Vaccine for prevention of mild and moderate-to-severe influenza in children.

BACKGROUND: Commonly used trivalent vaccines contain one influenza B virus lineage and may be ineffective against viruses of the other B lineage. We evaluated the efficacy of a candidate inactivated quadrivalent influenza vaccine (QIV) containing both B lineages. METHODS: In this multinational, phase 3, observer-blinded study, we randomly assigned children 3 to 8 years of age, in a 1:1 ratio, to receive the QIV or a hepatitis A vaccine (control). The primary end point was influenza A or B confirmed by real-time polymerase chain reaction (rt-PCR). Secondary end points were rt-PCR-confirmed, moderate-to-severe influenza and rt-PCR-positive, culture-confirmed influenza. The vaccine efficacy and the effect of vaccination on daily activities and utilization of health care resources were assessed in the total vaccinated cohort (2584 children in each group) and the per-protocol cohort (2379 children in the QIV group and 2398 in the control group). RESULTS: In the total vaccinated cohort, 62 children in the QIV group (2.40%) and 148 in the control group (5.73%) had rt-PCR-confirmed influenza, representing a QIV efficacy of 59.3% (95% confidence interval [CI], 45.2 to 69.7), with efficacy against culture-confirmed influenza of 59.1% (97.5% CI, 41.2 to 71.5). For moderate-to-severe rt-PCR-confirmed influenza, the attack rate was 0.62% (16 cases) in the QIV group and 2.36% (61 cases) in the control group, representing a QIV efficacy of 74.2% (97.5% CI, 51.5 to 86.2). In the per-protocol cohort, the QIV efficacy was 55.4% (95% CI, 39.1 to 67.3), and the efficacy against culture-confirmed influenza 55.9% (97.5% CI, 35.4 to 69.9); the efficacy among children with moderate-to-severe influenza was 73.1% (97.5% CI, 47.1 to 86.3). The QIV was associated with reduced risks of a body temperature above 39°C and lower respiratory tract illness, as compared with the control vaccine, in the per-protocol cohort (relative risk, 0.29 [95% CI, 0.16 to 0.56] and 0.20 [95% CI, 0.04 to 0.92], respectively). The QIV was immunogenic against all four strains. Serious adverse events occurred in 36 children in the QIV group (1.4%) and in 24 children in the control group (0.9%). CONCLUSIONS: The QIV was efficacious in preventing influenza in children. (Funded by GlaxoSmithKline Biologicals; ClinicalTrials.gov number, NCT01218308.).

Durability and cross-reactivity of immune responses induced by a plant-based virus-like particle vaccine for COVID-19.

As the SARS-CoV-2 pandemic evolves, vaccine evaluation needs to include consideration of both durability and cross-reactivity. This report expands on previously reported results from a Phase 1 trial of an AS03-adjuvanted, plant-based coronavirus-like particle (CoVLP) displaying the spike (S) glycoprotein of the ancestral SARS-CoV-2 virus in healthy adults (NCT04450004). Humoral and cellular responses against the ancestral strain were evaluated 6 months post-second dose (D201) as secondary outcomes. Independent of dose, all vaccinated individuals retain binding antibodies, and ~95% retain neutralizing antibodies (NAb). Interferon gamma and interleukin-4 responses remain detectable in ~94% and ~92% of vaccinees respectively. In post-hoc analyses, variant-specific (Alpha, Beta, Delta, Gamma and Omicron) NAb were assessed at D42 and D201. Using a live virus neutralization assay, broad cross-reactivity is detectable against all variants at D42. At D201, cross-reactive antibodies are detectable in almost all participants against Alpha, Gamma and Delta variants (94%) and the Beta variant (83%) and in a smaller proportion against Omicron (44%). Results are similar with the pseudovirion assay. These data suggest that two doses of 3.75 µg CoVLP+AS03 elicit a durable and cross-reactive response that persists for at least 6 months post-vaccination.

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).

Safety and immunogenicity of an AS03-adjuvanted plant-based SARS-CoV-2 vaccine in Adults with and without Comorbidities.

