Anti-influenza IgG Research Articles

Advax-SM™-Adjuvanted COBRA (H1/H3) Hemagglutinin Influenza Vaccines.

Adjuvants enhance immune responses stimulated by vaccines. To date, many seasonal influenza vaccines are not formulated with an adjuvant. In the present study, the adjuvant Advax-SM™ was combined with next generation, broadly reactive influenza hemagglutinin (HA) vaccines that were designed using a computationally optimized broadly reactive antigen (COBRA) methodology. Advax-SM™ is a novel adjuvant comprising inulin polysaccharide and CpG55.2, a TLR9 agonist. COBRA HA vaccines were combined with Advax-SM™ or a comparator squalene emulsion (SE) adjuvant and administered to mice intramuscularly. Mice vaccinated with Advax-SM™ adjuvanted COBRA HA vaccines had increased serum levels of anti-influenza IgG and IgA, high hemagglutination inhibition activity against a panel of H1N1 and H3N2 influenza viruses, and increased anti-influenza antibody secreting cells isolated from spleens. COBRA HA plus Advax-SM™ immunized mice were protected against both morbidity and mortality following viral challenge and, at postmortem, had no detectable lung viral titers or lung inflammation. Overall, the Advax-SM™-adjuvanted COBRA HA formulation provided effective protection against drifted H1N1 and H3N2 influenza viruses.

Abstract 5341: Novel FcγR humanized mouse model enables assessment of PK/PD of therapeutic antibodies

Abstract Therapeutic antibodies have revolutionized the way we treat cancer. Enhanced activity of therapeutic IgG can be achieved by the modulation of Fc binding to Fcγ receptors (FcγR), which will consequently modulate the Fc-effector functions triggered upon crosslinking of target and effector cells by these antibodies. Assessment of therapeutic antibodies pharmacodynamic (PD) in preclinical models is challenging, as the FcγR are different from mice to human, as well as their expression pattern. FcRn is involved in the recycling and transport of IgG and is a key determinant of the pharmacokinetics (PK) of IgG. We report here a novel mouse model expressing human FcγR with a human-like expression pattern, in which human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB are expressed and replace their mouse orthologs. The model was intercrossed with the hSA/hFcRn mouse model (Viuff et al., 2016), which has shown to improve translatability of PK assessment of therapeutics with extended half-life via FcRn recycling and/or HSA binding. Investigation of FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB expression on DC, monocytes, granulocytes, NK, T and B cells shows that expression pattern in hFcγR mice is consistent with their expression on human PBMC, with a few exceptions. These receptors are functional, as a human IgG1 targeting mouse CD20 induces approximately 50% of B-cell depletion in blood and liver, after a single iv administration in hFcγR mice. This depletion is Fc-mediated, as it is abrogated when hFcγR mice are treated with an anti-mouse CD20 human IgG1 with a Fc-null mutation. Assessment of antibody-dependent cell cytotoxicity (ADCC) ex vivo to investigate FcγR functionality in NK cells shows that Rituximab, an anti-human CD20 chimeric IgG1 with regular Fc, induces higher ADCC efficacy in NK cells from hFcγR than in NK cells from WT mice. Obinutuzumab, an anti-human CD20 featuring an optimized Fc portion to enhance binding to FcγRIII, shows higher tumor cell lysis than Rituximab in NK cells from hFcγR mice, suggesting this model enables ranking of antibodies. Moreover, assessment of activity of Fc-engineered anti-influenza IgG antibodies also shows that anti-Flu antibody with enhanced Fc binding outperforms the WT antibody, improves protective activity, and demonstrates effector function in influenza challenge model, following prophylactic treatment. Conversely, hFcγR mice treated with an Fc-null version of the anti-Flu antibody shows reduced protective activity and survival compared with the WT antibody. Therefore, the model reported here is a novel tool for testing efficacy of Fc-engineered therapeutic antibodies. We are currently investigating the PK profile of therapeutic antibodies in the hFcγR/hSA/hFcRn model. This novel tool is being improved to enable tolerability to human IgG1, biodistribution to the central nervous system, and flexibility of test of therapeutics through expression of human immune checkpoints. Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Rachel Courtois, Séverine Augier, Yacine Cherifi, Ariane Morel, Patricia Isnard-Petit, Kader Thiam. Novel FcγR humanized mouse model enables assessment of PK/PD of therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5341.

