Cross-reactive antibodies enhance live attenuated virus infection for increased immunogenicity

Abstract

Vaccination has achieved remarkable successes in the control of childhood viral diseases. To control emerging infections, however, vaccines will need to be delivered to older individuals who, unlike infants, probably have had prior infection or vaccination with related viruses and thus have cross-reactive antibodies against the vaccines. Whether and how these cross-reactive antibodies impact live attenuated vaccination efficacy is unclear. Using an open-label randomized trial design, we show that subjects with a specific range of cross-reactive antibody titres from a prior inactivated Japanese encephalitis vaccination enhanced yellow fever (YF) immunogenicity upon YF vaccination. Enhancing titres of cross-reactive antibodies prolonged YF vaccine viraemia, provoked greater pro-inflammatory responses, and induced adhesion molecules intrinsic to the activating Fc-receptor signalling pathway, namely immune semaphorins, facilitating immune cell interactions and trafficking. Our findings clinically demonstrate antibody-enhanced infection and suggest that vaccine efficacy could be improved by exploiting cross-reactive antibodies.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Clinical trial overview.

a, Consort diagram showing the flow of participants through each stage of the randomized trial. b, Diagrammatic representation of the study. Subjects in the treatment group were vaccinated with Ixiaro followed by Stamaril at 1 month (Group 1, red), 4 months (Group 2, blue) or 9 months (Group 3, cyan) apart, indicated by §. The control group (Group 4) received YF vaccination only. c, Anti-JE antibody titres of subjects from Group 1 (n = 23), Group 2 (n = 11) and Group 3 (n = 14) before YF vaccination as measured by PRNT. *P < 0.05, **P < 0.01 (unpaired t-test).

Figure 2. Cross-reactive antibodies enhance YF vaccine infection to produce prolonged viraemia and improved immunogenicity.

a,b, Correlation between anti-JE (day 0) (a) and anti-JE (1 month) (b) versus anti-YF (1 month) specific antibody titres for Groups 1–3 (n = 34) as measured by PRNT. Dotted lines indicate the 99% confidence interval of the mean titre of Group 4 and the enhancing group, defined as subjects with titre greater than the 99% confidence interval of Group 4. c,d, Pre-YF immune sera from the treatment group (Groups 1–3; n = 48) (c) or the control group (n = 20) (d) were diluted 1:10 and incubated with YF17D before infecting THP-1 monocytes. YF17D plaque titres, represented by pre-vaccination serum mediated YF17D infection, were correlated with the log-transformed YF antibody titres (1 month). e–g, YF RNA levels at days 1, 3 and 7 after YF vaccination for Group 4 subjects (n = 20) (e) and the non-enhancing group (n = 40) (f) and enhancing group (n = 8) (g). h,j, YF RNA levels across the different groups at day 3 (h) and day 7 (j) post-YF vaccination. Error bars indicate standard error of the mean. i,k, Correlation of log-transformed YF antibody titres (1 month) with log-transformed YF RNA levels observed on day 3 (i) and day 7 (k) (n = 48). *P < 0.05, **P < 0.01 (paired t-test for e–g, unpaired t-test for h,j).

Figure 3. Cross-reactive antibodies differentially regulate the production of inflammatory lipids and metabolites during YF vaccination.

a,c,e, Serum levels of arachidonic acid (a), linoleic acid (c) and 12-HETE (e) in Group 4 (black bars) (n = 7), the non-enhancing group (blue bars) (n = 16) and the enhancing group (red bars) (n = 7) at days 0, 1, 3 and 7 post-YF vaccination. Box plot whiskers indicate the range of the data. b,d,f, Pearson correlation (r) of anti-YF antibody titres (1 month), as measured by PRNT, with log2-transformed values of fold changes (day 7 versus day 0) of arachidonic acid (b), linoleic acid (d) and 12-HETE (f) (n = 23). *P < 0.05, **P < 0.01, ***P < 0.001 (paired t-test).

Figure 4. Cross-reactive antibodies upregulate immune semaphorins via activating FcγR ligation.

a, GSEA analysis of the enriched signalling pathways in enhancing group (n = 6) compared with subjects from non-enhancing and control groups (n = 23) at day 3 post YF-vaccination. Normalized enrichment scores were computed by GSEA and these pathways have gene-set nominal P values < 0.05 (n = 21). b, GSEA analysis of enriched signalling pathways at day 3 post YF-vaccination that correlate with YF antibody titres for subjects with prior JE vaccination (black bars) (n = 21). White bars indicate corresponding enrichment scores for the control group (n = 7). Nominal gene-set P values were <0.05 for black bars and >0.05 for white bars. PLC, phospholipase C. SHC, Src homology 2 containing transforming protein. c, Immune semaphorin genes (in red) identified by GSEA to be enriched in subjects from the enhancing group. d, Pearson correlation (r) of YF antibody titres, as measured by PRNT, with log2-transformed values of fold changes (day 3 versus day 0) of SEMA4A, SEMA6A, SEMA7A and ITGA1 (n = 25). P values indicate significance of the slope. e, Localization and expression of SEMA4A (green) in THP-1 exposed to media, h4G2, YF17D or h4G2-opsonized YF17D (ADE) for 24 h. DAPI staining is blue. Values in white indicate the corrected total cell fluorescence of SEMA4A, normalized to DAPI. Scale bars, 20 µm. f, SEMA4A protein expression levels in primary monocytes exposed to media, YF17D or YF17D opsonized with pre-YF vaccination serum (1:10) from an enhancing group subject for 24 h. Relative expression is normalized to GAPDH. g, SEMA4A expression when YF17D opsonized with pre-vaccination serum from an enhancing group subject is added to primary monocytes blocked with either isotype antibodies or anti-FcγRI and FcγRII antibodies. h, SEMA4A protein expression in THP-1 when FcγRI, FcγRII or both receptors are ligated for 6 h. i–k, Quantitative PCR analysis of SEMA4A and SEMA7A in primary monocytes (i) and dendritic cells (j,k) when FcγRI, FcγRII or both receptors are ligated for 6 h (n = 4). Values were corrected for expression of the control gene (GAPDH). *P < 0.05, **P < 0.01 (unpaired t-test). Error bars indicate standard deviation of the data.

Figure 5. Cross-reactive antibodies increase DC activation, migration and semaphorin expression for improved antigen presentation during YF vaccination.

a, Diagrammatic representation showing how cross-reactive antibodies from mice immunized with Ixiaro were transferred to non-immunized mice to study their effects on YF vaccination. b,c, Total (b, CD11c+) and activated DCs (c, CD11c+CD80+CD86+) in DLNs after YF vaccination with pretreatment of different doses of cross-reactive JE IgG (n = 6). d–f, Percentage of SEMA4A+ DCs (d), representative histograms (from n = 6) showing SEMA4A staining on DCs (e) and mean fluorescence intensity (MFI) of SEMA4A+ staining on DCs (f) with pretreatment of different doses of cross-reactive JE IgG (n = 6). g, SEMA4A MFI of LN cells (n = 6). *P < 0.05, **P < 0.01 (unpaired t-test). Error bars indicate standard deviation of the data.