Selecting Endogenous Promoters for Improving Biosynthesis of Squalene in Schizochytrium sp.

Squalene (C30H50) is an acyclic triterpenoid compound renowned for its myriad physiological functions, such as anticancer and antioxidative properties, rendering it invaluable in both the food and pharmaceutical sectors. Due to the natural resource constraints, microbial fermentation has emerged as a prominent trend. Schizochytrium sp., known to harbor the intact mevalonate acid (MVA) pathway, possesses the inherent capability to biosynthesize squalene. However, there is a dearth of reported key genes in both the MVA and the squalene synthesis pathways, along with the associated promoter elements for their modification. This study commenced by cloning and characterizing 13 endogenous promoters derived from transcriptome sequencing data. Subsequently, five promoters exhibiting varying expression intensities were chosen from the aforementioned pool to facilitate the overexpression of the squalene synthase gene squalene synthetase (SQS), pivotal in the MVA pathway. Ultimately, a transformed strain designated as SQS-3626, exhibiting squalene production 2.8 times greater than that of the wild-type strain, was identified. Finally, the optimization of nitrogen source concentrations and trace element contents in the fermentation medium was conducted. Following 120 h of fed-batch fermentation, the accumulated final squalene yield in the transformed strain SQS-3626 reached 2.2 g/L.

A novel amphiphilic squalene-based compound with open-chain polyethers reduces malignant melanoma metastasis in-vitro and in-vivo.

Squalene (SQ) is a well-known antioxidant and anti-inflammatory agent that provides promising anti-aging and UV-protective roles on human skin. However, its strong hydrophobic nature, accompanied by issues such as poor solubility and limited tissue permeation, has created challenges for scientists to investigate its untapped potential in more complex conditions, including cancer progression. The present study assessed the potent anti-metastatic properties of a newly synthesized amphiphilic ethylene glycol SQ derivative (SQ-diEG) in melanoma, the most fatal skin cancer. In vitro and in vivo experiments have discovered that SQ-diEG may exert its potential on melanoma malignancy through the mitochondria-mediated caspase activation apoptotic signaling pathway. The potent anti-metastatic effect of SQ-diEG was observed in vitro using highly proliferative and aggressive melanoma cells. Administration of SQ-diEG (25 mg/kg) significantly decreased the tumor burden on the lung and inhibited the metastasis-associated proteins and gene markers in B16F10 lung colonization mice model. Furthermore, global gene profiling also revealed a promising role of SQ-diEG in tumor microenvironment. We anticipated that the amphiphilic nature of the SQ compound bearing ethylene glycol oligomers could potentially augment its ability to reach the pathology site, thus enhancing its therapeutic potential in melanoma.

An innate immune signature induced by AS01- or AS03-adjuvanted vaccines predicts the antibody response magnitude and quality consistently over time.

Background: Antibody-mediated protection can depend on mechanisms varying from neutralization to Fc-dependent innate immune-cell recruitment. Adjuvanted vaccine development relies on a holistic understanding of how adjuvants modulate the quantity/titer and quality of the antibody response. Methods: A Phase 2 trial (ClinicalTrials.gov: NCT00805389) evaluated hepatitis B vaccines formulated with licensed adjuvants (AS01B, AS01E, AS03, AS04 or Alum) in antigen-naïve adults. The trial investigated the role of adjuvants in shaping antibody-effector functions, and identified an innate transcriptional response shared by AS01B, AS01E and AS03. We integrated previously reported data on the innate response (gene expression, cytokine/C-reactive protein levels) and on quantitative/qualitative features of the mature antibody response (Fc-related parameters, immunoglobulin titers, avidity). Associations between the innate and humoral parameters were explored using systems vaccinology and a machine-learning framework. Results: A dichotomy in responses between AS01/AS03 and AS04/Alum (with the former two contributing most to the association with the humoral response) was observed across all timepoints of this longitudinal study. The consistent patterns over time suggested a similarity in the impacts of the two-dose immunization regimen, year-long interval, and non-adjuvanted antigenic challenge given one year later. An innate signature characterized by interferon pathway-related gene expression and secreted interferon-γ-induced protein 10 and C-reactive protein, which was shared by AS01 and AS03, consistently predicted both the qualitative antibody response features and the titers. The signature also predicted from the antibody response quality, the group of adjuvants from which the administered vaccine was derived. Conclusion: An innate signature induced by AS01- or AS03-adjuvanted vaccines predicts the antibody response magnitude and quality consistently over time.

