Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens

A synthetic hemagglutinin (HA) gene from the highly pathogenic avian influenza (HPAI) virus A/chicken/Indonesia/7/2003 (H5N1) (Indo/03) was expressed in aquatic plant Lemna minor (rLemna-HA). In Experiment 1, efficacy of rLemna-HA was tested on birds immunized with 0.2 μg or 2.3 μg HA and challenged with 10⁶ mean chicken embryo infectious doses (EID₅₀) of homologous virus strain. Both dosages of rLemna-HA conferred clinical protection and dramatically reduced viral shedding. Almost all the birds immunized with either dosage of rLemna-HA elicited HA antibody titers against Indo/03 antigen, suggesting an association between levels of anti-Indo/03 antibodies and protection. In Experiment 2, efficacy of rLemna-HA was tested on birds immunized with 0.9 μg or 2.2 μg HA and challenged with 10⁶ EID₅₀ of heterologous H5N1 virus strains A/chicken/Vietnam/NCVD-421/2010 (VN/10) or A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Birds challenged with VN/10 exhibited 100% survival regardless of immunization dosage, while birds challenged with PWT/06 had 50% and 30% mortality at 0.9 μg HA and 2.2 μg HA, respectively. For each challenge virus, viral shedding titers from 2.2 μg HA vaccinated birds were significantly lower than those from 0.9 μg HA vaccinated birds, and titers from both immunized groups were in turn significantly lower than those from sham vaccinated birds. Even if immunized birds elicited HA titers against the vaccine antigen Indo/03, only the groups challenged with VN/10 developed humoral immunity against the challenge antigen. None (rLemna-HA 0.9 μg HA) and 40% (rLemna-HA 2.2 μg HA) of the immunized birds challenged with PWT/06 elicited pre-challenge antibody titers, respectively. In conclusion, Lemna-expressed HA demonstrated complete protective immunity against homologous challenge and suboptimal protection against heterologous challenge, the latter being similar to results from inactivated whole virus vaccines. Transgenic duckweed-derived HA could be a good alternative for producing high quality antigen for an injectable vaccine against H5N1 HPAI viruses.     

Rational design of medium supplementation strategy for improved influenza viruses production based on analyzing nutritional requirements of MDCK Cells

Influenza vaccine production using cell culture technology has become popular nowadays. However, to meet the ever increasing demand of influenza vaccine, it is prerequisite to improve the yield of influenza virus in cells. To achieve this, in the present study, the nutritional requirements of MDCK cells in the virus production process were analyzed and a nutrient-feeding strategy was developed accordingly. Based on the consumption rates and corresponding concentration optimization, glucose and fast metabolized amino acids were supplemented into the maintaining medium at the time of infection. Compared with the non-supplemented culture, the average cell specific death rate during 0–48 h post-infection was 0.013 h⁻¹, which was 40.91% lower in the nutrient-supplemented culture. Total virus titer, HA antigen protein concentration and cell-specific virus yield were (1.88 ± 0.23) × 10³ HA units/50 μL, 11.70 ± 0.22 μg/mL and (10.06 ± 1.16) × 10³ virions/cell, respectively, which were 84.04 ± 22.50%, 31.46 ± 2.87% and 86.64 ± 25.81% higher than those in the control, respectively. These data showed that the appropriate supplementation of nutrients during virus production process could reduce cell death, and improve cell-specific virus yield and total influenza virus output. This study laid foundation for the development of cell culture technology for influenza vaccine production.     

Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells

The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging.Here we present a production platform based on BHK-21 suspension (BHK-21_SUS) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1–48 PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKV^PE) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21_SUS cells with ZIKV^PE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9 × 10³ PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2 × 10⁷ cells/mL, and virus titers of 3.9 × 10⁷ PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3 PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards process development for manufacturing inactivated or live-attenuated ZIKV vaccines.     

