A neutralizing antibody protects Kunming mice against AKAV lethal challenge

Abstract

Background

Akabane virus (AKAV) infection is associated with arthrogryposis-hydranencephaly syndrome in ruminants. Current commercialized AKAV live attenuated vaccines have safety concerns and cannot fully protect against all genotypes or emerging strains. Thus, developing a new type of vaccine or treatment is urgently required. The neutralizing antibodies (NAbs) directed against the Gc protein can efficiently neutralize the corresponding Bunyavirales viruses. We previously generated three NAbs against the Gc protein of AKAV that collectively recognize a highly conserved epitope among diverse AKAV genotypes and established a mouse model of AKAV infection. Here, our objective was to evaluate the protective efficacy of one of the produced NAbs, 4F12, against AKAV infection using the mouse model.

Methods

Suckling Kunming mice were first intraperitoneally administered varying doses of the NAb 4F12, followed by intraperitoneal (IP) or intracerebral (IC) challenge with a lethal dose of AKAV. Clinical symptoms, body weight, and mortality were then monitored and recorded daily for 14 days. The AKAV RNA, viral particles, antigens distribution, and microscopic lesions in the brain tissues of the experimental mice were analyzed.

Results

All mice that did not receive 4F12 pretreatment died before the experimental endpoint, regardless of the AKAV challenge routes. While a dose-dependent survival increase (50% ~ 83.33%) was observed in 4F12-pretreated mice, with higher antibody concentrations conferring greater protection against both IC and IP AKAV challenges. Moreover, all mice that survived AKAV challenge due to 4F12 pretreatment showed complete absence of AKAV RNA, viral particles, and antigens in brain tissues, with no detectable virus-associated brain lesions.

Conclusions

We proved that the NAb 4F12 could reduce the AKAV-induced mortality in mice. 4F12 is a promising candidate suitable for clinical development as an AKAV therapeutic. The highly conserved epitope recognized by 4F12 provides critical insights for the design of new broadly protective AKAV vaccines.

Introduction

Akabane virus (AKAV) is the etiological agent of Akabane disease in ruminants, including cattle, sheep, and goats. Akabane disease is characterized by abortion, stillbirth, premature birth, and congenital deformities in newborns, which have caused considerable economic losses to the cattle industry. Large outbreaks of Akabane disease occurred in Japan and Australia in the 1970 s, causing abortion, stillbirth, and congenital arthrogryopsis and hydranencephaly (A-H syndrome) in more than 31,000 cases and more than 8000 cases, respectively [1, 2]. Since then, epidemics of this disease have been reported frequently in Japan and South Korea [3–6]. Sporadic cases of Akabane disease were also found in other countries [7–12]. Transmitted primarily by the bites of midges of the genus Culicoides, including the species Culicoides brevitarisis, Culicoides oxystoma, and Culicoides nebeculosus [13], AKAV is widely distributed throughout Australia, Southeast Asia, East Asia, the Middle East, and Africa [14]. Although the use of live attenuated vaccines and inactivated vaccines has reduced the prevalence of Akabane disease, cases still occurred in areas where vaccines are administered [15]. Besides, inactivated vaccines often provide insufficient protection, while live attenuated vaccines carry the risk of virulence reversion and recombination with field AKAV strains. Therefore, it is important to develop effective vaccines and novel antiviral strategies to control this disease.

AKAV is a Simbu serogroup virus belonging to the genus Orthobunyavirus within the family Bunyaviridae. AKAV harbors a tripartite, negative-strand RNA genome that contains the small (S), medium (M), and large (L) RNA segments. The S segment encodes a nucleocapsid (N) protein and a nonstructural protein NSs. The L segment encodes the RNA-dependent RNA polymerase (RdRp) in the ribonucleoprotein complex. The M segment encodes two viral envelope glycoproteins, Gn and Gc, derived from a single polyprotein precursor, and a nonstructural protein, NSm [16]. Glycoprotein Gc is a major AKAV-neutralizing antigen, which is important for the induction of the host’s immune response and is responsible for viral neutralization and attachment to cell receptors [17, 18]. Antibodies directed against the Gc protein efficiently neutralize the corresponding Bunyavirales viruses [19–21] and may play a role in protection from the lethal infection [22–25].

