Μg Hemagglutinin Research Articles

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.

Novel 3D integrated microfluidic device: Design, construction, and application to the preparation of liposomes for vaccine delivery

As a kind of nanovesicles, liposomes are considered to be the most promising delivery vehicles for drugs and vaccines. The preparation of liposomes using microfluidic chips is one of the most effective and feasible methods. Removal of solvents and non-encapsulated drugs is usually required after liposome formation, which will affect the storage stability and medical safety of liposomes, thus hindering their industrial production and clinical application. Therefore, innovative preparation techniques with multiple integrated functions are of practical significance to address the challenges of traditional and latest developed liposome preparation technologies. A novel three-dimensional integrated microfluidic device (3D-mf device) reported here can be used as a continuous process flow system for liposome preparation and purification. The microchannel array of the microdevice is 4 × 3, and the computational fluid dynamics simulation is used to determine that the ratio of the four filter area channels is 1: 0.75: 0.5: 0.2. Drug-loaded liposomes with an average particle size of 208 nm and polydispersity index (PDI) of 0.211 were prepared by adjusting the total flow rate (TFR) and flow rate ratio (FRR). The removal rates of ethanol and polyinosinic acid: polycytidylic acid [poly (I: C)] after a single cycle were 66.8 % and 56.3 %, respectively. The size distribution and zeta potential of liposomes keep stable within 28 days. Finally, in an immunization study in rats, hemagglutination inhibition titers (HI, ≥512) were found to be significantly higher than those required for protection after two rounds of immunization with 6 μg of hemagglutinin from a split influenza vaccine with poly (I: C) liposomes. This innovative integrated device shows the potential for industrial production of nano-pharmaceutical applications.

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.

Influenza A(H7N9) Pandemic Preparedness: Assessment of the Breadth of Heterologous Antibody Responses to Emerging Viruses from Multiple Pre-Pandemic Vaccines and Population Immunity.

Influenza A(H7N9) viruses remain as a high pandemic threat. The continued evolution of the A(H7N9) viruses poses major challenges in pandemic preparedness strategies through vaccination. We assessed the breadth of the heterologous neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses using the 1st wave vaccine sera from 4 vaccine groups: 1. inactivated vaccine with 2.8 μg hemagglutinin (HA)/dose + AS03A; 2. inactivated vaccine with 5.75 μg HA/dose + AS03A; 3. inactivated vaccine with 11.5 μg HA/dose + MF59; and 4. recombinant virus like particle (VLP) vaccine with 15 μg HA/dose + ISCOMATRIX™. Vaccine group 1 had the highest antibody responses to the vaccine virus and the 3rd/5th wave drifted viruses. Notably, the relative levels of cross-reactivity to the drifted viruses as measured by the antibody GMT ratios to the 5th wave viruses were similar across all 4 vaccine groups. The 1st wave vaccines induced robust responses to the 3rd and Pearl River Delta lineage 5th wave viruses but lower cross-reactivity to the highly pathogenic 5th wave A(H7N9) virus. The population in the United States was largely immunologically naive to the A(H7N9) HA. Seasonal vaccination induced cross-reactive neuraminidase inhibition and binding antibodies to N9, but minimal cross-reactive antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies to A(H7N9).

Prime-boost, double-dose influenza vaccine immunity in COPD: a pilot observational study.

COPD patients are more susceptible to viral respiratory infections and their sequelae, and have intrinsically weaker immune responses to vaccinations against influenza and other pathogens. Prime-boost, double-dose immunisation has been suggested as a general strategy to overcome weak humoral response to vaccines, such as seasonal influenza vaccination, in susceptible populations with weak immunity. However, this strategy, which may also provide fundamental insights into the nature of weakened immunity, has not been formally studied in COPD. We conducted an open-label study of seasonal influenza vaccination in 33 vaccine-experienced COPD patients recruited from established cohorts (mean age 70 (95% CI 66.9-73.2) years; mean forced expiratory volume in 1 s/forced vital capacity ratio 53.4% (95% CI 48.0-58.8%)). Patients received two sequential standard doses of the 2018 quadrivalent influenza vaccine (15 μg haemagglutinin per strain) in a prime-boost schedule 28 days apart. We measured strain-specific antibody titres, an accepted surrogate of likely efficacy, and induction of strain-specific B-cell responses following the prime and boost immunisations. Whereas priming immunisation induced the expected increase in strain-specific antibody titres, a second booster dose was strikingly ineffective at further increasing antibody titres. Similarly, priming immunisation induced strain-specific B-cells, but a second booster dose did not further enhance the B-cell response. Poor antibody responses were associated with male gender and cumulative cigarette exposure. Prime-boost, double-dose immunisation does not further improve influenza vaccine immunogenicity in previously vaccinated COPD patients. These findings underscore the need to design more effective vaccine strategies for COPD patients for influenza.

