Improving Consistency and Accuracy of Neonatal Amplitude-Integrated Electroencephalography.

OBJECTIVE: This study aimed to improve the utilization of amplitude-integrated electroencephalography (aEEG) in a neonatal unit by improving aEEG documentation, aEEG knowledge, and pattern recognition ability of neonatal staff. METHODS: A quality improvement (QI) program comprising the two Plan-Do-Study-Act (PDSA) cycles was conducted in a level-3 neonatal intensive care unit. The first cycle was focused on improving aEEG documentation with the primary outcome indicator being compliance with aEEG documentation. The second cycle was focused on aEEG interpretation in a health care professional education program with the outcome indicators being accuracy of seizure identification on aEEG and change in conventional EEGs (cEEG) performed. Other outcome indicators included accuracy in identification of background pattern, sleep-wake cycles and artifacts. Process indicators included improvement in aEEG-related knowledge. RESULTS: First PDSA cycle includes lectures on aEEG interpretation, a bedside key, and documentation form. Second PDSA cycle includes online aEEG education pack and detailed aEEG guideline. There was a significant improvement in aEEG documentation after the implementation of both PDSA cycles. Seven of the 46 patients (15.2%) had isolated electrographic seizures which would not have been identified in the pre-aEEG monitoring era. There was an increase in the number of patients with cEEGs done but a steady decrease in number of cEEGs per patient. CONCLUSION: With the successful application of standardized QI methods, improvements in outcome indicators, such as correct aEEG pattern recognition and improved coverage of at risk infants with cEEGs, were observed. Our QI measures were associated with improvement in aEEG pattern recognition. KEY POINTS: · Consistent and accurate use of aEEG is challenging.. · Standardized forms and guidelines improve aEEG interpretation consistency and documentation.. · Interactive self-paced online education packs can improve aEEG knowledge and pattern recognition..

A Single Intramuscular Dose of a Plant-Made Virus-Like Particle Vaccine Elicits a Balanced Humoral and Cellular Response and Protects Young and Aged Mice from Influenza H1N1 Virus Challenge despite a Modest/Absent Humoral Response.

Virus-like-particle (VLP) influenza vaccines can be given intramuscularly (i.m.) or intranasally (i.n.) and may have advantages over split-virion formulations in the elderly. We tested a plant-made VLP vaccine candidate bearing the viral hemagglutinin (HA) delivered either i.m. or i.n. in young and aged mice. Young adult (5- to 8-week-old) and aged (16- to 20-month-old) female BALB/c mice received a single 3-µg dose based on the HA (A/California/07/2009 H1N1) content of a plant-made H1-VLP (i.m. or i.n.) split-virion vaccine (i.m.) or were left naive. After vaccination, humoral and splenocyte responses were assessed, and some mice were challenged. Both VLP and split vaccines given i.m. protected 100% of the young animals, but the VLP group lost the least weight and had stronger humoral and cellular responses. Compared to split-vaccine recipients, aged animals vaccinated i.m. with VLP were more likely to survive challenge (80% versus 60%). The lung viral load postchallenge was lowest in the VLP i.m. groups. Mice vaccinated with VLP i.n. had little detectable immune response, but survival was significantly increased. In both age groups, i.m. administration of the H1-VLP vaccine elicited more balanced humoral and cellular responses and provided better protection from homologous challenge than the split-virion vaccine.

Low hemagglutinin antigen dose influenza vaccines adjuvanted with AS03 alter the long-term immune responses in BALB/c mice.

We investigated the long-term immune profiles of dose-sparing, AS03-adjuvanted vaccines compared to a traditional high-dose, unadjuvated influenza vaccine formulation. BALB/c mice received 2 IM injections of influenza A/Uruguay/716/2007 (H3N2) split vaccine antigen: high-dose (HD) (3 µg hemagglutinin (HA)/dose) or low-dose (LD) formulations (0.03 µg or 0.003 µg HA) with AS03 and were followed to 34 weeks post-boost (pb). We examined serologic responses, spleen and bone marrow (BM) HA-specific antibody-secreting cells (ASCs) by ELISpot, influenza-specific cytokine/chemokine production in re-stimulated splenocytes by multiplex ELISA, and antigen-specific CD4+ T cells that express cytokines (IL-2, IFNγ, TNFα and IL-5) by flow cytometry. All formulations elicited robust serum antibody titers that persisted for at least 34 weeks. The number of antigen-specific ASCs in the spleen and BM were higher in the 2 LD +AS03 groups, but despite having fewer ASCs, the average spot size in the HD-unadjuvanted group was larger at later time-points, suggesting greater antibody production per cell. Striking differences in the long-term profiles induced by the different vaccine formulations may contribute to these different ASC profiles. The HD-unadjuvanted vaccine elicited strong Th2 cytokines during the first 6 weeks pb but LD+AS03 groups generated broader, more durable responses at later timepoints. Finally, the 0.03 µg HA+AS03 group generated the greatest number of antigen-specific CD4+ T cells and the highest percentage of poly-functional cells that expressed 2 or more cytokines. Although all of the tested vaccines induced durable antibody responses, we show that different vaccine formulations (dose-sparing, adjuvant) generate distinct long-term immune profiles. Furthermore, our data suggest that the different profiles may be generated through unique mechanisms.

