Hemagglutinin Content Research Articles

Collaborative study on the cross-reactivity of two influenza B viral components in single radial immunodiffusion assay using quadrivalent influenza vaccines in Japan from 2015/16 to 2021–22 influenza season

A quadrivalent influenza vaccine (QIV) has been available in Japan since the 2015/2016 influenza season. Single radial immunodiffusion (SRID) assays are currently used worldwide to measure the hemagglutinin (HA) content of influenza vaccine components because they are simple, accurate, and the regulatory requirement, ensuring consistency in manufacture for the HA content. However, the cross-reactivity of antisera against the two lineages of the influenza B virus (IFVB) may cause inaccurate quantification of HA content in QIVs using the SRID assay. To examine cross-reactivity and develop an appropriate procedure for accurate measurement of vaccine potency, a collaborative study with four Japanese vaccine manufacturers was conducted to measure the HA contents of trivalent influenza vaccines (TIVs) and QIVs by SRID assay with a single and a mixture of reference antigens (refAgs) from each lineage of IFVB for seven influenza seasons from 2015/16 to 2021/22. The cross-reactivity of the two IFVB components in the SRID assay varied depending on the vaccine viruses. Our study demonstrated that it is useful to validate a suitable combination for each refAg and reference antiserum by selecting the combination showing similar HA contents between experimental TIV and QIV before lot release testing.

Antibody response to sequential vaccination with cell culture, recombinant, or egg-based influenza vaccines among U.S. adults

ABSTRACT The immune response to inactivated influenza vaccines (IIV) is influenced by multiple factors, including hemagglutinin content and egg-based manufacturing. Only two US-licensed vaccines are manufactured without egg passage: cell culture-based inactivated vaccine (ccIIV) and recombinant vaccine (RIV). We conducted a randomized open-label trial in central Wisconsin during the 2018–19 and 2019–20 seasons to compare immunogenicity of sequential vaccination. Participants 18–64 years old were randomized 1:1:1 to receive RIV, ccIIV or IIV in strata defined by number of influenza vaccine doses in the prior 3 years. They were revaccinated with the same product in year two. Paired serum samples were tested by hemagglutination inhibition against egg-adapted and cell-grown vaccine viruses. Serologic endpoints included geometric mean titer (GMT), mean fold rise, and percent seroconversion. There were 373 participants randomized and vaccinated in 2018–19; 332 were revaccinated in 2019–20. In 2018–19, RIV and ccIIV were not more immunogenic than IIV against A/H1N1. The post-vaccination GMT against the cell-grown 3C.2a A/H3N2 vaccine virus was higher for RIV vs IIV (p = .001) and RIV vs ccIIV (p = .001). The antibody response to influenza B viruses was similar across study arms. In 2019–20, GMT against the cell-grown 3C.3a A/H3N2 vaccine virus was higher for RIV vs IIV (p = .03) and for RIV vs ccIIV (p = .001). RIV revaccination generated significantly greater backboosting to the antigenically distinct 3C.2a A/H3N2 virus (2018–19 vaccine strain) compared to ccIIV or IIV. This study adds to the evidence that RIV elicits a superior immunologic response against A/H3N2 viruses compared to other licensed influenza vaccine products.

Analysing the Potency of a Seasonal Influenza Vaccine Using Reference Antisera from Heterologous Strains.

The potency of inactivated seasonal influenza vaccine is harmonised by establishing the haemagglutinin (HA) content using the compendial single radial diffusion (SRD) method. SRD reagents (antigens and antisera) are prepared, calibrated and distributed by regulatory agencies as standards for potency testing, following the biannual World Health Organization (WHO) announcements of the virus strains suitable for inclusion in the vaccine. The generation of a homologous hyperimmune sheep antiserum constrains the time to vaccine release. This study tests the application of heterologous antisera to determine the potency of influenza vaccine compared to that of a standard homologous antiserum. The results indicate that the selected heterologous sheep antisera directed to seasonal H1N1, H3N2 or B Victoria virus strains can be used to determine the accurate potency of inactivated seasonal influenza vaccines. Individually selected antisera could be useful for two to fourteen seasons. A limitation to the heterologous antiserum approach is the diversity of each individual serum, indicating that the empirical determination of a specific serum is required. This application has the potential to enable the earlier availability of a seasonal vaccine and reduce animal usage.