Effect of Repeat Vaccination on Immunogenicity of Quadrivalent Cell-Culture and Recombinant Influenza Vaccines Among Healthcare Personnel Aged 18-64 Years: A Randomized, Open-Label Trial.

Antibody responses to non-egg-based standard-dose cell-culture influenza vaccine (containing 15 µg hemagglutinin [HA]/component) and recombinant vaccine (containing 45 µg HA/component) during consecutive seasons have not been studied in the United States. In a randomized trial of immunogenicity of quadrivalent influenza vaccines among healthcare personnel (HCP) aged 18-64 years over 2 consecutive seasons, HCP who received recombinant-HA influenza vaccine (RIV) or cell culture-based inactivated influenza vaccine (ccIIV) during the first season (year 1) were re-randomized the second season of 2019-2020 (year 2 [Y2]) to receive ccIIV or RIV, resulting in 4 ccIIV/RIV combinations. In Y2, hemagglutination inhibition antibody titers against reference cell-grown vaccine viruses were compared in each ccIIV/RIV group with titers among HCP randomized both seasons to receive egg-based, standard-dose inactivated influenza vaccine (IIV) using geometric mean titer (GMT) ratios of Y2 post-vaccination titers. Y2 data from 414 HCP were analyzed per protocol. Compared with 60 IIV/IIV recipients, 74 RIV/RIV and 106 ccIIV/RIV recipients showed significantly elevated GMT ratios (Bonferroni corrected P < .007) against all components except A(H3N2). Post-vaccination GMT ratios for ccIIV/ccIIV and RIV/ccIIV were not significantly elevated compared with IIV/IIV except for RIV/ccIIV against A(H1N1)pdm09. In adult HCP, receipt of RIV in 2 consecutive seasons or the second season was more immunogenic than consecutive egg-based IIV for 3 of the 4 components of quadrivalent vaccine. Immunogenicity of ccIIV/ccIIV was similar to that of IIV/IIV. Differences in HA antigen content may play a role in immunogenicity of influenza vaccination in consecutive seasons. NCT03722589.

Antibody responses against heterologous A/H5N1 strains for an MF59-adjuvanted cell culture–derived A/H5N1 (aH5N1c) influenza vaccine in healthy pediatric subjects

BackgroundVaccines are the main prophylactic measure against pandemic influenza. Adjuvanted, cell culture–derived vaccines, which are not subject to limitations of egg-based vaccine production, have the potential to elicit an antibody response against heterologous strains and may be beneficial in the event of an A/H5N1 pandemic. MethodsA prespecified exploratory analysis of data from a phase 2, randomized, controlled, observer-blind multicenter trial (NCT01776554) to evaluate the immunogenicity of a MF59-adjuvanted, cell culture–based A/H5N1 influenza vaccine (aH5N1c), containing 7.5 µg hemagglutinin antigen per dose, in subjects 6 months through 17 years of age was conducted. Geometric mean titers (GMT) were determined using hemagglutination inhibition (HI) and microneutralization (MN) assays, and proportions of patients achieving seroconversion, HI and MN titers ≥ 1:40, and a 4-fold increase in MN titers against 5 heterologous strains (influenza A/H5N1 Anhui/2005, Egypt/2010, Hubei/2010, Indonesia/2005, and Vietnam/1203/2004) three weeks after administration of the second dose were assessed. ResultsAfter the second dose, HI GMTs against heterologous strains increased between 8- and 40-fold, and MN GMTs increased 13- to 160-fold on Day 43 vs Day 1. On Day 43, 32–72% of subjects had HI titers ≥ 1:40 and achieved seroconversion against the heterologous strains. Using the MN assay, 84–100% of subjects had MN titers ≥ 1:40 and 83–100% achieved an at least 4-fold increase in MN titers against the heterologous strains. The highest responses were consistently against A/H5N1 Egypt/2010. ConclusionsWhen given to children aged 6 months through 17 years, aH5N1c resulted in increased immunogenicity from baseline against all 5 heterologous A/H5N1 strains tested, demonstrating the potential of an MF59-adjuvanted, cell-derived A/H5N1 vaccine to provide cross-protection against other A/H5N1 strains (NCT01776554).