Evaluation of the attenuation,immunogenicity, and efficacy of a live virus vaccine generated by codon-pair bias de-optimization of the 2009 pandemic H1N1 influenza virus,in ferrets

Codon-pair bias de-optimization (CPBD) of viruses involves re-writing viral genes using statistically underrepresented codon pairs, without any changes to the amino acid sequence or codon usage. Previously, this technology has been used to attenuate the influenza A/Puerto Rico/8/34 (H1N1) virus. The de-optimized virus was immunogenic and protected inbred mice from challenge. In order to assess whether CPBD could be used to produce a live vaccine against a clinically relevant influenza virus, we generated an influenza A/California/07/2009 pandemic H1N1 (2009 pH1N1) virus with de-optimized HA and NA gene segments (2009 pH1N1-(HA + NA)^Min), and evaluated viral replication and protein expression in MDCK cells, and attenuation, immunogenicity, and efficacy in outbred ferrets. The 2009 pH1N1-(HA + NA)^Min virus grew to a similar titer as the 2009 pH1N1 wild type (wt) virus in MDCK cells (∼10⁶ TCID₅₀/ml), despite reduced HA and NA protein expression on western blot. In ferrets, intranasal inoculation of 2009 pH1N1-(HA + NA)^Min virus at doses ranging from 10³ to 10⁵ TCID₅₀ led to seroconversion in all animals and protection from challenge with the 2009 pH1N1 wt virus 28 days later. The 2009 pH1N1-(HA + NA)^Min virus did not cause clinical illness in ferrets, but replicated to a similar titer as the wt virus in the upper and lower respiratory tract, suggesting that de-optimization of additional gene segments may be warranted for improved attenuation. Taken together, our data demonstrate the potential of using CPBD technology for the development of a live influenza virus vaccine if the level of attenuation is optimized.     

Process intensification for high yield production of influenza H1N1 Gag virus-like particles using an inducible HEK-293 stable cell line

Influenza virus dominant antigens presentation using virus like particle (VLP) approach is attractive for the development of new generation of influenza vaccines. Mammalian cell platform offers many advantages for VLP production. However, limited attention has been paid to the processing of mammalian cell produced VLPs. Better understanding of the production system could contribute to increasing the yields and making large-scale VLP vaccine manufacturing feasible. In a previous study, we have generated a human embryonic kidney HEK-293 inducible cell line expressing Hemagglutinin (HA) and Neuraminidase (NA), which was used to produce VLPs upon transient transfection with a plasmid containing HIV-1 Gag. In this work, to streamline the production process, we have developed a new HEK-293 inducible cell line adapted to suspension growth expressing the three proteins HA, NA (H1N1 A/PR/8/1934) and the Gag fused to GFP for monitoring the VLP production. The process was optimized to reach higher volumetric yield of VLPs by increasing the cell density at the time of induction without sacrificing the cell specific productivity. A 5-fold improvement was achieved by doing media evaluation at small scale. Furthermore, a 3-L perfusion bioreactor mirrored the performance of small-scale shake flask cultures with sequential medium replacement. The cell density was increased to 14 × 10⁶ cells/ml at the time of induction which augmented by 60-fold the volumetric yield to 1.54 × 10¹⁰ Gag-GFP fluorescent events/ml, as measured by flow cytometry. The 9.5-L harvest from the perfusion bioreactor was concentrated by tangential flow filtration at low shear rate. The electron micrographs revealed the presence of VLPs of 100–150 nm with the characteristic dense core of HIV-1 particles. The developed process shows the feasibility of producing high quantity of influenza VLPs from an inducible mammalian stable cell line aiming at large scale vaccine manufacturing.     

