Figure 1.
Generation and characterization of candidate MVA-CHIK vaccine virus.
Diagram of the MVA-GFP genome and transfer vector used for homologous recombination for creation of MVA-CHIK. The different areas where deletion occurred in the vaccinia virus genome to create MVA are shown. MVA-CHIK was created by transfecting the plasmid containing CHIKV p62 under control of a se/l promoter which also expressed dsRed2 under control of a p11 promoter. This plasmid contains flanking regions to deletion III (DelIII) which is where the recombinant gene was inserted into the genome with a fluorescent marker (A). PCR analysis of the DelIII region. Viral DNA was extracted from purified, final virus stocks of MVA-CHIK, MVA-GFP and MVA-WT. PCR was performed using primers specific for the DelIII flanking regions. A DNA ladder is included for comparison of size (B). Monolayers of CEF cells were infected with recombinant MVA-CHIK viruses at MOI of 5 or 10 PFU/cell for Western Blot or Immunostaining, respectively. After 24 h post infection, cells were harvested and subjected to SDS-PAGE followed by western blot analysis (C) or fixed with 2% PFA as described in the methods. Both the cellular pellet and supernatant were analyzed for expression of CHIKV E3/E2. The order is the same for both and is as follows, wild-type CHIKV (1), MVA-CHIK (2), MVA-GFP (3), and mock infected cells (4). Following fixation, cells for immunostaining were either allowed to remain intact or permeabilized with triton X-100 (D). Anti-CHIKV polyclonal rabbit was used for primary staining for both assays. Green represents CHIKV E2 positive cells, red is dsRed protein produced by the virus and blue is nuclear staining with Hoechst.
Figure 2.
MVA-CHIK candidate vaccine protected A129 mice against CHIK-LR challenge.
A129 mice were vaccinated with either prime alone or a prime and boost (day 28) of each vaccine virus (107 TCID50 units of MVA-CHIK or MVA-GFP, or 104 TCID50 units of CHIK-IRES). Mice were then challenged with 102 TCID50 units of wild-type CHIKV intradermally (in the hind left footpad) eleven days post-prime or two weeks post-boost. Prior to boost (where applicable) and challenge, mice were bled and serum was monitored for both total Ig(G+M) and neutralizing activity by TCID50 micro-neutralization assay (A) and ELISA (B), respectively. Prime only MVA-GFP data is not shown because it does not differ with the prime & boost groups. Mice were monitored for 14 days following challenge for survival (C) and footpad swelling (D). The first three days following challenge viremia levels were measured via TCID50 (E). The dotted line indicates the limit of detection of the assay.
Figure 3.
MVA-CHIK vaccine protects against local inflammation following CHIK-LR challenge.
7(H&E). MVA-CHIK vaccinated mice showed low levels of inflammation (A) upon infection with 102 TCID50 units of wild-type CHIKV. MVA-GFP vaccinated mice developed massive inflammation with necrotic muscle degeneration and edema following challenge (B).
Figure 4.
MVA-CHIK candidate vaccine fully protects BALB/c mice against CHIKV viremia.
BALB/c mice were vaccinated with a prime and boost (day 28) of 107 TCID50 units of MVA-CHIK vaccine virus and then challenged with 104 TCID50 units of wild-type CHIKV intradermally (in the hind left footpad) two weeks post-boost. Prior to challenge, BALB/c mice were bled and serum was monitored for neutralizing activity by TCID50 micro-neutralization assay (A). Mice were bled two days following challenge and viremia levels were measured via TCID50 (B). For passive immunization, prime and boost vaccinated BALB/c and A129 mice were bled and serum taken from each mouse was pooled with equal volumes into separate pools from each strain. Pools of serum (100 µL for BALB/c and 200 µL for A129) were then injected i.p. into A129 mice which were challenged 24 hrs later with 102 TCID50 units of wild-type CHIKV. Viremia was measured 2 d.p.i (C) and survival was monitored for two weeks post-challenge (D). The dotted line indicates the limit of detection of the assay.
Figure 5.
MVA-CHIK elicited a CHIK-E2-specific CD4+ T cell response in A129 mice.
Two weeks following boost, splenocytes from MVA-GFP or MVA-CHIK immunized mice were stimulated with CHIKV NSP2, E2 or E3 peptide pools (1 µg/well). T-cells were then stained for intracellular IFNγ (A and C) or CD40L (B and D). Representative dot plots (for only one mouse per group) are shown for both cytokines with MVA-CHIK and MVA-GFP stimulated with E2 peptide pools or background media control (A and B). For stimulation with E2, E3 and NSP2 peptide pools data are presented as the mean+SD of the percentages cytokine-positive cells among gated CD4+ T cells (3 mice/group) with background subtracted (C and D). Stimulation was significantly higher for IFNγ and CD40L in the MVA-CHIK immune group, as compared to MVA-GFP control (p = 0.02 and p = 0.01, respectively). No cytokine production in CD8+ T cells was observed (data not shown).
Figure 6.
Depletion of MVA-CHIK immune CD4+ T cells, but not CD8+ T cells, abolished the protective immunity afforded by MVA-CHIK in A129 mice.
A129 mice were depleted of CD4+ or CD8+ T cells with anti-CD4 or CD8 mAbs and were then challenged with 500 TCID50 units of CHIK-LR i.d. via the left hind footpad on day 14 post boost. Mice were monitored for (A) footpad swelling; (B) survival and (C) Viremia (measured 2 d.p.i by TCID50 assay). The dotted line indicates the limit of detection of the assay.