Experimental Intranasal Immunization against Respiratory Viruses

The most common method of influenza prevention is intramuscular administration of vaccines, which causes a higher antibody response than subcutaneous. However, such routes of antigens administration result in the predominant formation of serum IgG against influenza viruses, while intranasal administration promotes higher titers of both IgG and IgA than intramuscular vaccination. Based on the fact that this infectious agent enters the body through the mucous membranes of the respiratory tract, we developed the concept of local etiologically adequate vaccination, based on the statement that the vaccine should be administered in the same way as the infection, i.e. in cases of respiratory infections it should be intranasal or oral administration of vaccine material. So, the aim of this work was to demonstrate the benefits of local vaccination against respiratory viruses, as well as the use of nanocarriers in such vaccination and possible cross-antigen reactions by hemagglutinin between antigens of influenza virus and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Methods. The study was performed using Wistar rats in 3 series of experiments. At first series we investigated the comparative immune response to influenza Influvac® vaccine (Abbott, The Netherlands) against A and B type influenza viruses, which was administered intranasally, per os and subcutaneously once at a dose of 1.5 μg of hemagglutinin. Animals from group 2were similarly administered with the same amount of vaccine with and without cerium dioxide nanoparticles (CeO2). Animals of group 3 intranasally received an identical volume of sodium chloride solution (placebo control). Rats were removed from the experiment by decapitation one week after the immunization. Tissue homogenates were prepared from the trachea of animals of all groups by homogenization at the rate of 100 mg/mL of 0.9% sodium chloride solution. The homogenates were kept at 2°C for 18 hours and then centrifuged at 120 g for 20 minutes (cold centrifuge NF800R, Turkey). The obtained extracts and sera were stored at a temperature of -20°C until the determination of antibodies titers to hemagglutinins of A and B influenza viruses in the reaction of hemagglutination inhibition and titers of interferons (IFN) -α and -γ and using enzyme-linked immunosorbent assay using Elabscience (USA) reagents and Stat Fax 2100 Microplate Reader (USA). In the 3rd series of experiments, the content of antihemagglutinins in the trachea and serum after immunization of animals with nucleocapsid antigen of SARS-Cov-2 coronavirus (recombinant antigen produced by PJSC SPC “Diaproph-Med”, Ukraine) at a dose of 2.5 μg in 0.2 mL of Hanks’ solution was determined. The antigen was administered intranasally or subcutaneously and then all other steps of the experiments were similar to those described below for the 1st series of experiments. Results. Conducted experimental studies aimed to develop new approaches and technologies for vaccination against respiratory viruses, which enter mainly through the upper respiratory tract, confirm the concept of the feasibility of local intranasal vaccination against influenza and other respiratory viruses. The data obtained during the research confirm more effective appearance of protective local immunity both in terms of humoral immune response and interferon protection of the respiratory tract during intranasal vaccination. The use of cerium dioxide nanoparticles in local vaccination may increase the effectiveness of this approach to stimulate the production of antibodies to influenza virus antigens in the upper respiratory tract. Finally, the advantages of local intranasal immunization with SARS-CoV-2 N-antigens over their systemic administration suggest that local intranasal vaccination against coronavirus antigens may also be more effective than systemic administration of antigens of this virus, which requires further research for clinical trials. Conclusions. Intranasal immunization of animals with influenza A and B virus antigens and N-antigen of SARS CoV-2 is more effective for creating local protective immunity in the respiratory system compared to parenteral administration of the antigen. The use of cerium dioxide nanoparticles together with the vaccine resulted in more effective local immune response to respiratory virus antigens.

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells.

Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10 (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.

Quadrivalent influenza vaccine (Sinovac Biotech) for seasonal influenza prophylaxis

ABSTRACT Introduction Quadrivalent Influenza Vaccine (Sinovac Biotech) is a quadrivalent split-virion-inactivated influenza vaccine approved in China in June 2020 for individuals ≥3 years of age. It contains 15 µg hemagglutinin per strain including A/H1N1, A/H3N2, B/Victoria, and B/Yamagata, which could potentially improve protection against influenza B viruses. Areas covered In this review, we summarize the development of quadrivalent influenza vaccines in China and foreign countries, and assess the immunogenicity and safety from the phase I and III clinical trials of Quadrivalent Influenza Vaccine in individuals ≥3 years of age. We also discuss the potential application of Quadrivalent Influenza Vaccine in young children 6–35 months of age according to the results of the phase III trial. Expert commentary The immunogenicity and safety profiles of Quadrivalent Influenza Vaccine containing two A and two B strains were comparable to the trivalent vaccines for the shared strains. The addition of a second B strain to the trivalent vaccine could induce superior immune responses for the alternate B strain. Since the two B strains co-circulated worldwide, the introduction of quadrivalent influenza vaccines has been expected to be a cost-effective strategy.