Plant-made virus-like particle vaccines bearing the hemagglutinin of either seasonal (H1) or avian (H5) influenza have distinct patterns of interaction with human immune cells in vitro

IntroductionThe recent emergence of avian influenza strains has fuelled concern about pandemic preparedness since vaccines targeting these viruses are often poorly immunogenic. Weak antibody responses to vaccines have been seen across multiple platforms including plant-made VLPs. To better understand these differences, we compared the in vitro responses of human immune cells exposed to plant-made virus-like particle (VLP) vaccines targeting H1N1 (H1-VLP) and H5N1 (H5-VLP).MethodsPeripheral blood mononuclear cells (PBMC) from healthy adults were stimulated ex vivo with 2-5 µg/mL VLPs bearing the hemagglutinin (HA) of either H1N1 (A/California/7/2009) or H5N1 (A/Indonesia/5/05). VLP-immune cell interactions were characterized by confocal microscopy and flow cytometry 30 min after stimulation with dialkylaminostyryl dye-labeled (DiD) VLP. Expression of CD69 and pro-inflammatory cytokines were used to assess innate immune activation 6 h after stimulation.ResultsH1- and H5-VLPs rapidly associated with all subsets of human PBMC but exhibited unique binding preferences and frequencies. The H1-VLP bound to 88.7 ± 1.6% of the CD19⁺ B cells compared to only 21.9 ± 1.8% bound by the H5-VLP. At 6 h in culture, CD69 expression on B cells was increased in response to H1-VLP but not H5-VLP (22.79 ± 3.42% vs. 6.15 ± 0.82% respectively: p < 0.0001). Both VLPs were rapidly internalized by CD14⁺ monocytes resulting in the induction of pro-inflammatory cytokines (i.e.: IL-8, IL-1β, TNFα and IL-6). However, a higher concentration of the H5-VLP was required to induce a comparable response and the pattern of cytokine production differed between VLP vaccines.ConclusionsPlant-made VLP vaccines bearing H1 or H5 rapidly elicit immune activation and cytokine production in human PBMC. Differences in the VLP-immune cell interactions suggest that features of the HA proteins themselves, such as receptor specificity, influence innate immune responses. Although not generally considered for inactivated vaccines, the distribution and characteristics of influenza receptor(s) on the immune cells themselves may contribute to both the strength and pattern of the immune response generated.     

Immunoglobulin GM and KM genes and measles vaccine-induced humoral immunity

Identifying genetic polymorphisms that explain variations in humoral immunity to live measles virus vaccine is of great interest. Immunoglobulin GM (heavy chain) and KM (light chain) allotypes are genetic markers known to be associated with susceptibility to several infectious diseases. We assessed associations between GM and KM genotypes and measles vaccine humoral immunity (neutralizing antibody titers) in a combined cohort (n = 1796) of racially diverse healthy individuals (age 18–41 years). We did not discover any significant associations between GM and/or KM genotypes and measles vaccine-induced neutralizing antibody titers. African-American subjects had higher neutralizing antibody titers than Caucasians (1260 mIU/mL vs. 740 mIU/mL, p = 7.10 × 10⁻¹³), and those titers remained statistically significant (p = 1.68 × 10⁻⁰⁹) after adjusting for age at enrollment and time since last vaccination. There were no statistically significant sex-specific differences in measles-induced neutralizing antibody titers in our study (p = 0.375). Our data indicate a surprising lack of evidence for an association between GM and KM genotypes and measles-specific neutralizing antibody titers, despite the importance of these immune response genes.     

A clinical phase I study of an EB66 cell-derived H5N1 pandemic vaccine adjuvanted with AS03

