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The chlorophyll fluorescence parameter Fv /Fm reflects the maximum quantum efficiency of photosystem II (PSII) photochemistry and has been widely used for early stress detection in plants. Previously, we have used a three-tiered approach of phenotyping by Fv /Fm to identify naturally existing genetic variation for tolerance to severe heat stress (3 days at 40°C in controlled conditions) in wheat (Triticum aestivum L.). Here we investigated the performance of the previously selected cultivars (high and low group based on Fv /Fm value) in terms of growth and photosynthetic traits under moderate heat stress (1 week at 36/30°C day/night temperature in greenhouse) closer to natural heat waves in North-Western Europe. Dry matter accumulation after 7 days of heat stress was positively correlated to Fv /Fm . The high Fv /Fm group maintained significantly higher total chlorophyll and net photosynthetic rate (PN ) than the low group, accompanied by higher stomatal conductance (gs ), transpiration rate (E) and evaporative cooling of the leaf (ΔT). The difference in PN between the groups was not caused by differences in PSII capacity or gs as the variation in Fv /Fm and intracellular CO2 (Ci ) was non-significant under the given heat stress. This study validated that our three-tiered approach of phenotyping by Fv /Fm performed under increasing severity of heat was successful in identifying wheat cultivars differing in photosynthesis under moderate and agronomically more relevant heat stress. The identified cultivars may serve as a valuable resource for further studies to understand the physiological mechanisms underlying the genetic variability in heat sensitivity of photosynthesis., (© 2014 Scandinavian Plant Physiology Society.)

In the recent decade, epidemic meningitis in the African meningitis belt has mostly been caused by Neisseria meningitidis of serogroups A, W and X (MenA, MenW and MenX, respectively). There is at present no licensed vaccine available to prevent MenX meningococcal disease. To explore a trivalent MenAWX vaccine concept, we have studied the immunogenicity in mice of MenX outer membrane vesicles (X-OMV) or MenX polysaccharide (X-PS) when combined with a bivalent A-OMV and W-OMV (AW-OMV) vaccine previously shown to be highly immunogenic in mice. The vaccine antigens were produced from three representative wild type strains of MenA (ST-7), MenW (ST-11) and MenX (ST-751) isolated from patients in the African meningitis belt. Groups of mice were immunized with two doses of X-OMV or X-PS combined with the AW-OMV vaccine or as individual components. All vaccine preparations were adsorbed to Al(OH)3. Sera from immunized mice were tested by ELISA and immunoblotting. Functional antibody responses were measured as serum bactericidal activity (SBA) and opsonophagocytic activity (OPA). Immunization of mice with X-OMV, alone or in combination with AW-OMV induced high levels of anti-X OMV IgG. Moreover, X-OMV alone or in combination with the AW-OMV vaccine induced high SBA and OPA titers against the MenX target strain. X-PS alone was not immunogenic in mice; however, addition of the AW-OMV vaccine to X-PS increased the immunogenicity of X-PS. Both AWX vaccine formulations induced high levels of IgG against A- and W-OMV and high SBA titers against the MenA and MenW vaccine strains. These results suggest that a trivalent AWX vaccine, either as a combination of OMV or OMV with X-PS, could potentially prevent the majority of meningococcal disease in the meningitis belt.

