Anti-NP antibody titers and Gc-specific T-cells persist for at least 1-year post-vaccination

To assess the durability of our repNP + repGc vaccination, we immunized mice with a prime-boost regimen, 4 weeks apart, with 1 µg total RNA at each immunization and evaluated immunogenicity at 1, 3-, 6-, 9-, and 12-months post-boost. As a control, each time point included mice vaccinated with a repRNA expressing the irrelevant green fluorescent protein (sham). As expected, 1-month after the boost, mice vaccinated with repNP + repGc had robust CCHVF-specific IgG antibody titers which were predominantly against the CCHFV NP (Fig. 1a, b). These titers peaked at 1-month post-boost (mean endpoint titer >1/755,000) and while they waned over time, were still detectable at 12 months post vaccination (mean endpoint titer >1/27,000) (Fig. 1a, b). Similar to early timepoints, at later timepoints antibodies remained predominantly against the CCHFV NP and responses against the CCHFV glycoprotein c (Gc) were minimal (Fig. 1b). As seen previously7, T-cell responses were primarily against the Gc protein peptide pool 10 (a.a. 1081–1211), which spans the N-terminal portion of Gc (Fig. 1c). CCHFV-specific T-cell responses peaked at 6-months post-vaccination (mean >230 SFCs/106 splenocytes) and waned thereafter but were still significantly increased compared to sham vaccinated animals 12 months post-vaccination (mean >60 SFCs/106 splenocytes) (Fig. 1d). NP-specific T-cell responses were minimal and did not significantly increase above sham vaccinated animals at any timepoint (Fig. 1c, d). Cumulatively, repNP + repGc vaccination induced CCHFV-specific antibody and T-cell responses which persisted up to 1-year post-vaccination and no sex-related differences were observed in immune responses for any group. Similar to shortly after vaccination, antibody responses were primarily against the CCHFV NP while T-cell responses were primarily against the CCHFV Gc.

Fig. 1: repNP + repGc vaccination elicits humoral and cellular immunity up to 1 year post-vaccination.

WT C57BL6/J mice were vaccinated prime boost with 1 µg repNP + repGc RNA 4 weeks apart. Immunogenicity of vaccination was measured 1-, 3-, 6-, 9-, and 12-months post-boost vaccination. At each time point, groups of six mice were evaluated for CCHFV specific immune responses via a whole virion IgG ELISA shown with endpoint titers and b recombinant antigen ELISA to the CCHFV nucleocapsid (NP) and glycoprotein c (Gc) proteins. Sham mice are combined from all timepoints. Dashed lines indicate background absorbance of wells. CCHFV-specific T-cell responses were measured via IFNγ ELISpot shown as c cumulative spot-forming cells (SFCs) against peptide pools spanning the entire NP or Gc as well as d cumulative responses to NP and Gc overtime. Statistical comparisons were calculated using a one-way ANOVA with Turkey’s multiple comparison test. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are shown as mean plus standard deviation.

repNP + repGc vaccination confers significant protection for at least 1-year post-vaccination

We next evaluated the efficacy of immune responses over time by challenging groups of mice with a lethal, heterologous CCHFV challenge at 3-, 6-, 9-, and 12-months post-boost vaccination. Our challenge model utilizes C57BL6/J mice treated with the type I IFN suppressing antibody MAR1-5A3 and infected with 100 TCID50 CCHFV strain UG3010. Our vaccine antigens are based on CCHFV strain Hoti and this represents a stringent heterologous challenge model. At indicated timepoints post-boost, mice were challenged on “day 0” relative to CCHFV challenge and assessed for control of viral loads, weight loss, and survival over the course of study (Fig. 2a). Sham vaccinated mice from all timepoints consistently began to lose weight at 3 days post infection (d.p.i.) and succumbed to disease by day 7 (Fig. 2b, c), demonstrating that this lethal challenge model remains lethal even in mice over 1 year in age. Compared to sham vaccinated mice, repNP + repGc vaccinated mice were significantly protected from lethal disease for at least 1-year post-vaccination (Fig. 2c). At 3 months, we saw no signs of clinical disease (Fig. 2b), similar to what we have previously reported at 1-month post-vaccination. At later timepoints post-vaccination, mice experienced mild weight loss, beginning at around 5 d.p.i. (Fig. 2b) suggesting breakthrough infection. However, vaccination conferred 100% survival up to 9 months post-vaccination and 80% survival (8/10 mice survived) in the 12-month group (Fig. 2c). Together, these data indicate that our vaccine administered as a prime-boost confers durable protection against lethal CCHFV infection for at least 1-year after vaccination.