Upper respiratory tract infections and influenza-like illnesses among healthcare workers: are serological tests useful in monitoring influenza and influenza-like illness?

Healthcare workers (HCWs) are at high risk for exposure to upper respiratory tract infections (URTIs) and influenza-like illnesses (ILIs). The present study aimed to surveil URTIs and ILIs and their impact among the Department of Pediatric Infectious Diseases in Wroclaw employees and evaluate their humoral response to influenza. Thirty-six HCWs participated in the first season and 32 HCWs in the second season during years of the study. The authors carried out a URTI/ILI surveillance, and all HCWs were asked to complete a weekly report during 2 influenza seasons: 2016/2017 (S1) and 2017/2018 (S2). In S1 both IgG and IgM antibodies against influenza A and B were assessed. The HCWs with symptoms of ILI were encouraged to undergo PCR tests for influenza. No significant differences in reporting URTI were found among vaccinated and non-vaccinated HCWs and HCWs and the control group. Depending on the year 5.5-17.2% of HCWs were treated with antibiotics because of URTI. In the study 58.7% of participants in S1 and 66.7% in S2 decide to work despite the URTI symptoms. There was no statistical relationship between the concentration of anti-influenza IgG and the number of URTIs and ILIs reported. Only vaccinated were willing to undergo voluntary influenza testing. The URTI and ILI occur commonly in HCWs, and HCWs contract URTIs as often as the control group. Despite their medical education, HCWs work with the symptoms of infection and overuse antibiotics to treat the URTI. Serology testing is not able to follow the infection's dynamics or identify the people immune to the influenza-like illness. The diagnostic value of IgM antibodies in acute influenza infection is negligible. Vaccinated HCWs are more focused on their health and are more willing to undergo influenza tests. Med Pr. 2022;73(6):441-7.

1270 nm near-infrared light as a novel vaccine adjuvant acts on mitochondrial photoreception in intradermal vaccines.

With the development of laser technology in the 1960s, a technique was developed to inject intradermal vaccines immediately after irradiating the skin with laser light to elicit an adjuvant effect, referred to as "laser adjuvant." We have been investigating the mechanism of laser adjuvant in influenza mouse models using noninvasive continuous-wave (CW) near-infrared (NIR) light mainly at a wavelength of 1064 nm, and have shown that the production of reactive-oxygen-species (ROS) in the skin and mast cells in the skin tissue plays an important role in the laser adjuvant effect. The new wavelength of 1270 nm NIR light is characterized by its ability to elicit the same vaccine adjuvant effect as other wavelengths at a lower energy, and may be suitable for clinical applications. In this study, we investigated the physiological activity of CW1270 nm NIR light in mast cells, its biological activity on mouse skin, and the durability of the vaccine adjuvant effect in influenza vaccine mouse models. We show that irradiation of mast cells with 1270 nm NIR light produced ROS and ATP, and irradiation of isolated mitochondria also produced ATP. In mouse skin, the relative expression levels of chemokine mRNAs, such as Ccl2 and Ccl20, were increased by irradiation with 1270 and 1064 nm NIR light at minimum safe irradiance. However, the relative expression of Nfkb1 was increased at 1064 nm, but not at 1270 nm. Serum anti-influenza IgG antibody titers increased early after immunization with 1064 nm, whereas with 1270 nm, there was not only an early response of antibody production but also persistence of antibody titers over the medium- to long-term. Thus, to our knowledge, we show for the first time that 1270 nm NIR light induces ROS and ATP production in mitochondria as photoreceptors, initiating a cascade of laser adjuvant effects for intradermal vaccines. Additionally, we demonstrate that there are wavelength-specific variations in the mechanisms and effects of laser adjuvants. In conclusion, CW1270 nm NIR light is expected to be clinically applicable as a novel laser adjuvant that is equivalent or superior to 1064 nm NIR light, because it can be operated at low energy and has a wavelength-specific adjuvant effect with medium- to long-lasting antibody titer.

