Key Points

  • Vaccination against influenza, with and without the adjuvant MF59, decreases the risk of inhibitor development in HA mice.

  • Decreased FVIII immunogenicity may be attributed to antigenic competition via T-cell chemotaxis toward the site of vaccination.

Abstract

Inflammatory signals such as pathogen- and danger-associated molecular patterns have been hypothesized as risk factors for the initiation of the anti–factor VIII (FVIII) immune response seen in 25% to 30% of patients with severe hemophilia A (HA). In these young patients, vaccines may be coincidentally administered in close proximity with initial exposure to FVIII, thereby providing a source of such stimuli. Here, we investigated the effects of 3 vaccines commonly used in pediatric patients on FVIII immunogenicity in a humanized HA murine model with variable tolerance to recombinant human FVIII (rhFVIII). Mice vaccinated intramuscularly against the influenza vaccine prior to multiple infusions of rhFVIII exhibited a decreased incidence of rhFVIII-specific neutralizing and nonneutralizing antibodies. Similar findings were observed with the addition of an adjuvant. Upon exposure to media from influenza- or FVIII-stimulated lymph node or splenic lymphocytes, naïve CD4+ lymphocytes preferentially migrated toward media from influenza-stimulated cells, indicating that antigen competition, by means of lymphocyte recruitment to the immunization site, is a potential mechanism for the observed decrease in FVIII immunogenicity. We also observed no differences in incidence or titer of rhFVIII-specific antibodies and inhibitors in mice exposed to the live-attenuated measles-mumps-rubella vaccine regardless of route of administration. Together, our results suggest that concomitant FVIII exposure and vaccination against influenza does not increase the risk of inhibitor formation and may in fact decrease anti-FVIII immune responses.

Introduction

The enigmatic development of factor VIII (FVIII)–neutralizing antibodies, known as inhibitors, remains the most serious treatment-related complication of hemophilia A (HA). Immune tolerance induction is currently the only proven strategy for eradicating antibodies toward FVIII but is costly and varies in efficacy.1,2  Prevention of inhibitor development would therefore be an ideal solution. However, why 25% to 30% of severely affected patients develop inhibitors is poorly understood.3,4  Clinical and epidemiological studies attribute this to a complex combination of genetic and treatment-related factors.

FVIII immunity likely begins with receptor-mediated endocytosis and degradation of FVIII by professional antigen-presenting cells (APCs) and the subsequent presentation of FVIII peptides on major histocompatibility complex (MHC) class II molecules.5,6  In the immature steady state, APCs are unable to prime FVIII-specific effector T-cell responses because of insufficient expression of costimulatory molecules such as CD80 and CD86. However, in a proinflammatory milieu, APCs mature and upregulate expression of these costimulators that enable priming of FVIII-specific effector CD4+ T cells and trigger a cascade of events ultimately leading to the differentiation of FVIII-specific B cells into antibody-producing plasma cells. Pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) can act as maturation and immunologic “danger signals” that enhance the quality and magnitude of the immune response against FVIII by activating APCs through pattern recognition receptors.7  A previous report has shown that rhFVIII, alone and in complex with von Willebrand factor (VWF), does not convey danger signals to dendritic cells (DCs).8  It is therefore reasonable to believe that the presence of “danger signals” influences the risk of inhibitor development in pediatric patients. We have recently shown in 2 mouse models of HA that surgery-associated DAMPs increase inflammatory cytokines interleukin-1 and interleukin-6, and also upregulate CD80 on APCs, but do not influence the magnitude or incidence of the anti-FVIII immune response.9 

Vaccination for infants occurs at around the time of initial FVIII replacement therapy and, as a result, may act as a danger signal.10  This concern is founded on the exposure to viral antigens and PAMPs, and in some cases adjuvants, that may enhance antibody responses. Indeed, studies in mice have implicated an amplification of the anti-FVIII immune response by lipopolysaccharide, a Toll-like receptor 2/4 (TLR2/4) agonist.11  No studies have reported a role of vaccination in inhibitor development, save for a single case report in which subcutaneous immunotherapy treating a pollen allergy reverted FVIII tolerance in a severe HA patient who had already undergone successful immune tolerance induction.12 

From a contrasting perspective, there is some concern that simultaneous exposure to multiple vaccines may reduce their overall effectiveness through interference or immune overload, rendering patients susceptible to other infections.10  In the context of FVIII, this antigenic competition may divert the immune system’s resources away from FVIII and result in a more anergic or tolerogenic response. Indeed, animal models have shown decreased antibody titers against FVIII when concurrently treated with a FVIII/VWF complex vs FVIII alone.13  However, there is conflicting evidence in the literature regarding concurrent vaccination. Recent studies have suggested that multiple concurrent vaccinations do not influence the efficacy of protection against each antigen, nor the susceptibility to other infections.14-16  Ultimately, the role of vaccination on FVIII immunogenicity or tolerance remains unclear and is the subject of this report.

Three commonly used vaccines were selected based, in part, on their recommended age of inoculation: the live-attenuated measles-mumps-rubella (MMR) vaccine and the seasonal inactivated subunit influenza vaccine with and without the adjuvant MF59.10  Here, we assessed the effects of vaccination on recombinant human FVIII (rhFVIII) immunogenicity in a humanized HA mouse model with a variable tolerance to intravenously administered rhFVIII.17 

Materials and methods

Murine models of HA

The 8- to 12-week-old male humanized F8 E17KO HLA-DRB1*1501 HA mice on a mixed S129/C57Bl/6 background were gifts from the Baxalta Corporation.17  These mice possess a complete knockout of the murine MHC class II locus and instead express a functional chimeric human-mouse HLA-DRB1*1501 allele. The 8- to 12-week-old male and female C57Bl/6 F8 E16KO HA mice were used for in vitro chemotaxis experiments.18  All mouse experiments were reviewed and approved by the Queen’s University Animal Care Committee.

