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.

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.Group II introns are large ribozymes that splice autocatalytically from their pre-mRNAs (1,14,17,22). Some selfsplicing group II introns are also mobile retroelements that invade new DNA sites in a duplicative process using an RNA intermediate, like retrotransposons and retroviruses (1). They can reinsert either in cognate intronless alleles (homing site [HS]) by retrohoming or in nonhomologous sites by retrotransposition (1,5,6,11,12). Mobile group II introns harbor a multifunctional open reading frame (ORF) that is directly involved in their mobility processes (1). Group II introns are found in eubacteria (7, 15), archaea (8, 25), and eukaryotic organelles derived from bacteria such as fungal and plant mitochondria and plant chloroplasts (1,14,17,22). Horizontal transfer of group II introns between organisms is a well-accepted model of intron dissemination and evolution. This model suggests that group II introns are not only mobile within cells but can also be transferred between species, where they can invade new sites (3,14).The Lactococcus lactis LtrB group II intron (Ll.LtrB) is the first bacterial group II intron that was shown to splice and mobilize in vivo (18,19,23). L. lactis, an industrially important low-GϩC gram-positive bacterium, is extensively used in the dairy industry. Ll.LtrB mobility via the retrohoming (5) and retrotransposition pathways (6, 11, 12) was studied in both L. lactis and Escherichia coli. The Ll.LtrB group II intron (2.5 kb) harbors an ORF called LtrA (599 amino acids) that exhibits reverse transcriptase, endonuclease, and RNA maturase activities (16). These three functions are essential for retrohoming of Ll.LtrB to intronless alleles (5). F...

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-g 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 splitvirion vaccine.KEYWORDS aged-mouse model, influenza, virus-like particles (VLPs), plant-made vaccines A ccording to the World Health Organization, influenza epidemics account for 250,000 to 500,000 deaths worldwide every year (http://www.who.int/mediacentre/factsheets/ fs211/en/). Although vaccines are widely recommended to protect against influenza, the elderly often respond poorly, in part due to prior experience with influenza virus antigens (Ag) (1), but also as a result of immunosenescence (2). The latter affects both innate and adaptive immune responses and has broad implications for both natural infection and vaccination (1, 2).Influenza vaccines for adults are administered by either intramuscular (i.m.) or intradermal injection of detergent-split virions at a fixed dose of 15 g hemagglutinin (HA)/strain (3). These vaccines typically elicit strong antibody responses in healthy young adults and achieve vaccine efficacy (VE) that varies between strains and years but averages 50 to 60% (4). These formulations work less well in the elderly (5

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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.