Comparison of AS03 and Alum on immune responses elicited by A/H3N2 split influenza vaccine in young, mature and aged BALB/c mice.

INTRODUCTION: There is great interest in developing more effective influenza vaccines for the elderly. Oil-in-water adjuvants can boost humoral responses to seasonal vaccines in elderly subjects but relatively little is known about their mechanism of action. METHODS: We compared humoral and cellular immune profiles in young adult (2 months), mature (11-12 months) or aged (16-17 month) female BALB/c mice following two doses of Alum or AS03-adjuvanted A/H3N2 split-virus antigen (A/Uruguay/716/2007) at 0.75 or 3 µg hemagglutinin (HA) per dose intramuscularly versus 3 µg HA without adjuvant. RESULTS: Overall, hemagglutination inhibition (HAI), microneutralization (MN) and end-point ELISA titres were higher in the young mice and when an adjuvant was used. Both adjuvants increased humoral responses in older animals but the highest titres across all groups were observed in the AS03-adjuvanted groups. Neither IgG avidity nor A/H3N2-specific splenocyte proliferation was influenced by age, antigen dose or adjuvant. In contrast, cytokine production by ex vivo-stimulated splenocytes differed widely between groups. Most cytokine levels in older mice vaccinated with antigen alone (3 µg HA/dose) were ≤ 50% of those in young animals. In young mice, cytokine levels increased modestly with Alum and significantly with AS03. Increases tended to be greatest at the lower antigen dose (0.75 µg versus 3 µg HA). In the older animals, Alum had little impact on cytokine production but responses in the AS03 groups paralleled those of the young mice (broad activation of Th1, Th2, and Th17-type cytokines) and the greatest increases were seen with the higher antigen dose (3 µg HA). CONCLUSIONS: In both young and aged mice, Alum and AS03 increased the magnitude of humoral and cellular responses to split influenza virus vaccination. Overall, these effects were most pronounced in the younger animals and the groups receiving AS03. These data support the use of oil-in-water adjuvants in influenza vaccines targeting the elderly.

Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP).

Medicago, Inc. has developed an efficient virus-like particle (VLP) vaccine production platform using the Nicotiana benthamiana expression system, and currently has influenza-based products targeting seasonal/pandemic hemagglutinin (HA) proteins in advanced clinical trials. We wished to generate a trackable HA-based VLP that would allow us to study both particle assembly in plants and VLP interactions within the mammalian immune system. To this end, a fusion protein was designed, composed of H5 (from influenza A/Indonesia/05/2005 [H5N1]) with enhanced green fluorescent protein (eGFP). Expression of H5-eGFP in N. benthamiana produced brightly fluorescent ∼160 nm particles resembling H5-VLPs. H5-eGFP-VLPs elicited anti-H5 serologic responses in mice comparable to those elicited by H5-VLPs in almost all assays tested (hemagglutination inhibition/IgG(total)/IgG1/IgG2b/IgG2a:IgG1 ratio), as well as a superior anti-GFP IgG response (mean optical density = 2.52 ± 0.16 sem) to that elicited by soluble GFP (mean optical density = 0.12 ± 0.06 sem). Confocal imaging of N. benthamiana cells expressing H5-eGFP displayed large fluorescent accumulations at the cell periphery, and draining lymph nodes from mice given H5-eGFP-VLPs via footpad injection demonstrated bright fluorescence shortly after administration (10 min), providing proof of concept that the H5-eGFP-protein/VLPs could be used to monitor both VLP assembly and immune trafficking. Given these findings, this novel fluorescent reagent will be a powerful tool to gain further fundamental insight into the biology of influenza VLP vaccines.