BackgroundWe conducted a phase I clinical trial of a cell culture-derived AS03-adjuvanted influenza vaccine containing HA antigen (A/Indonesia/05/2005(H5N1)/PR8-IBCDC-RG2) derived from EB66 cells (KD-295).MethodsHealthy male adult volunteers (20–40 years old, N = 60) enrolled in the study were divided into 3 groups, the MA group (3.8 μg of HA + AS03), HA group (7.5 μg of HA + AS03), and 1/2 MA group (half the volume of the MA group), and received KD-295 intramuscularly twice with a 21-day interval. After administration of KD-295, adverse events, clinical laboratory parameters, and immune response to the vaccine strain and heterologous virus strains were evaluated.ResultsNo severe adverse events leading to discontinuation of vaccine administration occurred. The vaccine was well-tolerated. There was no dose dependency in the rate, timing, or duration of the adverse events. Immunogenicity of the vaccines was evaluated by HI (hemagglutination inhibition) assay, which confirmed that the antibody response to the vaccine strain and heterologous strain in all groups met the three criteria for immunogenicity described in the Japanese guidelines for development of a pandemic prototype vaccine. We also measured the neutralizing antibody titers against several virus strains, and confirmed a significant rise in antibody levels to both the vaccine strain and heterologous strains.ConclusionThe EB66-derived H5N1 influenza vaccine adjuvanted with AS03 elicited a broad cross-reactive antibody response among H5N1 strains with acceptable reactogenicity. Therefore, KD-295 can be considered a useful pandemic and pre-pandemic influenza vaccine candidate.     

Baseline serum interleukin-6 to interleukin-2 ratio is associated with the response to seasonal trivalent influenza vaccine in solid organ transplant recipients

BackgroundThe analysis of pre- and post-vaccination B-cell-associated cytokines might be useful in predicting the immunogenicity of seasonal trivalent influenza vaccine (TIV) in solid organ transplant (SOT) recipients.MethodsWe performed a subanalysis of a clinical trial that compared the safety and efficacy of high-dose intradermal (ID) versus intramuscular (IM) TIV in SOT recipients. Serum levels of selected cytokines (interferon [IFN]-γ, interleukin [IL]-2, IL-4, IL-5, IL-6, IL-12 and IL-21, and tumor necrosis factor [TNF]-α) were measured pre- and one month post-vaccination in 155 patients (with 84 and 71 receiving the ID and IM vaccines, respectively). Cytokine profiles were compared according to vaccine response (seroconversion [≥4-fold increase in hemagglutination inhibition antibody titers] to ≥1 influenza vaccine antigen).ResultsMean baseline IL-6 levels were higher (1.20 versus 0.65 pg/mL; P-value = 0.021) and IL-2 levels were lower (0.01 versus 0.50 pg/mL; P-value = 0.051) in patients achieving vaccine response. After adjusting for clinical variables, baseline IL-6/IL-2 ratio remained predictive of vaccine response (odds ratio per 10-unit increment: 1.06; 95% confidence interval: 1.02–1.10; P-value = 0.002). Vaccination induced an increase in TNF-α (P-value <0.0001) and a decrease in IL-5 levels (P-value = 0.0007). There were no significant differences in cytokine kinetics between vaccine responders and non-responders. Mean baseline TNF-α levels were higher in patients experiencing moderate-to-severe adverse events after vaccination (1.93 versus 1.72 pg/mL; P-value = 0.009).ConclusionsBaseline serum IL-6 and IL-2 levels, two cytokines that modulate the role of CD4⁺ T follicular helper cells and the terminal differentiation of B-cells, predict vaccine response in SOT recipients.     

Plasma cell and serum antibody responses to influenza vaccine in preterm and full-term infants