Fig. 2: repNP + repGc vaccination is protective up to 1-year post-vaccination.

Groups of WT C57BL6/J mice vaccinated prime-boost with repNP + repGc were treated with MAR1-5A3 antibody to block the type I IFN response and infected with 100 TCID50 CCHFV strain UG3010 3-, 6-, 9-, and 12-months post-boost vaccination. At each timepoint, mice were a challenged on “day 0” and groups of mice were assessed for control of viral loads and survival on days 5 and to day 14, respectively, relative to CCHFV challenge. Mice (N = 8) were monitored for b weight loss and c survival over the course of infection. On day 5 post-infection, mice (N = 6) were evaluated for control of d viral genome copies via qRT-PCR and e infectious virus via TCID50 in the blood, liver, and spleen. Dashed lines indicate limit of detection. Statistical comparisons were calculated using a one-way ANOVA with Turkey’s multiple comparison test. ns >0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are shown as mean plus standard deviation.

We also evaluated viral loads in the blood, liver, and spleen by qRT-PCR and infectious titration. Similar to 1-month post-vaccination7, at 3-months post-vaccination, viral RNA was significantly reduced and little to no infectious virus was detectable in these tissues (Fig. 2d, e). Interestingly, although mice were protected from lethal disease out to 12-months, breakthrough in viral control was detected beginning 6-months post-vaccination (Fig. 2d, e). Although viral genome copies were significantly lower in the blood, liver, and spleen than sham vaccinated animals at 6- and 9-months post-vaccination, they were also significantly higher compared to the 3-month group (Fig. 2d). At 12-months, viral RNA remained significantly lower than sham vaccinated animals in the blood and liver but not the spleen (Fig. 2d). However, infectious virus in the blood, liver, and spleen was significantly decreased compared to sham vaccinated animals at all timepoints (Fig. 2e) although, titers were significantly higher in the liver and spleen in the 9-and 12-month groups compared to the 3-month group (Fig. 2e). Cumulatively, our data shows that repNP + repGc vaccination confers durable protection against lethal CCHFV challenge in mice out to 1-year post vaccination.

repNP + repGc vaccination protects mice from hepatic and splenic pathology 1-year post-vaccination

To further assess the durability of repNP + repGc vaccination, we evaluated spleen and liver samples, key tissues of CCHFV pathogenesis, from infected mice 5 d.p.i, during peak disease. The sham-vaccinated mice from each group developed histologic lesions and immunoreactivity results consistent with CCHFV infection in mice (Fig. 3a, b, Supplementary Fig. 1a, b). Hepatic lesions consist of multifocal to diffuse hepatocellular degeneration and necrosis with neutrophilic and histiocytic inflammation (Fig. 3a). Anti-CCHFV immunohistochemistry in sham vaccinated mice reveals near diffuse hepatocellular immunoreactivity (Fig. 3b). Splenic lesions consisted of necrosis and loss of white pulp with swollen red pulp macrophages and varying amounts of necrotic debris (Supplementary Fig. 1a). Immunohistochemistry revealed a near diffuse immunoreactivity of red pulp macrophages and occasional white pulp mononuclear cells believed to be macrophages, lymphocytes or follicular dendritic cells or any combination of the three (Supplementary Fig. 1b). These data suggest that older mice, in this study up to 56 weeks of age at time of challenge, develop similar CCHFV-induced pathology compared to younger mice.

Fig. 3: Histologic findings in the liver.

Groups of WT mice sham vaccinated (a, b) or vaccinated with repNP + repGc and challenged at 3mo (c, d), 6mo (e, f), 9mo (g, h), or 12mo (i, j) post-vaccination were euthanized on day 5 p.i. and liver collected, and formalin fixed. Sections were (left) H&E stained or (right) probed for the presence of viral antigen via immunohistochemistry (IHC). Representative images for each group are shown at 200× magnification. Complete findings are provided in Supplementary Table 1.