Previously, we prepared and characterized three monoclonal antibodies (mAbs), 4D1, 4E6, and 4F12, against the Gc protein of AKAV (TJ2016 strain, GenBank accession nos. MT761689, MT761688, and MT755621) and identified a broadly neutralizing epitope that is highly conserved across different genotypes of AKAV strains by using these mAbs [26]. We also established experimental mouse models infected with the TJ2016 strain [27]. In this study, we selected the NAb 4F12, with the highest neutralizing titers of the three mAbs, to evaluate its therapeutic protection against AKAV infection in a mouse model.

Materials and methods

Cells, antibodies, and virus

Baby hamster kidney cells (BHK-21) were purchased from Procell Life Science & Technology Co., Ltd. previously and are currently stored in our laboratory (the Institute of Animal Inspection and Quarantine, Chinese Academy of Quality and Inspection & Testing). BHK-21 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (SolarbioLife Sciences, Beijing, China). The hybridoma cells secreting the mAb 4F12 were prepared in our lab previously [26] and are stored in our lab now. The hybridoma cells were cultured in DMEM supplemented with 20% FBS and 1% penicillin–streptomycin. All the cells were cultured in a humidified incubator at 37 °C with 5% CO₂.

The mAb 2D3 specific to AKAV N protein and the rabbit polyclonal antibody (pAb) specific to AKAV were generated in our laboratory previously [28]. FITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG were purchased from SolarbioLife Sciences (Beijing, China).

The AKAV strain TJ2016 was isolated and maintained by our lab [28].

Animals

Specific pathogen-free (SPF) female BALB/C mice aged 8 weeks and SPF Kunming mice aged 7 days were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

Preparation and neutralizing identification of 4F12

To prepare enough NAb 4F12 for the animal trials, the hybridoma cells secreting the NAb 4F12 were cultured and injected into the abdominal cavity of the 8-week-old female BALB/C mice to generate mouse ascites as described previously [29]. The harvested ascites were mixed, aliquoted, and stored at −80℃.

The neutralization of NAb 4F12 against the TJ2016 strain was assessed in BHK-21 cells. Briefly, the ascites containing mAb 4F12 was serially diluted twofold (from 2¹ to 2¹²) with three replicates using DMEM supplemented with 2% FBS and 1% penicillin–streptomycin. The dilutions were mixed with 200 TCID₅₀ of AKAV in equal volumes and incubated at 37 °C for 1 h. Then the virus-mAb mixture was added to BHK-21 cells in 96-well plates and incubated at 37 °C with 5% CO₂. After 48 h, the supernatant was discarded, and the BHK-21 cells were processed for indirect immunofluorescence assay (IFA). In detail, the cells were fixed with ice-cold absolute ethyl alcohol for 30 min and then incubated with primary antibody rabbit pAb (1:1000 dilution) against AKAV at 4 °C overnight, followed by incubation with FITC-conjugated goat anti-rabbit IgG (1:500 dilution) for 45 min. All cells were washed with PBS three times after each incubation step. The fluorescence was observed using the Invitrogen EVOS FL cell fluorescence imaging system (Thermo Fisher Scientific, USA), and the percentage of uninfected cells per well was calculated based on cell numbers.

Animal trials

In a pre-trial, 48 seven-day-old Kunming mice of both sexes were randomly divided into 12 groups of 4 animals each. The TJ2016 strain was tenfold diluted in DMEM from 10⁷ down to 10⁰ TCID₅₀/ml. Seven of the groups received intracerebral (IC) inoculation (10 μL/mouse) with viral titers of 10^–2 ~ 10⁴ TCID₅₀ per dose. Four of the groups received intraperitoneal (IP) inoculation (100 μL/mouse) with viral titers of 10³ ~ 10⁶ TCID₅₀ per dose. The remaining group was kept as the control, in which two mice received IC inoculation of 10 μL DMEM and the other two were administered 100 μL DMEM via IP injection. Detailed information was summarized in Table 1. All mice were examined for clinical symptoms for 14 days. The mice showing severe paralysis were euthanized immediately and recorded as dead. The mice surviving to the end of the trial were euthanized. The minimum 100% lethal dose (LD100) of the TJ2016 strain in suckling Kunming mice by each inoculation route was recorded and used for the further experiments.

