Broad and Protective Influenza B Virus Neuraminidase Antibodies in Humans after Vaccination and their Clonal Persistence as Plasma Cells

Abstract

Although most seasonal inactivated influenza vaccines (IIV) contain neuraminidase (NA), the extent and mechanisms of action of protective human NA-specific humoral responses induced by vaccination are poorly resolved. Due to the propensity of influenza virus for antigenic drift and shift and its tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to waves of new strains of seasonal viruses and is at risk from viruses with pandemic potential for which limited or no immunity may exist. Here we demonstrate that the use of IIV results in increased levels of influenza B virus (IBV) NA-specific serum antibodies. Detailed analysis of the IBV NA B cell response indicates concurrent expansion of IBV NA-specific peripheral blood plasmablasts 7 days after IIV immunization which express monoclonal antibodies with broad and potent antiviral activity against both IBV Victoria and Yamagata lineages and prophylactic and therapeutic activity in mice. These IBV NA-specific B cell clonal lineages persisted in CD138⁺ long-lived bone marrow plasma cells. These results represent the first demonstration that IIV-induced NA human antibodies can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IBV NA-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development.IMPORTANCE Influenza virus infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets such as regions within the influenza neuraminidase protein. We have demonstrated that seasonal immunization stimulates neuraminidase-specific antibodies in humans that are broad and potent in their protection from influenza B virus when tested in mice. These antibodies further persist in the bone marrow, where they are expressed by long-lived antibody-producing cells, referred to here as plasma cells. The significance in our research is the demonstration that seasonal influenza immunization can induce a subset of neuraminidase-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.

Keywords: B cell responses; human; influenza vaccines; monoclonal antibodies; neuraminidase.

FIG 1

Increased levels of IBV NA-specific plasma antibodies and plasmablasts after IIV immunization. Peripheral blood was collected at baseline and at day 7 (D7) after immunization with IIV. (A) Plasma was serially diluted, IgG specific for NA, HA, and RSV F proteins was detected by ELISA, and area under the curve (AUC) data (n = 17 subjects) are presented. (B) IBV NA and IIV (Fluzone) IgG-specific ELISpot assays were performed on total PBMC (n = 11 subjects). Representative ELISpot assay from a single subject displayed (top). Symbols represent individual subjects. Significance determined by paired t test.

FIG 2

IBV NA-specific hMAbs recognize Victoria and Yamagata lineages. IBV NA-specific hMAbs were generated from plasmablasts following IIV immunization. (A) Increasing concentrations of hMAbs were tested for binding to the indicated NA proteins and IIV (Fluzone) by ELISA. RU, relative units. (B) hMAbs were tested for avidity for NA proteins at 1 μg/ml in increasing concentrations of urea. (C) MDCK cells were mock infected (Mock) or infected (MOI of 0.1) with the indicated viruses and 17 h later were fixed and stained with 1 μg/ml of the NA-specific hMAbs and NA protein expression evaluated by IFA. KPF1 is an H1-specific hMAb used as an internal control in this IFA. Bar, 100 µm. Designations used in the figure are as follows: Malaysia, B/Malaysia/2506/2004; Ohio, B/Ohio/01/2005; Brisbane, B/Brisbane/60/2008; Nevada, B/Nevada/03/2011; Yamagata, B/Yamagata/16/1988; Sydney, B/Sydney/507/2006; Wisconsin, B/Wisconsin/01/2010; Texas, B/Texas/06/2011; Lee, B/Lee/1940; pH1N1, A/California/4_NYICE_E3/2009.

FIG 3

Ability of IBV NA-specific hMAbs to inhibit viral infection and NA activity. (A) Fluorescence-based microneutralization assay. MDCK cells were infected with the indicated mCherry-expressing virus (B/Brisbane/60/2008 or reB/Yamagata/16/1988) and then incubated with 2-fold serial dilutions (starting concentration, 10 µg/ml) of the IBV NA-specific hMAbs. Virus neutralization was evaluated and quantified using a fluorescence microplate reader, and the percentage of infectivity was calculated using sigmoidal dose response curves. Mock-infected cells and viruses in the absence of hMAb were used as internal controls. Percentages of inhibition were normalized to infection in the absence of hMAb. Data show means of the results determined in triplicate. IC₅₀ data corresponding to the IBV NA hMAbs were determined using a fluorescence-based assay (FA) or a traditional viral neutralization assay (VN) and mCherry-expressing or WT viruses, respectively. (B) IBV NA-specific hMAbs inhibit NA enzymatic activity. B/Brisbane/60/2008 or B/Yamagata/16/1988 WT viruses were preincubated with 2-fold serial dilutions of the IBV NA-specific hMAbs, and NA activity was determined at 18 h postincubation on fetuin-coated plates. Data represent mean percentages of virus-alone NA activity from duplicate wells. The percentage of activity and the IC₅₀ were calculated using sigmoidal dose response curves. (C) IBV NA hMAbs recognize IBV oseltamivir resistance mutations. MDCK cells were infected (MOI of 0.1) with the indicated WT and NA (E117A and H273Y) viruses, and hMAb binding (1 μg/ml) was evaluated by IFA. Bar, 100 µm.

FIG 4

In vivo prophylactic and therapeutic activity of the IBV hMAbs. (A) Prophylactic activity. Female C57BL/6 mice (n = 3 per group/time point) were administered the indicated IBV NA hMAbs at 20 mg/kg i.p. or the irrelevant isotype control (IC) 1069 D6 hMAb at 20 mg/kg i.p. At 6 h after dosing, mice were inoculated i.n. with 1 × 10⁶ FFU B/Brisbane/60/2008. (B) Therapeutic activity. Female C57BL/6 mice (n = 3 per group) were inoculated i.n. with 1 × 10⁶ FFU B/Brisbane/60/2008 and 24 h later were administered the indicated IBV NA hMAb or the irrelevant isotype control (IC) 1069 D6 hMAb at 20 mg/kg i.p. Viral replication (A and B) was determined by measuring viral titers in the lungs of the infected mice at 2 and 4 days p.i. (dpi). Each symbol represents an individual mouse. The “&” symbol indicates that virus was detected in only one or two mice per group. “f” indicates that virus was not detected in any mouse in the group. *, P < 0.05 (using one-way ANOVA with multiple-outcome correction).

FIG 5

Clonal persistence of IBV NA hMAbs in bone marrow. Phylogenic analysis of 1092E10 (A) and 1092D4 and 1122C7 (B) IBV NA hMAb lineages was based on amino acid sequence. Lineage members are defined as exhibiting the same heavy chain V and J gene usage and HCDR3 lengths and ≥85% HCDR3 similarity. The germline sequence is represented by green diamonds, the MAb sequence is represented by blue squares, sequences obtained by MiSeq-based deep sequencing of bulk total peripheral blood B cells (D7) are represented by teal circles, bulk total bone marrow (BM) B cells (Y1) are represented by orange circles, CD138⁺ bone marrow plasma cells (Y1) are represented by red circles, sequences present in both bulk peripheral blood and bone marrow B cells are represented as purple circles, and inferred intermediate sequences are represented by gray circles. Sizes of symbols are proportional to the number of identical sequences obtained from an individual lineage member (n = 1 to 120), with the exception of the germline and hMAb sequences.