Vaccines

Mice were vaccinated either subcutaneously or intravenously with the MMR vaccine (Priorix; GlaxoSmithKline), and intramuscular or intravenously with the influenza vaccine in the presence and absence of the adjuvant MF59 (Fluad [2013-2014 season] and Agriflu [2012-2013 season], respectively; Novartis).

rhFVIII treatment regimen

For MMR experiments, naïve HA mice received 4 weekly intravenous infusions of 2 IU rhFVIII (0.2 μg or 80 IU/kg, Advate; Baxter BioScience), followed by 4 twice-weekly infusions of 6 IU rhFVIII (0.6 μg or 240 IU/kg). For influenza experiments, mice received 7 twice-weekly infusions of 6 IU rhFVIII. Experiments described represent aggregated cohorts of at least 3 independent experiments.

Blood sampling

Mice were anesthetized and blood was collected using capillary tubes via the retro-orbital plexus. Sodium citrate (3.2%) was added to the samples at one-tenth total volume. Plasma was isolated by centrifugation. Terminal samples were collected by cardiac puncture.

Anti-FVIII antibodies and FVIII inhibitor assays

Total FVIII-specific immunoglobulin G (IgG) titers were quantified by enzyme-linked immunosorbent assay (ELISA) as previously described.19  Total FVIII-specific IgG was detected using an horseradish peroxidase–conjugated goat anti-mouse IgG (Southern Biotech); positive results were defined as >0.1 optical density. FVIII-specific IgG isotypes were quantitated using the SBA Clonotyping System horseradish peroxidase (Southern Biotech). Wells coated with IgG capture antibodies were used with mouse reference serum (Bethyl Laboratories) to generate a standard curve.

FVIII inhibitors were assessed by a 1-stage FVIII clotting assay using an automated coagulometer STACompact (Stago). Samples were incubated at 56°C for 30 min to eliminate background FVIII activity in our experimental system and prepared as described previously.20  Dilutions were performed using FVIII-deficient human plasma (Affinity Biologicals). Positive inhibitor samples were defined as >0.5 BU.21 

Cell chemotaxis assays

Spleens and inguinal lymph nodes (LNs) were isolated from F8 E16KO HA mice. Splenocytes and LN lymphocytes were stimulated at 1 × 107 cells per mL for 18 hours with FVIII (1 μg/mL) or influenza vaccine (1 μg/mL), respectively. Total CD4+ T lymphocytes were isolated using the EasySep Mouse CD4+ T-cell Isolation Kit (StemCell Technologies). Purity ranged from 88% to 95%. Naïve splenocytes or CD4+ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (CellTrace CFSE Cell Proliferation Kit; Life Technologies) and suspended in a 1.5 μg/mL rat tail collagen matrix. Three-dimensional migration was imaged by confocal microscopy using μ-Slide Chemotaxis 3D (ibidi) slides. Positive controls used were 10 μg/mL CCL2 (R&D Systems) and 20 ng/mL CCL5 (eBioscience). Cell migration was automatically tracked and plotted using the MosaicSuite and Chemotaxis Tool Fiji plugins, respectively.

Results

MMR vaccination of HA mice is unsuccessful and does not influence anti-FVIII antibody generation

The live-attenuated MMR vaccine is typically administered as a series of 2 subcutaneous doses.10  In this study, we attempted to mimic this regimen with an additional goal of assessing the ability of the MMR vaccine to break FVIII-specific tolerance, as well as expose humanized E17KO mice to low- and high-intensity FVIII treatments (Figure 1A). The vaccine was administered at 10 times the weight-scaled standard human dose; preliminary studies using a standard dose were inconclusive (data not shown). Plasma levels of anti-FVIII IgG and inhibitory Bethesda units were measured at the indicated time points. Our results show that subcutaneous vaccination of mice with the MMR vaccine 24 hours before the first FVIII exposure does not influence the proportion of mice that develop FVIII-specific IgG (Figure 1B). Similarly, a second reexposure to the vaccine followed by a more intensive FVIII treatment regimen exhibited no effect on FVIII IgG or inhibitors (Figure 1B-C). We also quantified the titers of FVIII-specific IgG and inhibitors in FVIII responders, which showed negligible differences between vaccinated and nonvaccinated mice (Figure 1D-E).

Figure 1

The influence of subcutaneous and intravenous exposure to the MMR vaccine on FVIII immunogenicity. (A) HA mice were challenged with the MMR vaccine, at 10 times the standard scaled human dose, 24 hours before the first of 4 weekly infusions of 2 IU rhFVIII (∼80 IU/kg). Week 5 plasma was obtained at week 5 by retro-orbital sampling. Mice were subsequently rechallenged with the same dose of MMR vaccine followed by 4 biweekly infusions of 6 IU rhFVIII (∼240 IU/kg). Plasma samples at week 9 were obtained via cardiac puncture. (B) Incidence of FVIII-specific IgG in subcutaneous (SQ) and intravenous (IV) treatment arms at weeks 5 and 9, assessed by indirect ELISA. Total cohort sizes are indicated above the bars. (C) Incidence of FVIII inhibitors. (D) Comparison of FVIII-specific IgG titers among FVIII responders. (E) Comparison of inhibitory activity among FVIII responders. The horizontal lines and error bars represent the mean and standard error of the mean (SEM).

Figure 1

The influence of subcutaneous and intravenous exposure to the MMR vaccine on FVIII immunogenicity. (A) HA mice were challenged with the MMR vaccine, at 10 times the standard scaled human dose, 24 hours before the first of 4 weekly infusions of 2 IU rhFVIII (∼80 IU/kg). Week 5 plasma was obtained at week 5 by retro-orbital sampling. Mice were subsequently rechallenged with the same dose of MMR vaccine followed by 4 biweekly infusions of 6 IU rhFVIII (∼240 IU/kg). Plasma samples at week 9 were obtained via cardiac puncture. (B) Incidence of FVIII-specific IgG in subcutaneous (SQ) and intravenous (IV) treatment arms at weeks 5 and 9, assessed by indirect ELISA. Total cohort sizes are indicated above the bars. (C) Incidence of FVIII inhibitors. (D) Comparison of FVIII-specific IgG titers among FVIII responders. (E) Comparison of inhibitory activity among FVIII responders. The horizontal lines and error bars represent the mean and standard error of the mean (SEM).