Table 1.

Mortality of the suckling Kunming mice inoculated with AKAV

LgTCID50 per mouse	Inoculation route
	IC	IP
N/A	0/2	0/2
−2	0/4	N/A
−1	0/4	N/A
0	4/4	N/A
1	4/4	N/A
2	4/4	N/A
3	4/4	0/4
4	4/4	0/4
5	N/A	2/4
6	N/A	4/4

N/A Not applicable (the mice were inoculated with DMEM by IC or IP routes, or no mice were present/analyzed in these groups), IP Intraperitoneal, IC Intracerebral

For the main trial, 30 mice were randomly assigned to 5 groups of 6 animals each. The mice were administered NAb 4F12 by IP route and inoculated with TJ2016 by IP or IC routes after 24 h (Fig. 1A and B). For control groups, mice were inoculated as follows: four via IP route with TJ2016, four via IC route with TJ2016, two via IC route with DMEM, two via IP route with DMEM, and two via IP route with 4F12. Detailed information was summarized in Table 2. All mice were weighed and examined daily. The clinical symptoms of each mouse were monitored and scored according to the scoring system established previously [27]. The mice showing severe paralysis were euthanized immediately and recorded as dead. The mice surviving to the end of the trial, 14 days post-inoculation (dpi), were euthanized. Brain tissues were collected immediately after premature death or euthanasia for further analysis.

Fig. 1.

Experimental timeline for antibody delivery, virus challenge, and necropsy. A The NAb 4F12 was administered by IP route at the doses of 100 μL or 200 μL per mouse. After 24 h, the mice were challenged with AKAV (10⁶ TCID₅₀/mouse) by IP route. B The NAb 4F12 was administered by IP route at the doses of 50 μL, 100 μL, or 200 μL per mouse. After 24 h, the mice were challenged with AKAV (10⁰ TCID₅₀/mouse) by the IC route. As the controls, mice were only injected with AKAV or DMEM by IC route or injected with AKAV, DMEM, or 4F12 by IP route. Mice were monitored daily and euthanized at 14 days post inoculation of AKAV. NAb, neutralizing antibody; IP, intraperitoneal; IC, intracerebral

Table 2.

Animal trial design and the survival rate

Group	Survival rate	4F12		AKAV / DMEM
		Route	Volume (μL)per mouse	Route	TCID50 per mouse
IP-4F12-100	4/6	IP	100	IP	106
IP-4F12-200	5/6	IP	200	IP	106
IP-DMEM	2/2	N/A	N/A	IP	N/A
IP-AKAV	0/4	N/A	N/A	IP	106
IC-4F12-50	3/6	IP	50	IC	100
IC-4F12-100	4/6	IP	100	IC	100
IC-4F12-200	4/6	IP	200	IC	100
IC-DMEM	2/2	IP	N/A	IC	N/A
IC-AKAV	0/4	IP	N/A	IC	N/A
4F12-Control	2/2	IP	200	N/A	N/A

N/A Not applicable (Mice in the corresponding groups were not inoculated with 4F12 or AKAV), IP Intraperitoneal, IC Intracerebral

All efforts were made to minimize animal suffering. Euthanasia in this study was performed by CO₂ asphyxiation followed by cervical dislocation.

RNA extraction and RT-PCR amplification

Total RNA was extracted from brain tissues with the RNA Easy Fast Tissue/Cell Kit (TianGen Biotech, Beijing, China) following the manufacturer’s protocol. The extracted RNA was detected for the AKAV N gene by an RT-PCR assay using the HiScript II One Step RT-PCR Kit (Vazyme, Nanjing, China). The RT-PCR was conducted as previously described [27].

