Inactivated Rabies Virus Vectored MERS-Coronavirus Vaccine Induces Protective Immunity in Mice, Camels, and Alpacas

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emergent coronavirus that has caused frequent zoonotic events through camel-to-human spillover. An effective camelid vaccination strategy is probably the best way to reduce human exposure risk. Here, we constructed and evaluated an inactivated rabies virus-vectored MERS-CoV vaccine in mice, camels, and alpacas. Potent antigen-specific antibody and CD8⁺ T-cell responses were generated in mice; moreover, the vaccination reduced viral replication and accelerated virus clearance in MERS-CoV-infected mice. Besides, protective antibody responses against both MERS-CoV and rabies virus were induced in camels and alpacas. Satisfyingly, the immune sera showed broad cross-neutralizing activity against the three main MERS-CoV clades. For further characterization of the antibody response induced in camelids, MERS-CoV-specific variable domains of heavy-chain-only antibody (VHHs) were isolated from immunized alpacas and showed potent prophylactic and therapeutic efficacies in the Ad5-hDPP4-transduced mouse model. These results highlight the inactivated rabies virus-vectored MERS-CoV vaccine as a promising camelid candidate vaccine.

Keywords: MERS-CoV; alpaca; camel; mice; rabies virus vector; vaccine.

Figure 1

Characterization and validation of rSRV9-MERS_S1. (A) Schematic of the candidate vaccine rSRV9-MERS_S1. The MERS_S1 membrane-anchoring chimera protein gene, which contains MERS-CoV S1 gene fused to the gene of human CD4 transmembrane domain (TM) and RABV G protein cytoplasmic domain (CD), was amplified and subcloned into the enzyme cutting sites BsiWI/PmeI of the recombinant plasmid containing full-length RABV cDNA (pD-SRV9-PM-eGFP), generating the recombinant cDNA clone pD-SRV9-PM-MERS_S1. (B) TEM detection of rSRV9-MERS_S1. The samples of inactivated rRABV-MERS_S1 viral culture supernatants were stained with 1% sodium phosphotungstate. Bar = 100 nm. (C) Multiple-step growth curves of rSRV9-MERS_S1 and rSRV9 on BSR cells (multiplicity of infection (MOI) = 0.1). Cell culture supernatants were then harvested at 24, 48, 72, 96, and 120 h post-infection. Data were obtained using GraphPad Prism version 9.0 (GraphPad software). Data are shown as mean ± SD. (D) Validation of the expression of MERS-CoV S1 protein and RABV G protein in rSRV9-MERS_S1-infected NA cells by indirect immunofluorescence staining. NA cells infected with rSRV9 or mock-infected NA cells were used as controls. (E) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of viral protein expression in purified rSRV9-MERS_S1 virions and rSRV9 virions. (F) Western blotting detection of MERS-CoV S1 protein and RABV G protein expressions in purified recombinant virus particles rSRV9-MERS_S1 using mouse anti-MERS-S1 monoclonal antibodies and mouse anti-RABV-G monoclonal antibodies. Purified parental virus particles rSRV9 were used as control.

Figure 2

Humoral immune responses against MERS-CoV elicited by the inactivated rabies virus-vectored MERS-CoV vaccine in mice. (A) Evaluation of an appropriate vaccine from recombinant rabies virus-vectored MERS-CoV vaccine candidates (n = 9 per group). The immunogenicity of the vaccine candidates with different forms (live and inactivated) and delivery doses (one dose assayed for both live and inactivated vaccines, two doses assayed for inactivated vaccine) was evaluated by anti-MERS-CoV IgG antibody through indirect ELISA. Endpoint dilution titers were calculated at the indicated time points. (B) The total anti-MERS-CoV IgG antibody titers of sera from each group of mice (n = 8) were assessed on days 14, 28, and 35 after the first immunization and shown as endpoint dilution titers. (C) The dynamic changes and duration of serum antibodies from each group of mice (n = 8) were evaluated by indirect ELISA at the indicated time. (D) The cross-neutralizing activity against divergent MERS-CoV isolates (human MERS-CoV KOR/HIN strain and dromedary camel MERS-CoV D1271 strain) of sera from each group of mice (n = 3) were evaluated by a pseudovirus-based neutralization assay. Data are shown as mean ± SD. Data were obtained using GraphPad Prism version 9.0 (GraphPad software). Significance was calculated using a two-way ANOVA with multiple comparisons tests (not indicated in graph; *p < 0.05, **p < 0.01, ****p < 0.0001).

