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DNA and mRNA Vaccines Against Hantaviruses: Antigen Design, Delivery Strategies and Translational Progress

Submitted:

01 September 2026

Posted:

02 September 2026

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Abstract
Hantaviruses are members of the Hantaviridae family, which can cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). At present, no specific antiviral treatment is available for hantavirus infection, while licensed hantavirus vaccines are restricted to a limited number of countries in Asia. This review summarizes recent advances in hantavirus DNA and messenger RNA (mRNA) vaccines, focusing on antigen design, delivery systems, immunogenicity, safety and translational development. Peer-reviewed studies published primarily within the past 10 years were evaluated together with available hantavirus vaccine trials registered in ClinicalTrials.gov, with emphasis on Hantaan virus (HTNV), Puumala virus (PUUV) and Andes virus (ANDV). DNA vaccine candidates have demonstrated immunogenicity in preclinical and early clinical studies, with an acceptable safety profile. mRNA vaccines represent a newer approach with promising preclinical evidence of humoral and cellular immune responses. However, they have not yet been evaluated clinically. Studies have also reported the potential for varying degrees of cross-protection between hantavirus species, although protection remains limited by antigenic diversity. Overall, current evidence supports further development of nucleic acid-based hantavirus vaccines, particularly multivalent strategies, with emphasis on standardized evaluation and clinical efficacy.
Keywords: 
hantavirus infections
;  
Andes virus
;  
orthohantaviruses
;  
Puumala virus
;  
vaccines
;  
DNA
;  
mRNA vaccines
Subject: 
Biology and Life Sciences  -   Biochemistry and Molecular Biology

1. Introduction

Hantaviruses are zoonotic viruses and members of the Hantaviridae family. They are maintained in nature by small mammalian reservoirs, particularly rodents and, in some cases, bats [1,2,3]. Each hantavirus genotype is usually linked to specific rodent species. Populations of these animals fluctuate in response to ecological and environmental conditions such as food supply, climate availability with population expansions sometimes associated with increased human hantavirus cases [4]. Human infection occurs predominantly through the inhalation of aerosolized particles contaminated with excreta or secretions from infected rodents (via urine, saliva, feces); in contrast transmission through animal bites is uncommon [6]. Human-to-human transmission has been described for Andes virus (ANDV) [6,7,8], although its broader significance remains controversial [9]. Human hantavirus infections are generally classified into two major forms: hemorrhagic fever with renal syndrome (HFRS), including milder nephropathia epidemica caused by Puumala virus (PUUV), primarily linked to Old World hantaviruses in Europe and Asia and hantavirus cardiopulmonary syndrome (HCPS), primarily linked to New World hantaviruses in the Americas [10,11,12]. Pathogenic Old World hantaviruses are for example Hantaan virus (HTNV), Seoul virus (SEOV), PUUV, Dobrava-Belgrade virus (DOBV) and Amur virus (AMV) [13,14], whereas New World hantaviruses include ANDV, Juquitiba virus (JUQV), Laguna Negra virus (LANV), Sin Nombre virus (SNV) and Araucaria virus (ARAV) [15].
The importance of hantaviruses has become increasingly apparent in the context of contemporary global events, including climate change, environmental disturbance, urban expansion, changes in agricultural practices, global travel and the growing recognition of zoonotic spillover as a major source of emerging infectious diseases. Environmental and climatic changes can influence the distribution, population density and ecological behaviour of rodent reservoirs, thereby altering the frequency and geographical range of human exposure to hantaviruses [16]. At the same time, the COVID-19 pandemic has demonstrated the profound social, economic and healthcare consequences that can result from emerging viral pathogens and has substantially increased global awareness of the importance of pandemic preparedness and the rapid development of vaccine platforms. In this context, hantaviruses should be considered not only as established endemic pathogens but also as viruses of increasing strategic importance within the broader landscape of emerging zoonotic infections. The continuing risk of environmental change–driven shifts in reservoir ecology and the potential emergence of hantaviruses in previously unaffected regions further strengthen the rationale for developing flexible, rapidly adaptable vaccine platforms, including DNA vaccines, capable of targeting high-priority pathogens such as HTNV [17].
At present, hantavirus vaccination remains an important but unresolved problem of disease prevention. Although inactivated vaccines have been used locally in China and the Republic of Korea settings, there is still no broadly available, internationally licensed vaccine for hantavirus infection in Europe, the United States or Latin America [18,19]. The World Health Organization (WHO) currently states that there is no licensed specific antiviral treatment or vaccine for hantavirus infection and clinical management remains primarily supportive, focused on early recognition and treatment of respiratory, cardiovascular and renal complications [19].
This review provides an overview of current progress in hantavirus deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) vaccine development, highlighting the diversity of vaccine platforms, their proposed immunological mechanisms and the major challenges that hinder translation into widely available vaccines. Particular attention is given to the most extensively studied and clinically relevant hantaviruses, including HTNV, PUUV and ANDV.