Given this lack of effect on FVIII immunogenicity, we then asked whether the MMR vaccine inherently conveys danger signals to the immune system. To address this, we immunized mice intravenously with MMR to deliver both the vaccine and FVIII for immune processing in the spleen.22  Surprisingly, we observed no influence of the initial MMR vaccination, nor the reexposure, on the incidence of FVIII-specific IgG and inhibitors (Figure 1B-C). Among FVIII responders, the difference in titers of FVIII-specific IgG and inhibitors was again insignificant between vehicle and MMR-exposed groups. (Figure 1D-E).

To address the stark contrast between these data and our hypothesis, we considered the efficacy of the MMR vaccine in mice. Measles-specific IgG was undetectable in mice vaccinated either subcutaneously or intravenously, indicating unsuccessful immunization (data not shown), which may be required to observe an influence on FVIII immunogenicity. Furthermore, analysis of DC costimulatory molecule expression demonstrated that there are no accessible danger signals in the MMR vaccine that may modulate FVIII antigen presentation in mice (supplemental Figure 1, available on the Blood Web site).

Immunization against influenza decreases the incidence of anti-FVIII antibodies and inhibitors

We next investigated the potential effects of the influenza vaccine, a surface antigen vaccine composed of seasonal-specific hemagglutinin and neuraminidase. To assess its role on FVIII immunogenicity, humanized E17KO HA mice were immunized intramuscularly with a standard human dose (4.8 μg/kg), either 24 hours before, at the same time, or 24 hours after the first of 7 twice-weekly FVIII infusions (Figure 2A). Four weeks after the initial FVIII infusion, we observed significant decreases in the incidence of FVIII-specific antibodies regardless of when the mice were vaccinated (30%, 40%, 14% vs control: 80%; Figure 2B) We also observed similar decreases in the incidence of FVIII inhibitors (30%, 43%, 11% vs 80%; Figure 2C). Further serological analyses revealed that all mice exhibited high titers of anti-influenza IgG, suggesting successful immunization (Figure 2D). Titers of anti-FVIII antibodies and FVIII inhibitors were also measured in FVIII responders, and there were no changes in either outcome in response to influenza vaccination (Figure 2D-E). Furthermore, there was no correlation between the titer of influenza-specific antibodies and FVIII-specific antibodies or inhibitors (data not shown), indicating that influenza does not augment the anti-FVIII immune response. To examine the potential influence of influenza vaccination on the FVIII immune response profile, we analyzed anti-FVIII IgG subclasses IgG1, 2a, and 2b. We observed an increase in FVIII-specific IgG2a antibodies attributed to concurrent influenza and FVIII exposure (Figure 2F).

Figure 2

The effects of intramuscular immunization against influenza on FVIII immunogenicity. (A) Mice were immunized intramuscularly with a scaled standard human dose (4.8 μg/kg) within 24 hours of the first of 7 rhFVIII (6 IU, ∼240 IU/kg) infusions. Blood was collected by cardiac puncture 4 weeks later and plasma was isolated by centrifugation. (B) Incidence of FVIII-specific IgG assessed by indirect ELISA. Total cohort sizes are indicated above each bar. (C) Incidence of FVIII inhibitors. (D) Titers of influenza- and FVIII-specific IgG among FVIII responders. (E) Comparison of inhibitory activity among FVIII responders. (F) FVIII-specific IgG subclasses IgG1, IgG2a, and IgG2b among FVIII responders. The horizontal lines and error bars represent the mean and SEM. Fisher’s exact and Mann-Whitney U tests were used where appropriate. ND, not detectable; n.s., not significant.

Figure 2

The effects of intramuscular immunization against influenza on FVIII immunogenicity. (A) Mice were immunized intramuscularly with a scaled standard human dose (4.8 μg/kg) within 24 hours of the first of 7 rhFVIII (6 IU, ∼240 IU/kg) infusions. Blood was collected by cardiac puncture 4 weeks later and plasma was isolated by centrifugation. (B) Incidence of FVIII-specific IgG assessed by indirect ELISA. Total cohort sizes are indicated above each bar. (C) Incidence of FVIII inhibitors. (D) Titers of influenza- and FVIII-specific IgG among FVIII responders. (E) Comparison of inhibitory activity among FVIII responders. (F) FVIII-specific IgG subclasses IgG1, IgG2a, and IgG2b among FVIII responders. The horizontal lines and error bars represent the mean and SEM. Fisher’s exact and Mann-Whitney U tests were used where appropriate. ND, not detectable; n.s., not significant.

We then assessed the robustness of the apparent influenza-mediated reduction in anti-FVIII antibody responses. Following an initial intramuscular influenza immunization concurrent with the first of 7 FVIII infusions, mice were subjected to a 5-week period without FVIII exposure, after which they received 3 consecutive intravenous infusions of FVIII. Two weeks after this intensive reexposure, we observed an increase of 40% to 50% in the incidence of FVIII-specific IgG in the vaccination treatment arm (supplemental Figure 2). Moreover, this decrease in the FVIII immune response appears to be specific to the influenza vaccine, as mice immunized intramuscularly against a more inert antigen, ovalbumin, did not exhibit the same pattern of reduced FVIII immunoreactivity (supplemental Figure 3).

Intramuscular vaccination does not ensure its colocalization with FVIII. Therefore, we next evaluated the presence of a danger signal by inoculating mice intravenously with vaccine and FVIII at the same time points as described previously, with the assumption that the biodistribution of the 2 products is more likely to be similar. In this scenario, any potential danger signal would stimulate the same cells interacting with FVIII. When mice received FVIII at the same time as the intravenous vaccine, there was a 30% and 40% increase in the incidence of FVIII-specific IgG and inhibitors, respectively (Figure 3A-B). However, when vaccinated either 24 hours before or after the first FVIII infusion, the incidence of inhibitory antibodies was lower, although there was an increase in total anti-FVIII antibodies in mice vaccinated 24 hours before receiving FVIII. Intravenous immunization against influenza elicited similarly robust anti-influenza immune responses compared with intramuscular vaccination (Figure 3C). No differences in FVIII antibody or inhibitor titers were associated with influenza immunization compared with controls (Figure 3C-D). These data, albeit not statistically significant, suggest a trend of increased FVIII immunogenicity associated with danger signals in the influenza vaccine delivered to the same anatomic location as FVIII.

