Single dose recombinant VSV based vaccine elicits robust and durable neutralizing antibody against Hantaan virus

Zhang, Hui; Liu, He; Wei, Jing; Dang, Yamei; Wang, Yuan; Yang, Qiqi; Zhang, Liang; Ye, Chuantao; Wang, Bin; Jin, Xiaolei; Cheng, Linfeng; Ma, Hongwei; Dong, Yangchao; Li, Yinghui; Bai, Yinlan; Lv, Xin; Lei, Yingfeng; Xu, Zhikai; Ye, Wei; Zhang, Fanglin

doi:10.1038/s41541-024-00814-2

Download PDF

Article
Open access
Published: 10 February 2024

Single dose recombinant VSV based vaccine elicits robust and durable neutralizing antibody against Hantaan virus

Hui Zhang¹^na1,
He Liu¹^na1,
Jing Wei^1,2^na1,
Yamei Dang¹^na1,
Yuan Wang¹,
Qiqi Yang¹,
Liang Zhang¹,
Chuantao Ye¹,
Bin Wang³,
Xiaolei Jin⁴,
Linfeng Cheng¹,
Hongwei Ma¹,
Yangchao Dong¹,
Yinghui Li¹,
Yinlan Bai¹,
Xin Lv¹,
Yingfeng Lei¹,
Zhikai Xu¹,
Wei Ye ORCID: orcid.org/0000-0002-3980-8547¹ &
…
Fanglin Zhang¹

npj Vaccines volume 9, Article number: 28 (2024) Cite this article

1051 Accesses
3 Altmetric
Metrics details

Subjects

Live attenuated vaccines

Abstract

Hantaan virus (HTNV) is a pathogenic orthohantavirus prevalent in East Asia that is known to cause hemorrhagic fever with severe renal syndrome (HFRS), which has a high fatality rate. However, a Food and Drug Administration (FDA)-approved vaccine is not currently available against this virus. Although inactivated vaccines have been certified and used in endemic regions for decades, the neutralizing antibody (NAb) titer induced by inactivated vaccines is low and the immunization schedule is complicated, requiring at least three injections spanning approximately 6 months to 1 year. Replication-competent vesicular stomatitis virus (VSV)-based vaccines provide prolonged protection after a single injection. In this study, we successfully engineered the HTNV glycoprotein (GP) in the VSV genome by replacing the VSV-G open reading frame. The resulting recombinant (r) rVSV-HTNV-GP was rescued, and the immunogenicity of GP was similar to that of HTNV. BALB/c mice immunized with rVSV-HTNV-GP showed a high titer of NAb against HTNV after a single injection. Notably, the cross-reactive NAb response induced by rVSV-HTNV-GP against Seoul virus (an orthohantavirus) was higher than that induced by three sequential injections of inactivated vaccines. Upon challenge with HTNV, rVSV-HTNV-GP-immunized mice showed a profoundly reduced viral burden in multiple tissues, and inflammation in the lungs and liver was nearly undetectable. Moreover, a single injection of rVSV-HTNV-GP established a prolonged immunological memory status as the NAbs were sustained for over 1 year and provided long-term protection against HTNV infection. The findings of our study can support further development of an rVSV-HTNV-GP-based HTNV vaccine with a simplified immunization schedule.

Single dose rVSVΔG-JUNVGP vaccine protects guinea pigs against lethal Junin virus challenge

Article Open access 09 August 2021

Vesicular Stomatitis Virus-Based Vaccine Protects Mice against Crimean-Congo Hemorrhagic Fever

Article Open access 23 May 2019

The rVSV-EBOV vaccine provides limited cross-protection against Sudan virus in guinea pigs

Article Open access 10 June 2023

Introduction

Rodent-borne Orthohantavirus (hereafter referred to as hantavirus) infection can cause two severe clinical diseases, namely, hantavirus pulmonary syndrome (HPS) caused by the New World Hantavirus (NWH) and hemorrhagic fever with severe renal syndrome (HFRS) caused by the Old World Hantavirus (OWH), which have fatality rates greater than 35% and up to 5–15%, respectively¹. The causative agents of HFRS are Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava-Belgrade virus (DOBV), and Puumala virus (PUUV), which are found mainly in Europe and Asia. The clinical manifestations of HFRS caused by different hantavirus infections vary from subclinical, mild, and moderate to severe; among them, HTNV and DOBV are responsible for more severe symptoms². Annually, over 85% of the approximately 50,000 cases worldwide occur in China alone, and they are mainly caused by HTNV and SEOV and pose a significant threat to human health, especially in rural areas³. At present, cell culture-based HTNV and SEOV bivalent inactivated vaccines are used in areas endemic for the disease in China⁴. Although it is effective in inducing an antibody response after inoculation, the duration of the neutralizing antibodies (NAbs) is extremely short. Thus, developing a safe and effective prophylactic vaccine with a high titer and durable NAb response is urgently required to control hantavirus infections.

Belongs to the order Bunyavirales, family Hantaviridae, and genus Orthohantavirus, HTNV is an enveloped, trisegmented, negative-sense ribonucleic acid (RNA) virus. The small (S) segment encodes the nucleocapsid protein, the medium (M) segment encodes the glycoprotein precursor (GPC), and the large (L) segment encodes the RNA-dependent RNA polymerase (RdRp)⁵. GPC is cleaved by host proteases at the conserved WAASA pentapeptide site into two mature envelope glycoproteins, glycoprotein N (Gn) and glycoprotein C (Gc)⁶. Both Gn and Gc are type I transmembrane proteins and the sole glycoproteins on the virion surface. Thus, both Gn and Gc serve as protective antigens against NAbs in vivo. Other Gn/Gc-based vaccines have also been developed, such as virus-like particle (VLP)-based, deoxyribonucleic acid (DNA), and virus-vectored vaccines^{7,8,9,10,11,12,13}. However, the immunization schedule for VLP and DNA vaccines often requires three sequential injections and extends for approximately 6 months to 1 year, thus posing a great challenge for improving vaccination regime adherence, especially in rural areas. For vectored vaccines, pre-existing antibodies against the vectors often hinder vaccine efficacy. Generation of a vectored vaccine without pre-existing antibodies and with a single inoculation could serve as a potential solution, which will help to elevate the immunization rate and reduce the disease burden.

Vesicular stomatitis virus (VSV) is a non-segmented, negative-sense, single-stranded RNA virus that belongs to the family Rhabdoviridae and genus Vesiculovirus. The genome contains five sequential open reading frames (ORFs) that encode nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RdRp (L). VSV is an animal virus; its attenuated ability to infect humans and its ability to pseudotype different viral glycoproteins have made VSV a widely accepted choice of vector for vectored vaccines since the reverse genetics strategy was established^14,15. As replication-competent vectors, recombinant VSV (rVSV)-based vaccine candidates have been shown to strongly elicit NAbs and have been suggested to protect against multiple lethal pathogens, such as Ebola virus (EBOV), Marburg virus, Lassa virus, and Nipah virus^16,17,18,19. An rVSV expressing the Zaire Ebola virus glycoprotein (rVSV-ZEBOV) was generally well tolerated in vaccine recipients during clinical trials. The U.S. FDA and the European Medicines Agency licensed it for use in 2019 due to its high protection and effectiveness²⁰.

In this study, we constructed an infectious complementary DNA (cDNA) clone of the VSV and replaced the VSV-G segment with HTNV glycoprotein (GP), resulting in the generation of rVSV-HTNV-GP. We then characterized the immunological features of rVSV-HTNV-GP and evaluated its protection effects in a mouse model. We found that a single dose of rVSV-HTNV-GP elicited long-lasting NAbs and afforded protection against HTNV challenge. Our study paves the way for further development of a VSV-vectored HTNV vaccine and calls for future research.