2. Structure of Hantaviruses

Based on the International Committee on Taxonomy of Viruses (ICTV), hantaviruses are classified to the family Hantaviridae and the genus Orthohantavirus. They are enveloped RNA viruses with a negative polarity single-stranded genome with virions generally measuring approximately 80-100 nm in diameter [1,20]. As shown in Figure 1, hantaviruses genome consists of three RNA segments: small (S) segment encodes the nucleocapsid (N) protein and in some of hantaviruses a nonstructural protein (NSs), medium (M) segment encodes a glycoprotein precursor (GPC) and and large (L) segment encodes the viral RNA-dependent RNA polymerase (RdRp) [21,22]. GPC is processed by the cellular signal peptidase complex at the conserved WAASA sequence, into the two viral envelope glycoproteins, Gn and Gc [23]. The nucleotide length of the S, M and L genome segments varies among hantavirus species and even between individual strains or isolates [24].
The viral genome is contained within ribonucleoprotein complexes (RNP), in which each RNA segment is encapsidated by the nucleoprotein (N) [25,26]. Hantavirus particles are surrounded by a host-derived lipid envelope originating from Golgi membranes. The lipid membrane is lined with 10-nm-long glycoprotein spikes, composed of the viral envelope glycoproteins Gn and Gc [27].
Table 1. Hantavirus genome segments, encoded proteins and their functional roles in the viral replication cycle [28,29,30,31,32,33,34,35,36,37].
Table 1. Hantavirus genome segments, encoded proteins and their functional roles in the viral replication cycle [28,29,30,31,32,33,34,35,36,37].
Segment Encoded viral protein(s) Main function of protein References
S Nucleoprotein/Nucleocapsid protein (N) Encapsidates viral genomic RNA and forms vRNP complexes associated with the L protein; supports genome packaging, replication and transcription. [28,29]
Nonstructural protein (NSs) NSs may act as an antagonist of type I interferon induction and contribute to viral immune evasion (this function has been shown for ANDV NSs and for PUUV NSs). [30,31,32]
M Glycoprotein precursor GPC → Gn and Gc glycoproteins Gc and Gn form the spike complex responsible for receptor-binding.
Gc acts as a class II fusion protein mediating endosomal membrane fusion.
GN binds to N and RNA, supporting recruitment of vRNPs to sites of assembly and genome packaging into virions. They are the principal surface-exposed antigens and major targets of neutralizing antibodies. 		[33,34,35,36]
L L protein, which contain the RdRp and endonuclease Catalyzes viral genome replication and transcription within vRNP complexes composed of genomic RNA, N protein and L protein. Mediates cap-snatching to acquire 5′ capped host-derived primers for viral mRNA transcription. [37]
Abbreviations: ANDV - Andes virus; GPC - glycoprotein precursor; Gn - glycoprotein N; Gc - glycoprotein C; N - nucleocapsid protein; NSs - nonstructural protein; PUUV - Puumala virus; RdRp - RNA-dependent RNA polymerase; RNA - ribonucleic acid; vRNP - viral ribonucleoprotein complex.

3. DNA Vaccines Against Hantaviruses

3.1. Mechanism of Action of DNA Vaccines

DNA vaccines are a nucleic acid vaccine platform in which plasmid DNA encoding a selected antigen is delivered into host cells [38,39]. DNA vaccines work by delivering plasmid DNA containing a gene encoding a selected antigen into host cells. The plasmid enters the nucleus, where the antigen-encoding gene is transcribed, and the resulting mRNA is used to produce the antigen. The antigen can then be presented through both MHC class I and class II pathways, activating CD8+ and CD4+ T cells and inducing antigen-specific B-cell responses and antibody production. Thus, DNA vaccines can stimulate both cellular and humoral immune responses [40].

3.2. Design and Construction of the DNA Vaccine Plasmid

The first HTNV DNA vaccine constructs were designed to express the two envelope glycoproteins, Gn and Gc (historically referred as G1 and G2). These are the M-gene-based DNA vaccines. The viral M segment encodes a glycoprotein precursor [41]. Experimental studies have shown that it is processed in host cells and the resulting Gn and Gc glycoproteins are directed to the secretory pathway [41,42,43]. Mature Gn and Gc are major envelope antigens. Eventually, these can induce antibody responses, including virus-neutralizing antibodies [41].
The pWRG/HTN-M plasmid was generated using the pWRG7077 backbone [41]. The backbone provides regulatory elements necessary for gene expression in mammalian cells [41,42,43]. One of these elements is the human cytomegalovirus immediate-early (hCMV IE) promoter, which drives transcription of the inserted sequence in mammalian cells [41,42]. The construct also contains intron A CMV, which increases expression from the expression cassette [42]. The bovine growth hormone polyadenylation signal (BGH pA) participates in ending transcription and transcript polyadenylation [43,44]. Additionally, the plasmid backbone contains the kanamycin resistance gene (KanR) and a bacterial origin of replication type pUC19/pMB1. These are bacterial elements used for bacterial selection and plasmid propagation, respectively [45,46]. A BglII fragment containing the HTNV M-segment coding sequence was excised from pTZ19RHTNMm and inserted into the BamHI-cut site of pWRG7077. This construct subsequently served as the basis for further optimization of HTNV DNA vaccine candidates [41,47].
To refine the construct and eliminate non-essential sequences, a modified version was generated. The HTNV M-segment insert was PCR amplified from pWRG/HTN-M using primer 1-24 together with the reverse primer (HTNMX, 5′-GCGCGGATCCGTTTGTGGTTAGAAAGCTAC). The product was cut with NotI and BamHI and ligated into theNotI-BglII-cut pWRG7077 vector. Although pWRG/HTN-M(x) retained the same HTNV coding region as the parental plasmid, it lacked part of the 3′ untranslated region and the vector sequence between BamHI andBglII [41,48], with approximately 100 undesirable nucleotides derived from the gene nef SIV [41]. Hooper et al. demonstrated that the deletion of these fragments did not result in a detectable reduction in expression of the cloned gene [41].
The final stage of construct optimization involved replacing the native viral sequence with a synthetically produced, codon-optimized M-segment open reading frame (ORF) and plasmid designated pWRG/HTN-M(co). This modification was intended to eliminate sequence features that could reduce transcript stability or limit efficient expression in mammalian cells. The optimized sequence, encoding the HTNV envelope glycoproteins Gn and Gc, was inserted into the pWRG7077 expression vector through the NotI and BglII restriction sites [41].
The similar strategy was also amplified to DNA vaccines targeting other pathogenic Orthohantaviruses. pWRG/PUU-M(s2), the PUUV DNA vaccine plasmid, contained a synthetic cDNA sequence based on a consensus of several PUUV isolates, which are optimized for codon usage (Homo sapiens) and mRNA stability [49]. In preclinical studies, it induced high-titer neutralizing antibodies in hamsters and nonhuman primates and protected vaccinated hamsters against PUUV infection. Protection was not observed against all related HFRS-associated hantaviruses. Interestingly, pWRG/PUU-M(s2) also protected hamsters against lethal ANDV disease despite no discernible ANDV cross-neutralizing antibodies [49]. The ANDV construct, pWRG/AND-M(opt2), was prepared by inserting the optimized M-segment ORF from the Chile-9717869 strain into the pWRG7077 vector, following the same general approach used for the HTNV and PUUV vaccines [50]. These constructs were first evaluated in preclinical models - for example in rabbits and nonhuman primates. Vaccination induced high-titer neutralizing antibody responses [51].
These plasmid constructs were subsequently used as vaccine candidates in clinical studies [Table 2].