Voltammetric determination of hemagglutinin using triazolotriazine derivatives as agents for the biomolecule recognition

An original approach for rapid voltammetric determination of the hemagglutinin (HA) protein using original new triazoloazine-based compounds as agents of biomolecule recognition was developed. A pronounced redox activity of original molecules was established; a possible mechanism of electrochemical conversion was proposed. The peak current of the nitro group electroreduction was chosen as an analytical signal. An efficient method for immobilizing the molecules on the surface of carbon nanotubes using thiol-ene and thiol-ine reactions with the radical initiator azobisisobutyronitrile has been proposed. The binding constants of the “compound-hemagglutinin” interaction were determined experimentally without and with external potential and calculated by the molecular modeling method. Compound 2-methylthio-6-nitro-7-oxo-4-propylene-1,2,4-triazolo-4,7-dihydro[5,1-c]-1,2,4-triazine(1a) showed the best characteristics of selective binding to the biotarget. A dependence of the analytical signal on the concentration of hemagglutinin in probe I* = -(13.1 ± 0.5) lgC + (93±2) was obtained in the range 10−7–10−3 M, the limit of detection calculated by the 3-sigma criteria was 7.0 10−8 M. The proposed approach has been successfully tested on saliva model solutions and anti-influenza vaccines.

Immunogenicity and safety of varying dosages of a fifth-wave influenza A/H7N9 inactivated vaccine given with and without AS03 adjuvant in healthy adults

BackgroundHuman infections with the avian influenza A(H7N9) virus were first reported in China in 2013 and continued to occur in annual waves. In the 2016/2017 fifth wave, Yangtze River Delta (YRD) lineage viruses, which differed antigenically from those of earlier waves, predominated. MethodsIn this phase 2 double-blinded trial we randomized 720 adults ≥ 19 years of age to receive two injections of a YRD lineage inactivated A/Hong Kong/125/2017 fifth-wave H7N9 vaccine, given 21 days apart, at doses of 3.75, 7.5, and 15 µg of hemagglutinin (HA) with AS03A adjuvant and at doses of 15 and 45 µg of HA without adjuvant. ResultsTwo doses of adjuvanted vaccine were required to induce HA inhibition (HI) antibody titers ≥ 40 in most participants. After two doses of the 15 µg H7N9 formulation, given with or without AS03 adjuvant, the proportion achieving a HI titer ≥ 40 against the vaccine strain at 21 days after the second vaccination was 65 % (95 % CI, 57 %-73 %) and 0 % (95 % CI, 0 %-4%), respectively. Among those who received two doses of the 15 µg adjuvanted formulation the proportion with HI titer ≥ 40 at 21 days after the second vaccination was 76 % (95 % CI, 66 %-84 %) in those 19–64 years of age and 49 % (95 % CI, 37 %-62 %) in those ≥ 65 years of age. Responses to the adjuvanted vaccine formulations did not vary by HA content. Antibody responses declined over time and responses against drifted H7N9 strains were diminished. Overall, the vaccines were well tolerated but, as expected, adjuvanted vaccines were associated with more frequent solicited systemic and local adverse events. ConclusionsAS03 adjuvant improved the immune responses to an inactivated fifth–wave H7N9 influenza vaccine, particularly in younger adults, but invoked lower responses to drifted H7N9 strains. These findings may inform future influenza pandemic preparedness strategies.