Use of Adjuvant Compositions Based on Squalene Ensures Induction of Neutralizing Antibodies against SARS-CoV-2.

Squalene-based adjuvant compositions that can provide effective induction of specific humoral immune response have been developed. Recombinant receptor-binding domain (RBD) of surface S-protein of SARS-CoV-2 was used to evaluate the properties of the composition. Immunization of mice with the developed squalene-based compositions in combination with RBD allows obtaining high titers of specific antibodies: from 105 to 2×106. The blood sera from immunized mice exhibit neutralizing activity against SARS-CoV-2 Delta variant (B.1.617.2) with a titer up to 1:2000.

Preparation of a novel type I feline coronavirus virus-like particle vaccine and its immunogenicity in mice and cats.

Feline coronavirus (FCoV) infection is a leading cause of death in cats. In this study, we produced FCoV-I virus-like particles (VLPs) containing E, M, N, and S proteins using a baculovirus expression system and mixed VLPs with the adjuvants MF59 and CpG 55.2 to prepare an VLP/MF59/CpG vaccine. After immunization of mice with the vaccine, IgG specific antibodies titers against S and N proteins increased to 1:12,800, and IFN-γ+ and IL-4+ splenocytes were significantly increased. Following immunization of FCoV-negative cats, the S protein antibodies in immunized cats (5/5) increased significantly, with a peak of 1:12,800. Notably, after booster vaccination in FCoV-positive cats, a significant reduction in viral load was observed in the feces of partial cats (4/5), and the FCoV-I negative conversion was found in two immunized cats (2/5). Therefore, the VLP/MF59/CpG vaccine is a promising candidate vaccine to prevent the FCoV infection.

Relative vaccine effectiveness of MF59-adjuvanted vs high-dose trivalent inactivated influenza vaccines for prevention of test-confirmed influenza hospitalizations during the 2017-2020 influenza seasons.

OBJECTIVES: This study evaluated relative vaccine effectiveness (rVE) of MF59-adjuvanted trivalent inactivated influenza vaccine (aTIV) vs high-dose trivalent inactivated influenza vaccine (HD-TIV) for prevention of test-confirmed influenza emergency department visits and/or inpatient admissions ("ED/IP") and for IP admissions alone pooled across the 2017-2020 influenza seasons. Exploratory individual season analyses were also performed. METHODS: This retrospective test-negative design study included United States (US) adults age ≥65 years vaccinated with aTIV or HD-TIV who presented to an ED or IP setting with acute respiratory or febrile illness during the 2017-2020 influenza seasons. Test-positive cases and test-negative controls were grouped by vaccine received. The rVE of aTIV vs HD-TIV was evaluated using a combination of inverse probability of treatment weighting and logistic regression to adjust for potential confounders. RESULTS: Pooled analyses over the three seasons found no significant differences in the rVE of aTIV vs HD-TIV for prevention of test-confirmed influenza ED/IP (-2.5% [-19.6, 12.2]) visits and admissions or IP admissions alone (-1.6% [-22.5, 15.7]). The exploratory individual season analyses also showed no significant differences. CONCLUSIONS: Evidence from the 2017-2020 influenza seasons indicates aTIV and HD-TIV are comparable for prevention of test-confirmed influenza ED/IP visits in US adults age ≥65 years.

Kinetics of Squalene Quenching Singlet Oxygen and the Thermal Degradation Products Identification.