3.3. Immunogenicity of DNA Vaccines and Its Determinants

Immunogenicity is primarily assessed through the induction of neutralizing antibodies (NAbs), typically quantified using plaque reduction neutralization tests (PRNT) [52,53]. The PRNT, originally developed by Dulbecco and colleagues in 1956, is a functional assay that evaluates the capacity of serum antibodies to inhibit viral entry and replication. Initially, this method was employed to monitor the evolution of dengue viruses by measuring virus-specific neutralizing antibody responses. Subsequently, PRNT has been widely applied to correlate immune status with clinical outcomes, such as distinguishing asymptomatic from symptomatic dengue virus infections and to evaluate vaccine-induced immunogenicity. At present, PRNT is regarded as the gold standard for determining NAb titers in contexts where humoral immune responses are associated with protective immunity [53].
DNA vaccines encoding HTNV glycoproteins have consistently demonstrated the ability to elicit strong neutralizing antibody responses across multiple animal models, including hamsters, rabbits and nonhuman primates. Due to the absence of an ideal animal model that fully recapitulates human HFRS, preclinical evaluation has relied heavily on hamster infection models. In this system, vaccine efficacy is assessed indirectly by measuring infection following viral challenge [52]. In addition to inducing humoral immunity, these vaccines also stimulate cellular immune responses, notably CD4⁺ T-cell activation and cytokine production, which are essential for the establishment of effective and long-lasting immunity [52].
In vaccine studies, neutralizing antibodies targeting the envelope glycoproteins encoded by the M segment are quantified in sera from vaccinated hamsters using PRNT assays [54,55,56]. Following viral challenge, antibodies against the N protein are detected by enzyme-linked immunosorbent assay (ELISA) using recombinant N antigen produced in Escherichia coli [57]. Since the S genome segment encoding the N protein is not included in the vaccine construct, the absence of anti-N antibodies serves as an indicator of protection against infection with the challenge virus [58].
The pseudovirus neutralization test (pVNT) represents a variation of the PRNT, based on the same underlying principle but utilizing pseudotyped viral particles instead of live virus. These pseudoviruses are non-replicative and incorporate viral entry proteins, such as the spike protein found in SARS-CoV-2, enabling a single round of infection. The assay typically employs ACE2-expressing cell lines, most commonly HEK-293T cells, thereby emphasizing that viral entry is mediated through the interaction between the spike protein and the ACE2 receptor [59].
The delivery of the DNA vaccines can significantly influence immunogenicity. Previous studies have focused primarily on gene gun and intramuscular (IM) electroporation, whereas more recent studies have explored simpler delivery methods by using disposable syringe and needle-free injection [51,60,61,62,63]. Physical delivery methods, particularly electroporation, can enhance the immunogenicity of DNA vaccines compared with conventional needle-and-syringe administration. This conventional administration, done by needle and syringe, leads to the deposition of DNA in tissues [51,60,61,62].
Other delivery methods were developed to facilitate the administration [51,60,62]. The TriGrid Delivery System combines the delivery with electroporation, the method increasing the cellular uptake of plasmid DNA [18,64,65]. Upon the DNA injection, the electrical pulses are applied, causing the transient disruption and permeability of cell membranes [61].
An alternative method, that uses high-pressure fluid jet to deposit DNA in tissue, is PharmaJet Stratis® Needle-Free Jet Injection [50,61]. Unlike the TriGrid System, it does not use electroporation and is operationally simpler and does not require an electroporation pulse generator or electrode array. This method was used for example in a Phase I clinical trial of HTNV and PUUV DNA vaccines [61].
DNA vaccine immunogenicity is influenced not only by the delivery technology but also by the route of administration, dose, vaccine construct and vaccination schedule [51,60]. Kwilas et al. in a preclinical study on immunogenicity of an SNV DNA vaccine were comparing the IM and ID route using the PharmaJet System. In rabbits, the seroconversion after the first dose reached 100% (4/4) in both the 0,4 mg IM group and 0,4 mg ID group; after the third dose seroconversion remained at 100% in both groups. In rhesus macaques that received 1,0 mg IM seroconversion was 100% (3/3) after the first dose and 100% after the third dose. However, in rhesus macaques receiving 1.0mg ID only 1/3 animals have seroconverted after the first dose, but 3/3 animals reached 100% seroconversion after the third dose. It is important to note the presence of species-specific differences in skin structure which are a significant factor for ID route. Another consideration is injection volume of the liquid vaccine solution - the ID route required a smaller volume than the IM route [51].
Although it is not possible to compare directly the results of both methods, needle-free jet injection (PharmaJet Stratis) and electroporation-based delivery (TriGrid) both resulted in inducing neutralizing antibody responses in clinical trials [61,62,63,64]. The important element is not only the efficiency of DNA delivery, but also reproducibility and practicality of the vaccination procedure [61]. Electroporation requires electrical impulses application and another device, while the needle-free jet injection does not use electroporation and eliminates the conventional needle [61]. The progression from using gene gun method, IM electroporation and needle-free injection, reflects efforts to make simpler, more practical hantavirus DNA vaccine procedures, all while maintaining immunogenicity [61,62,63].
Preclinical studies have demonstrated that DNA vaccination against HTNV confers protective immunity not only against homologous HTNV challenge but also against related hantaviruses, including SEOV and DOBV, indicating the presence of cross-protective immune responses. However, protection against more antigenically divergent viruses, such as PUUV, remains limited, thereby necessitating the development of multivalent vaccine formulations to achieve broader coverage [18,52,65].
Despite these promising findings, several challenges have emerged in preclinical evaluations. A major limitation is the occurrence of immune interference in multivalent vaccine formulations. Specifically, when HTNV and PUUV DNA vaccines are co-administered, the immune response to HTNV is frequently diminished, whereas responses to PUUV predominate. This phenomenon presents a significant obstacle to the development of broadly protective hantavirus vaccines [52].
In addition to safety assessments, serum neutralizing antibody responses to HTNV and PUUV were evaluated in samples collected on days 0 and 57 from a cohort of eighty rabbits. PRNT analysis of day 57 sera demonstrated that 100% of rabbits vaccinated with HTNV DNA vaccine, PUUV DNA vaccine, or a combination of both developed neutralizing antibodies against HTNV and/or PUUV following a three-dose immunization regimen. Neutralizing antibody titers against HTNV in sera from rabbits vaccinated with either the HTNV DNA vaccine alone or the combined HTNV and PUUV vaccine ranged from <20 to 40,960. Similarly, PUUV-specific neutralizing antibody titers in sera from rabbits vaccinated with the PUUV DNA vaccine alone or in combination ranged from 40 to 40,960. The homotypic PRNT₅₀ geometric mean titers (GMTs) for the PUUV and HTNV vaccine groups were 2,388 and 7,828, respectively. Meaning, that both vaccines were highly effective at producing protective antibodies in rabbits when given on their own. The HTNV vaccine produced a higher average level of antibodies than the PUUV vaccine, but both results were considered strong indicators that the vaccines were "biologically active and immunogenic" [58].
To address the issue of vaccine interference observed in hamsters, rabbits and humans modified vaccine ratios were evaluated in hamster models. Animals were immunized three times at approximately three-week intervals via IM-electroporation (IM-EP) with a total dose of 100 μg DNA, administered either as individual vaccines or as mixtures. Tested HTNV:PUUV DNA ratios included 1:1, 2:1 and 10:1. Consistent with previous observations, the 1:1 vaccine mixture elicited neutralizing antibodies against PUUV but not HTNV. Increasing the proportion of HTNV DNA to 2:1 or 10:1 did not alter this outcome, as no detectable HTNV-specific neutralizing antibody response was observed by PRNT [58]. Recent advances in antigen design, particularly prefusion stabilization of glycoproteins, represent a significant step forward in improving vaccine efficacy. These structure-based approaches enhance the presentation of neutralizing epitopes and promote stronger and more durable immune responses. Furthermore, strategies such as codon optimization, synthetic gene design and prime–boost regimens are being explored to further enhance immunogenicity [18,66]. Continued optimization of antigen design and delivery methods is likely to be critical for successful translation into effective human vaccines.