Isolated specific IgA against respiratory viruses, Influenza or SARS-CoV-2, present in the saliva of a fraction of healthy and asymptomatic volunteers

ObjectivesDefense against respiratory viruses depends on an immune response present in the mucosa, as saliva IgA secretes antibodies. During the pandemic, such as influenza or SARS-CoV-2, most infected patients are asymptomatic but retain specific antibodies post-infection. The authors evaluated IgG and IgA antibodies against SARS-CoV-2 and influenza in the saliva of asymptomatic volunteers, validated with controls or vaccinated individuals. MethodsThe authors detected specific antibodies by validated conventional ELISA using natural SARS-CoV-2 antigens from infected Vero cells or capture-ELISA for influenza using natural antigens of the influenza vaccine. ResultsSaliva from influenza-vaccinated individuals had more IgA than paired serum, contrary to the findings for specific IgG. In COVID-19-vaccinated samples, specific IgA in saliva increased after vaccination, but IgG levels were high after the first dose. In saliva from the asymptomatic population (226), anti-Influenza IgG was found in 57.5% (130) of samples, higher than IgA, found in 35% (79) of samples. IgA results were similar for SARS-CoV-2, with IgA present in 30% (68) of samples, while IgG was less present, in 44.2% (100) of samples. The proportion of influenza IgG responders was higher than that for SARS-CoV-2 IgG, but both populations presented similar proportions of IgA responders, possibly due to variable memory B cell survival. For both viruses, the authors found an important proportion (> 10%) of IgA+IgG- samples, suggesting the occurrence of humoral immunity directed to the mucosa. ConclusionSpecific antibodies for respiratory viruses in saliva are found in either infection or vaccination and are a convenient and sensitive diagnostic tool for host immune response.

Anti-Influenza Virus-Specific Nasal Secretory IgA and Serum IgG Titers in a Japanese Adult Population and their Changes after Subcutaneous Vaccination

Anti-Influenza Virus-Specific Nasal Secretory IgA and Serum IgG Titers in a Japanese Adult Population and their Changes after Subcutaneous Vaccination

Formulation Approach that Enables the Coating of a Stable Influenza Vaccine on a Transdermal Microneedle Patch.

A trivalent influenza split vaccine was formulated at high concentration for coating on the transdermal microneedle system. Monovalent vaccine bulks of three influenza strains, two influenza A strains, and one B strain were diafiltrated, concentrated, and lyophilized. The lyophilized powder of each vaccine strain was separately reconstituted and subsequently combined into a coating formulation of high concentration trivalent vaccine. The formulation process converted the monovalent vaccine bulks with low hemagglutinin (HA) concentrations 0.1 mg/mL into a viscous, emulsion containing HA at ~50 mg/mL. This physically stable emulsion demonstrated viscosity 1 poise and 30° contact angle for effective, homogeneous coating on each microneedle. Evaluation of the vaccine antigen HA by SRID and SDS-PAGE/Western blot showed that HA remained stable throughout the vaccine transdermal microneedle system manufacturing process and 1-year ambient storage (25°C). Anti-influenza antibody responses were evaluated by ELISA and hemagglutination inhibition (HAI) assay after primary and booster immunization with the vaccine-coated transdermal microneedle systems at either 25-μg or 40-μg total HA. The results showed the induction of serum anti-influenza IgG and anti-HA neutralizing antibodies after primary immunization and significant titer rises after booster immunization for both doses, indicating the dry-coated trivalent vaccine delivered by transdermal microneedle system elicited both primary and recall antibody responses against all three antigen strains. The study demonstrates that the transdermal microneedle system provides an attractive alternative for influenza vaccine delivery with key advantages such as preservative-free and room-temperature storage.