Virus titration

Approximately 100 mg of brain tissue was homogenized in 1 mL DMEM, followed by centrifugation at 12,000 rpm for 15 min. The supernatant was collected for virus titration. All procedures were performed under low-temperature conditions. The homogenate supernatant was serially diluted tenfold in DMEM supplemented with 2% FBS and 2% penicillin–streptomycin. The dilutions (from 10^–2 to 10^–7) were then added to confluent BHK-21 cell monolayers in 96-well plates at 100 μL per well. Four replicates were performed for each dilution, with three independent repetitions for each homogenate supernatant. The culture was continued at 37 °C in a 5% CO₂ incubator for 48 h. Then the supernatant was discarded, and the cells were fixed with ice-cold absolute ethyl alcohol for IFA analysis. Briefly, the mAb 2D3 (1:1000 dilution) was used as the primary antibody, and the FITC-conjugated goat anti-mouse IgG (1:500 dilution) was used as the secondary antibody. The fluorescence was observed, and the virus titers were calculated by using the Reed-Muench method [30].

Histopathology and immunohistochemistry (IHC)

Half of each collected brain was fixed immediately in 4% paraformaldehyde and sent to Bioss (Beijing, China) for histopathological analysis. The fixed brain tissues were embedded in paraffin wax and sectioned into 4 µm sections. Each sample was examined in two sections. One section was stained with hematoxylin and eosin (HE) to observe pathological changes. The other section was used to detect the AKAV antigen-positive cells, with the rabbit pAb (1:1000 dilutions) specific to AKAV as the primary antibody and the goat anti-rabbit IgG H&L (HRP polymer) (Bioss, Beijing, China) as the secondary antibody. The severity of microscopic brain lesions and AKAV antigen levels were evaluated and scored from 0 to 4, as previously described [27].

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 8.0 software (GraphPad Software, USA). Multiple t test-one per row was used for assessing differences between two groups. For comparisons of the virus titers within the same group, a two-tailed unpaired t-test with Welch’s correction was used. A P value of less than 0.05 was considered statistically significant, and a P value of less than 0.01 was considered extremely significant.

Results

NAb 4F12 inhibits the AKAV infectionin vitro

To confirm the neutralizing activity of our newly prepared mouse ascites that containing mAb 4F12, we evaluated the ability of 4F12 to inhibit AKAV infection in BHK-21 cells. As a result, neutralizing activity of 4F12 was observed, and the inhibition was dependent on the dose of 4F12 when the AKAV amount was fixed. The ascites that containing mAb 4F12 exhibited 100% inhibition against AKAV infection in BHK-21 cells at dilutions up to 1:8, while maintaining > 50% inhibitory activity at dilutions as high as 1:64 (Fig. 2).

Fig. 2.

The neutralization efficiency of NAb 4F12 against AKAV in vitro. 100 μL of the AKAV strain TJ2016 and a series of two-fold serially diluted the ascites that containing mAb 4F12, ranging from 1:2 to 1:4096, were mixed in equal volumes and incubated at 37℃ for 60 min. The ascites-virus mixture was used to infect BHK-21 cells (n = 3) and incubated at 37 °C with 5% CO₂. After 48 h, the virus was then detected by IFA. Inhibition of infection was fitted to a dose–response curve

Determination of LD100 for AKAV infection in suckling Kunming mice

Previous study has shown that the AKAV strain TJ2016 can kill the suckling Kunming mice by IC or IP route [27]. In this study, we aim to study whether the NAb 4F12 can protect the Kunming mice against AKAV lethal challenge. Therefore, we did a pre-trial to determine the minimum LD100 of AKAV in 7-day-old Kunming mice by each inoculation route. As the results in Table 1 showed, the minimum LD100 of the TJ2016 strain in suckling Kunming mice was 10⁰ TCID₅₀/mouse by IC route or 10⁶ TCID₅₀/mouse by IP route, which was used for the subsequent animal experiment.

NAb 4F12 protects Kunming mice against AKAV lethal challenge

In preliminary experiments, we found that administering 4F12 at 24 h before AKAV infection provided stronger protection compared to administration at 24 h post-infection (data not shown). Therefore, only prophylactic administration was employed in this experiment. Here, 4F12 showed great protection from clinical symptoms and mortality. In the IP infection groups, 66.67% (4/6) of mice in the IP-4F12-100 group and 83.33% (5/6) of mice in the IP-4F12-200 group survived until the end of the trial (Table 2 and Fig. 3A). In the IC infection groups, survival rates of 50% (3/6), 66.67% (4/6), and 66.67% (4/6) were observed in AKAV-infected mice from groups IC-4F12-50, IC-4F12-100, and IC-4F12-200, respectively (Table 2 and Fig. 3B). The results demonstrate that as the dose of 4F12 increased, the survival rate of the AKAV-infected mice correspondingly rose.