Study of the immunotoxic properties of pegylated hyaluronidase

The development of safe drugs occupies a special place in the pharmaceutical industry. One of the main tasks of its preclinical phase is to evaluate possible toxic effects of the developed drug on the body and on various systems, including the immune system. The aim of our work was to study immunotoxic properties of pegylated hyaluronidase (PEG- HYAL). Material and methods. Mice F1(CBA/C57Bl/6) were divided into subgroups which were intragastric and intraperitoneally administered with PEG-HYAL in different dosages (50, 500, 1250, 2500 and 5000 U/kg). The number of antibody-producing cells in the spleen, the mass and cellularity of central and peripheral immune organs, phagocytic activity of peritoneal macrophages and neutrophils, delayed hypersensitivity reaction (DHR), level of hemagglutinin to sheep erythrocytes, spontaneous and mitogen-induced splenocyte proliferation were determined. Results. PEG-HYAL did not induce DHR, did not suppress phagocyte activity of peritoneal macrophages, at a dose of 50 ED/kg did not significantly affect the hemagglutinin content to erythrocytes, but at a dose of 500 ED/kg did statistically significantly reduce the titer of specific antibodies. When experimental animals were exposed to PEG-HYAL at doses of 50 and 500 U/kg, spontaneous and mitogen-induced proliferation of splenocytes decreased. Conclusions. The PEG-HYAL trial produced results that can be used to substantiate the administration of the PEG-HYAL-based medication.

Rapid determination of influenza vaccine potency by an SPR-based method using subtype or lineage-specific monoclonal antibodies.

Potency testing and release of annual influenza vaccines require preparation, calibration, and distribution of reference antigens (RAs) and antisera every year, which takes an average of 8 to 12 weeks, and can be a major limiting factor in pandemic situations. Here we describe for the first time a robust Surface Plasmon Resonance (SPR)-based method that employs influenza subtype or lineage hemagglutinin (HA) specific monoclonal antibodies (mAbs) to measure the HA concentration in influenza multivalent vaccines. Implementing such an advanced test method will at the very least eliminate the rate-limiting and laborious efforts of making antisera reagents annually, and thus expedite the influenza vaccine delivery to the public by at least 6 weeks. Results demonstrate that the SPR-based method, developed using Biacore, is robust and not influenced by the type of RAs (inactivated whole virus, split, or subunit vaccine-derived materials), whether they are used as monovalent or multivalent preparations. HA concentrations obtained for monovalent drug substances (DS) or quadrivalent drug products (DP) of inactivated influenza split vaccine showed a tight correlation (the best fit value for the slope is 1.001 with R2 of 0.9815 and P-value <0.0001) with the corresponding values obtained by the current potency assay, Single Radial Immunodiffusion (SRID). Supplementary analysis of the results by the Bland-Altman plot demonstrated good agreement between the SPR and SRID methods, with no consistent bias of the SPR versus SRID method. We further demonstrate that the SPR-based method can be used to estimate HA concentrations in intermediates of the influenza vaccine manufacturing process containing varying matrices and impurity levels. Further, the results demonstrate that the method is sensitive to detecting degradation of HA caused by elevated temperature, low pH, and freezing. It is evident from this report and other published work that the advancement of analytical techniques and the early findings are encouraging for the implementation of alternate potency assays with far-reaching benefits covering both seasonal and pandemic influenza.

Consideration on the usage of full-dose influenza vaccine for the infants aged 6-35 months old

Seasonal influenza has a high disease burden, and children infected with influenza are prone to multiple complications. Influenza vaccination is effective in preventing infection and reducing risks of severe diseases and complications. Influenza vaccines are trivalent and quadrivalent, depending on the components of the vaccine. According to the hemagglutinin content, it can be divided into full dose and half dose of influenza vaccine for children. The findings from clinical trials and real-world studies suggested, the full-dose influenza vaccine as in adults has the same safety profile and higher immunogenicity in children aged 6 to 35 months. The application of full-dose influenza vaccine in children aged 6 to 35 months can greatly improve the flexibility and convenience of vaccination, and help reduce the workload in the process.

Identification of the properties of H5 influenza vaccine viruses with high hemagglutinin yields.