Intrinsic antibody mediated enhancement of ZIKV infection in placental macrophages

Abstract Zika virus (ZIKV) vertical transmission from mother to child in utero is associated with a number of neonatal abnormalities, including congenital microcephaly. Yet, the mechanism by which ZIKV infects the placenta and subsequently the fetus remains poorly understood. ZIKV and Dengue virus (DENV) endemic regions are commonly overlapping, and DENV specific antibodies can cross-react with ZIKV. We and others have previously demonstrated that cross-reactive DENV IgG can mediate ZIKV infection of placental cells though increased viral entry via Fc receptors. Specifically, we found that infection with ZIKV immune complexes (ZIKV-IC) generated from cross-reactive anti-DENV monoclonal IgG was enhanced in human placental macrophages called Hofbauer cells, which are found within the chorionic villus stroma near the fetal vasculature. In this study, we aimed to characterize the intrinsic cellular changes that occur in Hofbauer cells as a result of ZIKV-IC infection. We performed RNA sequencing on Hofbauer cells infected with ZIKV complexed with different concentrations of anti-DENV monoclonal IgG. Principal component analysis revealed that while ZIKV incubated with anti-influenza IgG had a similar gene expression profile to ZIKV alone, ZIKV-IC infection with increasing anti-DENV IgG skewed gene expression towards a unique phenotype. We identified a number of transcriptional profiles related to anti-DENV IgG dose. Notably, we observed a pattern of decreased interferon stimulated genes with increasing anti-DENV IgG dose. Yet, increasing anti-DENV IgG also increased ZIKV viral RNA. These results may indicate that ZIKV-IC infection makes Hofbauer cells more permissive to ZIKV replication by modulating the innate antiviral response.

Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection.

Antibodies against viral pathogens represent promising therapeutic agents for the control of infection, and their antiviral efficacy has been shown to require the coordinated function of both the Fab and Fc domains1. The Fc domain engages a wide spectrum of receptors on discrete cells of the immune system to trigger the clearance of viruses and subsequent killing of infected cells1–4. Here we report that Fc engineering of anti-influenza IgG monoclonal antibodies for selective binding to the activating Fcγ receptor FcγRIIa results in enhanced ability to prevent or treat lethal viral respiratory infection in mice, with increased maturation of dendritic cells and the induction of protective CD8+ T cell responses. These findings highlight the capacity for IgG antibodies to induce protective adaptive immunity to viral infection when they selectively activate a dendritic cell and T cell pathway, with important implications for the development of therapeutic antibodies with improved antiviral efficacy against viral respiratory pathogens.

Probiotics: An Alternative Therapeutic Strategy for Covid-19

ABSTRACT: The COVID- 19 pandemic has made the scientists today all around the globe to look for its specific prevention and treatment modalities. The disease presents itself as asymptomatic to mild to severe respiratory symptoms along with lesser common gastrointestinal symptoms of diarrhea, nausea, and vomiting. The situation worsens due to lack of precise treatment strategy. The Healthcare system is being overwhelmed, emphasizing on the need to look for alternate supportive therapy that can not only enhance the immune status of people worldwide but at the same time, ensure better prognosis. The relationship between the gut microbiota and upper and lower respiratory tract viral infections are well studied. Oral administration of probiotic microorganisms of genera Lactobacillus, Bifidobacterium and Bacillusin mice suffering from influenza infection have shown increased TNFα, IFN-γ and NK cell responses along with the production of anti-Influenza IgG. At the same time they have shown immunomodulation by producing anti-inflammatory cytokines as well as cytotoxic T-cells and T-suppressor cells. Hence, probiotic strains of generaLactobacillus, Bifidobacterium and Bacillushave shown a probability to be used as preventive and therapeutic agents for SARS- CoV-2.

BBIQ, a pure TLR7 agonist, is an effective influenza vaccine adjuvant

ABSTRACT Better adjuvants are needed for vaccines against seasonal influenza. TLR7 agonists are potent activators of innate immune responses and thereby may be promising adjuvants. Among the imidazoquinoline compounds, 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (BBIQ) was reported to be a highly active TLR7 agonist but has remained relatively unexplored because of its commercial unavailability. Indeed, in silico molecular modeling studies predicted that BBIQ had a higher TLR7 docking score and binding free energy than imiquimod, the gold standard TLR7 agonist. To circumvent the availability issue, we developed an improved and higher yield method to synthesize BBIQ. Testing BBIQ on human and mouse TLR7 reporter cell lines confirmed it to be TLR7 specific with significantly higher potency than imiquimod. To test its adjuvant potential, BBIQ or imiquimod were admixed with recombinant influenza hemagglutinin protein and administered to mice as two intramuscular immunizations 2 weeks apart. Serum anti-influenza IgG responses assessed by ELISA 2 weeks after the second immunization confirmed that the mice that received vaccine admixed with BBIQ had significantly higher anti-influenza IgG1 and IgG2c responses than mice immunized with antigen alone or admixed with imiquimod. This confirmed BBIQ to be a TLR7-specific adjuvant able to enhance humoral immune responses.