To investigate the effect of the light spectrum on photosynthesis, growth, and secondary metabolites Rosa hybrida 'Scarlet', Chrysanthemum morifolium 'Coral Charm', and Campanula portenschlagiana 'BluOne' were grown at 24/18°C day/night temperature under purpose-built LED arrays yielding approximately 200 μmol m(-2)s(-1) at plant height for 16 h per day. The four light treatments were (1) 40% Blue/60% Red, (2) 20% Blue/80% Red, (3) 100% Red, and (4) 100% White (Control). The plant height was smallest in 40% Blue/60% Red in roses and chrysanthemums, while the biomass was smallest in the white control in roses and in 100% Red in chrysanthemums. The total biomass was unaffected by the spectrum in campanulas, while the leaf area was smallest in the 40% Blue/60% Red treatment. In 100% Red curled leaves and other morphological abnormalities were observed. Increasing the blue to red ratio increased the stomatal conductance though net photosynthesis was unaffected, indicating excess stomatal conductance in some treatments. With higher blue light ratio all phenolic acids and flavonoids increased. In view of the roles of these secondary metabolites as antioxidants, anti-pathogens, and light protectants, we hypothesize that blue light may predispose plants to better cope with stress., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The genotypic response of wheat cultivars as affected by two methods of heat stress treatment (treatment of intact plants in growth chambers versus treatment of detached leaves in test tubes) in a temperature controlled water bath were compared to investigate how such different methods of heat treatment affect chlorophyll fluorescence parameters. A set of 41 spring wheat cultivars differing in their maximum photochemical efficiency of photosystem (PS) II (Fv/Fm) under heat stress conditions was used. These cultivars were previously evaluated based on the heat treatment of intact plants. The responses of the same cultivars to heat stress were compared between the two methods of heat treatment. The results showed that in detached leaves, all of the fluorescence parameters remained almost unaffected in control (20°C at all durations tested), indicating that the detachment itself did not affect the fluorescence parameters. In contrast, heat induced reduction in the maximum photochemical efficiency of PSII of detached leaves occurred within 2h at 40°C and within 30min at 45°C, and the response was more pronounced than when intact plants were heat stressed for three days at 40°C. The proportion of total variation that can be ascribed to the genetic differences among cultivars for a trait was estimated as genetic determination. During heat treatment, the genetic determination of most of the fluorescence parameters was lower in detached leaves than in intact plants. In addition, the correlation of the cultivar response in intact plants versus detached leaves was low (r=0.13 (with expt.1) and 0.02 with expt.2). The most important difference between the two methods was the pronounced difference in time scale of reaction, which may indicate the involvement of different physiological mechanisms in response to high temperatures. Further, the results suggest that genetic factors associated with cultivar differences are different for the two methods of heat treatment., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Meningococcal meningitis is a significant global health challenge, especially for sub-Saharan area: the African meningitis belt. Neisseria meningitidis of serogroup A (MenA) is responsible for the large number of epidemics that have been recorded in these countries. To determine the level of antibodies against meningococcal A polysaccharide (APS) that correlates with protection against MenA disease in the African meningitis belt, it may be important to consider antibody avidity along with quantity. In this study, two ELISA methods using the chaotropic agent ammonium thiocyanate were compared and employed to measure avidity indexes (AI) of IgG antibodies against APS in controls and in acute and convalescent sera from Ethiopian meningococcal patients. High statistical correlations between the AIs determined by the two methods were observed. The geometric mean AI (GMAI) increased with time from acute to convalescent sera indicating affinity maturation. GMAI was significantly higher in convalescent sera from the MenA patients and in sera from the controls than in acute sera from patients with meningococcal disease. A significant correlation between serum bactericidal activity titres (SBA) and concentration of IgG antibodies against APS was observed; however, our results did not indicate that determination of antibody avidities by the thiocyanate elution method gave a better correlation with SBA than anti-APS IgG concentrations determined by the standard ELISA method., (© 2014 John Wiley & Sons Ltd.)

Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP) process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB) using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA), serogroup W (dOMVW), and serogroup X (dOMVX) were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC), Bordetella pertussis (dOMVBP), Mycobacterium smegmatis (dOMVSM), and BCG (dOMVBCG). The immunogenicity of the OMV has been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice has shown their protective potential. dOMVB has been evaluated with non-neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin, and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates.

Outer-membrane vesicles (OMVs) have inherent adjuvant properties, and many vaccines use OMV as vaccine components. Utilizing the adjuvant properties of OMV could lead to the formulation of vaccines that are less expensive and potentially more immunogenic than covalently conjugated polysaccharide vaccines. We evaluated the adjuvant effect in Balb/c mice of combinations of OMV from Neisseria meningitidis serogroup A and W135 as compared to that of the non-covalently conjugated capsular polysaccharide A. Both antigens were adsorbed onto aluminum hydroxide. The mice were given a booster dose of plain polysaccharide A to stimulate an immunologic memory response. Subclasses determination and cytokine assays demonstrated the capacity of OMV to induce a IgG2a/IgG2b isotype profile and IFN-γ production, suggesting the induction of a Th1 pattern immune response. Lymphoproliferative responses to OMVs were high, with affinity maturation of antibodies observed. Bactericidal titers after the booster dose were also observed. Memory B cells and long-term memory T cells were also detected. The results of this study indicate that combined meningococcal serogroup A and W135 OMV can activate cell-mediated immunity and induce a long-term memory response.