Manufactured influenza vaccines have to contain a defined amount of hemagglutinin (HA) antigen. Therefore, vaccine viruses with a high HA antigen yield (HAY) are preferable for manufacturing vaccines, particularly vaccines in response to a pandemic, when vaccines need to be rapidly produced. However, the viral properties associated with a high HAY have not yet been fully clarified. To identify the HAY-associated traits, we first propagated 26 H5 candidate vaccine viruses (CVVs) in eggs, which were previously developed based on genetic reassortment methods using master viruses, to determine their total protein yield (TPY), ratio of HA to total viral protein (%-HA content) and HAY. The results revealed that the HAY was correlated with the TPY but not with the %-HA content. We further found that altering the sequences of the 3' noncoding region of HA vRNA or replacing the master virus improved the HAYs and TPYs of the low-HAY CVVs to approximately double the values of the original CVVs but did not change the %-HA content, which a previous study suggested was associated with the HAY. Analyses based on real-time PCR assays and scanning electron microscopy revealed that the virus samples with an improved HAY contained more copies of the virus genome and viral particles than the original samples. The results suggest that an improvement in virus growth (i.e., an increase in the amount of viral particles) leads to an increase in the TPY and thus in the HAY, regardless of the %-HA content. The approximately twofold increase in the HAY shown in this study may not appear to represent a large improvement, but the impact will be significant given the millions of chicken eggs used to produce vaccines. These findings will be informative for developing high-HAY vaccine viruses.

Safety and Immunogenicity of Influenza A/H5N8 Virus Vaccine in Healthy Adults: Durability and Cross-reactivity of Antibody Responses.

Influenza A/H5N8 viruses infect poultry and wild birds in many countries. In 2021, the first human A/H5N8 cases were reported. We conducted a phase I, cohort-randomized, double-blind, controlled trial of inactivated influenza A/H5N8 vaccine (clade 2.3.4.4c) administered with or without adjuvant. Cohort 1 subjects received either two doses of AS03-adjuvanted vaccine containing 3.75 μg or 15 μg hemagglutinin (HA); two doses of 15 μg HA unadjuvanted vaccine; or one dose of AS03-adjuvanted vaccine (3.75 μg or 15 μg HA), followed by one dose of non-adjuvanted vaccine (same HA content). Cohort 2 subjects received two doses of MF59-adjuvanted vaccine containing 3.75 μg or 15 μg HA, or 15 μg HA of non-adjuvanted vaccine. Subjects were followed for 13 months for safety and immunogenicity. We enrolled 386 adult subjects in good health. Solicited adverse events were generally mild and more common among subjects who received adjuvanted vaccines. Antibody responses (hemagglutination inhibition or microneutralization assays) were highest in the two-dose AS03 group, followed by the one-dose AS03 group, the MF59 groups, and the non-adjuvanted groups. Antibody levels returned to baseline 12 months after the second vaccination in all groups except the 15 μg AS03-adjuvanted group. Cross-reactive antibodies to clade 2.3.4.4b strains isolated from recent human cases were demonstrated in a subset of both 15 μg adjuvanted groups. Two doses of influenza A/H5N8 vaccine were well-tolerated. Immunogenicity improved with receipt of two doses of adjuvanted vaccine and higher antigen content. (Funded by the National Institute of Allergy and Infectious Diseases.

Inter-batch variability of hemagglutinin content transiently expressed in &lt;i&gt;Nicotiana benthamiana&lt;/i&gt; plants grown under sole-source lighting and sunlight conditions before gene transfer

Inter-batch variability of hemagglutinin content transiently expressed in &lt;i&gt;Nicotiana benthamiana&lt;/i&gt; plants grown under sole-source lighting and sunlight conditions before gene transfer

Bivalent hemagglutinin and neuraminidase influenza replicon particle vaccines protect pigs against influenza a virus without causing vaccine associated enhanced respiratory disease