Results

Generation and characterization of replication-competent rVSV expressing HTNV GP

The infectious cDNA clone of VSV with replacement in the VSV-G region by a codon optimized HTNV GP (Fig. 1a) and green fluorescent protein (GFP) marker was recovered. It expressed GFP reporter (Fig. 1b, upper panel) and formed typical plaques on Vero E6 cells (Fig. 1b, lower panel). The viral growth kinetics of rVSV-HTNV-GP were compared to those of the parental rVSV, which also contains a GFP ORF between VSV-G and VSV-L ORFs, resulting in an attenuated phenotype, with the peak titer for rVSV nearly hundred-fold that of rVSV-HTNV-GP (Fig. 1c). Negative-stain electron microscopy of iodixanol-gradient purified virus particles revealed that rVSV-HTNV-GP shared similar particle sizes and shapes with the parental rVSV particles (Fig. 1d). On subsequent testing of cell tropisms of rVSV-HTNV-GP on different cell lines. Vero E6 and human hepatoma (Huh7) cells had the highest titer, followed by human umbilical vein endothelial cells (HUVEC), baby hamster kidney (BHK)-21, and A549 cells, while Henrietta Lacks (HeLa) cells had the lowest titer (Fig. 1e); this is consistent with authentic HTNV tropisms. Purified rVSV and rVSV-HTNV-GP on subjection to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining, the VSV-G band was not visible in the lanes of rVSV-HTNV-GP (Fig. 1f). Furthermore, HTNV Gn and Gc expression was confirmed after western blot analysis of the virions (Fig. 1g). These results confirm that rVSV-HTNV-GP-bearing HTNV GP, instead of VSV-G, was successfully rescued.

**Fig. 1: Characterization of the Replication-Competent, Infectious rVSV Chimera bearing HTNV GP.**

rVSV-HTNV-GP share surface antigenic similarity with authentic HTNV

To evaluate antigenic similarities between rVSV-HTNV-GP and HTNV, we stained HTNV-, rVSV-HTNV-GP-, or rVSV-infected Vero E6 cells with antibodies against HTNV-nucleocapsid protein (NP),-Gn/-Gc, or VSV-G, respectively. rVSV caused significant cytopathic effects in Vero E6 cells, and the remaining cells were positive for VSV-G, but negative for HTNV-NP, Gn, or Gc (Fig. 2a). In contrast, rVSV-HTNV-GP-infected cells were positive for HTNV Gn or Gc but negative for HTNV-NP, and Gn or Gc was only expressed in GFP-positive cells, indicating that the HTNV GP ORF was successfully expressed upon viral infection (Fig. 2a). Subsequently, 10 serum samples from the HFRS convalescent plasma were selected to determine their neutralization titer against HTNV and rVSV-HTNV-GP; the trends of the curves between the groups were very similar (Supplementary Fig. 1). Heatmaps of viral infectivity revealed similar dose-dependent neutralization patterns for rVSV-HTNV-GP and authentic HTNV from each antiserum donor (Fig. 2b). However, the serum dilutions at half-maximal neutralization derived from logistic curve fits (neutralization 50% or half maximal inhibition concentration [IC₅₀]) revealed a 2- to 10-fold shift toward reduced neutralization with rVSV-HTNV-GP compared with authentic HTNV (Fig. 2c). This discrepancy may be due to assay-specific differences between rVSV-HTNV-GP (half-GFP puncta formation reduction neutralization test, GRNT₅₀) and authentic HTNV (half-focus reduction neutralization test, FRNT₅₀) microneutralization assays. Nonetheless, the relative potencies of the antisera against rVSV-HTNV-GP and authentic HTNV were correlated (R² = 0.5288) (Fig. 2d). These results suggest that the surface antigenicities of rVSV-HTNV-GP and authentic HTNV were similar. Consequently, rVSV-HTNV-GP was demonstrated to be an authentic surrogate for HTNV and was thus suitable for eliciting the desired immune response.

**Fig. 2: Antigenic immunogenicity between HTNV and rVSV-HTNV-GP.**

Potent anti-HTNV humoral immune responses induced by rVSV-HTNV-GP vaccine candidates

Given that rVSV-HTNV-GP is a replication-competent vector, whether the vaccine candidates could induce specific immune responses against HTNV in a single-dose regime was examined. Six-week-old BALB/c mice were divided into four groups and immunized with three different doses of rVSV-HTNV-GP or control rVSV (Fig. 3a). An inactivated vaccine (licensed for HFRS prevention in China) inoculated with three sequential injections at 3-week intervals served as the positive control (Fig. 3a). Serum was isolated from the mice at 4 weeks post-priming with rVSV and rVSV-HTNV-GP or at 3 weeks post-final boost of the inactivated vaccine, and IgG titers against HTNV were determined, and were nearly undetectable in rVSV control group (Fig. 3b). All three rVSV-HTNV-GP groups showed significantly higher levels of IgG (mean titers of 72.0, 104.0, and 88.0 in the 2 × 10⁴, 2 × 10⁵, and 2 × 10⁶ plaque-forming units (PFUs) groups, respectively) compared with the inactivated vaccine group (mean titers of 28.0) (Fig. 3b). Although the levels of HTNV-specific IgG among the three rVSV-HTNV-GP groups were not significantly different, 2 × 10⁵ PFUs resulted in the best performance.

**Fig. 3: Immunogenicity of rVSV-HTNV-GP.**

NAb titers against HTNV were measured using the focus-reduction neutralization test (FRNT)²¹. Immunization with 2 × 10⁴, 2 × 10⁵, and 2 × 10⁶ PFUs of rVSV-HTNV-GP-induced NAbs (mean titers of 77.4, 80, and 78.6, respectively) was two-fold higher compared to that of the inactivated vaccine (mean titer of 37.2) (Fig. 3c). Moreover, all three doses of rVSV-HTNV-GP elicited higher SEOV NAbs (mean titers of 29.6, 44.6, and 39.4) than those of the inactivated vaccine (mean titer of 13.6) (Fig. 3c). These findings demonstrate the suitability of rVSV-HTNV-GP as an immunogenic vaccine platform that elicits high titers of HTNV-specific antibodies and NAbs and induces cross-OWH NAbs against SEOV. Collectively, these data suggest that rVSV-HTNV-GP is immunogenic and elicits high titers of antibodies that neutralize HTNV GP.

In addition to 2 × 10⁴ PFUs, we also used 2 × 10⁵ and 2 × 10⁶ PFUs in the rVSV group and tested the specific antibodies and NAbs against HTNV. The experimental results showed that the specific antibodies (mean titers of 2 × 10⁴, 2 × 10⁵, and 2 × 10⁶ PFUs in the rVSV groups were 4, 2, and 2, respectively) and NAbs (mean titers of 2 × 10⁴, 2 × 10⁵, and 2 × 10⁶ PFUs in the rVSV groups were 35.6, 33.6, and 37.6, respectively) induced by the three rVSV groups were similar (data not shown). Therefore, the 2 × 10⁴ PFU group of rVSV was selected as the control in the following experiment.

rVSV-HTNV-GP vaccine candidates induced protection against HTNV infection mainly attributed to humoral immunity

Effective vaccination involves the induction of CD4⁺ T helper (Th) cells that produce cytokines that shape subsequent humoral adaptive immune responses. To evaluate the rVSV-HTNV-GP vaccination-induced T cell responses, splenocytes collected from control and immunized mice were cultured with the HTNV GP overlapping peptide pool for examination of Th1 cytokines (interferon gamma [IFN-γ] and tumor necrosis factor alpha [TNFα]) and Th2 cytokines (interleukin [IL] IL-4) production within the cells (Supplementary Fig. 2). We observed low or no levels of Th1 and Th2 cytokines in the splenocytes of rVSV immunized mice, whereas variably high levels of Th cytokines were detected in mice immunized with rVSV-HTNV-GP vaccine candidates or the inactivated vaccine (Supplementary Fig. 3a, b), although the differences between the versatile vaccine groups were not significant. To further study vaccine-mediated cellular immunity, splenocytes collected from control and immunized mice were further examined of cytokines IL-2, IL-4, IL-10 and IFNγ production within the cells by cultured with the HTNV GP overlapping peptide pool. We observed low or no levels of cytokines IL-2, IL-4, IL-10 and IFNγ in the splenocytes of rVSV immunized mice, whereas variably high levels of Th cytokines were detected in mice immunized with rVSV-HTNV-GP vaccine candidates or the inactivated vaccine, although the differences between the versatile vaccine groups were not significant (Supplementary Fig. 4). Collectively, these results demonstrate that rVSV-HTNV-GP-induced T cell responses did not have an obvious advantage over inactivated vaccines.