Efficacy, immunogenicity, and safety of a plant-derived, quadrivalent, virus-like particle influenza vaccine in adults (18–64 years) and older adults (≥65 years): two multicentre, randomised phase 3 trials

Seasonal influenza remains a substantial public health threat despite the availability of egg-derived and other vaccines. Plant-based manufacturing might address some of the limitations of current vaccines. We describe two phase 3 efficacy studies of a recombinant quadrivalent virus-like particle (QVLP) influenza vaccine manufactured in plants, one in adults aged 18-64 years (the 18-64 study) and one in older people aged 65 years and older (the 65-plus study). We did two randomised, observer-blind, multinational studies in the northern hemisphere in the 2017-18 (the 18-64 study) and 2018-19 (the 65-plus study) influenza seasons. The 18-64 study was done at 73 sites and the 65-plus study was done at 104 sites, both across Asia, Europe, and North America. In the 18-64 study, inclusion criteria were body-mass index less than 40 kg/m2; age 18-64 years at screening visit; and good health. In the 65-plus study, inclusion criteria were body-mass index of maximum 35 kg/m2; aged 65 years or older at screening visit; not living in a rehabilitation centre or care home; and no acute or evolving medical problems. Participants in the 18-64 study were randomly assigned (1:1) to receive either QVLP vaccine (30 μg haemagglutinin per strain) or placebo. Participants in the 65-plus study were randomly assigned (1:1) to receive QVLP vaccine (30 μg haemagglutinin per strain) or quadrivalent inactivated vaccine (QIV; 15 μg haemagglutinin per strain). The primary outcome in the 18-64 study was absolute vaccine efficacy to prevent laboratory-confirmed, respiratory illness caused by antigenically matched influenza strains. The primary outcome in the 65-plus study was relative vaccine efficacy to prevent laboratory-confirmed influenza-like illness caused by any influenza strain. The primary analyses were done in the per-protocol population and safety was assessed in all participants who received the assigned treatment. These studies are registered with ClinicalTrials.gov (18-64 study NCT03301051; 65-plus study NCT03739112). In the 18-64 study, between Aug 30, 2017, and Jan 15, 2018, 10 160 participants were randomly assigned to receive either QVLP vaccine (5077 participants) or placebo (5083 participants). The per-protocol population consisted of 4814 participants in the QVLP group and 4812 in the placebo group. The study did not meet its primary endpoint of 70% absolute vaccine efficacy for the QVLP vaccine (35·1% [95% CI 17·9 to 48·7]) against respiratory illness caused by matched strains. 55 (1·1%) of 5064 participants in the QVLP group versus 51 (1·0%) of 5072 in the placebo group had a serious adverse event. Four (0·1%) and six [0·1%] participants had severe treatment-related treatment-emergent adverse events. In the 65-plus study, between Sept 18, 2018, and Feb 22, 2019, 12 794 participants were randomly assigned to receive either QVLP vaccine (6396 participants) or QIV (6398 participants). The per-protocol population consisted of 5996 participants in the QVLP group and 6026 in the QIV group. The study met its primary non-inferiority endpoint with a relative vaccine efficacy of the QVLP vaccine for the prevention of influenza-like illness caused by any strain of 8·8% (-16·7 to 28·7). 263 (4·1%) of 6352 participants in the QVLP group versus 266 (4·2%) of 6366 in the QIV group had serious adverse events (one [<0·1%] vs two [<0·1%] were considered treatment-related); one (<0·1%) versus three (<0·1%) participants had severe treatment-related treatment-emergent adverse events. These efficacy studies are the first large-scale studies of any plant-derived human vaccine. Together, they show that the plant-derived QVLP vaccine can provide substantial protection against respiratory illness and influenza-like illness caused by influenza viruses in adults. QVLP vaccine was well tolerated and no major safety signal arose in participants who received QVLP vaccine across the two studies. Medicago.

Non-neutralizing antibody responses following A(H1N1)pdm09 influenza vaccination with or without AS03 adjuvant system.