Meningococci of serogroups A and W (MenA and MenW) are the main causes of epidemic bacterial meningitis outbreaks in sub-Saharan Africa. In this study we prepared a detergent extracted outer membrane vesicle (dOMV) vaccine from representative African MenA and MenW strains, and compared the immunogenicity of this vaccine with existing meningococcal conjugate and polysaccharide (PS) vaccines in mice. NMRI mice were immunized with preclinical batches of the A+W dOMV vaccine, or with commercially available vaccines; a MenA conjugate vaccine (MenAfriVac(®), Serum Institute of India), ACYW conjugate vaccine (Menveo(®), Novartis) or ACYW PS vaccine (Mencevax(®), GlaxoSmithKline). The mice received 2 doses of 1/10 or 1/50 of a human dose with a three week interval. Immune responses were tested in ELISA, serum bactericidal activity (SBA) and opsonophagocytic activity (OPA) assays. High levels of IgG antibodies against both A and W dOMV were detected in mice receiving the A+W dOMV vaccine. High SBA titers against both MenA and MenW vaccine strains were detected after only one dose of the A+W dOMV vaccine, and the titers were further increased after the second dose. The SBA and OPA titers in mice immunized with dOMV vaccine were significantly higher than in mice immunized with the ACYW-conjugate vaccine or the PS vaccine. Furthermore, the A+W dOMV vaccine was shown to induce SBA and OPA titers against MenA of the same magnitude as the titers induced by the A-conjugate vaccine. In conclusion, the A+W dOMV vaccine induced high levels of functional antibodies to both MenA and MenW strains, levels that were shown to be higher or equal to the levels induced by licensed meningococcal vaccines. Thus, an A+W dOMV vaccine could potentially serve as an alternative or a supplement to existing conjugate and PS vaccines in the African meningitis belt., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Modern highly insulated greenhouses are more energy efficient than conventional types. Furthermore applying dynamic greenhouse climate control regimes will increase energy efficiency relatively more in modern structures. However, this combination may result in higher air and crop temperatures. Too high temperature affects the plant photosynthetic responses, resulting in a lower rate of photosynthesis. To predict and analyse physiological responses as stress indicators, two independent experiments were conducted, to detect the effect of high temperature on photosynthesis: analysing photosystem II (PSII) and stomatal conductance (gs). A combination of chlorophyll a fluorescence, gas exchange measurements and infrared thermography was applied using Chrysanthemum (Dendranthema grandiflora Tzvelev) 'Coral Charm' as a model species. Increasing temperature had a highly significant effect on PSII when the temperature exceeded 38 °C for a period of 7 (±1.8) days. High temperature decreased the maximum photochemical efficiency of PSII (Fv/Fm), the conformation term for primary photochemistry (Fv/Fo) and performance index (PI), as well as increased minimal fluorescence (Fo). However, at elevated CO2 of 1000 μmol mol(-1) and with a photosynthetic photon flux density (PPFD) of 800 μmol m(-2) s(-1), net photosynthesis (Pn) reached its maximum at 35 °C. The thermal index (IG), calculated from the leaf temperature and the temperature of a dry and wet reference leaf, showed a strong correlation with gs. We conclude that 1) chlorophyll a fluorescence and a combination of fluorescence parameters can be used as early stress indicators as well as to detect the temperature limit of PSII damage, and 2) the strong relation between gs and IG enables gs to be estimated non-invasively, which is an important first step in modelling leaf temperature to predict unfavourable growing conditions in a (dynamic) semi closed greenhouse., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The utility of wild-type outer membrane vesicle (wtOMV) vaccines against serogroup B (MenB) meningococcal disease has been explored since the 1970s. Public health interventions in Cuba, Norway and New Zealand have demonstrated that these protein-based vaccines can prevent MenB disease. Data from large clinical studies and retrospective statistical analyses in New Zealand give effectiveness estimates of at least 70%. A consistent pattern of moderately reactogenic and safe vaccines has been seen with the use of approximately 60 million doses of three different wtOMV vaccine formulations. The key limitation of conventional wtOMV vaccines is their lack of broad protective activity against the large diversity of MenB strains circulating globally. The public health intervention in New Zealand (between 2004-2008) when MeNZB was used to control a clonal MenB epidemic, provided a number of new insights regarding international and public-private collaboration, vaccine safety surveillance, vaccine effectiveness estimates and communication to the public. The experience with wtOMV vaccines also provide important information for the next generation of MenB vaccines designed to give more comprehensive protection against multiple strains.