Application of volumetric absorptive microsampling (VAMS) to measure multidimensional anti-influenza IgG antibodies by the mPlex-Flu assay.

Introduction: Recently, volumetric absorptive microsampling (VAMS) has been used for accurate sampling of a fixed peripheral blood volume (10 µL) on a volumetric swab, and long-term sample storage. The mPlex-Flu assay is a novel, high-throughput assay that simultaneously measures the concentration of antibodies against the hemagglutinin (HA) proteins from multiple influenza virus strains with ≤5 µL of serum. Here we describe combining these two methods to measure multidimensional anti-influenza IgG activity in whole blood samples collected by a finger stick and VAMS, with correction for serum volume based on simultaneous hemoglobin measurement. Methods: We compared capillary blood samples obtained from a finger stick using a VAMS device with serum samples collected by traditional phlebotomy from 20 subjects, with the influenza antibody profiles measured by the mPlex-Flu assay. Results: We found that results with the two sampling methods were highly correlated within subjects and across all influenza strains (mean R 2 = 0.9470). Adjustment for serum volume, based on hemaglobin measurement, was used to estimate serum volume of samples and improved the accuracy. IgG measurements were stable over 3 weeks when VAMS samples were stored at room temperature or transported using a variety of shipping methods. Additionally, when volunteers performed finger-stick VAMS at-home by themselves, the comparison results of anti-HA antibody concentrations were highly consistent with sampling performed by study personnel on-site (R 2 = 0.9496). Conclusions: This novel approach can provide a simple, accurate, and low-cost means for monitoring the IgG anti-influenza HA antibody responses in large population studies and clinical trials.

Salmonella Typhi Porins OmpC and OmpF Are Potent Adjuvants for T-Dependent and T-Independent Antigens.

Several microbial components, such as bacterial DNA and flagellin, have been used as experimental vaccine adjuvants because of their inherent capacity to efficiently activate innate immune responses. Likewise, our previous work has shown that the major Salmonella Typhi (S. Typhi) outer membrane proteins OmpC and OmpF (porins) are highly immunogenic protective antigens that efficiently stimulate innate and adaptive immune responses in the absence of exogenous adjuvants. Moreover, S. Typhi porins induce the expression of costimulatory molecules on antigen-presenting cells through toll-like receptor canonical signaling pathways. However, the potential of major S. Typhi porins to be used as vaccine adjuvants remains unknown. Here, we evaluated the adjuvant properties of S. Typhi porins against a range of experimental and clinically relevant antigens. Co-immunization of S. Typhi porins with ovalbumin (OVA), an otherwise poorly immunogenic antigen, enhanced anti-OVA IgG titers, antibody class switching, and affinity maturation. This adjuvant effect was dependent on CD4+ T-cell cooperation and was associated with an increase in IFN-γ, IL-17A, and IL-2 production by OVA-specific CD4+ T cells. Furthermore, co-immunization of S. Typhi porins with an inactivated H1N1 2009 pandemic influenza virus experimental vaccine elicited higher hemagglutinating anti-influenza IgG titers, antibody class switching, and affinity maturation. Unexpectedly, co-administration of S. Typhi porins with purified, unconjugated Vi capsular polysaccharide vaccine (Vi CPS)—a T-independent antigen—induced higher IgG antibody titers and class switching. Together, our results suggest that S. Typhi porins OmpC and OmpF are versatile vaccine adjuvants, which could be used to enhance T-cell immune responses toward a Th1/Th17 profile, while improving antibody responses to otherwise poorly immunogenic T-dependent and T-independent antigens.

Simulated Respiratory Secretion for Use in the Development of Influenza Diagnostic Assays.