Figure 3

CD8⁺ T-cell responses elicited by the inactivated rabies virus-vectored MERS-CoV vaccine. Splenocytes from each group of mice (n = 3) were harvested 1 and 4 weeks following the second immunization and evaluated for the vaccine-induced specific cellular immune responses. (A) The MERS-CoV-specific IFN-γ activities in splenocytes were measured using commercial ELISpot kits. Spot-forming cells (SFCs) secreting IFN-γ were enumerated in an automated ELISpot reader and shown as mean responses in each group at the indicated time points. (B) The frequencies of MERS-CoV-specific IFN-γ-, TNF-α-, and IL-2-secreting CD8⁺ T cells in splenocytes were evaluated using intracellular cytokine staining (ICS) assays. (C) Levels of IFN-γ, TNF-α, and IL-2 secreted by splenocytes were measured using commercial ELISA kits. Data are shown as mean ± SD. Data were obtained using the GraphPad Prism version 9.0 (GraphPad software). Significance was calculated using a two-way ANOVA with multiple comparisons tests (not indicated in graph; **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 4

Immunization with the rabies virus-vectored MERS-CoV vaccine provides protection in MERS-CoV-infected Ad5-hDPP4-transduced mice model. (A) The schematic of the live challenge experiment. Six- to eight-week-old specific pathogen-free female C57BL/6 mice (n = 8 per group) were intramuscularly (i.m.) injected with either the inactivated recombinant rabies virus MERS-CoV vaccine or inactivated parental rabies virus vaccine on days 0 and 14; sera from each group of mice (n = 8) were collected on day 21. All mice were transduced with Ad5-hDPP4 5 days as previously described before intranasal challenge with 1 × 10⁵ PFU MERS-CoV (EMC/2012 strain) on day 42, and then the lungs from each group of mice (n = 3) were harvested for virus titration. (B) Binding and (C) neutralizing activities of sera collected on day 21 were respectively measured by indirect ELISA and live MERS-CoV (EMC/2012 strain). (D) On day 3 post-infection, virus titers in the lungs were measured on Vero 81 cells and expressed as FFU/g tissue. Data are shown as mean ± SD. Data were obtained using GraphPad Prism version 9.0 (GraphPad software). An unpaired Student’s t-test (two-tailed) was used for statistical analysis, and the relevant p-values are indicated (not indicated in graph; ***p < 0.001, ****p < 0.0001).

Figure 5

Humoral immune responses elicited by the inactivated rabies virus-vectored MERS-CoV vaccine in camels. (A) The schematic of the camel immunization experiment. Four Bactrian camels were injected subcutaneously (s.c.) in the neck with 5 ml of inactivated recombinant rabies virus-vectored MERS-CoV vaccine (n = 2) or the inactivated parental rabies virus vaccine (n = 1) or phosphate-buffered saline (PBS) (n = 1) two times at 4-week intervals. Sera of camels from each group were harvested before the first immunization and collected at weeks 4 and 8 after the first immunization. (B) Sera cross-neutralizing activity against representative human and camel MERS-CoV strains (human MERS-CoV KOR/HIN strain and dromedary camel MERS-CoV D1271 strain) were evaluated by a pseudovirus-based neutralization assay. (C) The cross-neutralizing activity against live viruses among the known MERS-CoV clade A (EMC/2012 strain), clade B (ChinaGD01 strain), and clade C (dromedary/Nigeria/HKU NV1657 strain) were measured by plaque reduction neutralizing assay. (D) Prophylactic and therapeutic efficacies of passive transfer with the rabies virus-vectored MERS-CoV vaccine camel immune sera collected at 8 w.p.i in MERS-CoV-infected Ad5-hDPP4-transduced mice (n = 3 per group). A total of 200 μl of camel immune sera collected was intravenously transferred 1 day before or after MERS-CoV (EMC/2012 strain) infection. Mice in the control group (n = 3) intravenously received the same dose of negative sera from healthy camels at the same time points. (E) The RABV-specific neutralizing antibody titers of sera from each group of mice (n = 3) were evaluated using fluorescent antibody virus neutralization (FAVN). Data are shown as mean ± SD. Data were obtained using GraphPad Prism version 9.0 (GraphPad software). Significance was calculated using a one-way ANOVA with multiple comparisons tests (not indicated in graph; *p < 0.05, ****p < 0.0001).