Fig. 3.

NAb 4F12 protects the suckling Kunming mice when given before AKAV inoculation. A and B Survival curve, (C and D) scores of clinical signs, and (E and F) ADWG of the inoculated mice. The data were shown as means ± SD (error bars). Statistical differences were labeled according to multiple t test-one per row. Asterisks (*) indicate a significant difference between IP-4F12-100 and IP-AKAV, or IP-4F12-200 and IC-AKAV, or IC-4F12-100 and IC-AKAV, or IC-4F12-200 and IC-AKAV groups (*, P < 0.05; **, P < 0.01;***, P < 0.001; ****, P < 0.0001). IC, intracerebral; IP, intraperitoneal; DPI, days post infection

No clinical symptoms were observed in any mouse surviving until the end of the experiment in any group. However, neurological symptoms such as tremors, ataxia, paddling movements, or paralysis were observed in all the mice that died or were euthanized before the end of the experiment in all the groups. After performing clinical scoring for all mice in each group, we found that the average clinical symptom scores in groups IP-4F12-100 and IP-4F12-200 were significantly lower than those in group IP-AKAV at 7 ~ 14 dpi; the scores in group IC-4F12-100 were significantly lower than those in group IC-AKAV at 7 ~ 14 dpi; and the scores in group IC-4F12-200 were significantly lower than those in group IC-AKAV at 3 ~ 14 dpi (Fig. 3C and D). For average daily weight gain (ADWG), no significant differences were observed between the groups (Fig. 3E and F).

Detection of AKAV in brains of the inoculated mice

To determine whether AKAV was present in the brain tissues of the inoculated mice, we detected the AKAV-N gene in brain tissue samples using RT-PCR and quantified the viral load by a microtitration infectivity assay. RT-PCR results showed that positive bands with 649 bp were only observed in the brains of the mice that died or were euthanized before the end of the experiment in all the groups (Fig. 4A). However, no bands were found in the brains of the mice that survived until the end of the experiment (Fig. 4B). Consistent with the RNA detection results, viral titer determination demonstrated that replication-competent AKAV particles were detected exclusively in the brain tissues of the mice that died or were euthanized before the end of the experiment (Fig. 4C and D).

Fig. 4.

Detection and determination of AKAV in brains of the inoculated mice. A RT-PCR detection of the AKAV N gene in brains collected from the mice that died or were euthanized before the end of the experiment. B RT-PCR detection of the AKAV N gene in brains collected from the mice that survived until the end of the experiment. C and D Virus titers in brains of the mice in different groups. Virus titers in brain tissues were determined by microtitration infectivity assay in BHK-21 cells. A two-tailed unpaired t-test with Welch’s correction was used to compare the virus titers between two groups. No significant difference was observed between IP-4F12-100 and IP-AKAV, or IP-4F12-200 and IC-AKAV, or IC-4F12-50 and IC-AKAV, or IC-4F12-100 and IC-AKAV, or IC-4F12-200 and IC-AKAV groups. IC, intracerebral; IP, intraperitoneal; dead mice, the mice that died or were euthanized before the end of the experiment; survival mice, the mice that survived until the end of the experiment

NAb 4F12 protects against AKAV invasion into brain tissues and prevents virus-induced brain lesions in intraperitoneally infected mice

Since no AKAV was detected in the surviving mice of groups IP-4F12-100 and IP-4F12-200, we hypothesized that the pre-administration of 4F12 prevented the invasion of intraperitoneally injected AKAV into brain tissues. To further validate this hypothesis, brain tissues of these surviving mice were subjected to IHC analysis and HE staining. Brains of mice in groups IP-DMEM and 4F12-control were used as the negative control, and brains of mice in the IP-AKAV group were used as the positive control. As expected, neither AKAV antigens (Fig. 5A and B) nor microscopic brain lesions (Fig. 5C and D) were observed in the brain tissues of these surviving mice. While viral antigens in the neurons as well as the lesions, including perivascular cuffing (PVC) of lymphocytes and macrophages, glial nodules (GN) consisting of microglial cells, and tissue liquefaction with loose structure, were present in the brains of the mice in the IP-AKAV group (Fig. 5).