To test whether rVSV-HTNV-GP elicited higher levels of NAbs to protect against HTNV infection, the mice were challenged with HTNV, and euthanized 3 days post infection (Fig. 3a). The viral RNA burdens were determined using quantitative reverse transcription polymerase chain reaction (PCR) (RT–qPCR) in mouse lung, kidney, liver, and spleen tissues. Viral RNA was markedly reduced in the rVSV-HTNV-GP groups, compared to the rVSV and inactivated vaccine groups, and was undetectable in 2 × 10⁵ PFUs of rVSV-HTNV-GP groups, suggesting sterile protection (Fig. 4a–c, Supplementary Fig. 5g).

**Fig. 4: rVSV-HTNV-GP vaccination reduces viral loads in multiple tissues and protects from Virus-Induced Inflammation.**

The pro-inflammatory cytokine messenger RNA (mRNA) levels in homogenates of lung, kidney, liver, and spleen tissues from vaccinated animals at 3 dpi were measured using RT-qPCR to assess whether rVSV-HTNV-GP limited HTNV-induced inflammation (Fig. 3a). Mice immunized with different doses of rVSV-HTNV-GP had significantly lower levels of proinflammatory cytokine mRNA as opposed to rVSV-and inactivated vaccine-immunized mice. Specifically, TNFα (Fig. 4d–f, Supplementary Fig. 5h), IL-1β (Fig. 4g–i, Supplementary Fig. 5i), IL-6 (Supplementary Fig. 5a–c, k), IL-10 (Supplementary Fig. 5d–f, l), and type I interferons (IFN-β) (Fig. 4j–l, Supplementary Fig. 5j) decreased early during infection in all three regimes of mice immunization with rVSV-HTNV-GP. Although there were no detectable differences among the three rVSV-HTNV-GP groups, the 2 × 10⁵ PFUs group performed the best, followed by 2 × 10⁴ PFUs. Altogether, these data establish that immunization with rVSV-HTNV-GP protects against HTNV infection in mice, and that NAbs are a possible protective factor.

rVSV-HTNV-GP vaccination reduces pathological damage in multiple tissues against HTNV Infection

The extent of tissue damage in HTNV-challenged mice was determined by staining lung and liver sections with hematoxylin and eosin (H&E) (Fig. 5a, b). Lung sections from rVSV-HTNV-GP-immunized mice, especially the 2 × 10⁵ and 2 × 10⁶ PFUs groups, showed the lowest signs of inflammation (Fig. 5a), which matched the viral RNA levels in the corresponding tissues (Fig. 4a). Shrinkage of alveolar tissue and immune cell infiltration was observed in rVSV-immunized mice (Fig. 5a). Hepatocellular edema and dilated and congested hepatic sinusoids were documented in the rVSV-, inactivated vaccine- and 2 × 10⁶ PFUs of the rVSV-HTNV-GP-immunized mice groups, but not in the 2 × 10⁴ and 2 × 10⁵ PFUs of the rVSV-HTNV-GP groups (Fig. 5a). This was consistent with the viral burden in the corresponding tissues (Fig. 4c). Thus, immunization with rVSV-HTNV-GP generates a protective immune response, particularly at a dose of 2 × 10⁵ PFUs, thereby limiting HTNV-induced lung and liver inflammation in mice.

**Fig. 5: rVSV-HTNV-GP Protects Mice from HTNV induced pathological injury.**

Immunization with a single-dose regime of rVSV-HTNV-GP induced neutralizing antibodies is superior to the two-doses regime

Given that the immunization regime with 2 × 10⁵ PFUs of rVSV-HTNV-GP showed the best performance in inducing NAbs and limiting tissue inflammation response, the immunization regime had to be standardized for the number of doses. Therefore, a second booster dose of rVSV-HTNV-GP was administered to determine if NAb titer against HTNV improved significantly (Fig. 6a). HTNV-specific antibodies and NAb titers against HTNV were measured after priming or boosting. Out of expected, boosting failed to significantly enhance HTNV-specific antibodies or NAb titers (Fig. 6b, c). Immunization with rVSV-HTNV-GP induced higher levels of anti-GP-specific IgG compared to the rVSV control, with reciprocal median serum endpoint titers of 104.0 and 32.0 for one and two doses of the vaccine, respectively (Fig. 6b). The NAb titers of the single- and two-dose immunization regimes of rVSV-HTNV-GP were 68.0 and 71.0, respectively, whereas that of the inactivated vaccine was 41.0 (Fig. 6c). Boosting failed to increase the HTNV-specific antibody titer after the second dose of rVSV-HTNV-GP, whereas the NAbs titer enhancement was limited and not statistically significant. These data suggest that rVSV-HTNV-GP elicits high titers of both HTNV-specific antibodies and NAbs in a single dosage.

**Fig. 6: Prime-boost immunization regimen of rVSV-HTNV-GP didn’t improve the immunological index compared to a single dose.**

Single-dose of rVSV-HTNV-GP immunization elicits durable neutralizing antibodies response

As a single dose of rVSV-HTNV-GP immunization elicited high levels of NAbs and boosting failed to enhance the NAbs titer, we speculated that priming induces a durable NAbs response that compensates for the second dose of rVSV-HTNV-GP. To test this hypothesis, we immunized a group of BALB/c mice with a single dose of 2 × 10⁵ PFUs of rVSV-HTNV-GP and collected blood samples at 0, 0.5, 1, 2, 4, 6, 8, and 10 months post immunization, respectively (Fig. 7a). The NAbs were then measured using FRNT. The NAbs titer gradually increased after immunization and reached a peak (85.6) at 1 month post immunization, and was maintained at a high level between 2 and 4 months (82.0 and 72.2, respectively). This was followed by a gradual decrease (36.0 at 6 months) (Fig. 7b). Ten months post immunization, the NAb titer remained detectable at 17.0 (Fig. 7b). Thus, rVSV-HTNV-GP elicited high titers of NAb that were maintained at the peak level for 3 months and were sustained at a detectable level for over 10 months. This suggests that long-lived plasma cells were induced after rVSV-HTNV-GP immunization.

**Fig. 7: Single dose of rVSV-HTNV-GP immunization elicits long-lasting neutralizing antibodies response in mice.**

Single-dose of rVSV-HTNV-GP immunization provides long-term protection against HTNV infection

Twelve months post rVSV-HTNV-GP immunization, blood samples were collected from each group (Fig. 8a). The NAb titers were measured; they were undetectable in the rVSV group, whereas the NAb titer in the three sequential injections of inactivated vaccines was only 0.7832, as compared to 18.52 in the rVSV-HTNV-GP group (Fig. 8b). Additionally, the level of SEOV crossing NAb in the rVSV-HTNV-GP group was higher than that in the inactivated vaccine-immunized mice (12.97 and 9.383, respectively). Given that a single dose of rVSV-HTNV-GP immunization maintained a relatively high NAb titer after 1 year, whether this level of NAb provided protection against HTNV challenge was evaluated. Mice were challenged intramuscularly with 1 × 10⁶ FFUs of HTNV to evaluate vaccine protection. The viral loads in the lungs and kidneys were significantly lower than those in the rVSV group (Fig. 8d–f), whereas the viral load in the rVSV-HTNV-GP group was lower than that in the inactivated vaccine group, although the differences were not significant (Fig. 8d–f). Subsequently, inflammatory cytokine levels in different organs were measured using RT-qPCR. The IFN-β in the lung and kidney was not significantly different between inactivated vaccines, rVSV, and rVSV-HTNV-GP groups (Fig. 8g, h); however, the liver IFN-β was lower in rVSV-HTNV-GP group than in inactivated vaccines and rVSV groups (Fig. 8i). Moreover, the TNFα and IL-6 were both lower in rVSV-HTNV-GP group than in rVSV groups (Supplementary Fig. 6a–c, g–i). In addition, kidney IL-1β was lower in rVSV-HTNV-GP group, while inflammatory factors were not significantly different in lungs between groups (Supplementary Fig. 6a–l). Nonetheless, single dose of rVSV-HTNV-GP immunization provided better protection than the rVSV group and provided similar inflammatory cytokine responses compared to the three sequential injections of inactivated vaccines to some extent.