In view of the global climate change, heat stress is an increasing constraint for the productivity of wheat (Triticum aestivum L.). Our aim was to identify contrasting cultivars in terms of heat tolerance by mass screening of 1274 wheat cultivars of diverse origin, based on a physiological trait, the maximum quantum efficiency of PSII (Fv/Fm). A chlorophyll fluorescence protocol was standardised and used for repeated screening with increased selection pressure with a view to identifying a set of cultivars extreme for the trait. An initial mass screening of 1274 wheat cultivars with a milder heat stress of 38°C in 300µmolm-2s-1 for 2h with preheating at 33-35°C for 19h in 7-14µmolm-2s-1 light showed a genetic determination of 8.5±2.7%. A heat treatment of 40°C in 300µmolm-2s-1 for 72h in the second screening with 138 selected cultivars resulted in larger differentiation of cultivars with an increased genetic component (15.4±3.6%), which was further increased to 27.9±6.8% in the third screening with 41 contrasting cultivars. This contrasting set of cultivars was then used to compare the ability of chlorophyll fluorescence parameters to detect genetic difference in heat tolerance. The identification of a set of wheat cultivars contrasting for their inherent photochemical efficiency may aid future studies to understand the genetic and physiological nature of heat stress tolerance in order to dissect quantitative traits into simpler genetic factors.

The bacterium Neisseria meningitidis of serogroups A and W-135 has in the recent decade caused most of the cases of meningococcal meningitis in the African meningitis belt, and there is currently no efficient and affordable vaccine available demonstrated to protect against both these serogroups. Previously, deoxycholate-extracted outer membrane vesicle (OMV) vaccines against serogroup B meningococci have been shown to be safe and induce protection in humans in clonal outbreaks. The serogroup A and W-135 strains isolated from meningitis belt epidemics demonstrate strikingly limited variation in major surface-exposed protein structures. We have here investigated whether the OMV vaccine strategy also can be applied to prevent both serogroups A and W-135 meningococcal disease. A novel vaccine combining OMV extracted from recent African serogroup A and W-135 strains and adsorbed to aluminium hydroxide was developed and its antigenic characteristics and immunogenicity were studied in mice. The specificity of the antibody responses was analysed by immunoblotting and serum bactericidal activity (SBA) assays. Moreover, the bivalent A+W-135 vaccine was compared with monovalent A and W-135 OMV vaccines. The bivalent OMV vaccine was able to induce similar SBA titres as the monovalent A or W-135 OMV towards both serogroups. High SBA titres were also observed against a meningococcal serogroup C strain. These results show that subcapsular antigens may be of importance when developing broadly protective and affordable vaccines for the meningitis belt., (© 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.)

Background: Mushrooms are the only non-animal food source of vitamin D. Wild mushrooms have naturally high vitamin D(2) content, and cultivated mushrooms produce vitamin D(2) from ergosterol when exposed to supplementary UV-B during the post-harvest phase., Objectives: This study investigated the effects of providing supplementary UV-B during the growth phase on vitamin D(2) formation and the interactions with growth of mushrooms, as compared to supplementary UV-B during the post-harvest phase or exposure to sunlight for both cultivated and wild mushrooms., Methods: Experiments were carried out with exposure to supplementary UV-B just prior to harvest in the range of 0-2,400 mJ cm(-2). Mushrooms grew for 2 days with or without repeated UV-B exposure each day. Vitamin D(2) and growth rate were determined. In addition, some mushrooms were post-harvest treated by exposure at 200 mJ cm(-2) supplementary UV-B or natural sunlight, prior to vitamin D(2) determination., Results: The content of vitamin D(2) was 0.2-164 µg 100 g(-1) fresh weight, and there was a linear relationship between UV-dose up to 1,000 mJ cm(-2) and vitamin D(2) content. The fast growth rate of the mushrooms diluted the vitamin D(2) from 24 to 3 µg 100 g(-1) within 2 days of exposure at 200 mJ cm(-2). Following repeated UV-B exposure, vitamin D(2) increased to 33 µg vitamin D(2) 100 g(-1). Growth was unaffected by UV-B. Post-harvest exposure to supplementary UV-B resulted in a higher vitamin D(2) content of 32 µg 100 g(-1) compared to the 24 µg 100 g(-1) obtained from exposure to UV-B during the growth phase. In contrast, wild and cultivated mushrooms with and without exposure to sunlight had vitamin D(2) content in the range of 0.2-1.5 µg vitamin D(2) 100 g(-1)., Conclusions: This study showed that mushrooms with a well-defined content of vitamin D(2) can be obtained by exposure to supplementary UV-B just prior to harvest.