BackgroundPreterm (PT) infants are at greater risk for severe influenza infection and experience decrements in long-term antibody responses to vaccines. This may related to defects in antibody secreting cell (ASC) generation.ObjectiveTo investigate the relationships among the frequencies of influenza-specific antibody secreting cells, ASC numbers and subsets, and antibody responses to influenza vaccines (IV) among PT and full-term (FT) infants.Design/methodsWe enrolled 11 former PT (≤32 weeks′ gestation, ≤1500 g′ birth weight) and 11 FT infants, 6–17 months of age, receiving their first influenza immunizations. Infants received two doses of inactivated trivalent (T)IV or quadrivalent (Q)IV during the 2012–2013 and 2013–2014 influenza seasons, respectively, at 0 and 28 days, and blood was drawn at 0, 10, 35, and 56 days and 9 months. Vaccine-specific antibody was measured by hemagglutination inhibition (HAI) at 0 and 56 days and 9 months, vaccine-specific ASC numbers by enzyme linked immunospot (ELISPOT) at 10 and 35 days, and ASC subsets by flow cytometry at 0, 10 and 35 days.ResultsPT infants had post-vaccine HAI titers to all 4 vaccine strains at least equal to FT infants at 56 days and 9 months after beginning immunization. Influenza-specific ASC ELISPOT responses at 35 days were higher among PT than FT infants (median 100 v. 30 per 10⁶ PBMC, p = 0.04). ASC numbers at 35 days were positively correlated with serum HAI titers at 56 days (ρ = 0.50–0.80). There were no statistical differences between PT and FT infants in the frequency of five ASC subsets and no specific ASC subset correlated with durability of serum antibody titers.ConclusionsInfluenza-specific ASC numbers in both FT and PT infants correlated with peak antibody titers, but ASC subsets did not correlate with durability of antibody response.     

Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using homologous challenge

The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0 × 10⁸ to 2.1 × 10¹¹ particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R² = 0.97) and viremia (R² = 0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R² = 0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge.     

Vitamin D levels and influenza vaccine immunogenicity among HIV-infected and HIV-uninfected adults

BackgroundVaccination is the most important preventive strategy against influenza, however post-vaccination antibody responses are often inadequate especially among HIV-infected persons. Vitamin D deficiency has been suggested to adversely influence immune responses and is highly prevalent among HIV-infected adults. Therefore, we evaluated the association between 25-hydroxyvitamin D [25(OH)D] levels and post-influenza vaccination responses.MethodsWe conducted a prospective cohort study evaluating the immunogenicity of monovalent influenza A (H1N1) vaccination among both HIV-infected and HIV-uninfected adults (18–50 years of age) during the 2009–2010 influenza season. Antibody titers were evaluated at baseline, day 28, and 6 months post-vaccination using hemagluttination inhibition assays. Serum 25(OH)D levels were measured at day 28. Univariate and multivariate regression analyses examined the association between 25(OH)D levels [categorized as <20 ng/ml (deficiency) vs. ⩾20 ng/ml] with the primary outcome of seroconversion. Secondary outcomes included seroprotection; a ⩾4-fold increase in titers; and geometric mean titers post-vaccination. Analyses were repeated using 25(OH)D levels as a continuous variable.ResultsA total of 128 adults [64 HIV-infected (median CD4 count 580 cells/mm³) and 64 HIV-uninfected] were included. Seroconversion at day 28 post-vaccination was achieved in fewer HIV-infected participants compared with HIV-uninfected participants (56% vs. 74%, p = 0.03). Vitamin D deficiency was more prevalent among HIV-infected persons vs. HIV-uninfected persons (25% vs. 17%), although not significantly different (p = 0.39). There were no associations found between lower 25(OH)D levels and poorer antibody responses at day 28 or 6 months for any of the study outcomes among either HIV-infected or HIV-uninfected adults.ConclusionVitamin D deficiency was common among both HIV-infected and HIV-uninfected adults, but lower levels did not predict antibody responses after H1N1 (2009) influenza vaccination. Low 25(OH)D levels do not explain poorer post-vaccination responses among HIV-infected persons.     

Predictors of reported influenza vaccination in HIV-infected women in the United States, 2006–2007 and 2007–2008 seasons