BackgroundNon‐neutralizing antibodies inducing complement‐dependent lysis (CDL) and antibody‐dependent cell‐mediated cytotoxicity (ADCC) activity may contribute to protection against influenza infection. We investigated CDL and ADCC responses in healthy adults randomized to receive either non‐adjuvanted or AS03‐adjuvanted monovalent A(H1N1)pdm09 vaccine (containing 15 µg/3.75 μg of hemagglutinin, respectively) on a 2‐dose schedule 21 days apart.MethodsWe conducted an exploratory analysis of a subset of 106 subjects having no prior history of A(H1N1)pdm09 infection or seasonal influenza vaccination enrolled in a previously reported study (NCT00985673). Antibody responses against the homologous A/California/7/2009 (H1N1) vaccine strain and a related A/Brisbane/59/2007 (H1N1) seasonal influenza strain were analyzed up to Day 42.ResultsBaseline seropositivity determined with hemagglutination inhibition (HI), CDL and ADCC antibody titers against viral strains was high; A/California/7/2009 (HI [40.4‐48.1%]; CDL [34.6‐36.0%]; ADCC [92.1‐92.3%]); A/Brisbane/59/2007 (HI [73.1‐88.9%]; CDL [38.0‐42.0%]; ADCC [86.8‐97.0%]). CDL seropositivity increased following vaccination with both adjuvanted and non‐adjuvanted formulations (A/California/7/2009 [95.9‐100%]; A/Brisbane/59/2007 [75.5‐79.6%]). At Day 21, increases in CDL and ADCC antibody geometric mean titers against both strains were observed for both formulations. After 2 doses of AS03‐adjuvanted vaccine, vaccine responses of 95.8% (≥9‐fold increase from baseline in CDL titers) and 34.3% (≥16‐fold increase from baseline in ADCC titers) were seen against A/California/7/2009; and 22.4% and 42.9%, respectively, against A/Brisbane/59/2007. Vaccine responses after 2 doses of the non‐adjuvanted vaccine were broadly similar.ConclusionsBroadly comparable non‐neutralizing immune responses were observed following vaccination with non‐adjuvanted and AS03‐adjuvanted A(H1N1)pdm09 formulations; including activity against a related vaccine strain.

Safety and immunogenicity of an alum-adjuvanted whole-virion H7N9 influenza vaccine: a randomized, blinded, clinical trial

ObjectivesA case of H7N9 influenza virus infection was first identified in China in 2013. This virus is considered to have high pandemic potential. Here we developed an H7N9 influenza vaccine containing an aluminium adjuvant and evaluated the safety and immunogenicity of the vaccine. MethodsFrom October 2017 through August 2018 we conducted a randomized, double-blinded, single-centre phase I clinical trial in China among 360 participants aged ≥12 years. All participants received two doses of the vaccine (7.5, 15 or 30 μg haemagglutinin antigen) or placebo at an interval of 21 days. Adverse event data were collected for 30 days after vaccination. Serum samples were collected on days 0, 21 and 42 for the haemagglutinin inhibition (HI) antibody assay. ResultsA total of 347 participants (347/360, 96.4%) completed the study. The proportions of vaccine-related adverse events after one injection were 56.7% (34/60) in the 7.5-μg group, 86.7% (52/60) in the 15-μg group and 86.7% (52/60) in the 30-μg group. The proportions of adverse events after two injections were less than those reported after the first dose. None of the serious adverse events were related to the vaccine. After receiving two doses of the 7.5-μg vaccine, the proportion of participants achieving an HI titre of ≥40 was 98.2% (55/56, 95%CI 72.3~100.0%), with a geometric mean titre (GMT) of 192.6 (95%CI 162.9~227.8). ConclusionsThe alum-adjuvanted H7N9 whole-virion inactivated vaccine was safe and strongly immunogenic in a population aged ≥12 years.

GLYCOSYLATION SITE OCCUPANCY OF THE HEMAGGLUTININ GLOBULAR HEAD IS IMPLICATED IN THE IMMUNE RESPONSE TO INFLUENZA A VIRUS