**Fig. 8: Single dose of rVSV-HTNV-GP immunization provides long-lasting protection to mice.**

Discussion

Bunyavirales comprise multiple highly pathogenic viruses, including Crimean–Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), severe fever with thrombocytopenia syndrome virus (SFTSV), and hantaviruses^22,23,24. Among these, hantaviruses is the most widely distributed. HTNV caused HFRS, exhibiting the highest mortality among other OWHs¹. Suckling mouse brain-based HTNV monovalent inactivated vaccines (Hantavax^TM) have been licensed in South Korea for decades, while HTNV and SEOV bivalent inactivated vaccines have been used in endemic areas in China for years^25,26. Although protective, the NAbs induced by inactivated vaccines are quite low and quickly disappear¹⁰. Moreover, complicated vaccination regimes are associated with poor adherence in rural areas. Thus, there is an urgent need to develop an effective HTNV vaccine that can enhance its ability to induce NAbs and simplify its administration regime. Therefore, the development of a single-dose immunization regime that induces high levels of NAbs is urgently needed. This study showed that rVSV based rVSV-HTNV-GP elicited high levels of NAbs with only a single dose in a mouse model and provided sterile protection against HTNV challenge.

Numerous vectored platforms have been investigated in animal models against hantavirus infection. Vaccinia virus-, adenovirus-, and lentivirus-vectored HFRS vaccine candidates have been extensively investigated by different groups^12,13,27. However, improvements in NAbs induced by these candidates is limited. One reason for this is that the hantavirus GP is encoded within the viral vector genome and is expressed only upon vector infection. The pre-existing immunity against the vector also poses other obstacles to the clinical use of these candidates²⁸. rVSV is an ideal vaccine platform that can easily incorporate other viral GPs and has been investigated extensively^16,29,30,31. The most evident example is Merck’s certified ERVEBO, which is a replication-competent VSV that encodes the EBOV glycoprotein and shows the ability to induce a humoral immune response³².

As a replication-competent vector, an rVSV-based vaccine can be easily amplified for bulk manufacturing. Safety is also a key consideration in vaccine development, even though human infections with VSV are extremely rare, with only a limited number of reports^33,34,35. The main pathogenic factor of VSV is VSV-G, which is substituted with foreign GPs in case of the vectored vaccine. The negative-sense single-stranded RNA genome of VSV is not prone to integration into the host genome or genetic reassortment, enhancing its safety when used in humans. In addition, there is nearly no pre-existing immunity to VSV in humans; hence, it would not interfere with efficacy of foreign glycoproteins expressing-rVSV³⁶.

Furthermore, rVSV-HTNV-GP produced cross-reactive NAbs against both HTNV and SEOV. Previous studies have consistently revealed that this phenomenon exists within NWHs because rVSV-Andes orthohantavirus (ANDV) can provide cross-protection against both ANDV and Sin Nombre viruses, and vice versa³⁷. Testing of the long-term immunogenicity and protection provided by a single rVSV-HTNV-GP vaccination revealed that both HTNV and SEOV NAbs were detectable after 1 year. As there is a lack of a lethal small animal model for OWHs, the main limitation of this study was attributed to the inflammation and viral loads of the different tissues induced by HTNV challenge, instead of animals that succumbed to infection.

Notably, from days 30–120 post vaccination, these mice had similar HTNV-specific NAb titers which sustained at the peak titers, followed by a drop by half on day 180, suggesting that the second boost dose may achieve the best results at least 6 months after priming. Consistent with our two-dose regime data, in which the boost dose was administered at 4-weeks post priming, and no significant improvement was observed compared to that of the single-dose regime.

Although a single injection of rVSV-HTNV-GP provides long-term protection from HTNV infection, improvements compared to three sequential injections of inactivated vaccines is limited. HTNV NP in the formulation may lead to non-inferior protection of the inactivated vaccine. Numerous studies have suggested that hantavirus NP is a protective antigen and strong T-cell immunity activator^27,38,39,40. Nevertheless, a recent study inserted both a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike and NP into the rVSV genome and rescued the recombinant virus, which showed improved protective efficacy in a hamster model⁴¹. Further investigation is required to determine whether the addition of hantavirus NP to the VSV genome can augment rVSV-HTNV-GP-induced protection.

In summary, we determined that recombinant VSV expressing the HTNV GP could elicit robust, durable, and OWH cross-reactive NAbs in a mouse model (Fig. 9). It also provides long-term protection from HTNV infection, which is promising for further evaluation in non-human primate models and clinical development.

Methods

Cells, viruses, and antibodies

Baby hamster kidney cells (BHK-21), African green monkey kidney epithelial cells (Vero E6), human non-small-cell lung carcinoma cells (A549), human hepatoma cells (Huh7), and human cervical carcinoma (HeLa) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Corning, NY, USA) with 10% fetal bovine serum (FBS) (YEASEN, Shanghai, China) and 1% penicillin-streptomycin-gentamicin solution (Solarbio, Beijing, China). HUVECs were maintained in an endothelial cell medium (ECM) (ScienCell, CA, USA). All cells were maintained in humidified incubators at 37 °C supplemented with 5% CO₂. HTNV (strain 76-118) and SEOV (strain SR-11) were stored in our laboratory.

Mouse monoclonal antibody (mAb) 1A8 against HTNV NP, mAb Gn-1 against HTNV Gn, and mAb Gc-10 against HTNV Gc were produced in our lab as previously indicated⁴². The anti-VSV-G tag was purchased from abcam (Cambridge, UK). Infrared (IR)Dye680RD-conjugated goat anti-mouse and anti-rabbit secondary antibodies were purchased from Lambda Instruments Corporation (LI-COR) Biosciences (LI-COR, NE, USA). Cyanine3 (Cy3)-conjugated goat anti-rabbit IgG, goat anti-mouse IgG, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, and horseradish peroxidase (HRP)-conjugated anti-mouse IgG were purchased from Biotech & Bio Basic Inc. (BBI) (Sangon Biotech). Hoechst 33258 stain was purchased from YEASEN.

Monoclonal Antibodies used for flow cytometry analysis, eFluor 506-CD3, FITC-CD4, phycoerythrin (PE)-IL-4, PE-Cyanine7-CD8a, allophycocyanin (APC)-IFNγ, Peridinin-Chlorophyll-Protein (PerCP)-eFluo 710-TNFα and Fixable Viability Dye eFluo 780 organic dye (FVD-780) were all obtained from eBioscience (Massachusetts, USA).

Recombinant VSV recovery and titration

The plasmid-bearing VSV antigenome lacking VSV-G ORF but with an additional GFP ORF was synthesized at GenScript (Nanjing, China). The generation and production of rVSV and rVSV-HTNV-GP was performed as previously described⁴³. An infectious cDNA clone of VSV was engineered by replacing endogenous VSV-G with HTNV GP and addition of a fluorescent marker (GFP) between the HTNV GP and VSV-L ORFs. To facilitate rVSV rescue, the endoplasmic reticulum (ER) retention sequence in the HTNV Gc cytoplasmic tail was truncated to alter HTNV GP retargeting to the plasma membrane instead of the ER-Golgi intermediate compartment (ERGIC)⁴⁴. To further enhance rescue efficiency, we mutated leucine 532 to lysine (I532K) and serine 1094 to leucine (S1094L). S1094L was located within the viral membrane-proximal external region (MPER), which would increase the amphipathic character of Gc MPER (designated as rVSV-HTNV-GP)^45,46. For rVSV-HTNV-GP, the codon-optimized GPC gene of HTNV (NC_005219) was amplified using PCR and inserted into the VSV antigenome plasmid, resulting in the rVSV-HTNV-GP vector. BHK-21 cells were inoculated with a vaccinia virus bearing T7 RNA polymerase (VV-T7), kindly provided by the Wuhan Institute of Virology, CAS, followed by co-transfection of five plasmids: full-length rVSV-HTNV-GP, together with VSV accessory plasmids encoding VSV-N, VSV-P, VSV-L, and VSV-G proteins, all of which were under T7 promoter control. The culture supernatant was collected 72 h post transfection and used to infect Vero E6 cells. The construct was recovered following an established protocol^15,47. After several passages, the cytopathic effect (CPE) in the cell monolayer was noticeable, indicating a successful rescue. The rescued virus was referred to as rVSV-HTNV-GP and verified by HTNV GPC-specific antibodies.