ObjectiveTo estimate the cumulative incidence of self-reported influenza vaccination (“vaccination coverage”) and investigate predictors in HIV-infected women.MethodsIn an ongoing cohort study of HIV-infected women in five US cities, data from two influenza seasons (2006–2007 n = 1209 and 2007–2008 n = 1161) were used to estimate crude and adjusted prevalence ratios (aPR) and 95% confidence intervals ([,]) from Poisson regression with robust variance models using generalized estimating equations (GEE).ResultsIn our study, 55% and 57% of HIV-infected women reported vaccination during the 2006–2007 and 2007–2008 seasons, respectively. Using data from both seasons, older age, non-smoking status, CD4 T-lymphocyte (CD4) count ≥ 200 cells/mm³, and reporting at least one recent healthcare visit was associated with increased vaccination coverage. In the 2007–2008 season, a belief in the protection of the vaccine (aPR = 1.38 [1.18, 1.61]) and influenza vaccination in the previous season (aPR = 1.66 [1.44, 1.91]) most strongly predicted vaccination status.ConclusionInterventions to reach unvaccinated HIV-infected women should focus on changing beliefs about the effectiveness of influenza vaccination and target younger women, current smokers, those without recent healthcare visits, or a CD4 count < 200 cells/mm³.     

Comparison of lyophilized versus liquid modified vaccinia Ankara (MVA) formulations and subcutaneous versus intradermal routes of administration in healthy vaccinia-naïve subjects

BackgroundModified vaccinia Ankara (MVA) is being developed as a safer smallpox vaccine and is being placed in the US Strategic National Stockpile (SNS) as a liquid formulation for subcutaneous (SC) administration at a dose of 1 × 10⁸ TCID₅₀ in a volume of 0.5 mL. This study compared the safety and immunogenicity of the standard formulation, dose and route with both a more stable, lyophilized formulation and with an antigen-sparing intradermal (ID) route of administration.Methods524 subjects were randomized to receive either a full dose of Lyophilized-SC, a full dose of Liquid-SC or 20% (2 × 10⁷ TCID₅₀ in 0.1 mL) of a full dose Liquid-ID MVA on Days 0 and 28. Safety and immunogenicity were followed through 180 days post second vaccination.ResultsAmong the 3 groups, the proportion of subjects with moderate/severe functional local reactions was significantly different (P = 0.0013) between the Lyophilized-SC group (30.3%), the Liquid-SC group (13.8%) and Liquid-ID group (22.0%) only after first vaccination; and for moderate/severe measured erythema and/or induration after any vaccination (P = 0.0001) between the Lyophilized-SC group (58.2%), the Liquid-SC group (58.1%) and the Liquid-ID group (94.8%) and the reactions lasted longer in the Liquid-ID group. In the ID Group, 36.1% of subjects had mild injection site skin discoloration lasting ≥6 months.After second vaccination Day (42–208), geometric mean of peak neutralization titers were 87.8, 49.5 and 59.5 for the Lyophilized-SC, Liquid-SC and Liquid-ID groups, respectively, and the maximum number of responders based on peak titer in each group was 142/145 (97.9%), 142/149 (95.3%) and 138/146 (94.5%), respectively. At 180 days after the second vaccination, geometric mean neutralization titers declined to 11.7, 10.2 and 10.4 with only 54.3%, 39.2% and 35.2% of subjects remaining seropositive for the Lyophilized-SC, Liquid-SC and Liquid-ID groups, respectively. Both the Lyophilized-SC and Liquid-ID groups were considered non-inferior (primary objective) to the Liquid-SC group.ConclusionsTransitioning to a lyophilized formulation, which has a longer shelf life, will not negatively impact immunogenicity. In a situation where insufficient vaccine is available, ID vaccination could be used, increasing the number of available doses of vaccine in the SNS 5-fold (i.e., from 20 million to 100 million doses).     