3.4. Clinical Evaluation of Hantavirus DNA Vaccines

DNA vaccines targeting pathogenic orthohantaviruses have progressed into clinical evaluation with efforts concentrated primarily on HTNV and PUUV and more recently on ANDV. Table 2, Table 3 and Table 4 summarises clinical trials of DNA vaccines against HTNV, PUUV and ANDV registered in ClinicalTrials.gov from 2013 to 2026. The data was searched in 4 August 2026 using terms: “Puumala AND vaccine,” “Hantaan AND vaccine,” “Andes AND vaccine”as well as the corresponding abbreviations: “PUUV AND vaccine”, “HTNV AND vaccine and “ANDV AND vaccine”. Six clinical trials were identified: five evaluated monovalent or combined DNA vaccines against HTNV and PUUV, whereas one evaluated a DNA vaccine against ANDV.
Table 3 summarizes the local and systemic adverse events (AEs) about registered clinical trials evaluating DNA vaccines against HTNV, PUUV and ANDV. Clinical trials were excluded from the quantitative comparison when their results had not been reported in a peer-reviewed scientific publication. Accordingly, NCT03718130 was excluded because the available findings were limited to the ClinicalTrials.gov registry and had not been published in a peer-reviewed journal [64]. NCT04333459 was excluded because no study results had been publicly posted or published at the time of the literature search [70].
Because most of the studies were phase I or early phase II trials, safety and tolerability were the most analyzed elements. Overall, the evaluated hantavirus DNA vaccines shown on Table 3 have an acceptable safety profile. Injection-site pain was the most frequently reported local reaction, occurring in 77.8-96.2% of participants in the HTNV/PUUV studies. Other common local events included erythema, bruising, swelling, induration, tenderness and skin discoloration. The high frequency of local reactions in studies using electroporation or needle-free jet injection suggests that many of these reactions may be related not only to the plasmid vaccine itself but to the delivery method.
The most frequently reported systemic AEs were headache, fatigue, myalgia and malaise. In the HTNV/PUUV trials, these reactions were generally mild or moderate and self-limiting.
No vaccine- or procedure-related serious adverse events (SAEs) were reported in mentioned studies. Several unrelated SAEs, including hypoglycaemia and acute rhabdomyolysis, were recorded in individual trials but were not considered causally related to vaccination [50,62,67,70].
Table 4 summarizes the available clinical data on the humoral immune response elicited by DNA vaccines against HTNV, PUUV, and ANDV, including seroconversion rates and neutralizing antibody titers.
The available clinical data indicate that orthohantavirus DNA vaccines are capable of inducing measurable neutralizing-antibody responses against HTNV, PUUV and ANDV. The earliest HTNV/PUUV study, NCT01502345, demonstrated proof-of-concept immunogenicity, but response rates were moderate and heterogeneous, whereas the combined formulation produced a markedly stronger response to PUUV than to HTNV. This asymmetry suggests that simply combining two plasmids does not necessarily preserve the immunogenicity of each component and raises the possibility of antigenic or immunological interference within bivalent formulations [62,67].
Later studies demonstrated that repeated dosing and booster administration could increase both the proportion of responders and neutralizing-antibody titers, suggesting that vaccination schedule is an important determinant of immunogenicity and may be at least as relevant as the absolute plasmid dose [50,61,63,64,68,69,70].
A notable limitation of the current evidence is the lack of methodological uniformity across trials. Different studies used distinct plasmid constructs, dosing regimens, delivery devices, routes of administration, sampling schedules and neutralization assays. In addition, seropositivity and seroconversion were not defined consistently across the clinical development program. Earlier studies often used a detectable neutralizing-antibody threshold as the principal measure of response, whereas later trials applied more stringent seroconversion criteria based on higher post-vaccination titers or fold increases from baseline [50,62,63,64,67,68,69,70].
Taken together, the available clinical data support the continued development of DNA vaccines against orthohantaviruses, particularly with further optimization of multidose vaccination schedules. The results obtained so far indicate that these vaccines can induce neutralizing antibody responses and generally show an acceptable safety profile, although the magnitude and consistency of the immune response vary between studies and vaccination regimens. Further clinical studies are therefore needed to determine the most effective dose and dosing schedule, evaluate the durability of vaccine-induced immunity and confirm long-term safety.