Squalene has been proven to possess various bioactive functions that are widely present in vegetable oils. A more comprehensive understanding of the reaction behavior of squalene under oxidative conditions was achieved by studying its antioxidant capacity and thermal degradation products. The total singlet oxygen quenching rate constant (kr + kq) of squalene was 3.8 × 107 M-1 s-1, and both physical and chemical quenching mechanisms equally contribute to the overall singlet oxygen quenching. Fourteen degradation products of squalene were identified at 180 °C by using gas chromatography-mass spectrometry (GC-MS). Combining with DFT calculations, the thermal degradation pathway of squalene was proposed: the aldehydes, ketones, and alcohols, and epoxy compounds were formed by the homolytic cleavage of squalene hydroperoxides to form alkoxy radicals, followed by ß-scission of the alkoxyl radicals at adjacent C-C bonds or intramolecular cyclization.

Safety and Immunogenicity of a DNA Vaccine With Subtype C gp120 Protein Adjuvanted With MF59 or AS01B: A Phase 1/2a HIV-1 Vaccine Trial.

BACKGROUND: An effective vaccine is required to end the HIV pandemic. We evaluated the safety and immunogenicity of a DNA (DNA-HIV-PT123) vaccine with low- or high-dose bivalent (TV1.C and 1086.C glycoprotein 120) subtype C envelope protein combinations, adjuvanted with MF59 or AS01B. METHODS: HIV Vaccine Trials Network (HVTN)108 was a randomized, placebo-controlled, double-blind, phase 1/2a trial conducted in the United States and South Africa. HIV-negative adults were randomly assigned to 1 of 7 intervention arms or placebo to assess DNA prime with DNA/protein/adjuvant boosts, DNA/protein/adjuvant co-administration, and low-dose protein/adjuvant regimens. HVTN111 trial participants who received an identical regimen were also included. Outcomes included safety and immunogenicity 2 weeks and 6 months after final vaccination. RESULTS: From June 2016 to July 2018, 400 participants were enrolled (N = 334 HVTN108, N = 66 HVTN111); 370 received vaccine and 30 received placebo. There were 48 grade 3 and 3 grade 4 reactogenicity events among 39/400 (9.8%) participants, and 32 mild/moderate-related adverse events in 23/400 (5.8%) participants. All intervention groups demonstrated high IgG response rates (>89%) and high magnitudes to HIV-1 Env gp120 and gp140 proteins; response rates for AS01B-adjuvanted groups approached 100%. V1V2 IgG magnitude, Fc-mediated functions, IgG3 Env response rates, and CD4+ T-cell response magnitudes and rates were higher in the AS01B-adjuvanted groups. The AS01B-adjuvanted low-dose protein elicited greater IgG responses than the higher protein dose. CONCLUSIONS: The vaccine regimens were generally well tolerated. Co-administration of DNA with AS01B-adjuvanted bivalent Env gp120 elicited the strongest humoral responses; AS01B-adjuvanted regimens elicited stronger CD4+ T-cell responses, justifying further evaluation.ClinicalTrials.gov registration: NCT02915016, registered 26 September 2016.

Comparative Study of Camellia oleifera Seed Oil on Chemical Constituents, Antioxidant Activities and Physicochemical Characteristics from Southern China.

Eleven kinds of Camellia oleifera seed oils (CSOs) were evaluated in terms of chemical constituents, antioxidant activities, acid value (AV) as well as peroxide value (POV). These CSOs contained abundant ß-sitosterol, squalene, α-tocopherol and phenolics, in which the squalene was the distinct constituent with the content between 45.8±0.8 and 184.1±5.5 mg/kg. The ß-sitosterol ranging from 143.7±4.8 to 1704.6±72.0 mg/kg contributed a considerable content to total accompaniments. Palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid were present in these CSOs, in which the dominant fatty acid was oleic acid with the content between 59.66±0.72 and 82.89±2.16 g/100 g. The AV ranged from 0.1±0.0 to 1.3±0.0 mg KOH/g, and the POV was between 0.1±0.0 and 1.0±0.0 g/100 g. These CSOs showed antioxidant activity based on DPPH and ABTS radical scavenging assay. Both α-tocopherol and ß-sitosterol contents showed a positive correlation with DPPH and ABTS values, respectively, while the α-tocopherol content showed a negative correlation with AV. These results suggested that CSO can be categorized into high oleic acid vegetable oil with abundant active constituents, of which the quality presented variation among different origins. These accompaniments may contribute to the delay of its quality deterioration.

