Fig 1.
AdV-MAYV Vaccine Vector Expression of MAYV Structural Proteins.
(A) The full length MAYV structural protein ORF was inserted into the replication defective huAdV expression vector under the control of the MCMV-IE2 promoter containing a Lac repressor to specifically modulate expression during reconstitution and stock production of the virus. (B) AdV-MAYV expression of the structural protein was evaluated in THF-CAR cells infected with increasing MOIs. Cell lysates were analyzed at 72 hpi for capsid expression by western blotting for the viral capsid protein. Shown is a representative image of two independent experiments. (C) Electron microscopy image of 4% PFA fixed AdV-MAYV VLPs from clarified supernatants of AdV-MAYV infected THF-CAR cells (MOI = 100 PFU/cell). Representative images are shown of 500 nm (left) and 100 nm (right) magnifications. Shown is a representative image of three biological replicates.
Fig 2.
AdV-MAYV-Induced Antibody Response Neutralize Virus in A Pre-attachment Mechanism.
(A) WT C57BL/6N mice were vaccinated with 106, 107, or 108 PFU AdV-MAYV by intramuscular injection (n = 5 mice per group). A separate group of animals were infected with 104 PFU MAYVBeAr. At 14 days post vaccination, a subgroup of mice received a vaccine boost with the same vector and dosage as was used during the primary vaccination. Blood was collected from the vaccinated mice at day 28. (B) Sera from each mouse was tested for MAYVCH neutralization potential using a PRNT50 assay. Shown are the average PRNT50 values calculated for each group by non-linear regression at a 95% confidence interval (n = 5 mice per treatment group). PRNT50 values were calculated by variable slope non-linear regression. (C and D) Pre- and post-attachment neutralization assays were performed to explore the mechanism of inhibition. For pre-attachment neutralization assays, aliquots of a known concentration of virus were mixed with serial dilutions of serum for one hour prior to application to a confluent monolayer of Vero cells. For post-attachment treatments, virus was incubated with Vero cells at 4°C for one hour to allow binding and then serial dilutions of antibody were added for one additional hour at 4°C. Triplicate biological replicates and representative curves determined by variable slope non-linear regression are shown. Error bars represent SEM representative of 4 biological replicates.
Fig 3.
Characterization of Anti-MAYV Antibody Responses.
(A) Isotype specific ELISAs were performed to characterize and measure the MAYVBeAr binding antibodies in sera from naïve, AdV-MAYV prime vaccinated, AdV-MAYV prime + boost vaccinated, and MAYV infected mice. Preparations of heat inactivated whole MAYV stocks were bound to high affinity 96-well plates. Serial dilutions of mouse sera were plated in order to calculate binding dilution titer. Binding antibodies were detected by secondary antibodies specific for mouse IgG1, IgG2a, and IgG3 as well as a pan IgG/IgM. Error bars represent SD representative of quadruplicate biological replicates. Statistical analysis was performed on log transformed data by a one-way ANOVA (*P < 0.05, **P < 0.005, ***P = 0.0001, P < 0.0001). (B) Western blot analysis was used to determine antigen specificity of antibodies in serum from vaccinated mice. Protein lysates containing purified MAYVTrVl were separated by SDS-PAGE and transferred to immunoassay membranes for western blotting. Cross-reactive anti-CHIKV E1 (panel 1) and E2 (panel 2) monoclonal antibodies were used to identify MAYV envelope proteins. Serum derived from naïve mice (panel 3) and AdV-MAYV prime + boost vaccinated mice (panel 4) demonstrate the presence of envelope and capsid specific antibodies to MAYVTrVl following vaccination. The blots shown are representative images of 3 independent experiments.
Fig 4.
AdV-MAYV Vaccination Protects WT Mice from MAYV challenge.