Alphavirus-derived RNA replicon particle (RP) vaccines represent the next generation of swine influenza A virus (IAV) vaccines, as they were shown to be safe, effective, and offer advantages over traditional vaccine platforms. IAV is a significant respiratory pathogen of swine and there is a critical need to improve current commercial swine IAV vaccine platforms. Adjuvanted whole inactivated virus (WIV) IAV swine vaccines provide limited heterologous protection and may lead to vaccine-associated enhanced respiratory disease (VAERD). This study investigated the ability of RP IAV hemagglutinin (HA) vaccines to avoid VAERD and evaluated experimental multivalent HA and neuraminidase (NA) RP vaccines. RP vaccines were formulated with HA or NA heterologous or homologous to the challenge virus in monovalent HA or HA and NA bivalent combinations (HA/NA bivalent). Pigs were vaccinated with an HA RP, HA/NA bivalent RP, or heterologous HA WIV, followed by IAV challenge and necropsy 5 days post infection. RP vaccines provided homologous protection from challenge and induced robust peripheral and local antibody responses. The RP vaccine did not induce VAERD after challenge with a virus containing the heterologous HA, in contrast to the traditional WIV vaccine. The HA monovalent and HA/NA bivalent RP vaccines showed superior protection compared to traditional WIV. Additionally, the RP platform allows greater flexibility to adjust HA and NA content to reflect circulating IAV in swine antigenic diversity.

Development of the H3N2 influenza microneedle vaccine for cross-protection against antigenic variants

Due to the continuously mutating nature of the H3N2 virus, two aspects were considered when preparing the H3N2 microneedle vaccines: (1) rapid preparation and (2) cross-protection against multiple antigenic variants. Previous methods of measuring hemagglutinin (HA) content required the standard antibody, thus rapid preparation of H3N2 microneedle vaccines targeting the mutant H3N2 was delayed as a result of lacking a standard antibody. In this study, H3N2 microneedle vaccines were prepared by high performance liquid chromatography (HPLC) without the use of an antibody, and the cross-protection of the vaccines against several antigenic variants was observed. The HA content measured by HPLC was compared with that measured by ELISA to observe the accuracy of the HPLC analysis of HA content. The cross-protection afforded by the H3N2 microneedle vaccines was evaluated against several antigenic variants in mice. Microneedle vaccines for the 2019–20 seasonal H3N2 influenza virus (19–20 A/KS/17) were prepared using a dip-coating process. The cross-protection of 19–20 A/KS/17 H3N2 microneedle vaccines against the 2015–16 seasonal H3N2 influenza virus in mice was investigated by monitoring body weight changes and survival rate. The neutralizing antibody against several H3N2 antigenic variants was evaluated using the plaque reduction neutralization test (PRNT). HA content in the solid microneedle vaccine formulation with trehalose post-exposure at 40℃ for 24 h was 48% and 43% from the initial HA content by HPLC and ELISA, respectively. The vaccine was administered to two groups of mice, one by microneedles and the other by intramuscular injection (IM). In vivo efficacies in the two groups were found to be similar, and cross-protection efficacy was also similar in both groups. HPLC exhibited good diagnostic performance with H3N2 microneedle vaccines and good agreement with ELISA. The H3N2 microneedle vaccines elicited a cross-protective immune response against the H3N2 antigenic variants. Here, we propose the use of HPLC for a more rapid approach in preparing H3N2 microneedle vaccines targeting H3N2 virus variants.

Aptasensor for Detection of Influenza-A in Human Saliva.

Access to low-cost, rapid, individualized diagnostics at point-of-care and point-of-need is vital to minimize the impact of highly infectious viruses, such as influenza. Herein, a biosensor for detecting hemagglutinin (HA), an abundant capsid protein in H1N1 viruses, is demonstrated. A gold working electrode was functionalized with a thiol-modified, HA-binding aptamer derivatized with a methylene blue modification for redox reporting. The aptamer was characterized by surface plasmon resonance to confirm its biorecognition activity for HA. The aptasensor was characterized by square wave voltammetry to quantify the sensor's response to varying concentrations of HA. The sensor exhibited a lower limit of detection of 1.5 pM with linear detection of up to 1.2 nM in both Tris buffer and simulated human saliva, thus encompassing the clinically relevant HA range in saliva. Average sensitivity was measured at 21.083 nA·nM-1in Tris and 14.5 nA·nM-1in artificial saliva across clinically relevant HA titers. Sensor stability across time was also investigated, providing a preliminary understanding of the translational viability of the aptasensors for mobile and remote diagnostic applications.