4. mRNA Vaccines Against Hantaviruses

4.1. Mechanism of Action of mRNA Vaccines

The mRNA vaccines are a flexible nucleic acid vaccine platform in which mRNA encoding a selected antigen is delivered into host cells and translated into the corresponding protein. The resulting antigen stimulates both humoral and cellular immune responses, including antigen-specific B-cell and T-cell responses [72,73]. Nucleoside modifications, particularly N1-methylpseudouridine (m1Ψ), can improve mRNA translation and reduce unwanted innate immune activation, while lipid nanoparticles (LNPs) facilitate mRNA delivery into cells [72,73]. The clinical success of mRNA vaccines against COVID-19 has further demonstrated the potential of this platform for the development of vaccines against other infectious diseases [74,75].
In hantavirus vaccine development, the M genomic segment, which encodes the glycoprotein precursor giving rise to the Gn and Gc glycoproteins, represents an important target for mRNA-based vaccine design [18,76,77]. These glycoproteins are expressed following vaccination and induce antigen-specific immune responses [18,76,77].
In a 2024 study, Zhang et al. developed an mRNA vaccine encoding HTNV glycoproteins. The vaccine induced robust humoral and cellular immune responses and provided protective efficacy in mice, with a particularly strong virus-specific Th1 response [76]. Similarly, an mRNA-LNP vaccine encoding a prefusion-stabilized HTNV glycoprotein induced potent neutralizing antibody responses and protected mice against viral challenge [77]. These findings support the potential of mRNA vaccines as a strategy for the prevention of Hantaan virus infection.

4.2. Immunological Response Induced by mRNA Vaccines

The protective effect of mRNA vaccines against hantaviruses depends on the induction of both humoral and cellular immune responses [72,73]. Following antigen expression, viral glycoproteins such as Gn and Gc are recognized by the immune system and stimulate antigen-specific B and T cells [72,73]. Activation of B cells and germinal-center formation promotes the generation of high-affinity antibodies, including neutralizing antibodies capable of interfering with viral entry [73,77]. T-cell responses, particularly Th1 responses, may further contribute to viral clearance and the development of immunological memory [72,76]. In preclinical HTNV studies, mRNA vaccination induced both neutralizing antibody responses and strong Th1-associated cellular immunity, while prefusion-stabilized glycoprotein vaccination additionally enhanced germinal-center responses [76,77]. In ANDV models, mRNA vaccination similarly induced neutralizing antibodies and cellular immune responses associated with protection against viral challenge [79,80]. Together, these findings suggest that effective protection against hantaviruses may require coordinated humoral and cellular immunity rather than antibody responses alone [76,77,78,79].

4.3. Advantages and Limitations of mRNA Vaccines

mRNA vaccines offer several advantages over conventional vaccine platforms. Their synthetic production allows for rapid vaccine design and modification without the need to cultivate the pathogen, while the platform can induce both humoral and cellular immune responses [72,73]. In addition, mRNA-LNP formulations can be readily adapted to encode different antigens, making the platform particularly attractive for emerging infectious diseases [72].
On the other hand, mRNA vaccines present several limitations. The intrinsic instability of mRNA and its susceptibility to degradation create challenges related to formulation, storage and delivery [72,80]. Lipid nanoparticles can improve mRNA stability and intracellular delivery, but their formulation and physicochemical properties may influence the stability and performance of mRNA vaccines [72,80]. In the context of hantavirus vaccination, an important limitation is the limited preclinical evidence and the absence of advanced clinical evaluation of mRNA-based candidates. Nevertheless, studies in Hantaan virus models have demonstrated promising immunogenicity and protective efficacy, supporting further investigation of this platform [76,77].

4.4. Preclinical Studies of mRNA Vaccines Against Hantaviruses

Preclinical studies of mRNA vaccines against hantaviruses have so far focused mainly on HTNV and ANDV [76,77,78,79]. For HTNV, Zhang et al. compared an mRNA vaccine encoding HTNV glycoproteins with other nucleic acid vaccine candidates in mice. The mRNA vaccine induced robust and sustained humoral and cellular immune responses and showed protective efficacy comparable to that of an inactivated vaccine [76]. Notably, it induced a particularly strong HTNV-specific Th1 response, whereas higher neutralizing antibody levels were observed with the DNA-LNP formulation [76].
Further optimization of the HTNV vaccine focused on antigen design. Ye et al. developed a prefusion-stabilized HTNV glycoprotein (GP-C3) and incorporated it into an mRNA-LNP vaccine [77]. The vaccine induced potent neutralizing antibody responses and enhanced germinal-center formation and protected mice against high-dose HTNV challenge [77].
For ANDV, Kuzmin et al. compared conventional uridine-containing mRNA (U-mRNA) with N1-methylpseudouridine-modified mRNA (m1Ψ-mRNA), both encoding the viral Gn/Gc glycoprotein precursor [78]. The study evaluated glycoprotein expression, innate immune activation, antibody responses, germinal-center formation and protective efficacy [78]. Both formulations induced neutralizing antibodies and protected Syrian hamsters against lethal ANDV challenge at the higher dose, while m1Ψ-mRNA showed reduced innate immune activation [78].
More recently, Meyer et al. investigated a single-dose ANDV mRNA vaccine and demonstrated protection from overt disease in golden Syrian hamsters, including at the lowest tested dose of 1 μg [79]. These findings suggest that mRNA vaccines may provide effective protection against hantaviruses while allowing further optimization of antigen design, nucleoside modification and vaccination schedules [76,77,78,79].
Overall, the available evidence remains limited to preclinical studies, mainly involving HTNV and ANDV [76,77,78,79]. Comparable primary mRNA vaccine studies against PUUV have not been identified. Importantly, no clinical trials evaluating mRNA vaccines against HTNV, PUUV, or ANDV were identified in ClinicalTrials.gov [81]. Therefore, the safety, optimal dosing, durability of protection and efficacy of these vaccines in humans remain unknown.

5. Materials and Methods

A comprehensive literature search was conducted using PubMed, Scopus, and Google Scholar, focusing primarily on studies published within the past 10 years (2016–2026), while earlier pivotal publications were also included when considered relevant to the development of hantavirus vaccines. Core search terms included combinations of: (“Puumala” AND “vaccine”), (“Hantaan” AND “vaccine”), (“Andes” AND “vaccine”), (“HTNV” AND “vaccine”), (“PUUV” AND “vaccine”) and (“ANDV” AND “vaccine”). ClinicalTrials.gov was searched using the same terms. All identified relevant hantavirus vaccine trials are summarized in Table 2. For the more detailed analyses presented in Table 3 and Table 4, only clinical trials for which results had been published in peer-reviewed journals were included. Additional eligibility criteria and the specific parameters extracted from these studies are described in text. The Figure 1 was created using GoodNotes 6 for iPad (v.6.3.55)—all based on the authors’ interpretation of synthesized data from multiple studies.