Abstract Objective N-linked glycosylation of the hemagglutinin (HA) affects host immune responses to influenza A virus (IAV). This study determines the significance of N-linked glycosylation site occupancy of the HA globular head in the generation of effective antibody responses to IAV in mice. Methods We grew A(H3N2)2013 IAV in the presence of an inhibitor of N-linked glycosylation, NGI-1, which reduces glycosylation in mammalian cells by targeting the oligosaccharyltransferase. Mass spectrometry-based analysis of this virus revealed reduction of glycosylation in six out of twelve HA N-linked glycosylation sites, compared to virus grown without the inhibitor. The globular head of HA was affected by NGI-1 treatment. The ability of NGI-1-treated HA to generate immune responses was assessed by immunization of naïve mice with two 10 μg HA doses followed by measurement of serum antibody titers by hemagglutination inhibition and ELISA tests. Results Reduced occupancy of N-linked glycosylation sites IAV HA globular head correlated with a significant reduction in cross-strain antibody responses. Serum antibody titers directed toward recombinant HAs from A(H3N2) IAVs of 2003–2013 were from 2 to 4 times lower in mice immunized with HA from NGI-1-treated virus than in the mice immunized with HA from the fully glycosylated virus. Similarly, titers of the antibodies that are correlated with protection against influenza disease, measured by hemagglutination inhibition assays, were significantly lower in mice immunized with NGI-treated virus than those immunized with fully-glycosylated virus. Conclusion Glycosylation site occupancy of the HA globular head affects the immune response to IAV.

Identification of an Indiscriminate Peptide by Phage Display Technology

Phage display has been widely used to develop high‐affinity peptide ligands against proteins and cell lines. Such peptides are typically selected using biopanning to exhibit significant binding affinity and specificity against a molecular target. Rarely are the same peptides identified from separate phage display selections, since the presence of these ligands in a selection typically signify specific binding to the target. However, here, we report the identification of the same peptide (TG1) from two different phage display selections against the hemagglutinin (HA) epitope and human pancreatic cancer (Mia Paca‐2) cells.Phage display selections were conducted to identify a peptide with high affinity and specificity for the HA epitope. In brief, a pre‐cleared 15‐mer fUSE5 phage library was subjected to four rounds of selections against 0.04–4 μg HA. Following the last round of selection, the phage DNA was amplified using PCR and submitted to next‐generation sequencing. These results identified peptide TG1 (RNVPPIFNDVYWIAF) as the most prevalent ligand in the selection. Modified enzyme‐linked immunosorbent assay (ELISA) results showed that 109 nM TG1 bound significantly higher (p&lt;0.05) to 5 μg immobilized HA compared to a non‐relevant peptide J18 known to bind ovarian adenocarcinoma (SKOV‐3) cells. In a separate, previously reported phage display selection, the same 15‐mer fUSE5 phage library was subjected to selections against human pancreatic cancer (Mia‐Paca‐2) cells. Again, next‐generation sequencing results identified TG1 as the third most prevalent peptide. Modified ELISA results using 10 μM TG1, and a known pancreatic cancer‐targeting peptide MCA2, showed that both ligands bound significantly (p&lt;0.01) to Mia‐Paca‐2 cells compared to dimethyl sulfoxide (DMSO; vehicle). A literature search revealed that TG1 was identified by two separate research groups conducting phage display selections against caveolin protein and mouse embryos. These results indicate that the peptide is an indiscriminate binder. Thus, we sought to elucidate the binding properties of peptide TG1 and phage TG1 (pTG1).Modified ELISA with 10 μM TG1, MCA2, J18, or DMSO were carried out against SKOV‐3 and human normal pancreatic (hTERT‐HPNE) cells. Results showed that TG1 bound significantly higher (p&lt;0.01) to both SKOV‐3 and hTERT‐HPNE cells compared to DMSO. As expected MCA2 showed no binding to either cell line. Peptide J18 showed significant binding (p&lt;0.05) to SKOV‐3 cells.Phage qPCR analysis was performed to further elucidate the binding properties of the pTG1. 1010 V/mL pTG1, pancreatic cancer‐targeting pMCA1, or WT phage was incubated with Mia‐Paca‐2, SKOV‐3, or hTERT‐HPNE cells. After washing with Tris‐buffered saline (TBS), bound phage were eluted with 2.5% 3‐cholamidopropyl dimethylammonio 1‐propanesulfonate (CHAPS). Collected phage virions (5 μL) were quantified by qPCR using Fast SYBR Green Master Mix on an Applied Biosystems StepOnePlus Real‐Time qPCR System (Applied Biosystems, CA). The results revealed that pTG1 demonstrated significantly higher binding (p&lt;0.01) compared to WT for all cell lines. As expected, pMCA1 showed binding to Mia‐Paca‐2, and pJ18 exhibited binding to SKOV‐3 cells. Taken together, these results indicate that TG1 indiscriminately binds multiple targets.

