Broad and long-lasting immune protection against various Chikungunya genotypes demonstrated by participants in a cross-sectional study in a Cambodian rural community

Figures & data

Fig. 1 Study design and outcome.

The recruitment included a comparable number of individuals born before 1975 (n = 74) and after 1980 (n = 80), as well as numbers of infections (IgM positive) confirmed in 2012. Based on the IgM status from 2012 and the antibody status in 2016 (investigated by HIA and FRNT), the study participants could be classified as individuals who remained naive in 2016 (gray shaded boxes), individuals infected during the outbreak in 2012 (red shaded boxes), and study participants with CHIKV immunity acquired at an unknown time point (blue shaded boxes). All criteria allowed for a separate analysis of subgroups, designated subgroup 1 based on the age of the study participants, subgroup 2 based on the IgM status in 2012, and subgroup 3 including both age and 2012 IgM status

Results of the three chikungunya serological assays used (MAC-ELISA, HIA, and FRNT) among the study participants (n = 154)

	Born before 1975		Born after 1980		Comparison of age groups	Total
	n	(%)	n	(%)	p value^a	n	(%)
MAC-ELISA in 2012
Negative	40	(54)	40	(50)	0.73	80	(52)
Positive	34	(46)	40	(50)		74	(48)
HIA in 2016
Negative	5	(7)	23	(29)	0.0009	28	(18)
Positive	69	(93)	57	(71)		126	(82)
FRNT in 2016
Negative	4	(5)	24	(30)	0.0002	27	(17.5)
Positive	70	(95.5)	56	(70)		127	(82.5)
MAC-ELISA in 2012 & HIA in 2016
Both negative	5	(7)	21	(26)	0.0012	26	(17)
Both positive	34	(50)	38	(47.5)		72	(47)
Discrepant	35	(47)	21	(26)		56	(36)
MAC-ELISA in 2012 & FRNT in 2016
Both negative	4	(5)	22	(27.5)	0.0002	26	(17)
Both positive	34	(46)	38	(47.5)		72	(47)
Discrepant	36	(48)	20	(25)		56	(36)
Total	74	(48)	80	(52)	—	154	(100)

MAC-ELISA IgM antibody-capture ELISA, HIA hemagglutination inhibition assay, FRNT foci reduction neutralization test

^aTwo-tailed χ² test with Yates correction.

HIA and FRNT₉₀ titers among the study participant subgroups (n = 154)

Subgroup	Subdivisions	Mean HIA	Mean FRNT90 Thailand 1958	Mean FRNT90 Thailand 1975	Mean FRNT90 New Caledonia 2011	Mean FRNT90 Cambodia 2011
		(95% CI)	(95% CI)	(95% CI)	(95% CI)	(95% CI)
—	Total	378	1028	398	507	1033
	(n = 154)	(286–469)	(841–1216)	(311–486)	(413–601)	(847–1219)
1: Age	Born before 1975	320	1000	393	505	862
	(n = 74)	(199–442)	(726–1274)	(283–502)	(362–648)	(726–1274)
	Born after 1980	431	1054	404	509	1191
	(n = 80)	(294–567)	(791–1318)	(267–540.1)	(382–636)	(912–1471)
2: IgM status	IgM negative	192	701	285	348	537
	(n = 80)	(117–266)	(487–914)	(194–376)	(234–462)	(370–703)
	IgM positive	579	1383	520	679	1570
	(n = 74)	(416–741)	(1083–1682)	(371–670)	(534–824)	(1269–1871)
3: Age and IgM status	Born before 1975	183	838	366	437	477
	IgM negative (n = 40)	(127–239)	(534–1143)	(234–498)	(250–624)	(306–648)
	Born before 1975	482	1190	424	584	1316
	IgM positive (n = 34)	(230–733)	(703–1677)	(236–611)	(356–812)	(863–1769)
	Born after 1980	200	563	204	259	596
	IgM negative (n = 40)	(59–341)	(257–869)	(76–332)	(125–393)	(303–890)
	Born after 1980	661	1546	603	759	1786
	IgM positive (n = 40)	(444–879)	(1164–1928)	(372–834)	(569–950)	(1379–2194)

CI confidence interval, HIA hemagglutination inhibition assay, FRNT foci reduction neutralization test

Bold number means higher HIA or FRNT₉₀ titer within the subgroup; underlined number means higher FRNT₉₀ titer within the subdivision (p  < 0.05)

Fig. 2 Subgroup 1: Influence of age on antibody levels.

a Study participants who were not infected in 2012 (IgM negative) and were antibody positive in 2016 (positive FRNT₉₀ titer; black bars) as well as individuals remaining naive in 2016 (negative FRNT₉₀ titer; gray bars), stratified by age. Asterisks indicate statistically significant differences in the proportions of participants (***p < 0.001; χ² test). b Individual FRNT₉₀ titers (with geometric mean) of study participants with neutralizing antibodies (n = 126), stratified by age group (circles: born before 1975, n = 70; squares: born after 1980, n = 56). FRNT₉₀ titers against the three Asian genotype strains from Thailand (TH 35: light green; TH 1455-75: dark green) and New Caledonia (NC-2011-568: blue) as well as the Cambodian ECSA IOL strain from 2011 (V1024306_KH11_PVH: red). Asterisks indicate statistically significant differences in mean FRNT₉₀ titers between the distinct virus strains (*p < 0.05; ***p < 0.001; ****p < 0.0001; one-way ANOVA, Tukey’s multiple comparison test)

Fig. 3 Subgroup 2: Influence of infection in 2012 on neutralizing antibody levels.

Individual FRNT₉₀ titers (with geometric mean) of study participants with neutralizing antibodies (n = 126), stratified by participants infected in 2012 (IgM positive, n = 54; down-pointing triangle) and participants not infected in 2012 (IgM negative, n = 72; up-pointing triangle). FRNT₉₀ titers against the three Asian genotype strains from Thailand (TH 35: light green; TH 1455-75: dark green) and New Caledonia (NC-2011-568: blue) as well as the Cambodian ECSA IOL strain from 2011 (V1024306_KH11_PVH: red). Asterisks indicate statistically significant differences in mean FRNT₉₀ titers between the distinct virus strains (*p < 0.05, ****p < 0.0001; one-way ANOVA, Tukey’s multiple comparison test)

Fig. 4 Subgroup 3: Influence of age and infection in 2012 on neutralizing antibody levels.

Individual FRNT₉₀ titers (with geometric mean) of study participants with neutralizing antibodies (n = 126). a FRNT₉₀ titers of study participants born before 1975 divided by IgM status in 2012 (open symbols: negative; filled symbols: positive). b FRNT₉₀ titers of study participants born after 1980 stratified by IgM status in 2012 (open symbols: negative; filled symbols: positive). FRNT₉₀ titers against the three Asian genotype strains from Thailand (TH 35: light green; TH 1455-75: dark green) and New Caledonia (NC-2011-568: blue) as well as the Cambodian ECSA IOL strain from 2011 (V1024306_KH11_PVH: red). Asterisks indicate statistically significant differences in the mean FRNT₉₀ titers between the various virus strains (*p < 0.05; **p < 0.01; ****p < 0.0001; one-way ANOVA, Tukey’s multiple comparison test)

Comparison of E1 and E2 amino acid sequences of the CHIKV strains used for neutralization assay

			Amino acid in the respective position
Structural protein	Polyprotein position	Protein position	Thailand 1958	Thailand 1975	New Caledonia 2011	Cambodia 2011
E1	809	1	Met	Leu	Leu	Met
E1	1122	304	Pro	Ser	Ser	Pro
E2	579	254	Ile	Val	Val	Ile