Fig. 5.

NAb 4F12 prevents AKAV invasion into brains and prevents the brain lesions in intraperitoneally infected mice. A and B IHC examinations and average scores for AKAV antigen in the brains of the mice in the IP-AKAV group and the mice that survived until the end of the experiment in other groups. AKAV antigen is present in neurons with marked labeling intensity. Hollow triangles indicate positive signals. C and D Microscopic brain lesions stained with hematoxylin and eosin (HE) and average microscopic brain lesion scores from the dead mice in the IP-AKAV group and the surviving mice at the end of the experiment in other groups. Solid arrows indicate PVC of lymphocytes and macrophages. Hollow arrows indicate tissue liquefaction with loose structure. Solid triangles indicate GN consisting of microglial cells. IP, intraperitoneal

NAb 4F12 inhibits AKAV replication in brain tissues and prevents virus-induced brain lesions in intracranially infected mice

We next investigated whether 4F12 pretreatment could suppress AKAV replication and prevent virus-induced brain lesions following IC inoculation. Histopathology examinations were performed on the brain tissues of the surviving mice from groups IC-4F12-50, IC-4F12-100, and IC-4F12-200. Brains of mice in groups IC-DMEM and IC-AKAV served as the negative and positive controls, respectively. As a result, the AKAV antigen was only detected in brains from the IC-AKAV group (Fig. 6A and B). Besides, brain lesions such as PVC and tissue liquefaction with loose structure were observed exclusively in the IC-AKAV group (Fig. 6C and D). Histopathological analysis revealed no apparent abnormalities in the surviving mice from Groups IC-4F12-50, IC-4F12-100, and IC-4F12-200 relative to controls (Fig. 6).

Fig. 6.

NAb 4F12 inhibits AKAV replication in brain tissues and inhibits brain lesions in intracranially infected mice. A and B IHC examinations and average scores for AKAV antigen in the brains of the mice in the IC-AKAV group and the mice that survived until the end of the experiment in other groups. AKAV antigen is present in neurons with marked labeling intensity. Hollow triangles indicate positive signals. C and D Microscopic brain lesions stained with HE and average microscopic brain lesion scores from the dead mice in the IC-AKAV group and the surviving mice at the end of the experiment in other groups. Solid arrows indicate PVC of lymphocytes and macrophages. Hollow arrows indicate tissue liquefaction with loose structure. IC, intracerebral

Discussion

Vaccination with the live attenuated vaccines has proven to be the most effective way to prevent Akabane disease. However, there are still some safety concerns regarding these vaccines. Additionally, existing live attenuated vaccines might not cover newly emerged AKAV strains arising from genetic mutations or recombination. Developing a safe and broadly applicable passive antibody treatment strategy could be a promising alternative. Studies on various viruses, including SARS-CoV-2, MERS-CoV, and EBV, have demonstrated that NAbs exhibit excellent prophylactic or therapeutic efficacy in animal models [31–35].

The Gc protein, which can mediate cell attachment and membrane fusion, as well as induce the production of NAbs to neutralize the infectivity of corresponding viruses, is very closely related to the pathogenicity and immunogenicity of bunyaviruses. Recent studies on bunyavirus, such as Rift Valley fever virus (RVFV), Schmallenberg virus (SBV), Crimean-Congo Hemorrhagic Fever Virus (CCHFV), Hantaviruses, and Severe fever with thrombocytopenia syndrome virus (SFTSV), have proved that the NAbs directed against part or the whole of the Gc protein hold great promise for the development of bunyavirus antiviral therapies [24, 36–38].