6. Conclusions and Future Directions

Hantaviruses remain an important public health concern due to their broad geographic distribution, zoonotic nature and potential to cause severe clinical syndromes, including HFRS and HCPS. Although inactivated vaccines are available and have been used in endemic regions of Asia, no broadly available, internationally licensed vaccine against hantavirus infection currently exists. The development of nucleic acid vaccine platforms, particularly DNA and mRNA vaccines, has therefore emerged as a promising approach to address the limitations associated with conventional hantavirus vaccine strategies.
Preclinical studies have provided substantial evidence that nucleic acid vaccines can induce both humoral and cellular immune responses against pathogenic orthohantaviruses. Among the antigens evaluated, the viral envelope glycoproteins Gn and Gc encoded by the M segment represent the principal targets because of their critical role in viral entry and their accessibility to neutralizing antibodies. Early DNA vaccine studies demonstrated that glycoprotein-based vaccination can induce neutralizing antibodies and protect experimental animals against homologous viral challenge. Importantly, studies involving HTNV, SEOV, DOBV, PUUV and ANDV have also demonstrated varying degrees of cross-reactive and cross-protective immunity, although the breadth of protection is strongly influenced by the antigenic relatedness of the viruses. These findings highlight both the potential and the limitations of developing broadly protective hantavirus vaccines based on a single viral antigen.
Advances in bioinformatics and computational biology have markedly accelerated progress in this field [82,83,84]. Comprehensive computational analyses of orthohantavirus motifs and codon usage have yielded important insights into host immune regulation and viral evolutionary history, facilitating the identification of conserved epitopes and antigenic regions essential for eliciting robust immune responses [85,86]. In addition, molecular docking and dynamic simulation approaches are employed to assess the binding affinity between vaccine candidate proteins and host immune receptors [87], while immune simulation analyses enable the prediction of natural immune responses [84,88]. Several multi-epitope subunit vaccines designed to provide cross-protection against orthohantaviruses, including HTNV and PUUV, have been developed using immunoinformatics-based strategies [82,83,84,85,86,87,88,89]. Nevertheless, all computationally designed vaccines require thorough experimental validation prior to clinical application.
Recent advances in antigen design have further improved the preclinical performance of nucleic acid vaccines. Structure-guided stabilization of hantavirus glycoproteins in their prefusion conformation, incorporation of conserved or immunodominant epitopes and optimization of antigen sequences have demonstrated the potential to enhance neutralizing antibody responses, germinal-center formation and cellular immunity. Notably, recent HTNV studies indicate that rationally designed glycoprotein antigens can generate stronger and more durable immune responses than earlier vaccine constructs.
The clinical development of DNA vaccines has provided an important translational extension of these preclinical findings. Early-phase studies targeting HTNV, PUUV and ANDV have demonstrated that DNA vaccination can induce measurable neutralizing-antibody responses and has generally shown an acceptable safety and tolerability profile. However, the magnitude and consistency of immunogenicity have varied between studies, vaccine formulations and vaccination schedules. In particular, studies of combined HTNV and PUUV vaccines indicate that simultaneous targeting of multiple hantaviruses does not necessarily result in equivalent immune responses against each component, emphasizing the potential challenge of antigenic or immunological interference in multivalent formulations. Moreover, differences in neutralization assays, response definitions, dosing schedules and administration strategies complicate direct comparisons across clinical studies.
mRNA vaccine platforms offer an additional opportunity to overcome some limitations associated with conventional DNA vaccination. Their transient expression profile and flexibility in antigen design allow rapid adaptation to newly characterized viral strains and facilitate the incorporation of structurally optimized or multivalent antigens. Together with recent advances in lipid nanoparticle-based formulations and structure-guided antigen engineering, these characteristics make mRNA technology particularly attractive for the development of next-generation hantavirus vaccines. Nevertheless, the current evidence base for mRNA vaccines remains less mature than that for DNA vaccines and further preclinical studies are required to establish the durability, breadth and protective efficacy of mRNA-induced immunity against diverse pathogenic orthohantaviruses.
Several major challenges therefore remain before nucleic acid vaccines can be translated into broadly protective hantavirus vaccines. These include the considerable antigenic diversity among hantaviruses, incomplete cross-protection between Old World and New World viruses, uncertainty regarding correlates of protection, the absence of an animal model that fully reproduces human disease and the need to establish durable immunity. The development of multivalent or pan-hantavirus vaccine strategies will likely require careful selection and structural optimization of conserved antigenic regions while maintaining the ability to induce potent neutralizing and cellular responses. In addition, standardized preclinical and clinical evaluation criteria would facilitate comparison between vaccine candidates and improve the interpretation of immunogenicity data.
Overall, the accumulated evidence supports DNA and mRNA vaccination as promising platforms for hantavirus prevention. The progression from early proof-of-concept DNA vaccines to structure-guided and increasingly sophisticated nucleic acid vaccine candidates demonstrates substantial progress in the field. Future research should focus on defining the immunological correlates of protection, improving the breadth and durability of vaccine-induced immunity and developing multivalent or broadly protective formulations capable of targeting the major pathogenic orthohantaviruses. Continued integration of structural virology, immunology, reverse vaccinology and nucleic acid vaccine technology may ultimately enable the development of a safe, durable and broadly effective hantavirus vaccine.