ARHGAP1 Transported with Influenza Viral Genome Ensures Integrity of Viral Particle Surface through Efficient Budozone Formation.

ABSTRACTInfluenza viral particles are assembled at the plasma membrane concomitantly with Rab11a-mediated endocytic transport of viral ribonucleoprotein complexes (vRNPs). The mechanism of spatiotemporal regulation of viral budozone formation and its regulatory molecules on the endocytic vesicles remain unclear. Here, we performed a proximity-based proteomics approach for Rab11a and found that ARHGAP1, a Rho GTPase-activating protein, is transported through the Rab11a-mediated apical transport of vRNP. ARHGAP1 stabilized actin filaments in infected cells for the lateral clustering of hemagglutinin (HA) molecules, a viral surface membrane protein, to the budozone. Disruption of the HA clustering results in the production of virions with low HA content, and such virions were less resistant to protease and had enhanced antigenicity, presumably because reduced clustering of viral membrane proteins exposes hidden surfaces. Collectively, these results demonstrate that Rab11a-mediated endocytic transport of ARHGAP1 with vRNPs stimulates budozone formation to ensure the integrity of virion surface required for viral survival.

Anti-nutrient reduction and nutrient retention capacity of fermentation, germination and combined germination-fermentation in legume processing

Research has proven that the processing of legumes improves nutritional value. However, the extent of anti-nutrient reduction and nutrient retention of combined processing methods compared to single processing methods has not been extensively studied. Therefore, this study investigated the anti-nutrient reduction and nutrient retention potentials of fermentation, germination and combined germination/fermentation in pigeon pea flour. Pigeon pea seeds were naturally fermented, germinated, and further fermented for 24 and 48 h separately. The processed flour was analyzed for nutrients and anti-nutrients composition. Ranges of phytate, tannin and hemagglutinin contents of raw and processed pigeon pea flour were 97–1235 mg/100 g, 49–304 mg/100 g and 18.69–59.29 µg/mg, respectively. Fermentation (96%) and germination (67%) reduced significantly more anti-nutrients compared to the combined processes (42%). Calcium content of raw pigeon pea sample (22.93 mg/100 g) increased significantly with fermentation (56%), germination-48 h fermentation (16%) and germination (11%). Iron content (18.88 mg/100g) decreased significantly in germinated (7%) and germinated-48 h fermented (5%) samples. The treatments had significant (p < 0.05) negative effects on zinc and Vitamin A levels by 9–23% and 5–17%, respectively. Fermentation was effective in the processing of pigeon pea. However, enrichment of fermented pigeon pea flour with zinc and Vitamin A is recommended.

A flow-through chromatography purification process for Vero cell-derived influenza virus (H7N9)

Immunization is the most effective way to respond to an influenza epidemic. To produce Vero cell-derived influenza vaccines, a more efficient, stable and economical purification process is required. In this study, we purified the H7N9 influenza virus grown in Vero cells that were cultured in a serum-free medium by using a combination of anion exchange chromatography (AEC) and ligand-activated core chromatography (LCC), which avoids the virus capture step. After purification, 99.95 % host cell DNA (hcDNA) (final concentration: 28.69 pg/dose) and 98.87 % host cell protein (HCP) (final concentration: 28.28 ng/dose) were removed. The albumin content was 11.36 ng/dose. All these remnants met the current Chinese Pharmacopoeia and WHO requirements. The final virus recovery rate was 58.74 %, with the concentration of hemagglutinin recorded at 132.12 μg/mL. The flow-through chromatography purification process represents an alternative to the existing processes for cell-derived influenza viruses and might be suitable for the purification of other viruses as well.

Protective Efficacy of Recombinant Influenza Hemagglutinin Ectodomain Fusions.