The rapid spread of SARS-CoV-2 continues to impact humanity on a global scale with rising total morbidity and mortality. Despite the development of several effective vaccines, new products are needed to supply ongoing demand and to fight variants. We report herein a pre-specified interim analysis of the phase 2 portion of a Phase 2/3, randomized, placebo-controlled trial of a coronavirus virus-like particle (CoVLP) vaccine candidate, produced in plants that displays the SARS-CoV-2 spike glycoprotein, adjuvanted with AS03 (NCT04636697). A total of 753 participants were recruited between 25th November 2020 and 24th March 2021 into three groups: Healthy Adults (18-64 years: N = 306), Older Adults (≥65 years: N = 282) and Adults with Comorbidities (≥18 years: N = 165) and randomized 5:1 to receive two intramuscular doses of either vaccine (3.75 µg CoVLP/dose+AS03) or placebo, 21 days apart. This report presents safety, tolerability and immunogenicity data up to 6 months post-vaccination. The immune outcomes presented include neutralizing antibody (NAb) titres as measured by pseudovirion assay at days 21 and 42 as well as neutralizing antibody cross-reactivity to several variants of concern (VOCs): Alpha, Beta, Gamma, Delta, and Omicron (BA.1), up to 201 days post-immunization. Cellular (IFN-γ and IL-4 ELISpot) response data in day 21 and 42 peripheral blood are also presented. In this study, CoVLP+AS03 was well-tolerated and adverse events (AE) after each dose were generally mild to moderate and transient. Solicited AEs in Older Adults and Adults with Comorbidities were generally less frequent than in Healthy Adults and the reactogenicity was higher after the second dose. CoVLP+AS03 induced seroconversion in >35% of participants in each group after the first dose and in ~98% of participants, 21 days after the second dose. In all cohorts, 21-days after the second dose, NAb levels in sera against the vaccine strain were ~10-times those in a panel of convalescent sera. Cross-reactivity to Alpha, Beta and Delta variants was generally retained to day 201 (>80%) while cross-reactivity to the Gamma variant was reduced but still substantial at day 201 (73%). Cross-reactivity to the Omicron variant fell from 72% at day 42 to 20% at day 201. Almost all participants in all groups (>88%) had detectable cellular responses (IFN-γ, IL-4 or both) at 21 days after the second dose. A Th1-biased response was most evident after the first dose and was still present after the second dose. These data demonstrated that CoVLP+AS03 is well-tolerated and highly immunogenic, generating a durable (at least 6 months) immune response against different VOCs, in adults ≥18 years of age, with and without comorbidities.

Differential mechanisms of shedding of the glycosylphosphatidylinositol (GPI)-anchored NKG2D ligands.

Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our investigation was to explore the cellular mechanisms of release used by various members of the ULBP family. Using biochemical and cellular approaches in both transfectant systems and tumor cell lines, this paper shows that ULBP1, ULBP2, and ULBP3 are released from cells with different kinetics and by distinct mechanisms. Whereas ULBP2 is mainly shed by metalloproteases, ULBP3 is abundantly released as part of membrane vesicles known as exosomes. Interestingly, exosomal ULBP3 protein is much more potent for down-modulation of the NKG2D receptor than soluble ULBP2 protein. This is the first report showing functionally relevant differences in the biochemistry of the three members of the ULBP family and confirms that in depth study of the biochemical features of individual NKG2D ligands will be necessary to understand and manipulate the biology of these proteins for therapy.

Variation of inflammatory dynamics and mediators in primiparous cows after intramammary challenge with Escherichia coli.