In the absence of an affordable conjugate meningococcal vaccine, mass vaccination campaigns with polysaccharide vaccines are the means to control meningitis epidemics in sub-Saharan Africa. Facing global vaccine shortage, the use of reduced doses, which have been shown to be protective by serum bactericidal activity, can save many lives. In this study, we investigated the antibody responses and avidity of IgG antibodies evoked against the serogroup A capsule of Neisseria meningitidis by different doses of an A/C/Y/W135 polysaccharide vaccine. Volunteers in Uganda were vaccinated with 1/10, 1/5 or a full dose (50 μg) and revaccinated with a full dose after 1 year. Specific IgG geometric mean concentrations and geometric mean avidity indices (GMAI) were determined by a modified enzyme-linked immunosorbent assay (ELISA) using thiocyanate as a chaotropic agent. After vaccination with 1/10 or 1/5 doses, the GMAI increased from 1 month to 1 year. One year following the initial dose, the GMAI levels were higher in the arm receiving reduced doses than for the arm receiving a full dose. Following the second full dose, avidity indices equalized at approximately the same level in the three arms. Although there are practical challenges to the use of reduced doses in the field, our findings suggest that reduced doses of polysaccharide vaccine are able to elicit antibodies of as good avidity against serogroup A polysaccharide as a full dose., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

Meningococcal outer membrane proteins have been used for over 20 years in more than 80 million doses; either as carrier protein in a Haemophilus influenzae type b (Hib) polysaccharide conjugate vaccine or as vesicle vaccine formulations against meningococcal disease. Conventional wild-type outer membrane vesicle (wtOMV) vaccines are the only formulations that have shown efficacy against serogroup B meningococcal disease. This has been demonstrated in Cuba, Norway and New Zealand; where epidemics, dominated by one particular strain or clone, were causing high rates of disease and wtOMV vaccines have been used for epidemic control. The most significant limitation for widespread use of wtOMV is that the immune response is strain-specific in infants, mostly directed against the immuno-dominant porin protein, PorA. The natural orientation of surface-exposed membrane antigens and the preservation of good physico-chemical stability are key features of OMV vaccines. The efficacy, tolerability and safety of wtOMV vaccines have been well proven. The most recent experience from New Zealand demonstrated a vaccine effectiveness of 80% for children less than 5 years of age, over a period of 24 months. Such results are encouraging for the further use of "tailor-made" OMV vaccines for epidemic control. Moreover, it provides opportunities for development of OMV vaccines with various additional cross-protective potential. There is good reason to believe that in the coming few years the "OMV-concept" will be exploited further and that a number of cross-protective "universal" antigens will be included in vaccines against serogroup B meningococcal disease. The desire to have a global vaccine strategy that enables susceptible individuals to be protected against all the relevant serogroups of meningococcal disease may become a reality.

We investigated light acclimation in seedlings of the temperate oak Quercus petraea (Matt.) Liebl. and the co-occurring sub-Mediterranean oak Quercus pyrenaica Willd. Seedlings were raised in a greenhouse for 1 year in either 70 (HL) or 5.3% (LL) of ambient irradiance of full sunlight, and, in the following year, subsets of the LL-grown seedlings were transferred to HL either before leaf flushing (LL-HLBF plants) or after full leaf expansion (LL-HLAF plants). Gas exchange, chlorophyll a fluorescence, nitrogen fractions in photosynthetic components and leaf anatomy were examined in leaves of all seedlings 5 months after plants were moved from LL to HL. Differences between species in the acclimation of LL-grown plants to HL were minor. For LL-grown plants in HL, area-based photosynthetic capacity, maximum rate of carboxylation, maximum rate of electron transport and the effective photochemical quantum yield of photosystem II were comparable to those for plants grown solely in HL. A rapid change in nitrogen distribution among photosynthetic components was observed in LL-HLAF plants, which had the highest photosynthetic nitrogen-use efficiency. Increases in mesophyll thickness and dry mass per unit area governed leaf acclimation in LL-HLBF plants, which tended to have less nitrogen in photosynthetic components and a lower assimilation potential per unit of leaf mass or nitrogen than LL-HLAF plants. The data indicate that the phenological state of seedlings modified the acclimatory response of leaf attributes to increased irradiance. Morphological adaptation of leaves of LL-HLBF plants enhanced photosynthetic capacity per unit leaf area, but not per unit leaf dry mass, whereas substantial redistribution of nitrogen among photosynthetic components in leaves of LL-HLAF plants enhanced both mass- and area-based photosynthetic capacity.