(A) WT C57BL/6N mice were vaccinated with a AdV-MAYV or AdV-GFP prime by i.m. injection followed by a booster vaccination at 14 days. At day 28 or 84 post prime, mice were challenged with 104 PFU MAYVBeAr in the right footpad. Blood was collected at 2 dpi and tissues and blood were harvested at 4 or 7 dpi. The data represent a single experiment performed with an n = 10 mice per group. (B) Serum collected prior to challenge displayed robust neutralizing antibody titers for AdV-MAYV vaccinated mice at both 26 and 82 dpv compared to AdV-GFP controls. PRNT50 values calculated for each group by variable slope non-linear regression. Error bars represent SEM. (C and D) Serum viremia at 2 dpi was measured by limiting dilution plaque assay on Vero cells. Viral titers in the serum from AdV-MAYV vaccinated animals was below the detection limit (100 PFU/ml of serum) for all animals. Statistical analysis was performed on log-transformed data using an unpaired Mann-Whitney U test (**** P <0.0001). (E and F) Infectious viral loads in lysates derived from the ankles, calves, quads, spleen tissues and serum were measured by limiting dilution plaque assays at 4 dpi. Infectious viral loads in AdV-MAYV vaccinated animals were below the detection limit for the assay (100 PFU/ml of lysate). Statistical analysis was performed on log-transformed data using unpaired Mann-Whitney U tests (* P < 0.05, **** P < 0.0001). (G and H) Total RNA was extracted from mouse tissue lysates and viral RNA levels were measured by qRT-PCR using primers and probes directed against the virus. Statistical analysis was performed on log-transformed data using unpaired Mann-Whitney U tests (* P < 0.05, ** P < 0.005, *** P = 0.0001, **** P < 0.0001). Black dotted line indicates limit of detection (100 copies per μg of total RNA). Viral RNA was below the detection limit for most AdV-MAYV vaccinated animals following challenge. Error bars in panels C-H represent SD.
Fig 5.
MAYV-AdV Vaccination Reduces Inflammatory Mediators in the Joint.
Levels of cytokines and chemokines in control mice and mice challenged with MAYV following vaccination with AdV-GFP and AdV-MAYV. WT C57BL/6N mice were vaccinated with AdV-MAYV or AdV-GFP by i.m. injection followed by a booster vaccinated at 14 days. At day 28 post vaccination, mice were challenged with 1x104 PFU/ml MAYVBeAr in the right footpad (n = 10 mice per group). Ankle tissue homogenates collected from mice at 4 dpi were analyzed for cytokine and chemokines by a 26-plex cytokine multiplex kit and compared to naïve tissues. Statistical analysis was performed using Kruskal-Wallis tests and error bars represent SD (* P < 0.05, ** P < 0.005, *** P = 0.0001, **** P < 0.0001).
Fig 6.
Passive Transfer of AdV-MAYV Immune Sera Protects Against Pathological Effects of Infection.
Naïve or immune sera was passively transferred to groups of five-week-old female WT C57BL/6N mice one day prior to challenge with 104 PFU MAYVBeAr in the right hind limb footpad. A second group of naïve five-week-old female WT C57BL/6N were mock inoculated with PBS. (A) Footpad swelling was monitored by digital calipers throughout the experiment. Statistical analysis was performed by paired repeated measures ANOVA (* P < 0.05, **** P < 0.0001). (B) Whole hind legs were harvested at 7 dpi and sectioned for histopathology by hematoxylin and eosin staining. Tissue sections were scored on a 0–5 scale using the no infection control tissues as a baseline score: 0 absent (no lesions), 1 minimal (1~10% of tissues affected), 2 mild (11~25% affected), 3 moderate (26~50% affected), 4 marked (51~75% affected), 5 severe (>75% affected). Statistical analysis was performed by two-way ANOVA (* P <0.05, **** P < 0.0001). Error bars represent SD.
Fig 7.
AdV-MAYV Vaccination Protects IFNαR1-/- Mice from Lethal Challenge.
Male and female IFNαR1-/- mice were vaccinated with 108 PFU AdV-MAYV, AdV-GFP, or PBS by i.m. injection followed by a booster vaccination 14 days later. At day 28, mice were challenged with 1x104 PFU/ml MAYVBeAr in the right footpad. The data presented represents one independent experiment (n = 7 mice per vaccine). (A) Prior to challenge, blood was collected to measure neutralizing titers against MAYVCH by PRNT assay. AdV-MAYV elicited robust neutralizing antibodies in IFNαR1-/- mice. Error bars represent SEM. (B) Limiting dilution plaque assays were used to measure viremia for blood serum samples collected at 2 dpi. Black dotted line indicates limit of detection (100 PFU/ml). Statistical analysis was performed on log-transformed data using a Kruskal-Wallis test (** P < 0.005). Error bars represent SD. (C) Mouse morbidity and mortality was monitored daily for 7 days post infection with Kaplan-Meier survival curve analysis (**** P < 0.0001).