The objective of the current study was to investigate (i) the outcome of experimentally induced Escherichia coli mastitis in primiparous cows during early lactation in relation with production of eicosanoids and inflammatory indicators, and (ii) the validity of thermography to evaluate temperature changes on udder skin surface after experimentally induced E. coli mastitis. Nine primiparous Holstein Friesian cows were inoculated 24 ± 6 days (d) after parturition in both left quarters with E. coli P4 serotype O32:H37. Blood and milk samples were collected before and after challenge with E. coli. The infrared images were taken from the caudal view of the udder following challenge with E. coli. No relationship was detected between severity of mastitis and changes of thromboxane B2 (TXB2), leukotriene B4 (LTB4) and lipoxin A4 (LXA4). However, prostaglandin E2 (PGE2) was related to systemic disease severity during E. coli mastitis. Moreover, reduced somatic cell count (SCC), fewer circulating basophils, increased concentration of tumor necrosis factor-α (TNF-α) and higher milk sodium and lower milk potassium concentrations were related to systemic disease severity. The thermal camera was capable of detecting 2-3 °C temperature changes on udder skin surface of cows inoculated with E. coli. Peak of udder skin temperature occurred after peak of rectal temperature and appearance of local signs of induced E. coli mastitis. Although infrared thermography was a successful method for detecting the changes in udder skin surface temperature following intramammary challenge with E. coli, it did not show to be a promising tool for early detection of mastitis.

Brief residence at the plasma membrane of the MHC class I-related chain B is due to clathrin-mediated cholesterol-dependent endocytosis and shedding.

Recognition of MHC class I-related chain (MIC) molecules on the surface of target cells by the activating receptor NKG2D leads to their lysis by immune effector cells. Up-regulation of NKG2D ligands is broadly related to stress, although the detailed molecular mechanisms that control the presence of these molecules at the plasma membrane are unclear. To investigate the posttranslational mechanisms that control surface expression of the human NKG2D ligand MICB, we studied the subcellular localization and trafficking of this molecule. We found that in several cellular systems, the expression of MICB molecules on the cell surface is accompanied by an intracellular accumulation of the molecule in the trans-Golgi network and late endosome-related compartments. Surprisingly, MICB has a much shorter half-life at the plasma membrane than MHC molecules and this depends on both recycling to internal compartments and shedding to the extracellular medium. Internalization of MICB depends partially on clathrin, but importantly, the lipid environment of the membrane also plays a crucial role in this process. We suggest that the brief residence of MICB at the plasma membrane modulates, at least in part, the function of this molecule in the immune system.

Cutting edge: the metalloproteinase ADAM17/TNF-alpha-converting enzyme regulates proteolytic shedding of the MHC class I-related chain B protein.

MHC class I-related chain (MIC) A/B are transmembrane proteins expressed in pathological conditions that are ligands for the activating receptor NKG2D found on cytotoxic lymphocytes. Soluble NKG2D ligands are detected in sera of patients suffering from multiple types of cancer where they are associated with reduced levels of receptor expression and compromised function of NK and CTLs. In this study, we report the identification of a metalloproteinase involved in the cleavage process of MIC; inhibition and knockdown of ADAM17/TACE blocks the shedding of these proteins. Strikingly, the recruitment of both enzyme and substrate to detergent-resistant membrane microdomains is crucial for efficient proteolysis. These findings provide a novel insight into the molecular mechanisms of MIC shedding.

AS03-adjuvanted H7N9 inactivated split virion vaccines induce cross-reactive and protective responses in ferrets.

Human infections with avian H7N9 subtype influenza viruses are a major public health concern and vaccines against H7N9 are urgently needed for pandemic preparedness. In early 2013, novel H7N9 influenza viruses emerged in China that caused about 1600 human cases of infection with a high associated case fatality rate. In this study, two H7N9 split virion vaccines with or without AS03 adjuvant were tested in the naive ferret model. Serological analyses demonstrated that homologous hemagglutination inhibition and microneutralization antibody titers were detectable in the ferrets after the first immunization with the AS03-adjuvanted vaccines that were further boosted by the second immunization. In addition, heterologous antibody titers against older H7 subtype viruses of the North American lineage (H7N7, H7N3) and newer H7 subtype viruses of the Eurasian lineage (H7N9) were detected in the animals receiving the AS03-adjuvanted vaccines. Animals receiving two immunizations of the AS03-adjuvanted vaccines were protected from weight loss and fever in the homologous challenge study and had no detectable virus in throat or lung samples. In addition, microscopic examination post-challenge showed animals immunized with the AS03-adjuvanted vaccines had the least signs of lung injury and inflammation, consistent with the greater relative efficacy of the adjuvanted vaccines. In conclusion, this study demonstrated that the AS03-adjuvanted H7N9 vaccines elicited high levels of homologous and heterologous antibodies and protected against H7N9 virus damage post-challenge.