Figure 3

The effects of intravenous immunization against influenza on FVIII immunogenicity. Mice were immunized intravenously with a scaled standard human dose (4.8 μg/kg) of influenza vaccine within 24 hours of the first of 7 rhFVIII (6 IU, ∼200 IU/kg) infusions. Blood was collected by cardiac puncture 4 weeks later, and plasma was isolated by centrifugation. Plasma samples were assessed for the presence of FVIII-specific IgG (A) and inhibitors (B). Total cohort sizes are indicated above each bar. (C) Comparison of titers of influenza- and FVIII-specific IgG among FVIII responders. (D) Comparison of inhibitory activity among FVIII responders. The horizontal lines and error bars represent the mean and SEM.

Figure 3

The effects of intravenous immunization against influenza on FVIII immunogenicity. Mice were immunized intravenously with a scaled standard human dose (4.8 μg/kg) of influenza vaccine within 24 hours of the first of 7 rhFVIII (6 IU, ∼200 IU/kg) infusions. Blood was collected by cardiac puncture 4 weeks later, and plasma was isolated by centrifugation. Plasma samples were assessed for the presence of FVIII-specific IgG (A) and inhibitors (B). Total cohort sizes are indicated above each bar. (C) Comparison of titers of influenza- and FVIII-specific IgG among FVIII responders. (D) Comparison of inhibitory activity among FVIII responders. The horizontal lines and error bars represent the mean and SEM.

Immunization against influenza with the adjuvant MF59 decreases the incidence of anti-FVIII antibodies and inhibitors

We next investigated FVIII immunogenicity in response to the influenza vaccine with the additional of an oil-in-water squalene adjuvant, MF59, which may confer immune-stimulatory properties against FVIII as well as influenza antigens. Intramuscular vaccination of HA mice together with an intravenous infusion of FVIII resulted in a decreased incidence of FVIII-specific IgG (45%, 25%, 42% vs 80%; Figure 4A). In addition, the frequency of FVIII inhibitor positive mice significantly decreased across all immunization time points (36%, 31%, 33% vs 80%; Figure 4B). All mice developed high titer anti-influenza antibodies, but there was no observed influence of the vaccine and adjuvant on the titer of total FVIII-specific IgG or inhibitors (Figure 4C-D). Upon more detailed analysis of IgG subclasses, we observed an increase of IgG1 antibodies at all immunization time points (Figure 4E).

Figure 4

The effects of intramuscular immunization against influenza, with the adjuvant MF59, on FVIII immunogenicity. Mice were immunized intramuscularly with a scaled standard human dose (4.8 μg/kg) within 24 hours of the first of 7 rhFVIII (6 IU, ∼240 IU/kg) infusions. Blood was collected by cardiac puncture 4 weeks later and plasma was isolated by centrifugation. (A) Incidence of FVIII-specific IgG. Total cohort sizes are indicated above each bar. (B) Incidence of inhibitors. (C) Comparison of influenza- and FVIII-specific IgG titers among FVIII responders. (D) Comparison of inhibitory activity among FVIII responders. (E) Anti-FVIII IgG subclasses IgG1 and IgG2a among FVIII responders. The horizontal lines and error bars represent the mean and SEM.

Figure 4

The effects of intramuscular immunization against influenza, with the adjuvant MF59, on FVIII immunogenicity. Mice were immunized intramuscularly with a scaled standard human dose (4.8 μg/kg) within 24 hours of the first of 7 rhFVIII (6 IU, ∼240 IU/kg) infusions. Blood was collected by cardiac puncture 4 weeks later and plasma was isolated by centrifugation. (A) Incidence of FVIII-specific IgG. Total cohort sizes are indicated above each bar. (B) Incidence of inhibitors. (C) Comparison of influenza- and FVIII-specific IgG titers among FVIII responders. (D) Comparison of inhibitory activity among FVIII responders. (E) Anti-FVIII IgG subclasses IgG1 and IgG2a among FVIII responders. The horizontal lines and error bars represent the mean and SEM.

Interestingly, mice concurrently exposed to FVIII and the adjuvanted influenza vaccine through intravenous injection did not exhibit an increased incidence of FVIII-specific antibodies or inhibitors (Figure 5A-B). Moreover, no significant changes were detected in the titers of FVIII antibodies or inhibitors (Figure 5C-D). Intravenous immunization of mice to influenza with MF59 resulted in anti-influenza responses of a similar magnitude to the intramuscular route.

Figure 5

The effects of intravenous immunization against influenza, with the adjuvant MF59, on FVIII immunogenicity. Mice were immunized intravenously with a scaled standard human dose (4.8 μg/kg) within 24 hours of the first of 7 rhFVIII (6 IU, ∼240 IU/kg) infusions. Blood was collected by cardiac puncture 4 weeks later and plasma was isolated by centrifugation. Plasma samples were assessed for the presence of FVIII-specific IgG (A) and inhibitors (B). Total cohort sizes are indicated above each bar. (C) Titers of influenza- and FVIII-specific IgG among FVIII responders. (D) Comparison of inhibitory activity among FVIII responders. The horizontal lines and error bars represent the mean and SEM.

Figure 5

The effects of intravenous immunization against influenza, with the adjuvant MF59, on FVIII immunogenicity. Mice were immunized intravenously with a scaled standard human dose (4.8 μg/kg) within 24 hours of the first of 7 rhFVIII (6 IU, ∼240 IU/kg) infusions. Blood was collected by cardiac puncture 4 weeks later and plasma was isolated by centrifugation. Plasma samples were assessed for the presence of FVIII-specific IgG (A) and inhibitors (B). Total cohort sizes are indicated above each bar. (C) Titers of influenza- and FVIII-specific IgG among FVIII responders. (D) Comparison of inhibitory activity among FVIII responders. The horizontal lines and error bars represent the mean and SEM.

