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. 2015 Nov 25:7:124.
doi: 10.1186/s13073-015-0239-y.

Longitudinal analysis of the peripheral B cell repertoire reveals unique effects of immunization with a new influenza virus strain

Bernardo Cortina-Ceballos  1 , Elizabeth Ernestina Godoy-Lozano  1 , Juan Téllez-Sosa  1 , Marbella Ovilla-Muñoz  1 , Hugo Sámano-Sánchez  1 , Andrés Aguilar-Salgado  1 , Rosa Elena Gómez-Barreto  1 , Humberto Valdovinos-Torres  1 , Irma López-Martínez  2 , Rodrigo Aparicio-Antonio  2 , Mario H Rodríguez  1 , Jesús Martínez-Barnetche  3
Affiliations

Longitudinal analysis of the peripheral B cell repertoire reveals unique effects of immunization with a new influenza virus strain

Bernardo Cortina-Ceballos et al. Genome Med. .

Abstract

Background: Despite the potential to produce antibodies that can neutralize different virus (heterotypic neutralization), there is no knowledge of why vaccination against influenza induces protection predominantly against the utilized viral strains (homotypic response). Identification of structural patterns of the B cell repertoire associated to heterotypic neutralization may contribute to identify relevant epitopes for a universal vaccine against influenza.

Methods: Blood samples were collected from volunteers immunized with 2008/2009 trivalent inactivated vaccine (TIV), pandemic H1N1 (pdmH1N1) monovalent inactivated vaccine (MIV) and the 2014/2015 TIV. Neutralization was assessed by hemagglutination and microneutralization test. IgG V(H) amplicons derived from peripheral blood RNA from pre-immune and 7 days post vaccination were subjected to 454-Roche sequencing. Full reconstruction of the sampled repertoires was done with ImmunediveRsity.

Results: The TIV induced a predominantly homotypic neutralizing serologic response, while the 09 MIV induced a heterotypic neutralizing seroconversion in 17% of the individuals. Both the 08/09 and the 14/15 TIV were associated with a reduction in clonotypic diversity, whereas 09 MIV was the opposite. Moreover, TIV and MIV induced distinctive patterns of IGHV segment use that are consistent with B cell selection by conserved antigenic determinants shared by the pre-pandemic and the pandemic strains. However, low somatic hypermutation rates in IgG after 09 MIV immunization, but not after 08/09 and 14/15 TIV immunization were observed. Furthermore, no evidence of the original antigenic sin was found in the same individuals after vaccination with the three vaccines.

Conclusions: Immunization with a new influenza virus strain (2009 pdmH1N1) induced unique effects in the peripheral B cell repertoire clonal structure, a stereotyped response involving distinctive IGHV segment use and low somatic hypermutation levels. These parameters were contrastingly different to those observed in response to pre-pandemic and post-pandemic vaccination, and may be the result of clonal selection of common antigenic determinants, as well as germinal center-independent responses that wane as the pandemic strain becomes seasonal. Our findings may contribute in the understanding of the structural and cellular basis required to develop a universal influenza vaccine.