Author Contributions

Conceptualization, K.P. and E.P.; methodology, K.P.; software, M.Z. and E.N.; validation, E.N., A.F., E.P. and K.P.; formal analysis, K.P. and M.Z.; investigation, A.F., K.P. and M.Z.; resources, E.N. and E.P; data curation, A. F.; writing—original draft preparation, K.P, A.F, E.N., M.Z. and E.P.; writing—review and editing, E.P.; visualization, K.P.; supervision, E.P.; project administration, K.P and E.P.; funding acquisition, Y.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ANDV Andes virus
GPC Glycoprotein precursor
Gn Glucoprotein N
Gc Glucoprotein C
N Nucleocapsid protein
NSs Non structural protein
PUUV Puumala virus
RdRp RNA-dependent RNA polymerase
RNA Ribonucleic acid
vRNP Viral ribonucleoprotein complex
HTNV Hantaan virus
DNA Deoxyribnobucelic acid
IM Intramuscular
ID Infectious diseases
U.S. United States
mg milligram
AE Adverse event
SAEs Serious adverse events
PRNT50 50% plaque reduction neutralization test
PsVNA50 50% pseudovirion neutralization assay

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Figure 1. Schematic structure of hantaviruses [21,22]. The virion consists of a host-derived lipid bilayer containing surface glycoproteins Gn and Gc. The genome comprises three negative-sense RNA segments: L segment, M segment, and S segment. Each vRNA segment is associated with N protein and the viral RNA-dependent RNA polymerase (RdRp), forming ribonucleoprotein complexes enclosed within the viral envelope.
Figure 1. Schematic structure of hantaviruses [21,22]. The virion consists of a host-derived lipid bilayer containing surface glycoproteins Gn and Gc. The genome comprises three negative-sense RNA segments: L segment, M segment, and S segment. Each vRNA segment is associated with N protein and the viral RNA-dependent RNA polymerase (RdRp), forming ribonucleoprotein complexes enclosed within the viral envelope.
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Table 2. General characteristics of clinical trials evaluating DNA vaccines against HTNV, PUUV and ANDV.
Table 2. General characteristics of clinical trials evaluating DNA vaccines against HTNV, PUUV and ANDV.
Vaccine target Trial ID, phase and status Last posted update Sponsor Number of enrolled and analyzed participants Dose and vaccination schedule Delivery method Name of the used vaccine plasmid References
HTNV DNA vaccine, PUUV DNA vaccine, mixed HTNV/PUUV vaccine NCT01502345, phase I, completed 2013-01-31 U.S. Army Medical Research and Development Command 31 enrolled; 27 completed study procedures 2 mg on Days 0, 28 and 56 IM administration using the TriGrid Delivery System pWRG/HTN-M(x);pWRG/PUUV-M(s2) [67]
Mixed HTNV and PUUV DNA vaccine NCT02116205, phase IIa, completed 2021-02-12 U.S. Army Medical Research and Development Command 130 enrolled; 120 completed study procedure 1 or 2 mg: two-dose (Days 0 and 56) or three-dose (Days 0, 28 and 56) schedule, followed by a booster on Day 168. Cohort 1 (2 mg, four doses); Cohort 2 (2 mg, three doses); Cohort 3 (1 mg, four doses); Cohort 4 (1 mg, three doses). IM administration electroporation using the TriGrid Delivery System pWRG/HTN-M(co); pWRG/PUU-M(s2) [68]
HTNV DNA vaccine, PUUV DNA vaccine, mixed HTNV/PUUV DNA vaccines NCT02776761, phase I, completed 2021-02-16 U.S. Army Medical Research and Development Command 27 enrolled; 22 completed study procedure 2 mg on Days 0, 28, 56 and 168; 20 participants evaluable after the booster IM administration the Pharmajet Stratis® Needle-Free Jet Injection Delivery Device pWRG/HTN-M(co); pWRG/PUU-M(s2) [69]
HTNV vaccine, PUUV vaccine NCT03718130, phase I, Completed 2025-06-26 U.S. Army Medical Research and Development Command 61 enrolled, 35 completed study procedure
by day 220		 0.6 mg ID or 3.0 mg for monovalent HTNV or PUUV vaccine; 1.2 mg ID or 6.0 mg IM for the combined HTNV/PUUV vaccine, administered on Days 0, 28 and 56 IM or ID administration using the TriGrid Delivery System pWRG/HTN-M(co); pWRG/PUU-M(s2) (data from clinical trial form) [64]
HTNV vaccine, PUUV vaccine NCT04333459, phase II, Completed 2026-05-19 U.S. Army Medical Research and Development Command 132 enrolled 1 or 2 mg of HTNV or PUUV DNA on Days 1, 29, 57 and 169 IM administration using the Pharmajet Stratis® Needle-Free Jet Injection Delivery Device pWRG/HTN-M(co); pWRG/PUU-M(s2) [70]
ANDV vaccine NCT03682107, phase I, Completed 2022-11-22 NIAID 48 enrolled; 48 completed study procedure 2 or 4 mg administered in a three-dose schedule (Days 1, 29 and 169) or a four-dose schedule (Days 1, 29, 57 and 169) IM administration using the Pharmajet Stratis® Needle-Free Jet Injection Delivery Device pWRG/AND-M (opt2) [71]
Abbreviations: HTNV - Hantaan virus; PUUV - Puumala virus; ANDV - Andes virus; DNA - deoxyribonucleic acid; IM - intramuscular; ID - intradermal; NIAID - National Institute of Allergy and Infectious Diseases; U.S. - United States; mg - milligram.
Table 3. Summary of local and systemic adverse events (AEs) of DNA vaccines against HTNV, PUUV and ANDV tested in clinical trials.
Table 3. Summary of local and systemic adverse events (AEs) of DNA vaccines against HTNV, PUUV and ANDV tested in clinical trials.
Vaccine target Trial ID Local AEs Systemic AEs SAEs in clinical trial References
HTNV DNA vaccine, PUUV DNA vaccine, mixed HTNV/PUUV vaccine NCT01502345 Injection-site pain: 28/31 (90.3%); bruising: 7/31 (22.6%); erythema: 3/31 (9.7%). Pain was reported in 10/11 HTNV participants, 8/9 PUUV participants and 10/11 combined-vaccine participants. Headache: 8/31 (25.8%); myalgia: 7/31 (22.6%); fatigue: 6/31 (19.4%); muscle contractions and tachypnea were reported in two participants each. No SAEs related to the vaccine or study-related procedures were observed. One unrelated Grade 4 hypoglycaemia case was attributed to a previously unrecognized condition. [62,67]