T lymphocytes migrate preferentially in the direction of media from LN lymphocytes stimulated with influenza

FVIII immunity is thought to originate in the spleen, and the intramuscular influenza immunization likely initiates an immune response through the draining inguinal LNs.22  We hypothesized that the decrease in FVIII antibodies and inhibitors observed through intramuscular influenza immunization is attributed to antigenic competition by means of lymphocyte trafficking to local LNs at the site of vaccination. E16KO HA mice were used to account for the variability of E17KO mice in inhibitor development, thus providing a more controlled model. To directly assess the chemotactic-inducing ability of FVIII and the influenza vaccines, we used a μ-Slide Chemotaxis 3D in which a central imaging lane, flanked by 2 separate reservoirs, was seeded with naïve splenocytes, labeled with CFSE in a 3-dimensional rat-tail collagen matrix. We then separately stimulated splenocytes with FVIII, and lymphocytes from the inguinal LNs with the influenza vaccine, with and without MF59. After 18 hours, the cell supernatant was transferred into the flanking reservoirs, establishing 2 converging chemokine gradients within the collagen matrix. The central lane was imaged overnight by confocal microscopy. Cell migration and directionality were analyzed on the basis of angular nonuniformity among end points using Rayleigh’s test. We addressed the susceptibility of this test to random short migration paths by analyzing the total distance traveled in the direction of each gradient using the Student t test. Although not statistically significant, we observed that unsorted naïve splenocytes exhibited greater chemotaxis toward media from cells stimulated with the influenza (Figure 6A) and particularly that with influenza + MF59 at 12 hours (Figure 6B). For comparison, media from unstimulated splenocytes and LN-lymphocytes resulted in the random movement of splenocytes (Figure 6C), and addition of monocyte and T-cell chemokines CCL2 and CCL5, respectively, induced direction-specific chemotaxis (Figure 6D). Similarly, there was a trend suggesting increased displacement of cells toward influenza-induced chemokines with both forms of the vaccine (Figure 6E-F).

Figure 6

Comparison of naïve splenocyte chemotaxis in response to media from influenza vaccine-stimulated LN lymphocytes and FVIII-stimulated splenocytes. Chemokine gradients were generated by stimulating splenocytes and LN-lymphocytes with FVIII (1 μg/mL) or influenza with and without MF59 (1 μg/mL) for 18 hours. Chemotaxis competition of converging gradients were imaged overnight between FVIII and influenza (A) and FVIII and influenza with MF59 (B). (C) Media from unstimulated splenocytes and LN-lymphocytes were assessed as a negative control. (D) CCL2 (10 μg/mL) and CCL5 (20 μg/mL) gradients were assessed as a positive control. The vertical components traveled by T cells were analyzed for FVIII and influenza competition (E) and FVIII and influenza with MF59 (F) in a representative assay. Statistical analysis was conducted using Rayleigh’s test and Mann-Whitney U test for trajectory plots and distance traveled, respectively. Data representative of at least 3 independent experiments. Horizontal line and error bars represent mean and SEM, respectively.

Figure 6

Comparison of naïve splenocyte chemotaxis in response to media from influenza vaccine-stimulated LN lymphocytes and FVIII-stimulated splenocytes. Chemokine gradients were generated by stimulating splenocytes and LN-lymphocytes with FVIII (1 μg/mL) or influenza with and without MF59 (1 μg/mL) for 18 hours. Chemotaxis competition of converging gradients were imaged overnight between FVIII and influenza (A) and FVIII and influenza with MF59 (B). (C) Media from unstimulated splenocytes and LN-lymphocytes were assessed as a negative control. (D) CCL2 (10 μg/mL) and CCL5 (20 μg/mL) gradients were assessed as a positive control. The vertical components traveled by T cells were analyzed for FVIII and influenza competition (E) and FVIII and influenza with MF59 (F) in a representative assay. Statistical analysis was conducted using Rayleigh’s test and Mann-Whitney U test for trajectory plots and distance traveled, respectively. Data representative of at least 3 independent experiments. Horizontal line and error bars represent mean and SEM, respectively.

Despite the lack of statistical power, which may be attributed to the heterogeneous nature of the cells, these data suggest that there is a cellular subset exhibiting a higher responsiveness to influenza-induced chemokines. We hypothesized that CD4+ T cells would be the migratory subset considering the rationale that resident APCs would contribute to the majority of the chemokines produced. Indeed, using the same experimental system, CD4+ T cells preferentially migrated toward media from LN cells stimulated with influenza (Figure 7A) and influenza + MF59 (Figure 7B) over a period of 12 hours. For comparison, media from unstimulated splenocytes and LN-lymphocytes resulted in the random movement of T cells (Figure 7C), and addition of the CCL5 induced direction-specific chemotaxis (Figure 7D). Furthermore, T cells were found to migrate a significantly greater distance toward the influenza-induced chemokines vs that of FVIII at 1 and 12 hours (P = .0008 and .01, respectively; Figure 7E). We observed a similar increase in migratory distance of T cells toward influenza + MF59-induced chemokines (P = .003 and .006, respectively; Figure 7F). These findings suggest that migration of CD4+ T cells to local LNs at the site of vaccination may explain antigenic competition between vaccine antigens and FVIII.

Figure 7

Comparison of CD4+ T-cell chemotaxis in response to media from influenza vaccine-stimulated LN lymphocytes and FVIII-stimulated splenocytes. Chemokine gradients were generated by stimulating splenocytes and LN-lymphocytes with FVIII (1 μg/mL) or influenza with and without MF59 (1 μg/mL) for 18 hours. CD4+ T-cell chemotaxis competition of between converging gradients were imaged overnight between FVIII and influenza (A) and FVIII and influenza with MF59 (B). (C) Media from unstimulated splenocytes and LN-lymphocytes were assessed as a negative control. (D) A CCL5 (20 μg/mL) gradient was assessed as a positive control. The vertical components traveled by T cells were analyzed for FVIII and influenza competition (E) and FVIII and influenza with MF59 (F). Statistical analysis was conducted using Rayleigh’s test and Mann-Whitney U test for trajectory plots and distance traveled, respectively. Data representative of at least 3 independent experiments. *P < .05; **P < .01; ***P < .001.