The viruses were propagated and titrated on Vero E6 cells, and the titer was determined using plaque assays. Briefly, Vero E6 cells were inoculated into 12-well plates and grown overnight until the cells were more than 90% confluent. The stock of rVSV and rVSV-HTNV-GP were 10-fold serially diluted and used to infect Vero E6 cells at a concentration of 500 μL per well in triplicates. After incubation at 37 °C for 2 h, the supernatant was aspirated and 2 mL maintenance overlay DMEM supplemented with 2% FBS and 2% carboxymethyl cellulose (CMC) was added to each well. The plates were then incubated for the desired time (3 days for rVSV and 7 days for rVSV-HTNV-GP). The overlay medium was aspirated, and the cells were stained with crystal violet staining solution (Beyotime, Shanghai, China) for 30 min at room temperature. The number of plaques in each well was counted and the titers of rVSV and rVSV-HTNV-GP were calculated.

Growth kinetics of rVSVs

Vero E6 cells in 6-well plates at 90% confluency were infected in triplicates with rVSV or rVSV-HTNV-GP at a multiplicity of infection (MOI) of 0.0001. Following incubation at 37 °C for 2 h, the plates were washed three times and the culture media was replaced with DMEM supplemented with 2% FBS and cultured at 37 °C with 5% CO₂. The culture supernatant was collected from each well at 0, 12, 24, 48, 60 and 72 hours post infection (hpi), and subjugated to TCID₅₀ analysis. In short, ten-fold serial dilutions of each supernatant were added to 96-well plate in quadruplicate. After removing the virus, the maintenance overlay medium was added. At 7 dpi, the viscous overlay medium was discarded, the cells were stained with crystal violet solution, and the TCID₅₀ values were then calculated using the Reed-and-Muench method.

Virus purification and transmission electron microscopy

rVSV and rVSV-HTNV-GP were propagated in Vero E6 cells. The culture supernatant was harvested and clarified by centrifugation at 5000 × g for 30 min at 4 °C. The supernatant was then concentrated by mixing with four times the volume of 0.8 M NaCl buffered 5 × polyethylene glycol (PEG) 8000 (Sigma-Aldrich, St. Louis, USA) and incubated at 4 °C overnight, followed by centrifugation at 10,000 × g for 1 h at 4 °C. The pellet was then re-suspended in 1 mL Tris-HCl (Tris-HCl: 0.5 mM Tris-HCl, 1.5 mM NaCl, 0.05 mM ethylenediaminetetraacetic acid (EDTA), and H₂O 1 mL) at 4 °C overnight.

The resuspended solution was then loaded onto an iodixanol (Sigma-Aldrich) density gradient medium, which was formed by underlaying 4.2 mL of 15% iodixanol solutions onto 3.8 mL 35% iodixanol solutions. The viruses were banded by ultracentrifugation at 160,000 × g for 2 h at 4 °C using an SW41 Ti rotor (Beckman Coulter, Brea, USA). The purified virus adhered to glow-discharged carbon-coated copper grids. The samples were stained with 4% (w/v) phosphotungstic acid (pH 7.1) and viewed under a JEM-1400 Flash transmission electron microscope (JEOL, Japan).

SDS-PAGE and western blotting

Purified rVSV and rVSV-HTNV-GP were mixed with 4 × lithium dodecyl sulfate (LDS) sampling buffer (GenScript) and boiled at 100 °C for 10 min. Viral proteins were examined using SDS-PAGE followed by Coomassie Brilliant Blue G-250 (YEASEN) staining. For western blotting, viral proteins were separated and transferred onto a Poly (vinylidene fluoride) membrane, and incubated with mouse anti-Gn-1 (1:100), mouse anti-Gc-10 (1:1000), or rabbit anti-VSV-G (1:2000, abcam, 309106) antibody diluted in Tris-buffered saline containing 1% Tween-20 (TBST), followed by incubation with IRDye 680RD Goat anti-Mouse (1:10000, LI-COR, 926-68070) or IRDye 680RD Goat anti-Rabbit (1:10000, LI-COR, 926-32211), and imaged with an Odyssey Imager (LI-COR).

Immunofluorescence assay

Vero E6 cells were seeded in 24-well plates and incubated at 37 °C overnight. HTNV, rVSV, or rVSV-HTNV-GP was used to infect cells for 2 h, followed by medium change to DMEM containing 2% FBS and cultured for an additional 48 h. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min, permeabilized with 0.5% Triton X-100 for 10 min, and stained with 1A8 (HTNV NP) (1:1000), anti-Gn-1 (1:50), anti-Gc-10 (1:100), or anti-VSV-G (1:1000, abcam, 309106) antibodies at 4 °C overnight. After washing, Cy3-conjugated goat anti-mouse IgG (1:400, Sangon Biotech, D111024) or Cy3-conjugated goat anti-rabbit IgG (1:400, Sangon Biotech, D111018) were added to each well and incubated at room temperature for 1 h. The nuclei were visualized using Hoechst 33258 (1:1000, YEASEN, 40729ES10). The images were captured using a BX60 fluorescent cell imager (Olympus, Tokyo, Japan).

Focus-reduction neutralization test (FRNT)

The FRNT assay was performed according to a previously established protocol with mild modifications²¹. Vero E6 cells were seeded in 96-well plates and cultured until the cell confluency was greater than 90%. Ten convalescent serum samples from patients with HFRS were serially diluted (between 1:20 and 1:43,740) in 200 µL of infection medium, mixed with the same volume of 200 focus forming units (FFUs) of HTNV, and incubated at 37 °C for 1 h. The convalescent serum sample-related experiments were approved by the Ethics Committee of Tangdu Hospital, Air Force Medical University (approval number:202103-139). Cells were then infected with serum-virus complexes with a concentration of 100 μL/well in quadruplicates and adsorbed at 37 °C for 2 h. After removing the serum-virus complexes, each well was covered with a maintenance overlay medium. The overlay medium was discarded at 5 dpi, and the plate was fixed, permeabilized, and blocked with 3% bovine serum albumin (BSA) (YEASEN). mAb 1A8 (1:1000) was used to probe the HTNV NP at 4 °C overnight. After discarding 1A8, HRP-conjugated goat anti mouse IgG (1:1000, Sangon Biotech, D110087) was added, and the cells were incubated for 1 h at room temperature. After washing thrice, the plate was stained with 3, 3’, 5, 5’-tetramethylbenzidine substrate for blotting (Biokits, Beijing, China) for 30 min at 37 °C. The plate was then dried, and the foci were counted. The Nab titer was calculated. The FRNT₅₀ was defined as the maximum dilution of serum that inhibited HTNV infection in 50% of the cells. For mouse NAb detection, the same method was adopted, but mouse sera were diluted 1:10, 1:20, 1:40, 1:80, and 1:160 times.

GFP-based foci-reduction neutralization test

The indicated dilutions of HFRS convalescent serum samples were incubated with 100 plaque forming units (PFUs) of rVSV-HTNV-GP for 1 h at 37 °C. Serum-virus complexes were added to Vero E6 cells in 96-well plates and incubated at 37 °C for 7 h. Subsequently, cells were fixed in 4% PFA and stained with Hoechst 33258. Images were acquired with a BioTek Cytation 5 imaging reader (Agilent, California, USA) in FITC channel to visualize GFP-positive cells. Data were processed using Prism software (GraphPad 8.0, Inc., La Jolla, CA, USA).

