A live attenuated influenza A(H5N1) vaccine induces long-term immunity in the absence of a primary antibody response

Abstract

Background: Highly pathogenic avian influenza A(H5N1) causes severe infections in humans. We generated 2 influenza A(H5N1) live attenuated influenza vaccines for pandemic use (pLAIVs), but they failed to elicit a primary immune response. Our objective was to determine whether the vaccines primed or established long-lasting immunity that could be detected by administration of inactivated subvirion influenza A(H5N1) vaccine (ISIV).

Methods: The following groups were invited to participate in the study: persons who previously received influenza A(H5N1) pLAIV; persons who previously received an irrelevant influenza A(H7N3) pLAIV; and community members who were naive to influenza A(H5N1) and LAIV. LAIV-experienced subjects received a single 45-μg dose of influenza A(H5N1) ISIV. Influenza A(H5N1)- and LAIV-naive subjects received either 1 or 2 doses of ISIV.

Results: In subjects who had previously received antigenically matched influenza A(H5N1) pLAIV followed by 1 dose of ISIV compared with those who were naive to influenza A(H5N1) and LAIV and received 2 doses of ISIV, we observed an increased frequency of antibody response (82% vs 50%, by the hemagglutination inhibition assay) and a significantly higher antibody titer (112 vs 76; P = .04). The affinity of antibody and breadth of cross-clade neutralization was also enhanced in influenza A(H5N1) pLAIV-primed subjects.

Conclusions: ISIV administration unmasked long-lasting immunity in influenza A(H5N1) pLAIV recipients, with a rapid, high-titer, high-quality antibody response that was broadly cross-reactive across several influenza A(H5N1) clades.

Clinical trials registration: NCT01109329.

Trial registration: ClinicalTrials.gov NCT01443663 NCT01109329 NCT01443663.

Keywords: H5N1; avian influenza; live attenuated; vaccine.

Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Figures

Figure 1.

Study design. Abbreviations: H5, influenza A(H5N1); H5N1, influenza A(H5N1); H7N3, influenza A(H7N3); LAIV, live attenuated influenza vaccine.

Figure 2.

A, Kinetics of the hemagglutination inhibition (HAI) antibody response following vaccination with influenza A(H5N1) inactivated subvirion influenza vaccine (ISIV) in individuals primed with influenza A(H5N1) pandemic live attenuated influenza vaccine (pLAIV), in those primed with influenza A(H7N3) pLAIV, or in LAIV-naive individuals. HAI assay–based reciprocal geometric mean titers (GMTs) to wild-type A/VietNam/1203/2004(H5N1), using horse red blood cells. Dotted line indicates a HAI assay–based GMT of 1:40. Groups are represented by the following symbols: filled circle, group 1 (influenza A[H5N1] VN 04 pLAIV); square, group 2 (influenza A[H5N1] HK 03 pLAIV); triangle, group 3 (influenza A[H7N3] pLAIV); open circle, group 4 (single dose of influenza A[H5N1] ISIV); and X, group 5 (2 doses of influenza A[H5N1] ISIV). B, Influenza A(H5N1) pLAIV primes for a rapid antibody response to influenza A(H5N1) ISIV. Reverse cumulative frequency distribution of HAI titers for subjects in group 1 (primed with influenza A[H5N1] VN 2004 pLAIV) on day 7 after receipt of ISIV. C, Reverse cumulative frequency distribution of HAI titers from subjects in groups 1, 2, 4, and 5 on day 28 after receipt of the last dose of ISIV. Groups are represented by the following symbols: filled circle, group 1 (influenza A[H5N1] VN 04 pLAIV); square, group 2 (influenza A[H5N1] HK 03 pLAIV); open circle, group 4 (1 dose of ISIV); and X, group 5 (2 doses of influenza A[H5N1] ISIV).

Figure 3.