Nano-emulsion as a vaccine adjuvant can enhance the humoral immunity against influenza in aged and young mice

Objective To evaluate the immunogenicity of split influenza H1N1 vaccine formulated with an oil-in-water nano-emulsion adjuvant in aged mice and young mice. Methods A nano-emulsion adjuvant formulated split influenza H1N1 vaccine was used to immunize aged and young mice through intramuscular injection. Each mouse was immunized with 0.012 μg of hemagglutinin (HA) twice with an interval of 28 d. Hemagglutination inhibition (HI) titers in serum were measured 27 d after first immunization. Serum HI, IgG1 and IgG2a titers were detected 14 d after the last immunization. No adjuvant-formulated vaccine and normal saline (NS) were used to set up control groups. Virus challenge test was carried out using 10 times the median lethal dose (LD50) of A/Puerto Rico/8/34 (H1N1) strain two weeks after the last immunization and the protective effects were assessed through measuring the dynamic changes in body weight and survival rate. Results Higher levels of serum HI, IgG1 and IgG2a antibodies and higher HI antibody conversion rates were induced in the adjuvant groups, especially in the aged mice group, than in the control groups. Nano-emulsion adjuvant improved the immunogenicity of HA and mouse immunity to A/Puerto Rico/8/34 (H1N1). Conclusions Nano-emulsion adjuvant could enhance the immunogenicity of influenza antigens, especially in aged mice. Key words: Adjuvant; Influenza; Nano-emulsion adjuvant; Aged mice; Immunogenicity

Comparison of immunogenicity between intradermal and intramuscular injections of repeated annual identical influenza virus strains post-pandemic (2011-2012) in COPD patients

ABSTRACT We compared the antibody responses and persistence of the reduced-dose, 9 µg hemagglutinin (HA)/strain intradermal (ID) injection via the Mantoux technique and the 15 μg HA/strain intramuscular (IM) injection of the repeated annual identical trivalent, inactivated, split-virion vaccine 2011–2012 in chronic obstructive pulmonary disease (COPD) patients. Eighty patients were randomized to ID (n = 41) and IM (n = 39) groups. Four weeks post-vaccination, the antibody responses of the two groups were similar; those for influenza A(H1N1)pdm09 and influenza A(H3N2)–but not influenza B–met the criteria of the Committee for Proprietary Medicinal Products (CPMP). The antibody responses for influenza A(H1N1)pdm09 rapidly declined in both groups, especially with the ID injection, whereas those for influenza A(H3N2) maintained above the CPMP criteria throughout 12 months post-vaccination. The geometric mean titres for influenza A(H1N1)pdm09 persisted above the protective threshold (≥ 40) until 6 months post-vaccination in both the ID and IM groups. The seroprotection rates of the ID and IM groups were above 60% until 3 months and 6 months post-vaccination, respectively. In conclusion, the 9 μg HA/strain ID injection of vaccine 2011–2012 elicited antibody responses similar to the standard dose of 15 μg of the HA/strain IM injection at 4 weeks post-vaccination. However, the antibody responses for influenza A(H1N1)pdm09 rapidly declined, especially in the case of the ID injection, whereas they were comparable for influenza A(H3N2). Additional strategies for increasing vaccine durability should be considered, especially for new pandemic strains affecting elderly COPD patients.

Priming with MF59 adjuvanted versus nonadjuvanted seasonal influenza vaccines in children – A systematic review and a meta-analysis

BackgroundIdentifying optimal priming strategies for children <2 years could substantially improve the public health benefits of influenza vaccines. Adjuvanted seasonal influenza vaccines were designed to promote a better immune response among young vaccine-naïve children. MethodsWe systematically reviewed randomized trials to assess hemagglutination inhibition (HAI) antibody response to MF59-adjuvanted inactivated influenza vaccine (aIIV) versus nonadjuvanted IIV among children. We estimated pooled ratios of post-vaccination HAI geometric mean titer (GMT) for aIIV versus IIV and confidence intervals (CIs) using the pooled variances derived from reported CIs. ResultsMean age was 28 months (range, 6–72 months). Children received vaccines with either 7.5 μg (6–35 months) or 15 μg (≥36 months) hemagglutinin of each strain depending on age. Seven of eight trials administered trivalent vaccines and one used quadrivalent vaccine. Pooled post-vaccination GMT ratios against the three influenza vaccine strains were 2.5–3.5 fold higher after 2-dose-aIIV versus 2-dose-IIV among children 6–72 months, and point estimates were higher among children 6–35 months compared with older children. When comparing 1-dose-aIIV to 2-dose-IIV doses, pooled GMT ratios were not significantly different against A/H1N1 (1.0; 95% CI: 0.5–1.8; p = 0.90) and A/H3N2 viruses (1.0; 95% CI: 0.7–1.5; p = 0.81) and were significantly lower against B viruses (0.6; 95% CI: 0.4–0.8; p < 0.001) for both age groups. Notably, GMT ratios for vaccine-mismatched heterologous viruses after 2-dose-aIIV compared with 2-dose-IIV were higher against A/H1N1 (2.0; 95% CI: 1.1–3.4), A/H3N2 (2.9; 95% CI: 1.9–4.2), and B-lineage viruses (2.1; 95% CI: 1.8–2.6). ConclusionsTwo doses of adjuvanted IIV consistently induced better humoral immune responses against Type A and B influenza viruses compared with nonadjuvanted IIVs in young children, particularly among those 6–35 months. One adjuvanted IIV dose had a similar response to two nonadjuvanted IIV doses against Type A influenza viruses. Longer-term benefits from imprinting and cell-mediated immunity, including trials of clinical efficacy, are gaps that warrant investigation.