Figure 6

Humoral immune responses elicited by the inactivated rabies virus-vectored MERS-CoV vaccine in alpacas. (A) The schematic of the alpaca immunization experiment. Three alpacas received 3 ml of inactivated recombinant rabies virus-vectored MERS-CoV vaccine (n = 2) or phosphate-buffered saline (PBS) (n = 1) by multiple sites of subcutaneous (s.c.) injection in the neck two times at 3-week intervals. Sera of alpacas from each group were harvested before the first immunization and collected at 3, 6, and 12 weeks after the first immunization. (B) Sera cross-neutralizing activity against representative human and camel MERS-CoV strains (human MERS-CoV KOR/HIN strain and dromedary camel MERS-CoV D1271 strain) were evaluated by a pseudovirus-based neutralization assay. (C) Sera MERS-CoV-specific neutralizing antibodies measured 28 weeks after the first immunization by a pseudovirus-based neutralization assay. (D) The cross-neutralizing activity against live viruses among the known MERS-CoV clade A (EMC/2012 strain), clade B (ChinaGD01 strain), and clade C (dromedary/Nigeria/HKU NV1657 strain) were measured by focus reduction neutralizing assay. (E) Prophylactic and therapeutic efficacies of passive transfer with the rabies virus-vectored MERS-CoV vaccine alpaca immune sera collected at 6 w.p.i in MERS-CoV-infected Ad5-hDPP4-transduced mice. A total of 200 μl of alpaca immune sera collected were intravenously transferred 1 day before or after MERS-CoV (EMC/2012 strain) infection. Mice in the control group intravenously received the same dose of negative sera from healthy alpacas at the same time points. (F) Sera RABV-specific neutralizing antibodies were evaluated using fluorescent antibody virus neutralization (FAVN). Data are shown as mean ± SD. Data were obtained using GraphPad Prism version 9.0 (GraphPad software). An unpaired Student’s t-test (two-tailed) was used for statistical analysis, and the relevant p-values are indicated (not indicated in graph; *p < 0.05,).

Figure 7

MERS-CoV-specific alpaca VHHs protected mice from MERS-CoV infection. (A) Binding activity of VHH1-Fc with MERS-CoV RBD, S1, and S trimer protein were measured by indirect ELISA. (B) Neutralizing activity against MERS-CoV prototype strain EMC/2012 was evaluated by pseudovirus neutralization assay. (C) Prophylactic and (D) therapeutic administration of VHH1-Fc protected MERS-CoV-infected mice (n = 3 per group). The prophylactic and therapeutic efficacies of VHH1-Fc were evaluated in vivo using Ad5-hDPP4-transduced mice challenged with MERS-CoV. VHH1-Fc was intravenously transferred 1 day before or after MERS-CoV (EMC/2012 strain) infection. The lungs were harvested at 3 days post-infection for viral titer determination. Mice in the control group (n = 3) were negative control antibodies (anti-HIV antibody 2G12) at the same time points. Data are shown as mean ± SD. Data were obtained using GraphPad Prism version 9.0 (GraphPad software). An unpaired Student’s t-test (two-tailed) was used for statistical analysis, and the relevant p-values are indicated (not indicated in graph; *p < 0.05, ***p < 0.001).