Comparison of quantitative immunoturbidimetric and semiquantitative latex-agglutination assays for D-dimer measurement in canine plasma.

BACKGROUND: D-dimer measurement in dogs is considered the most reliable test for detecting disseminated intravascular coagulation or thromboembolism. OBJECTIVES: The purposes of this study were to compare 2 D-dimer assays, a quantitative immunoturbidimetric and a semiquantitative latex agglutination assay, and to assess the effect of hemolysis and storage conditions on D-dimer concentration using the quantitative assay. METHODS: The immunoturbidimetric assay was validated using canine citrated plasma samples containing different concentrations of D-dimer. The effect of storage at various temperatures and times was assessed. Hemolysis was produced by adding lysed RBCs to the samples for a final hemoglobin concentration of 0.35 g/dL. RESULTS: For clinically relevant values (>250 microg/L), intra-assay and interassay coefficients of variation were 6.8% and 7.2%. The assay was linear (r(2)=1.00), and the tests had good agreement (kappa=0.685, P<.001). Storage at 4 degrees C and -20 degrees C and hemolysis had no significant effect on D-dimer concentrations. In hemolyzed samples stored at room temperature for > or =48 hours, fine clots were noted and often resulted in falsely increased D-dimer concentrations. CONCLUSIONS: Our findings suggest that the immunoturbidimetric assay validated in this study is reliable and accurate for the measurement of D-dimer in canine plasma. Samples can be stored for up to 1 month at -20 degrees C and moderate hemolysis does not significantly affect the D-dimer concentration in frozen or refrigerated samples.

Heterologous prime-boost with A(H5N1) pandemic influenza vaccines induces broader cross-clade antibody responses than homologous prime-boost.

Highly pathogenic avian influenza (HPAI) A(H5Nx) viruses continue to pose a pandemic threat. US national vaccine stockpiles are a cornerstone of the influenza pandemic preparedness plans. However, continual genetic and antigenic divergence of A(H5Nx) viruses requires the development of effective vaccination strategies using stockpiled vaccines and adjuvants for pandemic preparedness. Human sera collected from healthy adults who received either homologous (2 doses of a AS03A-adjuvanted A/turkey/Turkey/1/2005, A/Turkey), or heterologous (primed with AS03A-adjuvanted A/Indonesia/5/2005, A/Indo, followed by A/Turkey boost) prime-boost vaccination regimens were analyzed by hemagglutination inhibition and microneutralization assays against 8 wild-type HPAI A(H5Nx) viruses from 6 genetic clades. Molecular, structural and antigenic features of the A(H5Nx) viruses that could influence the cross-clade antibody responses were also explored. Compared with homologous prime-boost vaccinations, priming with a clade 2.1.3.2 antigen (A/Indo) followed by one booster dose of a clade 2.2.1 antigen (A/Turkey) administered 18 months apart did not compromise the antibody responses to the booster vaccine (A/Turkey), it also broadened the cross-clade antibody responses to several antigenically drifted variants from 6 heterologous clades, including an antigenically distant A(H5N8) virus (A/gyrfalcon/Washington/410886/2014, clade 2.3.4.4) that caused recent outbreaks in US poultry. The magnitude and breadth of the cross-clade antibody responses against emerging HPAI A(H5Nx) viruses are associated with genetic, structural and antigenic differences from the vaccine viruses and enhanced by the inclusion of an adjuvant. Heterologous prime-boost vaccination with AS03A adjuvanted vaccine offers a vaccination strategy to use existing stockpiled vaccines for pandemic preparedness against new emerging HPAI A(H5Nx) viruses.

A phase 1, open-label safety and immunogenicity study of an AS03-adjuvanted trivalent inactivated influenza vaccine in children aged 6 to 35 months.