Many assays have been developed for the detection of influenza virus which is an important respiratory pathogen. Development of these assays commonly involves the use of human clinical samples for validation of their performance. However, clinical samples can be difficult to obtain, deteriorate over time, and be inconsistent in composition. The goal of this study was to develop a simulated respiratory secretion (SRS) that could act as a surrogate for clinical samples. To this end, we determined the effects major respiratory secretion components (Na+, K+, Ca2+, cells, albumin IgG, IgM, and mucin) have on the performance of influenza assays including both nucleic acid amplification and rapid antigen assays. Minimal effects on the molecular assays were observed for all of the components tested, except for serum derived human IgG, which suppressed the signal of the rapid antigen assays. Using dot blots we were able to show anti-influenza nucleoprotein IgG antibodies are common in human respiratory samples. We composed a SRS that contained mid-point levels of human respiratory sample components and studied its effect compared to phosphate buffered saline and virus negative clinical sample matrix on the Veritor, Sofia, CDC RT-PCR, Simplexa, cobas Liat, and Alere i influenza assays. Our results demonstrated that a SRS can interact with a variety of test methods in a similar manner to clinical samples with a similar impact on test performance.

Age and testosterone mediate influenza pathogenesis in male mice.

Influenza severity increases with age, with hospitalization and mortality rates during seasonal influenza epidemics being higher in older men than age-matched women. As it is known that with age, circulating testosterone levels decline in males, we hypothesized that reduced testosterone contributes to age-associated increases in influenza severity. A murine model was used to test this hypothesis. As in men, testosterone concentrations were lower in aged (18 mo) than young (2 mo) male C57BL/6 mice. Following inoculation with influenza A virus (IAV), aged males experienced greater morbidity, clinical disease, and pulmonary inflammation than young males, and had lower neutralizing and total anti-influenza IgG antibody responses. Peak titers of virus in the lungs did not differ between aged and young males, but virus clearance was delayed in aged males. In young males, removal of the gonads increased-whereas treatment of gonadectomized males with testosterone reduced-morbidity, clinical illness, and pulmonary pathology, but viral replication was not altered by hormone manipulation in young males. Treatment of aged males with testosterone improved survival following infection but did not alter either virus replication or pulmonary pathology. These results indicate that low concentrations of testosterone, whether induced surgically in young males or naturally occurring in aged males, negatively impact the outcome of influenza.

Dimeric FcγR Ectodomains as Probes of the Fc Receptor Function of Anti-Influenza Virus IgG.

Ab-dependent cellular cytotoxicity, phagocytosis, and Ag presentation are key mechanisms of action of Abs arising in vaccine or naturally acquired immunity, as well of therapeutic mAbs. Cells expressing the low-affinity FcγRs (FcγRII or CD32 and FcγRIII or CD16) are activated for these functions when receptors are aggregated following the binding of IgG-opsonized targets. Despite the diversity of the Fc receptor proteins, IgG ligands, and potential responding cell types, the induction of all FcγR-mediated responses by opsonized targets requires the presentation of multiple Fc regions in close proximity to each other. We demonstrated that such "near-neighbor" Fc regions can be detected using defined recombinant soluble (rs) dimeric low-affinity ectodomains (rsFcγR) that have an absolute binding requirement for the simultaneous engagement of two IgG Fc regions. Like cell surface-expressed FcγRs, the binding of dimeric rsFcγR ectodomains to Ab immune complexes was affected by Ab subclass, presentation, opsonization density, Fc fucosylation, or mutation. The activation of an NK cell line and primary NK cells by human IgG-opsonized influenza A hemagglutinin correlated with dimeric rsFcγRIIIa binding activity but not with Ab titer. Furthermore, the dimeric rsFcγR binding assay sensitively detected greater Fc receptor activity to pandemic H1N1 hemagglutinin after the swine influenza pandemic of 2009 in pooled human polyclonal IgG. Thus these dimeric rsFcγR ectodomains are validated, defined probes that should prove valuable in measuring the immune-activating capacity of IgG Abs elicited by infection or vaccination or experimentally derived IgG and its variants.