Influenza A(H5N1) pandemic live attenuated influenza vaccine (pLAIV) priming elicits neutralizing antibodies that show broad cross-reactivity against influenza A(H5N1) from antigenically distinct clades. The proportion of subjects from each group with a reciprocal microneutralization (MN) titer of ≥40 against any one of 5 influenza A(H5N1) viruses from 4 antigenically distinct clades of the Gs/gd lineage. MN assay was performed using reverse genetics–derived PR8 reassortant viruses bearing the hemagglutinin from the following viruses: A/VietNam/1203/2004 (clade 1), A/Indonesia/5/2005 (clade 2.1.3), A/Anhui/1/05 (clade 2.3.4), A/turkey/Turkey/1/05 (clade 2.2.1), and A/Egypt/3072/2101 (clade 2.2.1). Groups are represented by the following symbols: filled circle, group 1 (influenza A[H5N1] VN 04 pLAIV); square, group 2 (influenza A[H5N1] HK 03 pLAIV); triangle, group 3 (influenza A[H7N3] pLAIV); open circle, group 4 (single dose of influenza A[H5N1] inactivated subvirion influenza vaccine [ISIV]); and X, group 5 (2 doses of influenza A[H5N1] ISIV).

Figure 4.

A and B, Live attenuated influenza vaccine (LAIV) priming enhances antibody affinity (slower off-rates) to influenza A(H5N1) hemagglutinin polypeptide 1 (HA1; but not HA2). Surface plasmon resonance (SPR) analysis of postvaccination sera from all responders (R; MN ≥1:40) and nonresponders (NR; MN <1:40) from all 5 study groups was performed with properly folded functional oligomeric influenza A(H5N1) rHA1 (A) and rHA2 (B) domains from influenza A/VN/1203/2004. Antibody off-rate constants that describe the fraction of antibody-antigen complexes decaying per second were determined directly from the serum sample interaction with rHA1 (1–330) protein and rHA2 (331–480), using surface plasmon resonance in the dissociation phase. Serum antibody off-rate constants (each symbol represents 1 individual) were determined as described in Materials and Methods. Differences in mean off-rate constants were statistically significant between different vaccine groups, with a P value of < .05 (by the t test). The off-rate constants were determined from 2 independent SPR runs. Groups are represented by the colored circles as follows: group 1, red (influenza A[H5N1] VN 04 pLAIV); group 2, blue (influenza A[H5N1] HK 03 pLAIV); group 3, green (influenza A[H7N3] pLAIV); group 4, pink (single dose of influenza A[H5N1] inactivated subvirion influenza vaccine [ISIV]); and group 5, black (2 doses of influenza A[H5N1] ISIV). C–H, Serum antibody off-rate to VN04 rHA1 (but not rHA2) in influenza A(H5N1) pLAIV–primed individuals correlates with the in vitro neutralizing capacity against the homologous and heterologous influenza A(H5N1) from different clades. Neutralizing titer is expressed as the standardized end-point neutralizing antibody titer of sera obtained after vaccination. Serum antibody off-rate constants (each symbol represents 1 individual) of responders against rHA1 (C); but not rHA2 (D) of VN04 correlated with the in vitro homologous neutralizing influenza A(H5N1) capacity as determined by MN titers. E–H, Antibody affinity of responders against VN04 rHA1 correlated strongly with heterologous cross-neutralizing titers against A/Indonesia/5/2005 (clade 2.1.3; E), A/Anhui/1/05 (clade 2.3.4; F), A/turkey/Turkey/1/05 (clade 2.2.1; G), and A/Egypt/3072/2101 (clade 2.2.1; H). Responders in each group are represented by the colored circles as follows: group 1, red (influenza A[H5N1] VN 04 pLAIV); group 2, blue (influenza A[H5N1] HK 03 pLAIV); group 3, green (influenza A[H7N3] pLAIV); group 4, pink (single dose of influenza A[H5N1] ISIV); group 5, black (2 doses of influenza A[H5N1] ISIV).