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Figures

Fig. 1
Fig. 1
Experimental design timeline of immunizations and sequencing experiments. Six individuals naïve for 2009 pdmH1N1 were vaccinated with the 08/09 TIV and their peripheral blood IgG B cell repertoire was sequenced before vaccination (day 0) and 7 days after vaccination. Two years later, the IgG B cell repertoire of five to six individuals previously vaccinated plus eight additional participants vaccinated with 09 MIV (of 18 participants) were sequenced (day 0 and day 7). Finally, four of the same five participants vaccinated with 08/09 TIV and 09 MIV were vaccinated with 12/13 TIV. The same four participants were then vaccinated with 14/15 TIV and subjected to Rep-Seq (*), as in the previous trials. The overall experiment length from the first to the last vaccination trial was 5 years, 5 months
Fig. 2
Fig. 2
Vaccination with TIV induced homotypic seroconversion (a), while 09 MIV induced heterotypic seroconversion against pre-pandemic H1N1 (b). Homotypic and heterotypic seroconversion rates for vaccination with TIV and 09 MIV. TIV showed low seroconversion rates for vaccine strains due to pre-exposure (high pre-vaccination titers) (n = 6). The 09 MIV vaccine had a higher homotypic seroconversion (83 % and 75 %) and heterotypic seroconversion against 2008 H1N1 in 17 % of the immunized participants (n = 23) (arrow)
Fig. 3
Fig. 3
Vaccination with 08/09 TIV induced a reduction of the clonal and lineage diversity, while 09 MIV induced an increase in the diversity. A rarefaction analysis (a-d) was performed with progressive subsamples of 1,000 reads (axis x), plotted against the average standard error of clonal groups (a-c) and lineages (d-f) for 08/09 TIV (a and d), 09 MIV (b, e), and 14/15 TIV (c, f) vaccination trials. The empty symbols represent the values for day 0 (pre-immune), while the black symbols stand for those for day 7 after vaccination (two-way ANOVA. *P <0.05; **P <0.01; ***P <0.001). Fluctuations above 7,000 reads in pre-immmune are due to insufficient sequencing depth in two individuals (see also Additional file 3)
Fig. 4
Fig. 4
Effect of immunization with TIV and 09 MIV on the repertoire of peripheral B-lymphocytes. The change in the relative frequency of IGHV segment use (Δ day 7 – day 0) for TIV (red dots, n = 6) and with 09 MIV (blue dots, n = 12), for 47 IGHV segments (a), and, in detail, for IGHV1-69 (b), for IGHV3-7 (c), and for IGHV4-39 (d). The dotted lines represent ± 2 standard deviations (±6.7) from the mean (0) (Mann–Whitney test. ***P <0.001, *P <0.05)
Fig. 5
Fig. 5
Longitudinal changes in IGHV segment usage upon influenza vaccination. The change in the relative VH clonotype frequency of IGHV segment use (Δ day 7 – day 0) in the same four individuals vaccinated with 08/09 TIV, 09 MIV, and 14/15 TIV for (a) IGHV1-69, (b) IGHV4-39, and (c) IGHV3-7
Fig. 6
Fig. 6
Somatic hypermutation analysis in response to 08/09 TIV, 09 MIV, and 14/15 TIV. SHM rates (% of mutations in VH region) are shown according to vaccination trial. In the first approach, 670 randomly sampled lineages per individual were selected and plotted (a-c). (a) 08/09 TIV; (b) 09 MIV; and (c) 14/15 TIV. A second sampling approach was used based on selecting the single largest lineage of 250 VH clonotypes (d-f) (Kruskal–Wallis test. Dunn’s correction for multiple testing. **P <0.01, ***P <0.001)
Fig. 7
Fig. 7
Principal component analysis of influenza vaccinees according to each trial. Principal Component biplot showing clustering of vaccinees according to both TIVs or 09 MIV immunization. PCA was constructed using results of clonotype and lineage rarefaction and entropy analysis, IGHV1-69, IGHV3-7, and IGHV4-39 use and SHM rates per individual and trial. 08/09 TIV (green), 09 MIV (red), and 14/15 TIV (blue)
Fig. 8
Fig. 8
Effect of immunization with 09 MIV on the repertoire of peripheral B-lymphocytes with homo- and heterosubtypic seroconversion. Changes in the relative frequency of use of IGHV segments (Δ day 7 – day 0) for individuals with homotypic seroconversion (n = 7) (a, c), and with heterosubtypic seroconversion (n = 4) (b, d) for 47 IGHV segments. IGHV usage is expressed as relative transcription (unclustered reads) (a, b), and as the proportion of clonotypes using a particular IGHV segment (c, d). The dotted line indicates three standard deviations, which corresponds to 18.5 for relative transcription and 10.3 for clonotypic frequency above the means, 0.0014 and 0.003, respectively. Arrows indicate clonotypes selected for experimental validation. IGHV segments in bold letters indicate relevant expansions
Fig. 9
Fig. 9
B cell clonal expansions associated with heterotypic seroconversion are influenza-specific. (a) Structural features of recombinant monoclonal antibodies in terms of VDJ segment use and successful VL pair. (b) Enzyme-linked immunosorbent assay of three recombinant monoclonal antibodies derived from in silico repertoire mining of clonal expansions in segments IGHV1-69 and IGHV3-23 against TIV antigens, and (c) against MIV antigen.
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