Dissecting the specificities of human antibody responses following disease caused by serogroup A meningococci may be important for the development of improved vaccines. We performed a study of Ethiopian patients during outbreaks in 2002 and 2003. Sera were obtained from 71 patients with meningitis caused by bacteria of sequence type 7, as confirmed by PCR or culture, and from 113 Ethiopian controls. Antibody specificities were analyzed by immunoblotting (IB) against outer membrane antigen extracts of a reference strain and of the patients' own isolates and by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) levels against lipooligosaccharide (LOS) L11 and the proteins NadA and NspA. IB revealed that the main antigens targeted were the proteins PorA, PorB, RmpM, and Opa/OpcA, as well as LOS. MenA disease induced significant increases in IgG against LOS L11 and NadA. The IgG levels against LOS remained elevated following disease, whereas the IgG anti-NadA levels returned to acute-phase levels in the late convalescent phase. Among adults, the anti-LOS IgG levels were similar in acute-phase patient sera as in control sera, whereas anti-NadA IgG levels were significantly higher in acute-phase sera than in controls. The IgG antibody levels against LOS and NadA correlated moderately but significantly with serum bactericidal activity against MenA strains. Future studies on immune response during MenA disease should take into account the high levels of anti-MenA polysaccharide IgG commonly found in the population and seek to clarify the role of antibodies against subcapsular antigens in protection against MenA disease.

Background: Neisseria meningitidis serogroup A is the main causative pathogen of meningitis epidemics in sub-Saharan Africa. In recent years, serogroup W135 has also been the cause of epidemics. Mass vaccination campaigns with polysaccharide vaccines are key elements in controlling these epidemics. Facing global vaccine shortage, we explored the use of fractional doses of a licensed A/C/Y/W135 polysaccharide meningococcal vaccine., Methods and Findings: We conducted a randomized, non-inferiority trial in 750 healthy volunteers 2-19 years old in Mbarara, Uganda, to compare the immune response of the full dose of the vaccine versus fractional doses (1/5 or 1/10). Safety and tolerability data were collected for all subjects during the 4 weeks following the injection. Pre- and post-vaccination sera were analyzed by measuring serum bactericidal activity (SBA) with baby rabbit complement. A responder was defined as a subject with a > or =4-fold increase in SBA against a target strain from each serogroup and SBA titer > or =128. For serogroup W135, 94% and 97% of the vaccinees in the 1/5- and 1/10-dose arms, respectively, were responders, versus 94% in the full-dose arm; for serogroup A, 92% and 88% were responders, respectively, versus 95%. Non-inferiority was demonstrated between the full dose and both fractional doses in SBA seroresponse against serogroups W135 and Y, in total population analysis. Non-inferiority was shown between the full and 1/5 doses for serogroup A in the population non-immune prior to vaccination. Non-inferiority was not shown for any of the fractionate doses for serogroup C. Safety and tolerability data were favourable, as observed in other studies., Conclusions: While the advent of conjugate A vaccine is anticipated to largely contribute to control serogroup A outbreaks in Africa, the scale-up of its production will not cover the entire "Meningitis Belt" target population for at least the next 3 to 5 years. In view of the current shortage of meningococcal vaccines for Africa, the use of 1/5 fractional doses should be considered as an alternative in mass vaccination campaigns., Trial Registration: ClinicalTrials.gov NCT00271479.

The molecular mechanisms underlying differentiation and lineage commitment in neural stem cells are just beginning to be understood, however the molecules involved in this process and their functions remain largely unknown. Here we studied the effects and downstream signals of apoptosis signal-regulating kinase 1 (ASK1) together with all-trans retinoic acid (ATRA) on neuronal differentiation in adult hippocampus-derived progenitor (AHP) cells. Following ASK1 over-expression and ATRA treatment in AHPs, a larger number of cells differentiated into neurons and the MASH1 promoter became activated. Analyzing downstream effector molecules of ASK1 or ATRA targeting the MASH1 promoter revealed that the myocyte enhancer factor 2C (MEF2C) mediated ASK1 signalling, while activation of Sp1 was involved in ATRA signalling. Chromatin immunoprecipitation assay on the promoter revealed that ASK1 induced binding of MEF2C and Ca(2+)/calmodulin-dependent kinase II to the MASH1 promoter. Taken together, ASK1 and ATRA activate MEF2C and Sp1, respectively, and up-regulate MASH1 protein expression.