Dynamic application of microprojection arrays to skin induces circulating protein extravasation for enhanced biomarker capture and detection

Surface modified microprojection arrays are a needle-free alternative to capture circulating biomarkers from the skin in vivo for diagnosis. The concentration and turnover of biomarkers in the interstitial fluid, however, may limit the amount of biomarker that can be accessed by microprojection arrays and ultimately their capture efficiency. Here we report that microprojection array insertion induces protein extravasation from blood vessels and increases the concentration of biomarkers in skin, which can synergistically improve biomarker capture. Regions of blood vessels in skin were identified in the upper dermis and subcutaneous tissue by multi-photon microscopy. Insertion of microprojection array designs with varying projection length (40–190 μm), density (5000–20,408 proj.cm−2) and array size (4–36 mm2) did not affect the degree of extravasation. Furthermore, the location of extravasated protein did not correlate with projection penetration to these highly vascularised regions, suggesting extravasation was not caused by direct puncture of blood vessels. Biomarker extravasation was also induced by dynamic application of flat control surfaces, and varied with the impact velocity, further supporting this conclusion. The extravasated protein distribution correlated well with regions of high mechanical stress generated during insertion, quantified by finite element models. Using this approach to induce extravasation prior to microprojection array-based biomarker capture, anti-influenza IgG was captured within a 2 min application time, demonstrating that extravasation can lead to rapid biomarker sampling and significantly improved microprojection array capture efficiency. These results have broad implications for the development of transdermal devices that deliver to and sample from the skin.

Immunization Against Influenza but Not MMR Modulates the Anti-Factor VIII Immune Response in Hemophilia Α Mice

Introduction: The etiology of inhibitory factor VIII (FVIII) antibodies in 25-30% of hemophilia A (HA) patients remains poorly understood. It is possible that concurrent exposure to inflammatory stimuli, or 'danger signals’, with FVIII increases the risk of inhibitor formation. HA patients generally begin FVIII replacement therapy around 12 months of age, and the median age of inhibitors onset is between 15-21 months. During this time frame, patients may be exposed to vaccines, such as the mumps-measles-rubella (MMR) and seasonal influenza vaccines. Our investigation is the first to address the concern that these vaccines may serve as danger signals that augment FVIII immunogenicity.Methods: Our studies used 8-12 week-old FVIII E17KO C57Bl6/S129 HA mice, which carry a complete knockout of all murine MHC class II molecules, and instead express a chimeric human-mouse HLA-DRB1*1501 allele associated with increased inhibitor risk. Our preliminary studies show that 40-80% of these animals develop FVIII inhibitors following treatment.HA mice were immunized with 10x the standard human dose of live-attenuated MMR vaccine (Priorix) subcutaneously or intravenously 24 hrs prior to 4 weekly intravenous infusions of 2 IU recombinant human FVIII (rhFVIII, Advate). Mice were subsequently re-challenged with MMR 24 hrs prior to 4 biweekly infusions of 6 IU rhFVIII. Blood samples were collected retro-orbitally or via cardiac puncture. Plasma from weeks 5 and 9 was assessed for anti-FVIII IgG by ELISA; inhibitor concentrations were assessed by a Bethesda assay on week 9.The inactivated influenza vaccine (Agriflu) was administered at standard human doses either intramuscularly or intravenously 24 hrs before, after, or concurrently with the first of 7 biweekly infusions of 6 IU rhFVIII. Week 5 plasma samples were subjected to anti-FVIII and anti-influenza IgG ELISA and Bethesda assays. Statistical comparisons were made using the Fisher’s exact Mann-Whitney U tests, as appropriate.Results: Subcutaneous MMR vaccination exhibited no significant differences in the incidence or titres of anti-FVIII IgG compared to HBSS-injected controls at weeks 5 and 9 (n=13). Similarly, no differences in the incidence or concentration of inhibitors were detected at week 9. We next evaluated the effects of intravenous MMR immunization on FVIII immunogenicity. Surprisingly, we again observed no differences in the incidence or magnitude of the anti-FVIII immune response at weeks 5 and 9 (n=28-30).Importantly, we found a significant decrease in the incidence of FVIII-specific IgG in mice that were immunized intramuscularly with the influenza vaccine 24 hrs after and at the same time as the first infusion of rhFVIII (t=-24 hrs: 30%, t=0 hrs: 20%, t=+24 hrs: 26% vs control: 67%; p=0.11, 0.06, 0.04; n=10-15). Similarly, there was a significant decrease in the incidence of inhibitors at all immunization time points (t=-24 hrs: 30%, t=0 hrs: 43%, t=+24 hrs: 11% vs control: 80%; p=0.03, 0.02, 0.0022). No differences in IgG titres or inhibitor concentrations were detected. When immunized intravenously with the influenza vaccine, we observed an increase in the presence of FVIII-specific IgG, but no differences in titres. However, these differences were not statistically significant and need confirmation (t=-24 hrs: 80%, t=0 hrs: 90%, t=+24 hrs 40% vs control: 50%; p=0.58, 0.14, 1.00; n=5-10).Conclusion: These are the first experimental studies to address vaccination as a potential danger signal in the development of an anti-FVIII immune response. Our results suggest that both subcutaneous and intravenous immunization of HA mice with the MMR vaccine do not influence the incidence or magnitude of the anti-FVIII immune response. In contrast, our data suggest that intramuscular immunization with the inactivated influenza vaccine modulates the anti-FVIII immune response and may enhance tolerance induction to FVIII, possibly through antigen competition. However, this proposal awaits further confirmation. Finally, a trend in increased antibody and inhibitor incidence in intravenously immunized mice suggests that the influenza vaccine can serve as a danger signal, but is dependent on the route of administration. Our findings contradict current vaccination concerns in the treatment of young HA patients, and instead suggest an inhibitor-protective effect from influenza immunization. DisclosuresMoorehead:Baxter: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bayer: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Honoraria. Steinitz:Baxter: Employment. Reipert:Baxter: Employment. Hough:Bayer: Research Funding. Lillicrap:Baxter: Research Funding; Bayer: Research Funding; CSL Behring: Research Funding; Biogen Idec: Research Funding.