Mouse experiments and ethics statement

Six-to-eight-week-old female BALB/c mice were provided by the Laboratory Animal Centre of Air Force Medical University. All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. The protocols for animal studies were approved by the Committee on the Ethics of Laboratory Animal Centre of Air Force Medical University (Approval number: 20200403). HTNV challenge experiments were performed in a biosafety level 3 containment facility.

Short-term immunization regime

The BALB/c mice were randomly divided into five groups (n = 5 per group). Mice were intraperitoneally (i.p.) injected with the HFRS-inactivated vaccine, rVSV, or rVSV-HTNV-GP, and 200 μL (100 μg) HFRS inactivated vaccine was administered three times at 3 week intervals. Then, 2 × 10⁴ PFUs of rVSV were administered. For rVSV-HTNV-GP, three different doses of 2 × 10⁴, 2 × 10⁵, or 2 × 10⁶ PFUs were administered. Tail bleeds were taken from on 4 weeks after rVSV or rVSV-HTNV-GP administration, and 3 weeks after the vaccination regime was completed. HTNV-specific antibody and NAb levels were determined based on serum samples. HTNV-specific antibody titers were determined by immunofluorescence assay (IFA). Specifically, HTNV-infected Vero E6 cells in 48-well plates were probed with two-fold serial dilutions of immunized mice sera, and FITC-conjugated anti-mouse IgG (1:400, Sangon Biotech, D110105) was applied. The titer of specific antibodies was calculated using the maximum valid double dilution, while still being positive for HTNV. The titer of the HTNV-neutralizing antibody was determined based on FRNT. After bleeding, the animals were euthanized by cervical vertebra dislocation following isoflurane (RWD, R510-22-10) anesthesia, spleen cells were collected, and cytokines in CD4⁺ T cells were evaluated using flow cytometry.

To assess the protective effect, mice in each group were intramuscularly (i.m.) challenged with 1 × 10⁶ FFUs of HTNV. Animals were sacrificed at 3 dpi and viral loads in the liver, lung, spleen, and kidney were determined using qRT-PCR. The mRNA levels of inflammatory cytokines, including IL-1β, IL-6, IL-10, TNFα, and IFNβ, were also determined in these tissues.

Prime-boost immunization regime

The BALB/c mice were randomly divided into four groups (n = 5 per group). Mice were i.p. injected with HFRS-inactivated vaccine, rVSV, or rVSV-HTNV-GP. For one dose regime, 2 × 10⁵ PFUs of rVSV or rVSV-HTNV-GP were administered. For two dose regime, 2 × 10⁵ PFUs of rVSV-HTNV-GP were administered twice at a 4-week interval. After vaccination, tail bleeds were taken, and HTNV-specific antibodies and NAbs were evaluated.

Long-term immunization regime

Mice were immunized with 2 × 10⁵ PFUs of rVSV or rVSV-HTNV-GP via an i.p. route. In the inactivated vaccine group, the vaccines were administered thrice at a 3-week interval. Tail bleeds were taken from the rVSV-HTNV-GP-vaccinated mice at 0, 14, 30, 60, 120, 160, 240, and 300 dpi. One year after immunization, tail bleeds were taken from all mice, and NAbs were measured. The mice were intramuscularly injected with 1 × 10⁶ FFUs of HTNV and euthanized at 3 dpi. Viral loads and inflammatory cytokine mRNA levels in the liver, lung, and kidney were evaluated.

RT-qPCR-based viral burden measurement and cytokine analysis

Mouse tissues were weighed and homogenized using sterile stainless-steel beads in a TissueLyser II instrument (QIAGEN, Germany) in 1 mL of DMEM supplemented with 2% FBS. Total RNA was extracted from clarified tissue homogenates using Total RNA Isolation (TRizol) Universal RNA Extraction kit (TIANGEN, China). Further, 1 μg of total RNA was used to generate cDNA using the Hifair II 1st Strand cDNA Synthesis Kit (YEASEN) as per manufacturer’s instructions. Viral RNA levels were determined by RT-qPCR using Hieff qPCR SYBR Green Master Mix (YEASEN), as described previously, using the primer pair HTNV-S (forward:5′-GAGCCTGGAGACCATCTG-3′; reverse:5′-CGGGACGACAAAGGATGT-3′)⁸.

The mRNA levels of the inflammatory cytokine were determined using specific primer pairs for IL-1β, IL-6, IL-10, TNFα, and IFNβ (Supplementary Table 1). All results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and the fold-change for each was determined using the 2^−ΔΔCt method comparing to HTNV uninfected mice naive controls (n = 5).

Flow cytometry analysis of cytokines

Spleen cells were prepared, suspended, and maintained in Roswell Park Memorial Institute (RPMI) 1640 (Corning, Corning, NY, USA) supplemented with 10% FBS. Cells were stimulated with 15-mer peptides containing an overlap of 8 amino acid residues that covered the full-length HTNV GP for 8 h, along with a protein transport inhibitor cocktail (added 3 h later)^11,48. After stimulation, 1 μL of FVD-780 was added, and the cells were incubated for 30 min at 2–8 °C. They were then washed and stained with fluorophore-conjugated antibodies against the surface markers CD3 Monoclonal Antibody (17A2) (eFluor 506, 2.5 μL/100 μL, eBioseience, 69-0032-82), CD4 Monoclonal Antibody (GK1.5) (FITC, 1.25 μL/100 μL, eBioseience, 11-0041-82), and CD8a Monoclonal Antibody (53-6.7) (PE-Cyanine7, 2.5 μL/100 μL, eBioseience, 25-0081-82) for 60 min at room temperature. The cells were then treated with Intracellular Fixation & Permeabilization Buffer (eBioscience) for 30 min, centrifuged at 1000 × g for 3 min, and re-suspended. Further, fluorophore conjugated-antibodies against intracellular TNF alpha Monoclonal Antibody (MP6-XT22) (PerCP-eFluor™ 710, 2.5 μL/100 μL, eBioseience, 46-7321-82), IL-4 Monoclonal Antibody (11B11) (PE, 0.625 μL/100 μL, eBioseience, 12-7041-82), and IFN gamma Monoclonal Antibody (XMG1.2) (APC, 0.625 μL/100 μL, eBioseience, 17-7311-82) were added, and incubated for 30 min. After washing, spleen cells were analyzed using a flow cytometer (Agilent).

Enzyme-linked immunospot assay of cytokines

The cytokines IL-2, IL-4, IL-10 and IFN-γ were determined using ELISpot kits (MABTECH, NackaStrand, Sweden) following the manufacturer’s instructions. The mice were euthanized and their spleens were dissected and ground into a monocyte suspension. The splenocytes were washed and re-suspended following erythrocyte lysis. After blocking the ELISpot plates for 30 min with RPMI-1640 containing 10% FBS at room temperature, 1 × 10⁶ cells/well were transferred to the ELISpot plates and stimulated with 10 μg/mL HTNV Gn/Gc peptide pool for 24 h at 37 °C. Concanavalin A (1 μg/mL) was added as a positive control. Unstimulated cells were used as negative controls. After 24 h of incubation, streptavidin-HRP and fresh substrates were added to the plates. The reaction was stopped by rinsing the plate with deionized water. After drying, the number of spots was determined using an ImmunoSpot® analyzer (Cellular Technology Ltd., Santa Rosa, California, USA).

Histopathology

For H&E histopathology evaluation, mice were euthanized and their livers and lungs were rapidly isolated and fixed in 4% PFA at 4 °C overnight, followed by paraffin embedding. Sections were stained with H&E to analyze the histopathological changes. Images were captured using a Panoramic MIDI instrument (3D HISTECH, Hungary) and analyzed using SlideViewer V2.6.0 software.

Statistical analysis

Statistical significance was assigned at P-values less than 0.05 using Prism Version 8 (GraphPad Software). For the correlation analysis, Spearman’s correlation coefficients (R²) and P-values are indicated. Analysis of specific antibody and neutralization titers in mice after vaccination was performed using one-way Analysis of Variance (ANOVA) with Dunnett’s post-hoc test. Differences in viral loads or cytokine levels after HTNV challenge in immunized mice were determined using the Kruskal–Wallis test with Dunn’s post-hoc test.