MenBvac and MeNZB are safe and efficacious vaccines against serogroup B meningococcal disease. MenBvac is prepared from a B:15:P1.7,16 meningococcal strain (strain 44/76), and MeNZB is prepared from a B:4:P1.7-2,4 strain (strain NZ98/254). At 6-week intervals, healthy adults received three doses of MenBvac (25 microg), MeNZB (25 microg), or the MenBvac and MeNZB (doses of 12.5 microg of each vaccine) vaccines combined, followed by a booster 1 year later. Two-thirds of the subjects who received a monovalent vaccine in the primary schedule received the other monovalent vaccine as a booster dose. The immune responses to the combined vaccine were of the same magnitude as the homologous responses to each individual vaccine observed. At 6 weeks after the third dose, 77% and 87% of the subjects in the combined vaccine group achieved serum bactericidal titers of > or = 4 against strains 44/76 and NZ98/254, respectively, and 97% and 93% of the subjects achieved a fourfold or greater increase in opsonophagocytic activity against strains 44/76 and NZ98/254, respectively. For both strains, a trend of higher responses after the booster dose was observed in all groups receiving at least one dose of the respective strain-specific vaccine. Local and systemic reactions were common in all vaccine groups. Most reactions were mild or moderate in intensity, and there were no vaccine-related serious adverse events. The safety profile of the combined vaccine was not different from those of the separate monovalent vaccines. In conclusion, use of either of the single vaccines or the combination of MenBvac and MeNZB may have a considerable impact on the serogroup B meningococcal disease situation in many countries.

This study presents detailed analyses of total and specific serum antibody levels among 26 and 24 adult volunteers before vaccination and after the third dose of the meningococcal serogroup B outer membrane vesicle (OMV) vaccines MeNZB and MenBvac, respectively, in a clinical trial in New Zealand (V. Thornton, D. Lennon, K. Rasanathan, J. O'Hallahan, P. Oster, J. Stewart, S. Tilman, I. Aaberge, B. Feiring, H. Nokleby, E. Rosenqvist, K. White, S. Reid, K. Mulholland, M. J. Wakefield, and D. Martin, Vaccine 24:1395-1400, 2006). With the homologous vaccine strains as targets, both vaccines induced significant increases in serum bactericidal and opsonophagocytic activities and in the levels of immunoglobulin G (IgG) to OMV antigens in an enzyme-linked immunosorbent assay (ELISA) and to live meningococci by flow cytometry. They also induced high levels of activity against the heterologous strains, particularly in terms of opsonophagocytic activity and IgG binding to live bacteria. The antibody levels with the homologous and heterologous strains in the four assays showed high and significant positive correlations. Specific IgG binding to 10 major OMV antigens in each vaccine was measured by scanning of immunoblots; ELISAs for two antigens, lipopolysaccharide and Neisseria surface protein A (NspA), were also performed. Both vaccines elicited significant increases in IgG binding to all homologous and heterologous OMV antigens except NspA. The total IgG band intensity on the blots correlated significantly with the IgG levels determined by the OMV ELISA and flow cytometry. In conclusion, the results of the various immunological assays showed that both OMV vaccines gave rise to high levels of specific and cross-reacting antibodies.

To elucidate critical components of protective immune responses induced during the natural course of serogroup A meningococcal disease, we studied acute-, early-convalescent-, and late-convalescent-phase sera from Ethiopian patients during outbreaks in 2002 to 2003. Sera were obtained from laboratory-confirmed patients positive for serogroup A sequence type 7 (ST-7) meningococci (A:4/21:P1.20,9) (n = 71) and from Ethiopian controls (n = 113). The sera were analyzed using an enzyme-linked immunosorbent assay to measure levels of immunoglobulin G (IgG) against serogroup A polysaccharide (APS) and outer membrane vesicles (OMVs) and for serum bactericidal activity (SBA) using both rabbit and human complement sources. Despite relatively high SBA titers and high levels of IgG against APS and OMVs in acute-phase patient sera, significant increases were seen in the early convalescent phase. Antibody concentrations returned to acute-phase levels in the late convalescent phase. Considering all patients' sera, a significant but low correlation (r = 0.46) was observed between SBA with rabbit complement (rSBA) using an ST-5 reference strain and SBA with human complement (hSBA) using an ST-7 strain from Ethiopia. While rSBA demonstrated a significant linear relation with IgG against APS, hSBA demonstrated significant linear relationships with IgG against both APS and OMV. This study indicates that antibodies against both outer membrane proteins and APS may be important in providing the protection induced during disease, as measured by hSBA. Therefore, outer membrane proteins could also have a role as components of future meningococcal vaccines for the African meningitis belt.

