Live Zika virus chimeric vaccine candidate based on a yellow fever 17-D attenuated backbone

Figures & data

Fig. 1 Schematic representation of the design and recovery strategies used to generate chimeric viruses. We recovered infectious virus only with construct C.

The two cleavage sites are enlarged in boxes, with the amino acid alignment shown with separations between different proteins

Fig. 2 Evolution of viral production of chimeric viruses during serial passage that followed cell transfection.

A mix of BHK-21/HEK-293 cells was transfected. Cell supernatant medium was subsequently passaged 4–8 times in Vero-E6 cells. Viral production in cell supernatant medium was assessed using a real-time quantitative RT-PCR assay

Mutations detected during the passages that followed cell transfection of chimeric viruses

Chimeric virus	Nucleotide position	Frequency at #P2	Frequency at #P4	Frequency at #P8	Frequency at #P10	Region	Nucleotide change	aa change
CH-17-D/ZIKV	291	100%	100%	100%	100%	C	A>G	–
	1625	66%	90%	92%	94%	E	T>C	V>A
	1706	100%	100%	100%	100%	E	G>T	G>V
	2514	100%	100%	100%	79%	NS1	A>G	–
	4482	100%	100%	100%	100%	NS2B	A>G	–
CH-17-D/ZIKV*	291	n.a	n.d	70%	n.a	C	A>G	–
	1303	n.a	100%	n.d	n.a	E	C>T	H>Y
	1625	n.a	n.d	68%	n.a	E	T>C	V>A
	1706	n.a	n.d	69%	n.a	E	G>T	G>V
	2514	n.a	n.d	69%	n.a	NS1	A>G	–
	4482	n.a	n.a	56%	n.a	NS2B	A>G

Only consensus mutations (frequency >50%) are shown. n.a. not available, n.d. not detected

Fig. 3 CH-17-D/ZIKV in cellulo characterization.

a Expression of the ZIKV E protein in Vero-E6 was confirmed at day 2 and 5 post-infection using an indirect immunofluorescence assay with a specific ZIKV immune serum as the primary antibody. Uninfected cells (mock) and cells infected by ZIKV and the 17-D vaccine strain were used as controls. b–d Comparative growth kinetics of the CH-17-D/ZIKV and ZIKV 17-D vaccine strains in HUH7.5 (b), HEK-293 (c), and Vero-E6 cells (d). e,f Comparative growth kinetics of the CH-17-D/ZIKV and ZIKV 17-D vaccine strains in Vero cells. Cell supernatant medium was harvested at different time points after infection to assess the amount of viral RNA present using a real-time quantitative RT-PCR assay (e; expressed as the means ± SD) and the infectious titers using a TCID₅₀ assay (f; expressed as the means ± SD)

Fig. 4 Neutralizing antibody titers in transitory immunocompromised mice at day 21 post-immunization.

a Experimental timeline. b, c Groups of four mice were immunized with two doses of CH-17-D/ZIKV, ZIKV, and the 17-D vaccine strain (from 10e4 to 10e6 TCID₅₀). Twenty-one days later, sera from mice were tested for the presence of antibodies to ZIKV and YFV using a viral RNA Yield Reduction Neutralization Test. The results are expressed as individual log of YRNT50 (b) and YRNT90 titers (c) with mean values ±SD represented by black lines with error bars, respectively

Fig. 5 Amounts of viral RNA detected in brain and spleen samples collected during challenge experiments.

a Experimental timeline. b Amounts of viral RNA in brain and spleen samples collected during challenge experiments (cf. Table 2) measured using a real-time quantitative RT-PCR assay. Mean values ± SD are represented by black lines and error bars, respectively. The results from both doses of viruses are pooled

Protection of transitory immunocompromised mice challenged with a heterologous strain of ZIKV

Viral strain	Spleens	Brains
CH-17D/ZIKV (both doses)	10% (1/10)	10% (1/10)
17-D vaccine strain (both doses)	100% (8/8)	87.5% (7/8)
Unvaccinated	100% (4/4)	100% (4/4)
ZIKV PF	0% (0/4)	0% (0/4)

Groups of mice were immunized with two doses (10e4 and 10e5 TCID₅₀) of CH-17-D/ZIKV, the 17-D vaccine strain or PBS (unvaccinated). Twenty-one days later, mice were challenged with 10e6 TCID₅₀ of an African ZIKV strain. The proportion of mice testing positive for ZIKV in spleen/brain samples at day 10 post-challenge was expressed as a percentage. The results from both doses of viruses are pooled (the results for individual groups are provided in Supplemental Table 2). Viral RNA was detected using a real-time RT-PCR assay. The amounts of viral RNA detected in samples are shown in Fig. 5