Figure 7

Comparison of CD4+ T-cell chemotaxis in response to media from influenza vaccine-stimulated LN lymphocytes and FVIII-stimulated splenocytes. Chemokine gradients were generated by stimulating splenocytes and LN-lymphocytes with FVIII (1 μg/mL) or influenza with and without MF59 (1 μg/mL) for 18 hours. CD4+ T-cell chemotaxis competition of between converging gradients were imaged overnight between FVIII and influenza (A) and FVIII and influenza with MF59 (B). (C) Media from unstimulated splenocytes and LN-lymphocytes were assessed as a negative control. (D) A CCL5 (20 μg/mL) gradient was assessed as a positive control. The vertical components traveled by T cells were analyzed for FVIII and influenza competition (E) and FVIII and influenza with MF59 (F). Statistical analysis was conducted using Rayleigh’s test and Mann-Whitney U test for trajectory plots and distance traveled, respectively. Data representative of at least 3 independent experiments. *P < .05; **P < .01; ***P < .001.

Discussion

The immunogenic potential of APCs is influenced by maturation induced by inflammatory signals in the surrounding microenvironment such as PAMPs and DAMPs. It has been previously demonstrated that FVIII by itself, as well as in complex with VWF, does not induce APC maturation, and thus the immunogenic capability of human monocyte-derived DCs.8  This suggests that the microenvironment surrounding APCs in previously untreated HA patients is an essential regulator of whether immunity against FVIII develops. Indeed, a survey conducted across 42 centers showed a significant concern that administering FVIII in the presence of inflammatory stimuli, particularly surgery, would increase the risk of inhibitor development.23  Results from the recent Concerted Action on Neutralizing Antibodies in Severe Hemophilia A (CANAL) and Research of Determinants of Inhibitor Development (RODIN) studies have respectively shown 3.7- and 2-fold increases in inhibitor risk associated with surgical procedures that are linked with moments of intensive high-dose FVIII treatment.3,24  These findings suggest that proinflammatory signals, either foreign or endogenous, may potentiate the development of FVIII inhibitors.

We posed the question of whether vaccination would elicit an increased risk of FVIII antibody and inhibitor development with the hypothesis that vaccine-associated PAMPs would trigger APC activation. In a humanized murine model of HA, our data demonstrate that intramuscular immunization against both preparations of the influenza vaccine, with and without the adjuvant MF59, decreases the risk of developing FVIII-specific IgG antibodies and FVIII inhibitors. Furthermore, in FVIII-responding mice, we detected no differences in the titers of FVIII-specific IgG and their inhibitory activity suggesting that vaccination influences the initial decision of whether to mount an immune response but does not affect the magnitude of the immune response. However, mice immunized against influenza without an adjuvant showed increased levels of IgG2a antibodies suggesting that the normal antiviral T helper 1 (Th1) response expected exerts its influence on the immunoglobulin profile of the anti-FVIII response. As a single-stranded RNA virus, influenza has the potential to stimulate TLR7, and our data support the idea that TLR ligands, specifically TLR7 and TLR9, polarize anti-FVIII response toward Th1.25,26  Furthermore, the adjuvanted-vaccine modulation of the anti-FVIII response with the associated increase in plasma levels of FVIII-specific IgG1 suggest that the addition of the M59 adjuvant alters the Th profile to a predominately Th2 response, a role that has previously been suggested.27,28  However, these results may be strain specific as it has been suggested that the role of MF59 is to simply augment the default immune profile rather than biasing the profile.29 

The presence of danger signals in the vaccine preparation is irrelevant if they do not stimulate FVIII-presenting APCs. In mice intravenously immunized against influenza without an adjuvant, we observed a trend of increased FVIII immunogenicity, without a decrease in the immune response against influenza. Although not statistically significant, these findings confirm that the vaccine preparation contains danger signals, but that the effect on FVIII immunogenicity is dependent on the route of administration. Surprisingly, with the addition of the adjuvant, we observed no influence on the FVIII immune response. These findings may be attributed to a lack of interaction between FVIII and the adjuvant. In this scenario, the effect of the adjuvant on the antigen “depot” effect and lymphocyte recruitment may not be extended to FVIII.

In this study, we attribute the apparent decrease in FVIII immunogenicity to antigenic competition. The vaccine, administered intramuscularly, likely encounters the resident APCs in the muscle, followed by the subsequent migration of the APCs to the draining LN. Indeed, the MF59 preparation of the vaccine has been previously shown to provide antigen retention in the LN and induce lymphocyte recruitment.30,31  In contrast, the intravenous infusion of FVIII likely elicits immunity in the spleen.22  We hypothesized that concurrent vaccination and exposure to FVIII attracts immune cells toward the site of vaccination and thus decreases the effective immune response against FVIII. To directly address this hypothesis, we developed a novel in vitro antigenic competition assay allowing time-lapse 3-dimensional tracking of lymphocytes, toward media from either FVIII-stimulated splenocytes or influenza-stimulated LN cells. Following the documentation of unsorted splenocytes to migrate preferentially toward influenza-induced chemokines, we subsequently identified 1 candidate cell type as CD4+ T lymphocytes. Migration of helper CD4+ T cells away from the spleen may decrease the probability of MHC-bound FVIII peptides interacting with their corresponding antigen-specific T cell. Additionally, this trafficking may be attributed to the critical role of CD4+ T cells in the initiation and maintenance of influenza immunity.32,33  Although the specific chemokines responsible for these observations were not identified, we present here, as proof of concept, the potential for vaccination to divert immune resources away from the site of anti-FVIII immune responses. It should be cautioned that the extent of this effect may be magnified in this mouse model in which APC–T cell interactions are restricted to a single human MHC class II molecule. Thus, theoretically, only a fraction of the potential FVIII peptides can be presented to T cells compared with wild-type mice.11  The probability of an APC encountering an antigen-specific T cell is therefore less, and the inability of an APC to find an antigen-specific T cell may promote tolerance or anergy. Additional studies are needed to better elucidate this phenomenon, particularly in relation to the potential of FVIII-specific T-cell suppression in vivo.