Enhanced pulmonary immunization with aerosolized inactivated influenza vaccine containing delta inulin adjuvant

Vaccination is the primary intervention to contain influenza virus spread during seasonal and pandemic outbreaks. Pulmonary vaccination is gaining increasing attention for its ability to induce both local mucosal and systemic immune responses without the need for invasive injections. However, pulmonary administration of whole inactivated influenza virus (WIV) vaccine induces a Th2 dominant systemic immune response while a more balanced Th1/Th2 vaccine response may be preferred and only induces modest nasal immunity. This study evaluated immunity elicited by pulmonary versus intramuscular (i.m.) delivery of WIV, and tested whether the immune response could be improved by co-administration of delta (δ)-inulin, a novel carbohydrate-based particulate adjuvant. After pulmonary administration both unadjuvanted and δ-inulin adjuvanted WIV induced a potent systemic immune response, inducing higher serum anti-influenza IgG titers and nasal IgA titers than i.m. administration. Moreover, the addition of δ-inulin induced a more balanced Th1/Th2 response and induced higher nasal IgA titers versus pulmonary WIV alone. Pulmonary WIV alone or with δ-inulin induced hemagglutination inhibition (HI) titers>40, titers which are considered protective against influenza virus. In conclusion, in this study we have shown that δ-inulin adjuvanted WIV induces a better immune response after pulmonary administration than vaccine alone.

8. Stress, insomnia and influenza vaccine response

The current analyses were part of a larger two-year study examining the relationship between perceived stress and influenza vaccine antibody responses in a healthy young adult population by comparing people with insomnia to people without insomnia. Self-reported stress levels were assessed in all individuals using the Perceived Stress Scale (PSS). Influenza strains included H1N1, H3N2 and B. Participants gave a blood sample for antibody assessment both before and four weeks after the vaccine. Anti-influenza IgG antibody amounts were measured via ELISA. Correlations were examined between PSS scores (assessed prior to vaccination) and antibody amounts among the various influenza strains both pre- and post-vaccine. Overall, self-reported stress was negatively correlated with antibody levels pre-vaccine for the H1N1 (−.214, p = .009) and H3N2 (−.243, p = .003) strains and post-vaccine for H3N2 (−.244, p = .004) and B (−.186, p = .028) strains. Results indicated that overall, PSS scores were negatively correlated with anti-influenza antibody (IgG) responses both pre- and post-vaccine for some strains/years, a result that is consistent with a number of studies examining stress and vaccine responses. Interestingly, while people with insomnia self-reported more stress than those without (p = .000), covariate analyses examining stress as a predictor of antibody responses pre- to post-vaccine was not significant (p > .05).