In our previous study, three mAbs targeting the truncated Gc protein (aa991 ~ 1232) of AKAV were generated and were identified to possess in vitro neutralizing activity. All of the mAbs were mapped to the same linear epitope, ¹¹³⁴SVQSFDGKL¹¹⁴², which is highly conserved across different genotypes of AKAV strains [26]. In this present study, we selected one of the NAbs, 4F12, with the highest neutralizing titer, to analyze its ability to protect against AKAV infection in a mouse model. The NAb 4F12 was shown to reduce and prevent AKAV-induced morbidity and mortality (from 100% to 16.67% ~ 50%) via IC or IP inoculation in suckling Kunming mice upon prophylactic administration (Fig. 3A and B). The brain tissues of all the surviving mice showed no detectable AKAV RNA (Fig. 4B), viral particles (Fig. 4D), or antigens (Figs. 5A and 6A) and exhibited no AKAV-induced pathological alterations (Figs. 5B and 6B). Given that 4F12 recognizes a highly conserved linear epitope, we speculate that this NAb may confer broad-spectrum protection against different AKAV genotypes, possibly even against emerging variants, and this epitope could serve as a target for the development of new broadly protective AKAV vaccines. However, since we do not have other AKAV strains in stock, it is currently impossible to conduct the relevant animal experiments to verify this. Although the mouse model is a classic and commonly used system for evaluating drug or vaccine efficacy, validation in large animal models (such as cattle or sheep) remains necessary before 4F12 can be applied clinically.

Our recent study [27], as well as the positive controls in this study, showed that the AKAV strain TJ2016 can kill the suckling Kunming mice by both IC and IP routes, and AKAV particles can be detected in the brains of the dead mice. However, in this study, we found that preemptive administration of 4F12 not only prevented intraperitoneally injected AKAV from invading the brain but also suppressed the replication of intracranially inoculated AKAV viral particles, ultimately achieving viral clearance. Whether 4F12 employs the same mechanism to counteract AKAV invading through different routes remains unclear. The mechanistic basis for these observations requires further investigation. Some of the antibodies can prevent viral entry into cells or induce lysis of infected cells through antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) [39]. The ADCC and CDC effects of NAb 4F12 should be further explored.

In addition to Gc-specific NAbs, Gn-derived NAbs represent an equally crucial component of the immune response. NAbs targeting SFTSV Gn protein can elicit a robust humoral response and inhibit the viral infection, which can serve as promising therapeutic drugs for treating SFTSV infection [25, 38, 40]. Future studies should further investigate (1) the therapeutic efficacy of AKAV Gn-specific NAbs, (2) the combinatorial effects of Gc- and Gn-targeting NAbs, and (3) the underlying protective mechanisms mediated by these antibodies.

Our study revealed a positive correlation between 4F12 ascites administration and improved survival rates in AKAV-infected mice. However, the unpurified nature of the ascites fluid precluded precise antibody dosage determination, representing a key limitation. Future studies should employ purified antibodies with graded dose regimens to obtain more accurate results. Furthermore, although the increased survival rate strongly demonstrates 4F12's protective efficacy, we were unable to analyze antibody levels or viral load dynamics in blood due to the impracticality of collecting blood samples from 7-day-old Kunming suckling mice.

Altogether, our study demonstrates that the NAb 4F12, targeting a highly conserved epitope, provides potent protection against lethal AKAV infection in a mouse model. These findings not only offer a novel therapeutic option for AKAV infection but also provide critical insights for the design of new broadly protective AKAV vaccines.

Authors’ contributions

JJ.W.: Conceptualization, methodology, formal analysis, funding acquisition, writing—original draft preparation. RY.Y. and F.W.: investigation, methodology. CY.F. and XM.L.: funding acquisition, formal analysis. DJ.C. and SQ.W.: supervision, funding acquisition, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by National Key Research and Development Program of China (2022YFD18002000 and 2022YFD1800505), the Fundamental Research Funds of Chinese Academy of Quality and Inspection & Testing (2024JK002), and the Beijing Natural Science Foundation (6254044).

Data availability

All data associated with this study are included in the paper.

Declarations

Ethics approval and consent to participate

Animal studies were carried out in strict accordance with the experimental animal care and use guidelines of Beijing Animal Control Committee. All experimental protocols were reviewed and approved by the Animal Welfare Ethics Committee of Beijing MDKN Biotechnology Co., LTD., with approval No. MDKN-2024–104.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.