Fig 8.
Passive Transfer of Immune Serum Protects IFNαR1-/- Mice from Lethal MAYV Challenge.
(A) Six WT C57BL/6N mice were vaccinated with 108 PFU of AdV-MAYV following the prime + boost regimen; at 28 days post-prime total blood was collected and serum pooled from all mice. A bolus of 200 μl of pooled serum from AdV-MAYV vaccinated or naive mice was administered to IFN⍺R1-/- mice by intraperitoneal injection 1 day before challenge with 1x104 PFU MAYVBeAr. The data represents one experiment with n = 7 per condition. (B) Blood collected at 2 days post-challenge was used to measure viremia by limiting dilution plaque assays. While all 7 animals receiving control serum had high levels of virus, only three of the seven animals receiving passive transfer of immune sera had detectable virus, which was 5–6 logs lower than controls. Statistical analysis was performed on log-transformed data using a Mann-Whitney test (** P < 0.005). (C) Mice were weighed daily after challenge until experiment endpoint at 7 dpi. Statistical analysis was performed using multiple repeated measures mixed-effects ANOVA (*** P = 0.0001). (D) Mouse survival following MAYV challenge was graphed. Statistical analysis was performed using Kaplan-Meier survival curve analysis (**** P < 0.0001). (E) Tissue viral RNA levels were determined by qRT-PCR for mice that survived until 7 dpi (those animals receiving AdV-MAYV vaccine sera only). Virus was detected in the ipsilateral ankles of challenged mice but very little was detected in other tissues. Black dotted line indicates limit of detection (100 viral RNA copies/μg of total RNA). Error bars represent SD.
Fig 9.
AdV-MAYV Elicits Cross Neutralizing Antibodies Against Una and Chikungunya Viruses.
In order to determine whether the AdV-MAYV elicited cross-protection against related alphaviruses, PRNT50 assays were performed on serum from WT C57BL/6N mice vaccinated with AdV-MAYV, AdV-GFP vaccinated, and compared to serum from mice infected with MAYV. Shown are the average PRNT50 values calculated for each group (n = 5). PRNT50 assays for (A) Una virus and (B) CHIKV indicate cross-species neutralization is elicited in serum from mice vaccinated with AdV-MAYV as well as mice infected with MAYV but not for serum collected from AdV-GFP vaccinated controls. Curves were calculated using a variable slope non-linear regression analysis. Error bars representing SEM from 5 biological replicates.
Fig 10.
AdV-MAYV Vaccination Cross-Protects IFNAR1-/- Mice Against Lethal Challenge with CHIKV and Una Virus.
IFN⍺R1-/- mice vaccinated by intramuscular injection of AdV-MAYV, using the prime-boost vaccination regimen, were challenged with 1x104 PFU CHIKV or UNAV in the right footpad. Serum was collected at 2 dpi and animals were monitored for clinical signs for 7 days. Experimental timeline is similar to that in Fig 4A. Data represents one independent experiment performed with n = 5 mice per group for vaccinated animals and n = 6 for controls. (A) Viremia was measured by limiting dilution plaque assay on confluent Vero cells. Statistical analysis was performed within groups by Mann-Whitney tests (** P < 0.005). Following challenge mice were monitored daily until experiment endpoint for (B) weight loss (C) morbidity and mortality (D) changes in right posterior footpad swelling as measure by caliper. Statistical analysis was performed using multiple T-tests and Kaplan-Meier survival curve analysis. Error bars represent SD (* P < 0.05, ** P < 0.005, **** P < 0.0001). At 7 dpi tissues were harvested and ankle, calf, and spleen infectious viral loads for Una virus (E) and CHIKV virus (F) were measured in tissue homogenates by limiting dilution plaque assays. Black dotted line indicates limit of detection (100 copies of viral RNA/ μg of total RNA). Total RNA was extracted from ankle and calf tissue homogenates to quantify Una virus (G) and CHIKV virus (H) RNA loads by qRT-PCR. Error bars represent SD.