Reporting summary

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

Data availability

All data that support the findings of this study are available from the corresponding author upon reasonable request.

References

Jonsson, C. B., Figueiredo, L. T. & Vapalahti, O. A global perspective on hantavirus ecology, epidemiology, and disease. Clin. Microbiol. Rev. 23, 412–441 (2010).
Article CAS PubMed PubMed Central Google Scholar
Jiang, H. et al. Hantavirus infection: a global zoonotic challenge. Virol. Sin. 32, 32–43 (2017).
Article PubMed PubMed Central Google Scholar
Tariq, M. & Kim, D. M. Hemorrhagic Fever with Renal Syndrome: Literature Review, Epidemiology, Clinical Picture and Pathogenesis. Infection Chemotherapy 54, 1–19 (2022).
Article CAS PubMed PubMed Central Google Scholar
Brocato, R. L. & Hooper, J. W. Progress on the Prevention and Treatment of Hantavirus Disease. Viruses 11, 610 (2019).
Article CAS PubMed PubMed Central Google Scholar
Deng, X. et al. Distinct Genotype of Hantavirus Infection in Rodents in Jiangxi Province, China, in 2020–2021. Zoonoses 2, 31 (2022).
Article Google Scholar
Guardado-Calvo, P. & Rey, F. A. The surface glycoproteins of hantaviruses. Curr. Opin. Virol. 50, 87–94 (2021).
Article CAS PubMed Google Scholar
Li, C. et al. Hantavirus-like particles generated in CHO cells induce specific immune responses in C57BL/6 mice. Vaccine 28, 4294–4300 (2010).
Article CAS PubMed Google Scholar
Cheng, L. F. et al. Incorporation of GM-CSF or CD40L Enhances the Immunogenicity of Hantaan Virus-Like Particles. Front. Cell. Infect. Microbiol. 6, 185 (2016).
Article PubMed PubMed Central Google Scholar
Hooper, J. W., Custer, D. M., Thompson, E. & Schmaljohn, C. S. DNA vaccination with the Hantaan virus M gene protects Hamsters against three of four HFRS hantaviruses and elicits a high-titer neutralizing antibody response in Rhesus monkeys. J. Virol. 75, 8469–8477 (2001).
Article CAS PubMed PubMed Central Google Scholar
Zhang, F. L. et al. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments. Biochem. Biophys. Res. Commun. 354, 858–863 (2007).
Article CAS PubMed Google Scholar
Jiang, D. B. et al. Recombinant DNA vaccine of Hantavirus Gn and LAMP1 induced long-term immune protection in mice. Antiviral Res. 138, 32–39 (2017).
Article CAS PubMed Google Scholar
Schmaljohn, C. S., Hasty, S. E. & Dalrymple, J. M. Preparation of candidate vaccinia-vectored vaccines for haemorrhagic fever with renal syndrome. Vaccine 10, 10–13 (1992).
Article CAS PubMed Google Scholar
Yu, L. et al. A recombinant pseudotyped lentivirus expressing the envelope glycoprotein of hantaan virus induced protective immunity in mice. Virol. J. 10, 301 (2013).
Article PubMed PubMed Central Google Scholar
Lawson, N. D., Stillman, E. A., Whitt, M. A. & Rose, J. K. Recombinant vesicular stomatitis viruses from DNA. Proc. Natl Acad. Sci. USA. 92, 4477–4481 (1995).
Article ADS CAS PubMed PubMed Central Google Scholar
Whelan, S. P., Ball, L. A., Barr, J. N. & Wertz, G. T. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc. Natl Acad. Sci. USA. 92, 8388–8392 (1995).
Article ADS CAS PubMed PubMed Central Google Scholar
Foster, S. L. et al. A recombinant VSV-vectored vaccine rapidly protects nonhuman primates against lethal Nipah virus disease. Proc. Natl Acad. Sci. USA. 119, e2200065119 (2022).
Article CAS PubMed PubMed Central Google Scholar
Geisbert, T. W. et al. Single-injection vaccine protects nonhuman primates against infection with marburg virus and three species of ebola virus. J. Virol. 83, 7296–7304 (2009).
Article CAS PubMed PubMed Central Google Scholar
Mire, C. E. et al. Single-dose attenuated Vesiculovax vaccines protect primates against Ebola Makona virus. Nature 520, 688–691 (2015).
Article ADS CAS PubMed PubMed Central Google Scholar
Safronetz, D. et al. A recombinant vesicular stomatitis virus-based Lassa fever vaccine protects guinea pigs and macaques against challenge with geographically and genetically distinct Lassa viruses. PLoS Negl. Tropical Dis. 9, e0003736 (2015).
Article Google Scholar
Henao-Restrepo, A. M. et al. Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial (Ebola Ça Suffit!). Lancet 389, 505–518 (2017).
Article CAS PubMed PubMed Central Google Scholar
Ye, C. et al. An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection. Front. Pharmacol. 10, 1203 (2019).
Article CAS PubMed PubMed Central Google Scholar
Li, J. C. et al. Dynamics of neutralizing antibodies against severe fever with thrombocytopenia syndrome virus. Int. J. Infect Dis. 134, 95–98 (2023).
Article PubMed Google Scholar
Teng, A. Y. et al. Mapping the viruses belonging to the order Bunyavirales in China. Infect. Dis. Poverty 11, 81 (2022).
Article PubMed PubMed Central Google Scholar
Wu, Y. et al. Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope. Proc. Natl Acad. Sci. USA. 114, E7564–e7573 (2017).
Article CAS PubMed PubMed Central Google Scholar
Cho, H. W., Howard, C. R. & Lee, H. W. Review of an inactivated vaccine against hantaviruses. Intervirology 45, 328–333 (2002).
Article CAS PubMed Google Scholar
Zheng, Y. et al. Persistence of immune responses to vaccine against haemorrhagic fever with renal syndrome in healthy adults aged 16-60 years: results from an open-label2-year follow-up study. Infect. Dis. 50, 21–26 (2018).
Article ADS CAS Google Scholar
Li, K. et al. Induction of Hantaan virus-specific immune responses in C57BL/6 mice by immunization with a modified recombinant adenovirus containing the chimeric gene, GcS0.7. Int. J. Mol. Med. 32, 709–716 (2013).
Article ADS CAS PubMed Google Scholar
McClain, D. J., Summers, P. L., Harrison, S. A., Schmaljohn, A. L. & Schmaljohn, C. S. Clinical evaluation of a vaccinia-vectored Hantaan virus vaccine. J. Med. Virol. 60, 77–85 (2000).
Article CAS PubMed Google Scholar
Cao, W. et al. The rVSV-EBOV vaccine provides limited cross-protection against Sudan virus in guinea pigs. NPJ Vaccines 8, 91 (2023).
Article CAS PubMed PubMed Central Google Scholar
Emanuel, J. et al. A VSV-based Zika virus vaccine protects mice from lethal challenge. Sci. Rep. 8, 11043 (2018).
Article ADS PubMed PubMed Central Google Scholar
Yahalom-Ronen, Y. et al. A single dose of recombinant VSV-∆G-spike vaccine provides protection against SARS-CoV-2 challenge. Nat. Commun. 11, 6402 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Huttner, A. et al. Determinants of antibody persistence across doses and continents after single-dose rVSV-ZEBOV vaccination for Ebola virus disease: an observational cohort study. Lancet Infect. Dis. 18, 738–748 (2018).
Article CAS PubMed PubMed Central Google Scholar
Cline, B. L. Ecological associations of vesicular stomatitis virus in rural Central America and Panama. Am. J. Tropical Med. Hygiene 25, 875–883 (1976).
Article CAS Google Scholar
Johnson, K. M., Vogel, J. E. & Peralta, P. H. Clinical and serological response to laboratory-acquired human infection by Indiana type vesicular stomatitis virus (VSV). Am. J. Tropical Med. Hygiene 15, 244–246 (1966).
Article CAS Google Scholar
Roberts, A., Buonocore, L., Price, R., Forman, J. & Rose, J. K. Attenuated vesicular stomatitis viruses as vaccine vectors. J. Virol. 73, 3723–3732 (1999).
Article CAS PubMed PubMed Central Google Scholar
Marzi, A., Feldmann, F., Geisbert, T. W., Feldmann, H. & Safronetz, D. Vesicular stomatitis virus-based vaccines against Lassa and Ebola viruses. Emerging infect. Dis. 21, 305–307 (2015).
Article CAS Google Scholar
Warner, B. M. et al. Vesicular Stomatitis Virus-Based Vaccines Provide Cross-Protection against Andes and Sin Nombre Viruses. Viruses 11, 645 (2019).
Article CAS PubMed PubMed Central Google Scholar
Yoshimatsu, K. et al. Protective immunity of Hantaan virus nucleocapsid and envelope protein studied using baculovirus-expressed proteins. Arch. Virol. 130, 365–376 (1993).
Article CAS PubMed Google Scholar
Van Epps, H. L., Schmaljohn, C. S. & Ennis, F. A. Human memory cytotoxic T-lymphocyte (CTL) responses to Hantaan virus infection: identification of virus-specific and cross-reactive CD8(+) CTL epitopes on nucleocapsid protein. J. Virol. 73, 5301–5308 (1999).
Article PubMed PubMed Central Google Scholar
Li, J. et al. Heat shock protein 70 fused to or complexed with hantavirus nucleocapsid protein significantly enhances specific humoral and cellular immune responses in C57BL/6 mice. Vaccine 26, 3175–3187 (2008).
Article CAS PubMed Google Scholar
O'Donnell, K. L., Gourdine, T., Fletcher, P., Clancy, C. S. & Marzi, A. Protection from COVID-19 with a VSV-based vaccine expressing the spike and nucleocapsid proteins. Front. Immunol. 13, 1025500 (2022).
Article CAS PubMed PubMed Central Google Scholar
Cheng, L. F. et al. Incorporation of GM-CSF or CD40L Enhances the Immunogenicity of Hantaan Virus-Like Particles. Front. Cell Infect. Microbiol. 6, 185 (2016).
Article PubMed PubMed Central Google Scholar
Wang, B. et al. Dihydropyridine-derived calcium channel blocker as a promising anti-hantavirus entry inhibitor. Front. Pharmacol. 13, 940178 (2022).
Article CAS PubMed PubMed Central Google Scholar
Ramanathan, H. N. & Jonsson, C. B. New and Old World hantaviruses differentially utilize host cytoskeletal components during their life cycles. Virology 374, 138–150 (2008).
Article CAS PubMed Google Scholar
Serris, A. et al. The Hantavirus Surface Glycoprotein Lattice and Its Fusion Control Mechanism. Cell 183, 442–456.e416 (2020).
Article CAS PubMed PubMed Central Google Scholar
Slough, M. M., Chandran, K. & Jangra, R. K. Two Point Mutations in Old World Hantavirus Glycoproteins Afford the Generation of Highly Infectious Recombinant Vesicular Stomatitis Virus Vectors. mBio 10, e02372–02318 (2019).
Article CAS PubMed PubMed Central Google Scholar
Zhao, Y. et al. An algal lectin griffithsin inhibits Hantaan virus infection in vitro and in vivo. Front. Cell. Infect. Microbiol. 12, 881083 (2022).
Article CAS PubMed PubMed Central Google Scholar
Jiang, D. B. et al. Hantavirus Gc induces long-term immune protection via LAMP-targeting DNA vaccine strategy. Antiviral Res. 150, 174–182 (2018).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

