing experimental clarity and coherence is significantly needed and would substantially improve the manuscript to be considered for a publication in Nature Communications."}, "author_response": "Point-by-point response to the reviewers’ comments\nWe thank the reviewers for their time and constructive comments, which have significantly\nimproved our manuscript. Below, we provide a point-by-point response to all comments. All\nchanges in the manuscript have been highlighted in yellow for ease of review.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\n[...]\nResponse to the comments of Reviewer #1:\nWe sincerely thank you for your positive assessment of our work and the valuable feedback.\nWe greatly appreciate your constructive suggestions that help us improve our manuscript.\nWe have carefully considered all comments and have revised the manuscript accordingly.\nOur point-by-point responses to the specific points are detailed below.\n[...]\nResponse to the comments of Reviewer #2:\nWe are deeply grateful for your positive and encouraging assessment of our work. We\nsincerely appreciate your remarks that our “data are based on a solid hypothesis,”\n“conclusions are supported by experimental data,” and “methods are well-described.”\n[...]\nResponse to the comments of Reviewer #3:\nWe would like to express our sincere gratitude for your thorough evaluation of our manuscript\nand for providing these insightful and constructive comments. We have carefully considered\neach point and, where applicable, performed additional experiments and analyses to address\nthem. We believe these revisions have substantially strengthened our study, and we are\ngrateful for the time and expertise you have dedicated to improving our work. Our point-by-\npoint responses are detailed below."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have responded very carefully to the reviewers’ comments, and the revised manuscript has become a very valuable. I look forward to seeing this vaccine advance toward practical application. In particular, the GP-C3 mRNA vaccine achieved an FRNT50 of approximately 1:250, suggesting that its protective efficacy is likely to be sufficient in vivo.\nIn this study, post-immunization sera neutralized SEOV; however, it will be important in future work to determine whether cross-neutralization extends to other Old World hantavirus species. In addition, it will be essential to demonstrate that vaccination does not lead to antibody-dependent enhancement upon subsequent infection with heterologous hantaviruses.", "reviewer_3": "Thanks for the revised manuscript and the detailed replies. The part I cared most about was the potential overlap with your previous HTNV vaccine work and the other closely related paper, and I think you handled that well. Your explanation makes it clear why this is a different story and not just a repeat, especially because the core advance here is the structure guided prefusion stabilization approach and the GP C3 design, and you also make a clear distinction between a true mRNA LNP platform and other delivery setups. What helped is that you also reflected this in the manuscript text, so readers will understand the novelty without having to rely on the rebuttal.\nThe present revision reads cleaner and the flow is easier to follow. It is now straightforward to see how the DNA experiments were used to identify the lead construct and then how the same lead was tested in the mRNA LNP format. I also appreciate that you acknowledged the B cell level limitation and framed it as a real technical bottleneck rather than ignoring it. At this point, I feel my major concerns are addressed and I do not have additional substantive requests."}, "author_response": "Point-by-point response to the reviewers’ comments\nWe sincerely thank all reviewers for their time and valuable feedback on our manuscript. We are\ndelighted that the reviewers found the revised version substantially improved and the responses\nsatisfactory. We have carefully considered all comments and have incorporated corresponding\nrevisions into the manuscript, as detailed in the point-by-point responses below. All changes in the\nmanuscript text are highlighted in red for ease of reference.\n[...]"}]

Decision letter