Effect of seasonal vaccination on the selection of influenza A/H3N2 epidemic variants

The effect of vaccination on the dynamics of influenza virus variants remains largely unknown in humans, unlike in poultry. In this study, we compared influenza hemagglutinin (HA) gene sequences isolated from vaccinated and unvaccinated populations with the yearly vaccine strains. In total, 181 influenza A/H3N2 virus samples isolated from 82 vaccinated and 99 unvaccinated patients (2011–15, four Japanese influenza seasons) were genetically analyzed using a next-generation sequencer. Amino acid (AA) differences from corresponding vaccine strains were found in 74 of 329 HA1 sites. There was a maximum of four AA differences within the epitopes in the former three seasons (2011–14) and fifteen in the latter season (2014–15). Deviation to a greater number of AA differences was found more significantly in the isolates from vaccinated patients as compared to unvaccinated patients (P = 0.0005 in 2011–14; P = 0.0096 in 2014–15). AA difference rates within epitopes were also significantly higher in the isolates from vaccinated patients than from unvaccinated patients (2.64% vs. 2.14% for 2011–14, P = 0.033; 7.78% vs. 6.59% for 2014–15, P = 0.058). The AA differences at seven sites (48I-278K, 128A-142G, 145S, 158K, and 193S) became dominant in the following seasons. In all of these sites, the dominance was retained during the mismatch of isolates with the vaccine strains and was lost after vaccine match. Our data suggest that in humans, immune pressure induced by vaccination works to select influenza variants genetically distant from vaccine strains.     

Levels and sources of volatile organic compounds including carbonyls in indoor air of homes of Puertollano,the most industrialized city in central Iberian Peninsula. Estimation of health risk

Twenty nine organic air pollutants including carbonyl compounds, alkanes, aromatic hydrocarbons and terpenes were measured in the indoor environment of different houses together with the corresponding outdoor measurements in Puertollano, the most industrialized city in central Iberian Peninsula. VOCs were sampled during 8 weeks using Radiello^® passive samplers, and a questionnaire on potential VOCs sources was filled out by the occupants. The results show that formaldehyde and hexanal was the most abundant VOCs measured in indoor air, with a median concentration of 55.5 and 46.4 μg m⁻³, respectively followed by butanal (29.1 μg m⁻³), acetone (28.4 μg m⁻³) and acetaldehyde (21.4 μg m⁻³). After carbonyls, n-dodecane (13.1 μg m⁻³) and terpenes (α-pinene, 13.4 μg m⁻³ and limonene, 13.4 μg m⁻³) were the compounds with higher median concentrations. The indoor/outdoor (I/O) ratios demonstrated that sources in the indoor environment are prevailing for most of the investigated VOCs especially for limonene, α-pinene, hexanal, formaldehyde, pentanal, acetaldehyde, o-xylene, n-dodecane and acetone with I/O ratio >6. Multiple linear regressions were applied to investigate the indoor VOC determinants and Spearman correlation coefficients were used to establish common sources between VOCs. Finally, the lifetime cancer risk associated to formaldehyde, acetaldehyde and benzene exposure was estimated and they varied from 7.8 × 10⁻⁵ to 4.1 × 10⁻⁴ for formaldehyde, from 8.6 × 10⁻⁶ to 3.5 × 10⁻⁵ for acetaldehyde and from 2.0 × 10⁻⁶ to 1.5 × 10⁻⁵ for benzene. For formaldehyde, the attributed risk in most sampled homes was two orders of magnitude higher than the one (10⁻⁶) proposed as acceptable by risk management bodies.     

Rubella specific cell-mediated and humoral immunity following vaccination in college students with low antibody titers