This study was supported by the National Natural Science Foundation of China (82072268, 82002132), the Key Research and Development Project of Shaanxi Province (2019ZDLSF02-04), and University Support Grants (2018JSTS03, 2021JSTS10, 2020SWAQ09, 2022ZZXM044). The funders had no role in the study design, data collection, analysis, publication decisions, or manuscript preparation. Helper plasmids and vaccinia viruses used to generate recombinant vesicular stomatitis were gifts from the Wuhan Institute of Virology, Chinese Academy of Sciences (CAS). We thank the Editage for the language editing service.

Author information

These authors contributed equally: Hui Zhang, He Liu, Jing Wei, Yamei Dang.

Authors and Affiliations

Department of Microbiology, School of Preclinical Medicine, Airforce Medical University: Fourth Military Medical University, Xi’ an, Shaanxi, China
Hui Zhang, He Liu, Jing Wei, Yamei Dang, Yuan Wang, Qiqi Yang, Liang Zhang, Chuantao Ye, Linfeng Cheng, Hongwei Ma, Yangchao Dong, Yinghui Li, Yinlan Bai, Xin Lv, Yingfeng Lei, Zhikai Xu, Wei Ye & Fanglin Zhang
Center for Disease Control and Prevention of Shaanxi Province, Xi’an, Shaanxi, China
Jing Wei
Center of Clinical Aerospace Medicine, Airforce Medical University: Fourth Military Medical University, Xi’ an, Shaanxi, China
Bin Wang
Student Brigade, School of Preclinical Medicine, Airforce Medical University: Fourth Military Medical University, Xi’ an, Shaanxi, China
Xiaolei Jin

Authors

Hui Zhang
View author publications
You can also search for this author in PubMed Google Scholar
He Liu
View author publications
You can also search for this author in PubMed Google Scholar
Jing Wei
View author publications
You can also search for this author in PubMed Google Scholar
Yamei Dang
View author publications
You can also search for this author in PubMed Google Scholar
Yuan Wang
View author publications
You can also search for this author in PubMed Google Scholar
Qiqi Yang
View author publications
You can also search for this author in PubMed Google Scholar
Liang Zhang
View author publications
You can also search for this author in PubMed Google Scholar
Chuantao Ye
View author publications
You can also search for this author in PubMed Google Scholar
Bin Wang
View author publications
You can also search for this author in PubMed Google Scholar
Xiaolei Jin
View author publications
You can also search for this author in PubMed Google Scholar
Linfeng Cheng
View author publications
You can also search for this author in PubMed Google Scholar
Hongwei Ma
View author publications
You can also search for this author in PubMed Google Scholar
Yangchao Dong
View author publications
You can also search for this author in PubMed Google Scholar
Yinghui Li
View author publications
You can also search for this author in PubMed Google Scholar
Yinlan Bai
View author publications
You can also search for this author in PubMed Google Scholar
Xin Lv
View author publications
You can also search for this author in PubMed Google Scholar
Yingfeng Lei
View author publications
You can also search for this author in PubMed Google Scholar
Zhikai Xu
View author publications
You can also search for this author in PubMed Google Scholar
Wei Ye
View author publications
You can also search for this author in PubMed Google Scholar
Fanglin Zhang
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization, W YE and FL ZHANG; methodology, H LIU, W YE, J WEI and YM DANG; validation, H ZHANG, H LIU, J WEI, QQ YANG and Y WANG; formal analysis, H ZHANG, W YE, YM DANG and XL JIN; investigation, J WEI, H LIU, W YE and Y WANG; resources, W YE, H LIU, YF LEI, L ZHANG, Y WANG, CT YE, LF CHENG, YH LI, YL BAI, X LV, HW MA, B WANG and XL JIN; writing, original draft preparation, W YE and H ZHANG; writing, review and editing, W YE and FL ZHANG; supervision, W YE and FL ZHANG; project administration, FL ZHANG, W YE, H LIU and ZK XU; funding acquisition, FL ZHANG, W YE, H LIU and ZK XU. H ZHANG, H LIU, J WEI, and YM DANG were considered as co-first authors. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Zhikai Xu, Wei Ye or Fanglin Zhang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

SUPPLEMENTARY DATA

REPORTING SUMMARY

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Zhang, H., Liu, H., Wei, J. et al. Single dose recombinant VSV based vaccine elicits robust and durable neutralizing antibody against Hantaan virus. npj Vaccines 9, 28 (2024). https://doi.org/10.1038/s41541-024-00814-2

Download citation

Received: 04 September 2023
Accepted: 11 January 2024
Published: 10 February 2024
DOI: https://doi.org/10.1038/s41541-024-00814-2