We further assessed the role of a third pediatric vaccine, the MMR vaccine, and did not find any influence on the incidence or magnitude of the FVIII immune response. Mice in these studies did not mount an immune response against the vaccine delivered by either mode of administration. Incidentally, murine MMR immunization has not been reported in the literature, and mice are not known reservoirs for its viral constituents. Thus, mice may lack the necessary receptor-mediated processes required to elicit infection from the live-attenuated vaccine, and the viruses are simply degraded rather than presented to the immune system. Based on intravenous exposure of MMR and in vitro stimulation of DCs, it appears that the vaccine preparation does not contain accessible danger signals for mice.

Here, we have shown that concurrent intramuscular immunization with the influenza vaccine, with and without an adjuvant, reduces the immune response against rhFVIII in a humanized HA mouse model. A potential mechanism may be the redirection of immune resources and the recruitment of CD4+ T cells to the site of vaccination. Decreased anti-FVIII immunity in influenza-vaccinated mice was maintained upon intensive reexposure to rhFVIII after 5 weeks without FVIII administration. Furthermore, this phenomenon may have some specificity for the influenza vaccine given that mice immunized intramuscularly with ovalbumin did not exhibit a similar decrease in FVIII immunity. These data suggest that vaccination of young HA patients may not increase FVIII-specific antibody responses. Interestingly, an independent epidemiological study, by Hashemi et al, simultaneously observed that receiving vaccinations in close proximity to FVIII exposure in previously untreated severe HA patients did not increase inhibitor development.34  Taken together with our findings, these data provide evidence that vaccination may not always increase the risk of inhibitor development and may in fact result in reduced antibody responses to FVIII. Caution should be used in extrapolating this information as this protective effect may be dependent on the properties of vaccine and the time of immunization relative to FVIII exposure. Further identification of specific T-cell subsets that migrate and remain at the site of FVIII immunity may provide insight into the critical linkage of innate and adaptive immunity required for the development of FVIII inhibitors. The inhibition of T-cell chemotaxis by fingolimod, a sphingosine-1-phosphate receptor modulator, and maraviroc, a CCR5 antagonist, have shown promising results in treating multiple sclerosis and graft-versus-host disease, respectively.35,36  This diversion of immune resources may represent a novel avenue for FVIII immunotherapy that bypasses immunosuppression.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

The authors thank Jeff Mewburn for confocal microscopy assistance and Maria Schuster for organizing breeding and shipment of humanized F8 E17KO HLA-DRB1*1501 HA mice. Research animals were provided in part by Baxalta.

This work was supported in part by an operating grant from the Canadian Institutes of Health Research (MOP-10912). D.L. is the recipient of a Canada Research Chair in Molecular Hemostasis.

Authorship

Contribution: J.D.L. designed, performed, and interpreted the research and wrote the manuscript; P.C.M., K.N.S., B.M.R., C.H., and D.L. designed the study and edited the manuscript; and K.S. performed the animal experiments.

Conflict-of-interest disclosure: K.N.S. and B.M.R. are employees of Baxalta. The remaining authors declare no competing financial interests.

Correspondence: David Lillicrap, 88 Stuart St, Richardson Laboratory, Queen’s University, Kingston, ON K7L 3N6, Canada; e-mail: david.lillicrap@queensu.ca.

References

References
1
Brackmann
HH
Gormsen
J
Massive factor-VIII infusion in haemophiliac with factor-VIII inhibitor, high responder.
Lancet
1977
, vol. 
310
 
8044
pg. 
933
 
2
DiMichele
DM
Hoots
WK
Pipe
SW
Rivard
GE
Santagostino
E
International workshop on immune tolerance induction: consensus recommendations.
Haemophilia
2007
, vol. 
13
 
suppl 1
(pg. 
1
-
22
)
3
Gouw
SC
van den Berg
HM
Fischer
K
, et al. 
PedNet and Research of Determinants of INhibitor development (RODIN) Study Group
Intensity of factor VIII treatment and inhibitor development in children with severe hemophilia A: the RODIN study.
Blood
2013
, vol. 
121
 
20
(pg. 
4046
-
4055
)
4
Wight
J
Paisley
S
The epidemiology of inhibitors in haemophilia A: a systematic review.
Haemophilia
2003
, vol. 
9
 
4
(pg. 
418
-
435
)
5
Wroblewska
A
Reipert
BM
Pratt
KP
Voorberg
J
Dangerous liaisons: how the immune system deals with factor VIII.
J Thromb Haemost
2013
, vol. 
11
 
1
(pg. 
47
-
55
)
6
van Haren
SD
Wroblewska
A
Fischer
K
Voorberg
J
Herczenik
E
Requirements for immune recognition and processing of factor VIII by antigen-presenting cells.
Blood Rev
2012
, vol. 
26
 
1
(pg. 
43
-
49
)
7
Matzinger
P
The danger model: a renewed sense of self.
Science
2002
, vol. 
296
 
5566
(pg. 
301
-
305
)
8
Pfistershammer
K
Stöckl
J
Siekmann
J
Turecek
PL
Schwarz
HP
Reipert
BM
Recombinant factor VIII and factor VIII-von Willebrand factor complex do not present danger signals for human dendritic cells.
Thromb Haemost
2006
, vol. 
96
 
3
(pg. 
309
-
316
)
9
Moorehead
PC
Waters
B
Sponagle
K
Steinitz
KN
Reipert
BM
Lillicrap
D
Surgical injury alone does not provoke the development of factor VIII inhibitors in mouse models of hemophilia A [abstract].
Blood
2012
, vol. 
120
 
21
 
Abstract 627
10
Committee on Infectious Diseases, American Academy of Pediatrics
Recommended childhood and adolescent immunization schedule--United States, 2014.
Pediatrics
2014
, vol. 
133
 
2
(pg. 
357
-
363
)
11
Steinitz
KN
van Helden
PM
Binder
B
, et al. 
CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice.
Blood
2012
, vol. 
119
 
17
(pg. 
4073
-
4082
)
12
Bermejo
N
Martín Aguilera
C
Carnicero
F
Bergua
J
Allergenic vaccines administration and inhibitor development in haemophilia.
Haemophilia
2012
, vol. 
18
 
5
(pg. 
e392
-
e393
)
13
Qadura
M
Waters
B
Burnett
E
, et al. 
Recombinant and plasma-derived factor VIII products induce distinct splenic cytokine microenvironments in hemophilia A mice.
Blood
2009
, vol. 
114
 