In current seasonal influenza vaccines, neutralizing antibody titers directed against the hemagglutinin surface protein are the primary correlate of protection. These vaccines are, therefore, quantitated in terms of their hemagglutinin content. Adding other influenza surface proteins, such as neuraminidase and M2e, to current quadrivalent influenza vaccines would likely enhance vaccine efficacy. However, this would come with increased manufacturing complexity and cost. To address this issue, as a proof of principle, we have designed genetic fusions of hemagglutinin ectodomains from H3 and H1 influenza A subtypes. These recombinant H1-H3 hemagglutinin ectodomain fusions could be transiently expressed at high yield in mammalian cell culture using Expi293F suspension cells. Fusions were trimeric, and as stable in solution as their individual trimeric counterparts. Furthermore, the H1-H3 fusion constructs were antigenically intact based on their reactivity with a set of conformation-specific monoclonal antibodies. H1-H3 hemagglutinin ectodomain fusion immunogens, when formulated with the MF59 equivalent adjuvant squalene-in-water emulsion (SWE), induced H1 and H3-specific humoral immune responses equivalent to those induced with an equimolar mixture of individually expressed H1 and H3 ectodomains. Mice immunized with these ectodomain fusions were protected against challenge with heterologous H1N1 (Bel/09) and H3N2 (X-31) mouse-adapted viruses with higher neutralizing antibody titers against the H1N1 virus. Use of such ectodomain-fused immunogens would reduce the number of components in a vaccine formulation and allow for the inclusion of other protective antigens to increase influenza vaccine efficacy.

IFITM3 incorporation sensitizes influenza A virus to antibody-mediated neutralization.

The disease severity of influenza is highly variable in humans, and one genetic determinant behind these differences is the IFITM3 gene. As an effector of the interferon response, IFITM3 potently blocks cytosolic entry of influenza A virus (IAV). Here, we reveal a novel level of inhibition by IFITM3 in vivo: We show that incorporation of IFITM3 into IAV particles competes with incorporation of viral hemagglutinin (HA). Decreased virion HA levels did not reduce infectivity, suggesting that high HA density on IAV virions may be an antagonistic strategy used by the virus to prevent direct inhibition. However, we found that IFITM3-mediated reduction in HA content sensitizes IAV to antibody-mediated neutralization. Mathematical modeling predicted that this effect decreases and delays peak IAV titers, and we show that, indeed, IFITM3-mediated sensitization of IAV to antibody-mediated neutralization impacts infection outcome in an in vivo mouse model. Overall, our data describe a previously unappreciated interplay between the innate effector IFITM3 and the adaptive immune response.

Simulation for Production of Syariah-Compliant Influenza Vaccine using Trypsin and Acutase in Batch Reactor via SuperPro Designer

The halal status of the vaccine is being questionable among the Muslim community around the world. Some of them even took a risk not to take the vaccine when this issue is resurrected. This is due to the uses of porcine trypsin that comes from the prohibited animals which is pancreas (pig). The main aim of this study is to find the effectiveness of alternative enzyme (Acutase™) that comes from invertebrate sources in replacing the trypsin™ enzyme. Two modes of operation which are batch and continuous mode were compared to determine the high production rate of the vaccines. The production of syariah-compliant vaccine mainly focused on the bioreactor and simulated by using a software called SuperPro® Designer. The different reactions of time ranging from 1-5 days were tested to each enzyme. The hemagglutinin (HA) concentration gained will form the growth curves of the cell. The HA concentration data from the simulation indicates the number of cell growth in the cell culture process. Result shows a batch reactor can achieve until 60% of the yield production. The curves show the highest concentration of cell by using Acutase™ is at day 3 which is 1.17 X 106 cells/mL. Meanwhile, 0.98 x 106 cells/mL produced by trypsin enzyme at day 4. Total hemagglutinin produced is calculated by using the following standard condition, 1 HA unit corresponds to 104 cells per mL. Therefore, the hemagglutinin (HA) concentration produced by using acutase™ is 117 Unit HA compared to the trypsin™ which is 98.8 Unit HA. The results prove that the production of syariah compliant influenza vaccine can be produced in the future and enhance the uses of the vaccine among Muslim community.