Metabolic engineering of Saccharomyces cerevisiae for high-level production of (+)-ambrein from glucose.

(+)-Ambrein is the primary component of ambergris, a rare product found in sperm whales (Physeter microcephalus). Microbial production using sustainable resources is a promising way to replace animal extraction and chemical synthesis. We constructed an engineered yeast strain to produce (+)-ambrein de novo. Squalene is a substrate for the biosynthesis of (+)-ambrein. Firstly, strain LQ2, with a squalene yield of 384.4 mg/L was obtained by optimizing the mevalonate pathway. Then we engineered a method for the de novo production of (+)-ambrein using glucose as a carbon source by overexpressing codon-optimized tetraprenyl-ß-curcumene cyclase (BmeTC) and its double mutant enzyme (BmeTCY167A/D373C), evaluating different promoters, knocking out GAL80, and fusing the protein with BmeTC and squalene synthase (AtSQS2). Nevertheless, the synthesis of (+)-ambrein is still limited, causing low catalytic activity in BmeTC. We carried out a protein surface amino acid modification of BmeTC. The dominant mutant BmeTCK6A/Q9E/N454A for the first step was obtained to improve its catalytic activity. The yield of (+)-ambrein increased from 35.2 to 59.0 mg/L in the shake flask and finally reached 457.4 mg/L in the 2 L fermenter, the highest titer currently available for yeast. Efficiently engineered strains and inexpensive fermentation conditions for the industrial production of (+)-ambrein. The metabolic engineering tools provide directions for optimizing the biosynthesis of other high-value triterpenes.

Evaluating Possible Formation Mechanisms of Criegee Intermediates during the Heterogeneous Autoxidation of Squalene.

Organic molecules in the environment oxidatively degrade by a variety of free radical, microbial, and biogeochemical pathways. A significant pathway is heterogeneous autoxidation, in which degradation occurs via a network of carbon and oxygen centered free radicals. Recently, we found evidence for a new heterogeneous autoxidation mechanism of squalene that is initiated by hydroxyl (OH) radical addition to a carbon-carbon double bond and apparently propagated through pathways involving Criegee Intermediates (CI) produced from ß-hydroxy peroxy radicals (ß-OH-RO2•). It remains unclear, however, exactly how CI are formed from ß-OH-RO2•, which could occur by a unimolecular or bimolecular pathway. Combining kinetic models and multiphase OH oxidation measurements of squalene, we evaluate the kinetic viability of three mechanistic scenarios. Scenario 1 assumes that CI are formed by the unimolecular bond scission of ß-OH-RO2•, whereas Scenarios 2 and 3 test bimolecular pathways of ß-OH-RO2• to yield CI. Scenario 1 best replicates the entire experimental data set, which includes effective uptake coefficients vs [OH] as well as the formation kinetics of the major products (i.e., aldehydes and secondary ozonides). Although the unimolecular pathway appears to be kinetically viable, future high-level theory is needed to fully explain the mechanistic relationship between CI and ß-OH-RO2• in the condensed phase.

A squalene analog 4,4'-diapophytofluene from coconut leaves having antioxidant and anti-senescence potentialities toward human fibroblasts and keratinocytes.