Mixed HTNV and PUUV DNA vaccine NCT02116205 Injection-site pain: 125/130 (96.2%); erythema: 18/130 (13.8%); bruising: 9/130 (6.9%); swelling: 4/130 (3.1%). Pain was the predominant AE in every dose and schedule cohort. Fatigue: 38/130 (29.2%); headache: 31/130 (23.8%); myalgia: 23/130 (17.7%). Less common systemic findings included axillary discomfort, lymphadenopathy, transient muscle contractions and tachypnea. No SAEs related to the vaccine or study-related procedures were observed. [63,68]
HTNV DNA vaccine, PUUV DNA vaccine, mixed HTNV/PUUV DNA vaccines NCT02776761 Injection-site pain: 21/27 (77.8%); bruising: 11/27 (40.7%); erythema: 8/27 (29.6%). Local-event frequencies did not differ significantly among the three vaccine groups. Headache: 13/27 (48.1%); fatigue: 10/27 (37.0%). Overall, 23/27 participants (85.2%) reported at least one solicited related AE. No SAEs related to the vaccine or study-related procedures were observed. [61,69]
ANDV vaccine NCT03682107 At least one local solicited AE occurred in 47/48 participants (97.9%). Across all study groups, erythema and induration occurred in 43/48 (89.6%) each, tenderness in 39/48 (81.3%), pain in 32/48 (66.7%), bruising in 20/48 (41.7%) and skin discoloration in 13/48 (27.1%). High local-event frequencies were also influenced by the jet-injection procedure. At least one systemic solicited AE occurred in 28/40 vaccine participants (70.0%), compared with 3/8 placebo participants (37.5%). The most frequent systemic symptoms overall were headache (52%), fatigue (48%) and malaise (38%). Mild fever occurred in 3/40 vaccine participants. No SAEs related to the vaccine or study-related procedures were observed. One SAEs - acute rhabdomyolysis - occurred in 1/48 participants and was considered unrelated to vaccination. [50,71]
Abbreviations: HTNV - Hantaan virus; PUUV - Puumala virus; ANDV - Andes virus; DNA - deoxyribonucleic acid; AE - adverse event; SAEs - serious adverse events.
Table 4. Summary of serological response and neutralizing antibody titers of DNA vaccines against HTNV, PUUV and ANDV tested in clinical trials.
Table 4. Summary of serological response and neutralizing antibody titers of DNA vaccines against HTNV, PUUV and ANDV tested in clinical trials.
Vaccine target Trial ID Groups of participants Immunogenicity Assay & Definition of Response Major Outcomes References
HTNV DNA vaccine, PUUV DNA vaccine, mixed HTNV/PUUV vaccine NCT01502345 Group 1: HTNV DNA vaccine, 2 mg, 3-dose regimen - Days 0, 28 and 56 Group 2: PUUV DNA vaccine, 2 mg, 3-dose regimen - Days 0, 28 and 56 Group 3: combined HTNV/PUUV DNA vaccine, 2 mg total (1 mg HTNV + 1 mg PUUV), 3-dose regimen - Days 0, 28 and 56. PRNT₅₀; A detectable response was defined as a PRNT₅₀ titer ≥20. 0 HTNV: 7/11 responders (64%); PUUV: 6/8 (75%); combined vaccine: 7/9 (78%) responded to PUUV and 3/9 (33%) to both viruses after at least two doses. [62,67]
Mixed HTNV and PUUV DNA vaccine NCT02116205 Cohort 1: 2 mg, 4-dose regimen - Days 0, 28, 56, 168
Cohort 2: 2 mg, 3-dose regimen - vaccine on Days 0, 56, 168; placebo on Day 28
Cohort 3: 1 mg, 4-dose regimen - Days 0, 28, 56, 168
Cohort 4: 1 mg, 3-dose regimen - vaccine on Days 0, 56, 168; placebo on Day 28		 PsVNA₅₀ and PRNT₅₀ on Day 84. Seropositivity was generally defined as a neutralizing-antibody titer ≥20. Cumulative HTNV seropositivity in Cohorts 1-4 was 80.0%, 90.0%, 90.0% and 83.3%, respectively. Corresponding PUUV seropositivity was 86.7%, 80.0%, 83.3% and 73.3%. Responses to both viruses at one or more post-vaccination time points were observed in 76.7%, 73.3%, 80.0% and 66.7% of participants, respectively. [63,68]
HTNV DNA vaccine, PUUV DNA vaccine, mixed HTNV/PUUV DNA vaccines NCT02776761 Group 1: HTNV DNA vaccine, 2 mg/vaccination; Group 2: PUUV DNA vaccine, 2 mg/vaccination; Group 3: combined HTNV/PUUV DNA vaccine, 2 mg/vaccination total PsVNA₅₀ and PRNT₅₀; Seropositivity was defined as a titer ≥20, whereas seroconversion in initially seronegative participants was defined as a titer ≥40. By PsVNA₅₀, 7/7 HTNV vaccine participants and 6/6 PUUV vaccine participants seroconverted against the homologous virus. In the combined-vaccine cohort, 8/9 developed PUUV-neutralizing antibodies, 7/9 developed HTNV-neutralizing antibodies and 7/9 were seropositive to both viruses at one or more time points. By PRNT₅₀, homologous seroconversion occurred in 7/7 HTNV participants and 5/6 PUUV participants. In the combined group, 4/9 were seropositive to both viruses, but only 3/9 achieved titers ≥40 against both viruses. [61,69]
ANDV vaccine NCT03682107 Cohort 1: ANDV DNA 2 mg, 3-dose regimen - Days 1, 29, 169
Cohort 2: ANDV DNA 2 mg, 4-dose regimen - Days 1, 29, 57, 169
Cohort 3: ANDV DNA 4 mg, 3-dose regimen - Days 1, 29, 169
Cohort 4: ANDV DNA 4 mg, 4-dose regimen - Days 1, 29, 57, 169		 PsVNA₅₀ as the principal immunogenicity assay and confirmed using PRNT₅₀; Seropositivity (titer ≥20 ) and seroconversion (≥40 if the baseline was <20, or a minimum 4-fold rise compared to baseline if the baseline titer was ≥20) Cohorts 2-4 achieved at least 80% seropositivity by Day 197, which was sustained through Day 337. At the final assessment, seropositivity was 67% in Cohort 1 and approximately 88-90% in Cohorts 2-4. The highest reported final seroconversion frequencies were 88% in Cohort 2 and 89% in Cohort 4. [50,61]
Abbreviations: HTNV-Hantaan virus; PUUV-Puumala virus; DNA-deoxyribonucleic acid; PRNT₅₀-50% plaque reduction neutralization test; PsVNA₅₀-50% pseudovirion neutralization assay; mg-milligram.
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