4
(pg. 
871
-
880
)
14
Lum
LCS
Borja-Tabora
CF
Breiman
RF
, et al. 
Influenza vaccine concurrently administered with a combination measles, mumps, and rubella vaccine to young children.
Vaccine
2010
, vol. 
28
 
6
(pg. 
1566
-
1574
)
15
Stowe
J
Andrews
N
Taylor
B
Miller
E
No evidence of an increase of bacterial and viral infections following measles, mumps and rubella vaccine.
Vaccine
2009
, vol. 
27
 
9
(pg. 
1422
-
1425
)
16
Hilton
S
Petticrew
M
Hunt
K
‘Combined vaccines are like a sudden onslaught to the body’s immune system’: parental concerns about vaccine ‘overload’ and ‘immune-vulnerability’.
Vaccine
2006
, vol. 
24
 
20
(pg. 
4321
-
4327
)
17
Reipert
BM
Steinitz
KN
van Helden
PM
, et al. 
Opportunities and limitations of mouse models humanized for HLA class II antigens.
J Thromb Haemost
2009
, vol. 
7
 
suppl 1
(pg. 
92
-
97
)
18
Bi
L
Lawler
AM
Antonarakis
SE
High
KA
Gearhart
JD
Kazazian
HH
Jr
Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A.
Nat Genet
1995
, vol. 
10
 
1
(pg. 
119
-
121
)
19
Hausl
C
Ahmad
RU
Sasgary
M
, et al. 
High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors.
Blood
2005
, vol. 
106
 
10
(pg. 
3415
-
3422
)
20
Waters
B
Qadura
M
Burnett
E
, et al. 
Anti-CD3 prevents factor VIII inhibitor development in hemophilia A mice by a regulatory CD4+CD25+-dependent mechanism and by shifting cytokine production to favor a Th1 response.
Blood
2009
, vol. 
113
 
1
(pg. 
193
-
203
)
21
Miller
CH
Platt
SJ
Rice
AS
Kelly
F
Soucie
JM
Hemophilia Inhibitor Research Study Investigators
Validation of Nijmegen-Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance.
J Thromb Haemost
2012
, vol. 
10
 
6
(pg. 
1055
-
1061
)
22
Navarrete
A
Dasgupta
S
Delignat
S
, et al. 
Splenic marginal zone antigen-presenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A.
J Thromb Haemost
2009
, vol. 
7
 
11
(pg. 
1816
-
1823
)
23
van den Berg
HM
Chalmers
EA
Clinical prediction models for inhibitor development in severe hemophilia A.
J Thromb Haemost
2009
, vol. 
7
 
suppl 1
(pg. 
98
-
102
)
24
Gouw
SC
van der Bom
JG
Marijke van den Berg
H
Treatment-related risk factors of inhibitor development in previously untreated patients with hemophilia A: the CANAL cohort study.
Blood
2007
, vol. 
109
 
11
(pg. 
4648
-
4654
)
25
Diebold
SS
Kaisho
T
Hemmi
H
Akira
S
Reis e Sousa
C
Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.
Science
2004
, vol. 
303
 
5663
(pg. 
1529
-
1531
)
26
Allacher
P
Baumgartner
CK
Pordes
AG
Ahmad
RU
Schwarz
HP
Reipert
BM
Stimulation and inhibition of FVIII-specific memory B-cell responses by CpG-B (ODN 1826), a ligand for Toll-like receptor 9.
Blood
2011
, vol. 
117
 
1
(pg. 
259
-
267
)
27
Valensi
JP
Carlson
JR
Van Nest
GA
Systemic cytokine profiles in BALB/c mice immunized with trivalent influenza vaccine containing MF59 oil emulsion and other advanced adjuvants.
J Immunol
1994
, vol. 
153
 
9
(pg. 
4029
-
4039
)
28
Qadura
M
Waters
B
Burnett
E
, et al. 
Immunoglobulin isotypes and functional anti-FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice.
Haemophilia
2011
, vol. 
17
 
2
(pg. 
288
-
295
)
29
O’Hagan
DT
Wack
A
Podda
A
MF59 is a safe and potent vaccine adjuvant for flu vaccines in humans: what did we learn during its development?
Clin Pharmacol Ther
2007
, vol. 
82
 
6
(pg. 
740
-
744
)
30
Cantisani
R
Pezzicoli
A
Cioncada
R
, et al. 
Vaccine adjuvant MF59 promotes retention of unprocessed antigen in lymph node macrophage compartments and follicular dendritic cells.
J Immunol
2015
, vol. 
194
 
4
(pg. 
1717
-
1725
)
31
Seubert
A
Monaci
E
Pizza
M
O’Hagan
DT
Wack
A
The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells.
J Immunol
2008
, vol. 
180
 
8
(pg. 
5402
-
5412
)
32
Wilkinson
TM
Li
CK
Chui
CS
, et al. 
Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans.
Nat Med
2012
, vol. 
18
 
2
(pg. 
274
-
280
)
33
Miller
MA
Ganesan
AP
Luckashenak
N
Mendonca
M
Eisenlohr
LC
Endogenous antigen processing drives the primary CD4+ T cell response to influenza.
Nat Med
2015
, vol. 
21
 
10
(pg. 
1216
-
1222
)
34
Hashemi
SM
Fischer
K
Gouw
SC
, et al. 
Do vaccinations influence the risk of inhibitor development in patients with severe hemophilia A? [abstract].
J Thromb Haemost
2015
, vol. 
13
 
suppl 2
pg. 
147
  
Abstract OR141
35
Kappos
L
Antel
J
Comi
G
, et al. 
FTY720 D2201 Study Group
Oral fingolimod (FTY720) for relapsing multiple sclerosis.
N Engl J Med
2006
, vol. 
355
 
11
(pg. 
1124
-
1140
)
36
Reshef
R
Luger
SM
Hexner
EO
, et al. 
Blockade of lymphocyte chemotaxis in visceral graft-versus-host disease.
N Engl J Med
2012
, vol. 
367
 
2
(pg. 
135
-
145
)