Coconut (Cocos nucifera) leaves, an unutilized resource, enriched with valuable bioactive compounds. Spectral analysis of purified pentane fraction of coconut leaves revealed the presence of a squalene analog named 4,4'-diapophytofluene or in short 4,4'-DPE (C30H46). Pure squalene standard (PSQ) showed cytotoxicity after 8 µg/ml concentration whereas 4,4'-DPE exhibited no cytotoxic effects up to 16 µg/ml concentration. On senescence-induced WI38 cells, 4,4'-DPE displayed better percentage of cell viability (164.5% at 24 h, 159.4% at 48 h and 148% at 72 h) compared to PSQ and BSQ (bio-source squalene) with same time duration. Similar trend of result was found in HaCaT cells. SA-ß-gal assay showed that number of ß-galactosidase positive cells were significantly decreased in senescent cells (WI38 and HaCaT) after treated with 4,4'-DPE than PSQ, BSQ. Percentage of ROS was increased to 60% in WI38 cells after olaparib treatment. When PSQ, BSQ and 4,4'-DPE were applied separately on these oxidative-stress-induced cells for 48 h, the overall percentage of ROS was decreased to 39.3%, 45.6% and 19.3% respectively. This 4,4'-DPE was found to be more effective in inhibiting senescence by removing ROS as compared to squalene. Therefore, this 4,4'-DPE would be new potent senotherapeutic agent for pharmaceuticals and dermatological products.

Biosynthesis of carotenoids in Azospirillum brasilense Cd is mediated via squalene (C30) route.

Azospirillum brasilense is a non-photosynthetic α-Proteobacteria, belongs to the family of Rhodospirillaceae and produces carotenoids to protect itself from photooxidative stress. In this study, we have used Resonance Raman Spectra to show similarity of bacterioruberins of Halobacterium salinarum to that of A. brasilense Cd. To navigate the role of genes involved in carotenoid biosynthesis, we used mutational analysis to inactivate putative genes predicted to be involved in carotenoid biosynthesis in A. brasilense Cd. We have shown that HpnCED enzymes are involved in the biosynthesis of squalene (C30), which is required for the synthesis of carotenoids in A. brasilense Cd. We also found that CrtI and CrtP desaturases were involved in the transformation of colorless squalene into the pink-pigmented carotenoids. This study elucidates role of some genes which constitute very pivotal role in biosynthetic pathway of carotenoid in A. brasilense Cd.

Integrating population and single-cell variations in vaccine responses identifies a naturally adjuvanted human immune setpoint.

Multimodal single-cell profiling methods can capture immune cell variations unfolding over time at the molecular, cellular, and population levels. Transforming these data into biological insights remains challenging. Here, we introduce a framework to integrate variations at the human population and single-cell levels in vaccination responses. Comparing responses following AS03-adjuvanted versus unadjuvanted influenza vaccines with CITE-seq revealed AS03-specific early (day 1) response phenotypes, including a B cell signature of elevated germinal center competition. A correlated network of cell-type-specific transcriptional states defined the baseline immune status associated with high antibody responders to the unadjuvanted vaccine. Certain innate subsets in the network appeared "naturally adjuvanted," with transcriptional states resembling those induced uniquely by AS03-adjuvanted vaccination. Consistently, CD14+ monocytes from high responders at baseline had elevated phospho-signaling responses to lipopolysaccharide stimulation. Our findings link baseline immune setpoints to early vaccine responses, with positive implications for adjuvant development and immune response engineering.

AS03 adjuvant enhances the magnitude, persistence, and clonal breadth of memory B cell responses to a plant-based COVID-19 vaccine in humans.

Vaccine adjuvants increase the breadth of serum antibody responses, but whether this is due to the generation of antigen-specific B cell clones with distinct specificities or the maturation of memory B cell clones that produce broadly cross-reactive antibodies is unknown. Here, we longitudinally analyzed immune responses in healthy adults after two-dose vaccination with either a virus-like particle COVID-19 vaccine (CoVLP), CoVLP adjuvanted with AS03 (CoVLP+AS03), or a messenger RNA vaccination (mRNA-1273). CoVLP+AS03 enhanced the magnitude and durability of circulating antibodies and antigen-specific CD4+ T cell and memory B cell responses. Antigen-specific CD4+ T cells in the CoVLP+AS03 group at day 42 correlated with antigen-specific memory B cells at 6 months. CoVLP+AS03 induced memory B cell responses, which accumulated somatic hypermutations over 6 months, resulting in enhanced neutralization breadth of monoclonal antibodies. Furthermore, the fraction of broadly neutralizing antibodies encoded by memory B cells increased between day 42 and 6 months. These results indicate that AS03 enhances the antigenic breadth of B cell memory at the clonal level and induces progressive maturation of the B cell response.