AS03-Adjuvanted, Very-Low-Dose Influenza Vaccines Induce Distinctive Immune Responses Compared to Unadjuvanted High-Dose Vaccines in BALB/c Mice.

During the 2009-2010 influenza pandemic, an adjuvanted, dose-sparing vaccine was recommended for most Canadians. We hypothesize that differences exist in the responses to AS03-adjuvanted, low antigen (Ag) dose versus unadjuvanted, full-dose vaccines. We investigated the relationship between Ag dose and the oil-in-water emulsion Adjuvant System AS03. BALB/c mice received two IM doses of AS03A or AS03B with exaggerated dilutions of A/Uruguay/716/2007 H3N2 split virion vaccine Ag. Immune responses were assessed 3 weeks after the booster. Unadjuvanted "high" (3 µg) and low-dose (0.03-0.003 µg) vaccines generated similar serum antibody titers and cytokine secretion patterns in restimulated splenocytes. Compared to unadjuvanted "high-dose" vaccination, both AS03A and AS03B-adjuvanted low-dose vaccines tended to elicit higher serum antibody titers, broader induction of cytokine secretion and generated more influenza-specific antibody secreting cells and cytokine-secreting CD4 and CD8 T cells in splenocytes. We show that varying Ag and/or AS03 dose in this influenza vaccination mouse model can strongly influence both the magnitude and pattern of the immune response elicited. These findings are highly relevant given the likelihood of expanded use of adjuvanted, dose-sparing vaccines and raise questions about the use of "standard" doses of vaccines in pre-clinical vaccine studies.

High level antibody avidity is achieved in HIV-seropositive recipients of an inactivated split adjuvanted (AS03A) influenza vaccine.

PURPOSE: More severe influenza disease and poor vaccine immunogenicity is reported in HIV-infected patients. We measured antibody avidity after influenza vaccination in HIV patients to assess vaccine efficacy. METHODS: Two dosing strategies (Group1: single dose, n = 28. Group2: single dose plus booster, n = 36) with an AS03A-adjuvanted H1N12009 pandemic influenza vaccine (Arepanrix, GSK) were assessed in HIV patients. Serum hemagglutination inhibition (HAI) titers and antibody avidity reported as an avidity index (AI) were measured at days 21 and 42 and at 6 months. RESULTS: Baseline HIV parameters were similar among all participants. Eighteen participants had measurable baseline HAI titers. In these subjects, AI was at ~9 at baseline and was not significantly increased by one or two vaccine doses. In those without detectable baseline antibodies, immunization induced modest antibody titers [Group1 HAI, 61 (26-144); Group2 HAI, 46 (28-76)] with high AI after one dose at day 21 [Group1 AI, 8.8 (7.3-10.7); Group2 AI, 8.9 (7.8-10.1)]. A second dose of vaccine generated significantly higher HAI titers at day 42 [Group1 HAI, 41 (18-90); Group2 HAI, 92 (64-132)] and persisted to 6 months [Group1 HAI, 9 (6-13); Group2 HAI, 19 (13-30)]. All subjects who produced detectable HAI titers after vaccination generated high antibody avidity (AI, 9-10), which persisted up to 6 months. CONCLUSION: In participants initially seronegative, two doses of vaccine enabled a greater percentage of subjects to respond to the vaccine and elicited higher HAI titers. All subjects who produced detectable HAI titers also rapidly generated high AI in the short and long term. We demonstrate that high avidity antibodies can be achieved after vaccination and support a two-dose immunization strategy for HIV-positive subjects.

Unusual patterns of IgG avidity in some young children following two doses of the adjuvanted pandemic H1N1 (2009) influenza virus vaccine.

During the 2009-2010 H1N1 influenza pandemic, an adjuvanted monovalent vaccine containing ∼25% of the normal antigen dose and AS03 adjuvant was widely used in Canada. This vaccine was found to be well-tolerated and immunogenic in young children (D. W. Scheifele et al., Pediatr. Infect. Dis. J. 30:402-407, 2011). We report here additional analyses to further characterize the humoral response to this vaccine. We measured standard hemagglutination inhibition (HAI) and microneutralization (MN) titers, as well as influenza virus-specific IgG avidity and subclass distribution by enzyme-linked immunosorbent assay in 73 subjects. Sera were collected before (day 0) and 3 weeks after each dose of vaccine (days 21 and 42). Most children (55/73) had undetectable HAI and MN titers at day 0 (presumed to be antigen naive) and mounted good responses at days 21 and 42. The majority of these children (43/55) had the expected pattern of an increasing IgG avidity index (AI) after each dose of vaccine (not detected [ND], 0.30, and 2.97 at days 0, 21, and 42, respectively). The avidity responses in the remaining children (12/55) were quite different, with AIs increasing abruptly after the first dose and then declining after the second dose of vaccine (ND, 8.83, and 7.15, respectively). These children also had higher concentrations of influenza virus-specific IgG1 and IgG3 antibodies at day 21. Although the antibody titers were similar, some antigen-naive children demonstrated an unusual pattern of avidity maturation after two immunizations with AS03-adjuvanted, low-dose influenza virus vaccine. These data suggest the presence of subtle differences in the quality of the antibodies produced by some subjects in response to this vaccine.

Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling.

BACKGROUND: Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. METHODOLOGY/PRINCIPAL FINDINGS: We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H)1 CD4(+) and CD8(+) T cells and a systemic LACK-specific T(H)1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H)1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H)1 response. CONCLUSIONS/SIGNIFICANCE: This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.

Generation and evaluation of A2-expressing Lactococcus lactis live vaccines against Leishmania donovani in BALB/c mice.

Leishmaniasis is a parasitic disease affecting over 12 million individuals worldwide. As current treatments are insufficient, the development of an effective vaccine is a priority. This study generated and assessed the efficacy of Leishmania vaccines engineered from the non-colonizing, non-pathogenic Gram-positive bacterium Lactococcus lactis. A truncated, codon-optimized version of the A2 antigen from Leishmania donovani was engineered for expression in Lactococcus lactis in three different subcellular compartments: in the cytoplasm, secreted outside the cell or anchored to the cell wall. These three A2-expressing Lactococcus lactis strains were tested for their ability to generate A2-specific immune responses and as live vaccines against visceral Leishmania donovani infection in BALB/c mice. Subcutaneous immunization with live Lactococcus lactis expressing A2 anchored to the cell wall effectively induced high levels of antigen-specific serum antibodies. It was demonstrated that Lactococcus lactis-based vaccines are a feasible approach in the generation of live vaccines against leishmaniasis. The Lactococcus lactis strains generated in this study provide an excellent foundation for further studies on live bacterial vaccines against leishmaniasis and other pathogens.

Innate inflammatory responses to the Gram-positive bacterium Lactococcus lactis.

Lactococcus lactis is a non-pathogenic and non-colonizing Gram-positive bacterium commonly used in the dairy industry. To support the potential applications of this bacterium, such as use as an oral live vaccine, it is of interest to investigate the adjuvant properties of L. lactis. We compared the proinflammatory effects of L. lactis with two non-pathogenic Gram-negative bacteria: Escherichia coli and Salmonella typhi, a widely studied live vaccine. The gene expression profiles of chemokines induced by the three bacteria were examined in macrophages in vitro and in cells recruited into murine air-pouches in vivo. In addition, we studied the effect of co-incubating bacteria with dendritic cells (DCs) generated from mice bone marrow. We demonstrate that L. lactis exhibits proinflammatory effects, which indicates a capacity for adjuvanticity by this bacterium.

Conjugative transfer of the Lactococcus lactis chromosomal sex factor promotes dissemination of the Ll.LtrB group II intron.

The Ll.LtrB group II intron from the low-G+C gram-positive bacterium Lactococcus lactis was the first bacterial group II intron shown to splice and mobilize in vivo. This retroelement interrupts the relaxase gene (ltrB) of three L. lactis conjugative elements: plasmids pRS01 and pAH90 and the chromosomal sex factor. Conjugative transfer of a plasmid harboring a segment of the pRS01 conjugative plasmid including the Ll.LtrB intron allows dissemination of Ll.LtrB among L. lactis strains and lateral transfer of this retroelement from L. lactis to Enterococcus faecalis. Here we report the dissemination of the Ll.LtrB group II intron among L. lactis strains following conjugative transfer of the native chromosomally embedded L. lactis sex factor. We demonstrated that Ll.LtrB dissemination is highly variable and often more efficient from this integrative and conjugative element than from an engineered conjugative plasmid. Cotransfer among L. lactis strains of both Ll.LtrB-containing elements, the conjugative plasmid and the sex factor, was detected and shown to be synergistic. Moreover, following their concurrent transfer, both mobilizable elements supported the spread of their respective copies of the Ll.LtrB intron. Our findings explain the unusually high efficiency of Ll.LtrB mobility observed following conjugation of intron-containing plasmids.