In contrast to sham-vaccinated mice, 3-months post vaccination repNP + repGc vaccinated mice had no evidence of pathology or CCHFV antigen in the liver or spleen (Fig. 3c, d and Supplementary Fig. 1c, d). At 6- and 9- months post-vaccination, and consistent with breakthrough infection seen in weight loss and control of viral replication, vaccinated mice developed minimal to moderate histologic lesions and immunoreactivity consistent with CCHFV infection in 5/6 mice (Fig. 3e–h, Supplementary Fig. 1e–h). At 12-months post vaccination all 8 repNP + repGc vaccinated mice developed mild histologic lesions and minimal to marked immunoreactivity consistent with CCHFV infection and similar to the 6- and 9- month groups (Fig. 3i, j and Supplementary Fig. 1i, j). Despite evidence of pathology and immunoreactivity in vaccinated mice at later time points post-vaccination, at all timepoints the histologic lesions and immunoreactivity of the vaccinated mice were less than that of the sham vaccinated control mice.

Rapid anamnestic immune responses at 6-, 9-, and 12-months post vaccination

Previously, we showed that prime-boost vaccination with repNP + repGc was protective against lethal CCHFV challenge in mice one month post vaccination and surviving mice from these studies did not develop CCHFV-specific anamnestic responses7. However, since we observed breakthrough viral replication at 6-months post-vaccination, yet animals remained significantly protected against CCHFV disease, we hypothesized that rapid anamnestic humoral or cellular immunity may contribute to protection from CCHFV challenge at later timepoints post-vaccination. In surviving mice challenged at 6-, 9- and 12-months post-vaccination, we evaluated antibody responses using recombinant antigens, including Gn and GP38 antigens that were not present in the vaccine. We also measured T-cell responses in surviving vaccinated mice challenged at 9- and 12-months post-vaccination with peptides spanning the NP and entire GPC, including regions not present in the repGc vaccine. Two-weeks after CCHFV challenge, surviving mice challenged at 6-, 9-, and 12-months post-vaccination, had increases in both anti-NP and anti-Gc antibody titers compared to day 0 (Fig. 4a), consistent with breakthrough viral replication and transient clinical disease (Figs. 2b–e and 4a). Interestingly, although we failed to detect antibodies against the Gc prior to infection, after infection, surviving mice had increased antibody response to the Gc that was significant at 6- and 12-month post-vaccination (Fig. 4a). Previously we have shown that anti-Gc anamnestic antibody responses post-repNP and repGc vaccination and CCHFV challenge do not result in neutralizing activity7. No group had a response to the CCHFV Gn or GP38 proteins (Fig. 4a). In addition, anamnestic T-cell responses were observed against the GPC peptide pool 10, similar to the specificity of T cells prior to challenge, while mice also developed significant responses against GPC pool 13 and NP peptide pool 2 (Fig. 4b) suggesting a broadening of the CCHFV-specific T-cell response after infection. Together, these data support a hypothesis that repNP + repGc vaccination confers durable immunity against CCHFV challenge through both preexisting and rapid recall responses upon viral challenge.

Fig. 4: Anamnestic responses to CCHFV infection.

Surviving mice from repNP + repGc 6-, 9-, and 12-months groups were evaluated on day 14 p.i. for CCHFV-specific anamnestic responses. Sera was collected and evaluated for CCHFV-specific antibodies using a recombinant antigen ELISA to the CCHFV NP, Gn, GP38, and Gc. Responses are shown for the 6-, 9-, and 12-month groups as well as comparisons between D0 and D14 for CCHFV-NP and Gc responses. Further, anamnestic CCHFV-specific T-cell responses were measured via b IFNγ ELISpot shown as cumulative SFCs against peptide pools spanning the entire GPC or NP as well as a comparison between D0 and D15 responses to the GPC peptide pool 10. Data from day 0 is duplicated from Fig. 1 and shown here for comparison. Dashed lines indicate limit of detection. Statistical comparisons were calculated using a one-way ANOVA with Turkey’s multiple comparison test. ns >0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are shown as mean plus standard deviation.