BACKGROUND:  There is a need for better vaccines and vaccine strategies to reduce the burden of influenza in very young children.   METHODS:  This phase 1, open-label study assessed the reactogenicity, safety, and immunogenicity of an inactivated trivalent influenza vaccine (TIV) containing low doses of hemagglutinin antigen (7.5 µg each strain), adjuvanted with a tocopherol-based oil-in-water emulsion Adjuvant System (AS03). Influenza vaccine-naïve children aged 6-35 months were sequentially enrolled to receive TIV-AS03D (1.48 mg tocopherol) or TIV-AS03C (2.97 mg tocopherol), then a 6-month booster of conventional TIV. The primary endpoint was the incidence of fever (axillary temperature >38 °C) for 7 days post-vaccination. Immune responses were assessed by hemagglutination-inhibition (HI) assay. RESULTS:  Forty children were sequentially enrolled into the TIV-AS03D or the TIV-AS03C group. Fever >38.0 °C was reported in 5/20 (25.0%) and 7/20 (35.0%) children after the first and second doses of TIV-AS03D, respectively, and in 7/20 (35.0%) children after 1 dose of TIV-AS03C; the latter fulfilled the holding rule for safety, and the second dose of TIV-AS03C was cancelled. HI immune responses exceeded adult European licensure criteria for the immunogenicity, and all children had HI antibody titers ≥ 1:40 after 1 dose of TIV booster against booster strains. CONCLUSIONS:  One dose of primary vaccine containing a low dose of antigen and AS03 may be a possible influenza vaccination strategy for young children. The relatively high frequency of fever warrants further investigation, although the generalizability of the findings are uncertain given that many of the children had antibody evidence suggesting recent infection with A(H1N1)pdm09.

Natural killer cell cytotoxicity is suppressed by exposure to the human NKG2D ligand MICA*008 that is shed by tumor cells in exosomes.

The MHC class I-related chain (MIC) A and MICB ligands for the activating receptor NKG2D can be shed from tumor cells, and the presence of these soluble molecules in sera is related with compromised immune response and progression of disease. Recently, thiol disulphide isomerases and members of the ADAM (a disintegrin and metalloproteinase) gene family were identified as key enzymes in mediating MICA/B shedding from cells. Here, we report shedding of the most frequently expressed MICA allele in human populations (MICA*008) into exosomes, small membrane vesicles that are secreted upon fusion with the plasma membrane. Although similar to other MICA/B molecules in the extracellular domain, the predicted transmembrane and cytoplasmic domains of MICA*008 are quite different, and this difference seemed to be critical for the mode of release from tumor cells. Treatment of natural killer (NK) cells with exosomes containing MICA*008 molecules not only triggered downregulation of NKG2D from the cell surface but also provoked a marked reduction in NK cytotoxicity that is independent of NKG2D ligand expression by the target cell. Our findings reveal a mechanism of NK suppression in cancer that may facilitate immune escape and progression.

STAT5 is an ambivalent regulator of neutrophil homeostasis.

BACKGROUND: Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrophilia. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that in STAT5-/- mice, liver endothelial cells (LECs) autonomously secrete high amounts of G-CSF, allowing myeloid progenitors to overcompensate for their intrinsic survival defect. However, when injected with pro-inflammatory cytokines, mutant mice cannot further increase neutrophil production, display a severe deficiency in peripheral neutrophil survival, and are therefore unable to maintain neutrophil homeostasis. In wild-type mice, inflammatory stimulation induces rapid STAT5 degradation in LECs, G-CSF production by LECs and other cell types, and then sustained mobilization and expansion of long-lived neutrophils. CONCLUSION: We conclude that STAT5 is an ambivalent factor. In cells of the granulocytic lineage, it exerts an antiapoptotic function that is required for maintenance of neutrophil homeostasis, especially during the inflammatory response. In LECs, STAT5 negatively regulates granulopoiesis by directly or indirectly repressing G-CSF expression. Removal of this STAT5-imposed brake contributes to induction of emergency granulopoiesis.