Relative Effectiveness of the MF59®-Adjuvanted Influenza Vaccine Versus High-Dose and Non-Adjuvanted Influenza Vaccines in Preventing Cardiorespiratory Hospitalizations During the 2019-2020 US Influenza Season.

BACKGROUND: Adults ≥ 65 years of age have suboptimal influenza vaccination responses compared to younger adults due to age-related immunosenescence. Two vaccines were specifically developed to enhance protection: MF59-adjuvanted trivalent influenza vaccine (aIIV3) and high-dose egg-based trivalent influenza vaccine (HD-IIV3e). METHODS: In a retrospective cohort study conducted using US electronic medical records linked to claims data during the 2019-2020 influenza season, we compared the relative vaccine effectiveness (rVE) of aIIV3 with HD-IIV3e and a standard-dose non-adjuvanted egg-based quadrivalent inactivated influenza vaccine (IIV4e) for the prevention of cardiorespiratory hospitalizations, including influenza hospitalizations. We evaluated outcomes in the "any" diagnosis position and the "admitting" position on the claim. A doubly robust methodology using inverse probability of treatment weighting and logistic regression was used to adjust for covariate imbalance. rVE was calculated as 100 * (1 - ORadjusted). RESULTS: The study included 4,299,594 adults ≥ 65 years of age who received aIIV3, HD-IIV3e, or IIV4e. Overall, aIIV3 was associated with lower proportions of cardiorespiratory hospitalizations with diagnoses in any position compared to HD-IIV3e (rVE = 3.9% [95% CI, 2.7-5.0]) or IIV4e (9.0% [95% CI, 7.7-10.4]). Specifically, aIIV3 was more effective compared with HD-IIV3e and IIV4e in preventing influenza hospitalizations (HD-IIV3e: 9.7% [95% CI, 1.9-17.0]; IIV4e: 25.3% [95% CI, 17.7-32.2]). Consistent trends were observed for admitting diagnoses. CONCLUSION: Relative to both HD-IIV3e and IIV4e, aIIV3 provided improved protection from cardiorespiratory or influenza hospitalizations.

Promiscuous Oxidosqualene Cyclases from Neoalsomitra integrifoliola Catalyzing the Formation of Tetracyclic, Pentacyclic, and Heterocyclic Triterpenes.

Six oxidosqualene cyclases (NiOSC1-NiOSC6) from Neoalsomitra integrifoliola were characterized for the biosynthesis of diverse triterpene scaffolds, including tetracyclic and pentacyclic triterpenes from the 2,3-oxidosqualene (1) and oxacyclic triterpenes from the 2,3:22,23-dioxidosqualene (2). NiOSC1 showed high efficiency in the production of naturally rare (20R)-epimers of oxacyclic triterpenes. Mutagenesis results revealed that the NiOSC1-F731G mutant significantly increased the yields of (20R)-epimers compared to the wild type. Homology modeling and molecular docking elucidated the origin of the (20R)-configuration in the epoxide addition step.

Remodeling the Homologous Recombination Mechanism of Yarrowia lipolytica for High-Level Biosynthesis of Squalene.