ObjectiveThis study measured cell-mediated immunity (CMI) and antibodies to clarify the basis of rubella reinfection after vaccination.MethodsIn a pool of 65 college students, 39 who exhibited hemagglutination–inhibition (HI) antibody titers against rubella of ≤1:16 were vaccinated with a rubella vaccine. The CMI was assessed with interferon-gamma release assay.ResultsThere was low correlation (r = 0.24) between the antibody titers and interferon-gamma levels at pre-vaccination status. Preexisting interferon-gamma levels were low in some subjects with low HI antibody titers of 1:8 and 1:16. Fifty-seven percent (4/7) of the subjects who were antibody-negative with past history of rubella vaccination at entry onto the study exhibited CMI. And 57% (4/7) of the subjects remained antibody-negative following a second vaccination, despite exhibiting CMI. HI antibody titers increased significantly after vaccination, whereas post-vaccination interferon-gamma levels did not exhibit significant increases. When subjects were divided (based on their past history of vaccination and antibody values) into natural infection and vaccination groups, HI antibody titers (mean ± SD) increased to 1:2^4.4 ± 1.4 from 1: 2^3.2 ± 0.4 (p = 0.065) in the natural infection group and to 1:2^4.4 ± 1.0 from 1:2^3.0 ± 0.8 (p < 0.00001) in the vaccination group following vaccination. The same classification revealed that interferon-gamma values did not increase significantly in either group following vaccination, but the interferon-gamma values at pre- and post-vaccination in the natural infection group were significantly higher than those at pre- and post-vaccination in the vaccination group (p < 0.05 and p < 0.05, respectively).ConclusionPre-vaccination interferon-gamma levels in each HI antibody titer group were similar. And there were some subjects with antibody-positive exhibited CMI-negative. These data may explain why rubella reinfection can occur in vaccinated seropositive individuals.     

Humoral,T-cell and B-cell immune responses to seasonal influenza vaccine in solid organ transplant recipients receiving anti-T cell therapies

BackgroundWe analyzed the impact of the anti-T-cell agents basiliximab and antithymocyte globulins (ATG) on antibody and cell-mediated immune responses after influenza vaccination in solid-organ transplant recipients.Methods71 kidney and heart transplant recipients (basiliximab [n = 43] and ATG [n = 28]) received the trivalent influenza vaccine. Antibody responses were measured at baseline and 6 weeks post-vaccination by hemagglutination inhibition assay; T-cell responses were measured by IFN-γ ELISpot assays and intracellular cytokine staining (ICS); and influenza-specific memory B-cell (MBC) responses were evaluated using ELISpot.ResultsMedian time of vaccination from transplantation was 29 months (IQR 8–73). Post-vaccination seroconversion rates were 26.8% for H1N1, 34.1% for H3N2 and 4.9% for influenza B in the basiliximab group and 35.7% for H1N1, 42.9% for H3N2 and 14.3% for influenza B in the ATG group (p = 0.44, p = 0.61, and p = 0.21, respectively). The number of influenza-specific IFN-γ-producing cells increased significantly after vaccination (from 35 to 67.5 SFC/10⁶ PBMC, p = 0.0007), but no differences between treatment groups were observed (p = 0.88). Median number of IgG-MBC did not increase after vaccination (H1N1, p = 0.94; H3N2 p = 0.34; B, p = 0.79), irrespective of the type of anti-T-cell therapy.ConclusionsAfter influenza vaccination, a significant increase in antibody and T-cell immune responses but not in MBC responses was observed in transplant recipients. Immune responses were not significantly different between groups that received basiliximab or ATG.     

Determination of trace amounts of germanium in food and fruit by spectrophotometry with p-methybenzeneazosalicylflurone

A novel spectrophotometric method was developed for determination of germanium in food and fruit using p-methybenzeneazosalicylflurone as a new chromogenic reagent. Under optimum reaction conditions, the reagent reacts with germanium(IV) to form a red complex with a maximum absorption at 502 nm. The apparent molar coefficient of the complex was 2.0×10⁵ L mol⁻¹ cm⁻¹, and a linear absorbance was obtained for 0–7 μg of germanium in 10 mL solution. The reaction completed rapidly and the absorbance of the complex remains stable for at least 24 h at room temperature. The effect of various substances on the determination of germanium was investigated in succession. The results indicated that all studied co-existing substances can be tolerated in considerable amounts, e.g., 4000-fold molybdenum(VI) and tin(II) do not interfere with the determination of germanium; its selectivity is obviously better than other reagents in the literature. The detection limit, calculated using three times the standard error of estimate of the calibration graph, is 0.006 μg g⁻¹of germanium with the pre-concentration of germanium in digested solution using ferric hydroxide as the co-precipitator.