Preliminary evaluation of adjuvant effects of cyclic dinucleotides (CDN) on immune responses to nasal spray influenza vaccine

Objective To analyze the immunostimulatory effects of cyclic dinucleotides (CDN) on immune responses to a nasal spray influenza split virus vaccine and to evaluate its potential as a mucosal adjuvant. Methods A H1N1 influenza split virus vaccine combined with different CDN was used for mouse immunization. Each mouse was intranasally immunized twice with 4.5 μg of hemagglutinin (HA) and 10 μg of CDN with an interval of 21 d. Titers of hemagglutination inhibition (HI) antibodies in serum, secretory IgA (sIgA) in bronchoalveolar lavage fluid and IgG in serum were detected 21 d after the last immunization. Immunostimulatory activities of different CDN were compared. Effects of cyclic di-GMP (c-di-GMP) and chitosan (CSN) on the immunogenicity of H1N1 and H7N9 influenza split vaccines were analyzed and compared. H1N1 influenza split vaccine combined with c-di-GMP or CSN was used to immunize mice. Three weeks after the last immunization, these mice were challenged with 10 times the median lethal dose (LD50) of A/Puerto Rico/8/34 (H1N1) influenza virus. Survival rates of the mice were observed for 14 d. Results All three CDN induced high levels of HI antibodies and IgG in serum and sIgA in BALF. HI antibody seroconversion rates were also higher than those of the control groups. c-di-GMP was superior to CSN in enhancing the immunogenicity of H1N1 and H7N9 antigens as higher titers of HI antibodies in serum and sIgA in BALF were induced. Conclusions CDN could enhance the immunogenicity of influenza antigens with better efficacy than CSN adjuvant. Key words: Cyclic dinucleotide; Adjuvant; Chitosan; Immunogenicity

Efficacy of A/H1N1/2009 split inactivated influenza A vaccine (GC1115) in mice and ferrets.

To evaluate the efficacy of a non-adjuvant A/H1N1/2009 influenza A vaccine (GC1115), we demonstrated the immunogenicity and protective efficacy of GC1115 in mouse and ferret models. The immunogenicity of GC1115 was confirmed after intramuscular administration of 1.875, 3.75, 7.5, and 15 μg hemagglutinin antigen (HA) in mice and 7.5, 15, and 30 μg HA in ferrets at 3-week intervals. A single immunization with GC1115 at HA doses > 7.5 μg induced detectable seroconversion in most mice, and all mice given a second dose exhibited high antibody responses in a dose-dependent manner. The mice in the mock (PBS) and 1.875 μg HA immunized groups succumbed by 13 days following A/California/ 04/09 infection, while all mice in groups given more than 3.75 μg HA were protected from lethal challenge with the A/California/04/09 virus. In ferrets, although immunization with even a single dose of 15 or 30 μg of HA induced detectable HI antibodies, all ferrets given two doses of vaccine seroconverted and exhibited HI titers greater than 80 units. Following challenge with A/California/04/09, the mock (PBS) immunized ferrets showed influenza-like clinical symptoms, such as increased numbers of coughs, elevated body temperature, and body weight loss, for 7 days, while GC1115- immunized ferrets showed attenuated clinical symptoms only for short time period (3-4 days). Further, GC1115-immunized ferrets displayed significantly lower viral titers in the upper respiratory tract (nasal cavity) than the mock vaccinated group in a dose-dependent manner. Taken together, this study demonstrates the immunogenicity and protective efficacy of GC1115 as a non-adjuvanted vaccine.