Squalene is a high-value antioxidant with many commercial applications. The use of microbial cell factories to produce squalene as an alternative to plant and animal extracts could meet increasing market demand. Yarrowia lipolytica is an excellent host for squalene production due to its high levels of acetyl-CoA and a hydrophobic environment. However, the need for precise and complicated gene editing has hindered the industrialization of this strain. Herein, the rapid construction of a strain with high squalene production was achieved by enhancing the homologous recombination efficiency in Y. lipolytica. First, remodeling of the homologous recombination efficiency resulted in a 10-fold increase in the homologous recombination rate. Next, the whole mevalonate pathway was integrated into the chromosome to enhance squalene production. Then, a higher level of squalene accumulation was achieved by increasing the level of acetyl coenzyme A and regulating the downstream steroid synthesis pathway. Finally, the squalene production reached 35 g/L after optimizing the fermentation conditions and performing a fed-batch culture in a 5 L jar fermenter. This is the highest squalene production ever reported to date by de novo biosynthesis without adding any inhibitors, paving a new path toward the industrial production of squalene and its downstream products.

Squalene Depletion in Skin Following Human Exposure to Ozone under Controlled Chamber Conditions.

A major component of human skin oil is squalene, a highly unsaturated hydrocarbon that protects the skin from atmospheric oxidants. Skin oil, and thus squalene, is continuously replenished on the skin surface. Squalene is also quickly consumed through reactions with ozone and other oxidants. This study examined the extent of squalene depletion in the skin oils of the forearm of human volunteers after exposure to ozone in a climate chamber. Temperature, relative humidity (RH), skin coverage by clothing, and participants' age were varied in a controlled manner. Concentrations of squalene were determined in skin wipe samples collected before and after ozone exposure. Exposures to ozone resulted in statistically significant decreases in post-exposure squalene concentrations compared to pre-exposure squalene concentrations in the skin wipes when squalene concentrations were normalized by concentrations of co-occurring cholesterol but not by co-occurring pyroglutamic acid (PGA). The rate of squalene loss due to ozonolysis was lower than its replenishment on the skin surface. Within the ranges examined, temperature and RH did not significantly affect the difference between normalized squalene levels in post-samples versus pre-samples. Although not statistically significant, skin coverage and age of the volunteers (three young adults, three seniors, and three teenagers) did appear to impact squalene depletion on the skin surfaces.

A Phase 2 Clinical Trial to Evaluate the Safety, Reactogenicity, and Immunogenicity of Different Prime-Boost Vaccination Schedules of 2013 and 2017 A(H7N9) Inactivated Influenza Virus Vaccines Administered With and Without AS03 Adjuvant in Healthy US Adults.

INTRODUCTION: A surge of human influenza A(H7N9) cases began in 2016 in China from an antigenically distinct lineage. Data are needed about the safety and immunogenicity of 2013 and 2017 A(H7N9) inactivated influenza vaccines (IIVs) and the effects of AS03 adjuvant, prime-boost interval, and priming effects of 2013 and 2017 A(H7N9) IIVs. METHODS: Healthy adults (n = 180), ages 19-50 years, were enrolled into this partially blinded, randomized, multicenter phase 2 clinical trial. Participants were randomly assigned to 1 of 6 vaccination groups evaluating homologous versus heterologous prime-boost strategies with 2 different boost intervals (21 vs 120 days) and 2 dosages (3.75 or 15 µg of hemagglutinin) administered with or without AS03 adjuvant. Reactogenicity, safety, and immunogenicity measured by hemagglutination inhibition and neutralizing antibody titers were assessed. RESULTS: Two doses of A(H7N9) IIV were well tolerated, and no safety issues were identified. Although most participants had injection site and systemic reactogenicity, these symptoms were mostly mild to moderate in severity; injection site reactogenicity was greater in vaccination groups receiving adjuvant. Immune responses were greater after an adjuvanted second dose, and with a longer interval between prime and boost. The highest hemagglutination inhibition geometric mean titer (95% confidence interval) observed against the 2017 A(H7N9) strain was 133.4 (83.6-212.6) among participants who received homologous, adjuvanted 3.75 µg + AS03/2017 doses with delayed boost interval. CONCLUSIONS: Administering AS03 adjuvant with the second H7N9 IIV dose and extending the boost interval to 4 months resulted in higher peak antibody responses. These observations can broadly inform strategic approaches for pandemic preparedness. Clinical Trials Registration. NCT03589807.