In southern Uganda, only sporadic cases of serogroup A meningococcal disease have been reported since 2000. As part of an immunogenicity study of the tetravalent meningococcal polysaccharide vaccine, nasopharyngeal swab samples were collected twice, 4 weeks apart, from 2-19-year-old healthy individuals in Mbarara, Uganda. Only 15 (2.0%) of the 750 individuals carried meningococci asymptomatically. Most of the strains were non-serogroupable and none were serogroup A. However, two individuals carried a serogroup W135 strain, sequence type (ST)-11, similar to the clone that was responsible for the epidemic in Burkina Faso in 2002. Our study further demonstrates the geographical spread of serogroup W135 ST-11 strain and thus the potential epidemic risk.

MenBvac is an outer membrane vesicle vaccine against systemic meningococcal disease caused by serogroup B Neisseria meningitidis. In this placebo-controlled double-blind study including 374 healthy adolescents, the safety and immunogenicity of a schedule of three primary doses 6 weeks apart followed by a fourth dose a year later were evaluated. Antibody responses to the vaccine strain and heterologous strains (non-vaccine-type strains) and the persistence of these antibodies were measured by the serum bactericidal assay (SBA) and enzyme-linked immunosorbent assay up to 1 year after the last dose. The proportion of subjects with SBA titers of > or = 4 against the vaccine strain increased from 3% prevaccination to 65% after the third dose. Ten months later, this proportion had declined to 28%. The fourth dose induced a booster response demonstrated by 93% of subjects achieving a titer of > or = 4. One year after the booster dose, 64% still showed SBA titers of > or = 4. Cross-reacting antibodies were induced against all heterologous strains tested, although the magnitude of SBA titers differed widely between the different strains. All four doses of MenBvac were safe. Both MenBvac and the placebo had reactogenicity profiles of mild to moderate local and systemic reactions. Pain, the most common reaction, was reported with similar frequencies in both groups. No serious adverse events occurred in the MenBvac group. This study confirmed the good immunogenicity of the primary course of MenBvac and demonstrated prolonged persistence and increased cross-reactivity of functional antibodies elicited by a booster dose.

The objectives of this study were to collect and characterize epidemic meningococcal isolates from Ethiopia from 2002 to 2003 and to compare them to 21 strains recovered during the previous large epidemic of 1988 to 1989. Ninety-five patients in all age groups with clinical signs of meningitis and a turbid cerebrospinal fluid (CSF) sample were included in the study of isolates from 2002 to 2003. Seventy-one patients (74.7%) were confirmed as having Neisseria meningitidis either by culture (n = 40) or by porA PCR (n = 31) of their CSF. The overall case fatality rate (CFR) was 11.6%; the N. meningitidis-specific CFR was 4.2%. All 40 strains were fully susceptible to all antibiotics tested except sulfonamide, were serotyped as A:4/21:P1.20,9, and belonged to sequence type 7 (ST-7). The strains from 1988 to 1989 were also equally susceptible and were characterized as A:4/21:P1.20,9, but they belonged to ST-5. Antigenic characterization of the strains revealed differences in the repertoire of lipooligosaccharides and Opa proteins between the old and the recent strains. PCR analysis of the nine lgt genes revealed the presence of the lgtAHFG genes in both old and recent strains; lgtB was present in only some of the strains, but no correlation with sequence type was observed. Further analysis showed that in addition to their pgm alleles, the Ethiopian ST-5 and ST-7 strains also differed in their tbpB, opa, fetA, and lgtA genes. The occurrence of new antigenic structures in strains sharing the same serogroup, PorA, and PorB may help explain the replacement of ST-5 by ST-7 in the African meningitis belt.

As the first step towards control of a strain specific epidemic of meningococcal disease in New Zealand (NZ), this study, an observer-blind, randomised controlled trial in 75 healthy adults, evaluated safety and immunogenicity of two different dosages of a meningococcal group B vaccine administered in a three dose regime. The "tailor-made" outer membrane vesicle (OMV) vaccine (candidate vaccine) developed using a New Zealand meningococcal group B strain (B:4:P1.7b,4) was well tolerated with no vaccine related serious adverse events. Similar local and systemic reactions were observed in those receiving the New Zealand candidate vaccine and the control parent Norwegian vaccine (MenBvac). A four-fold rise in serum bactericidal antibodies (SBAb) against the vaccine strain 4-6 weeks after the third vaccination was achieved in 100% of New Zealand candidate vaccine 2,519 microg participants and in 87% of 50 microg participants. The safety and immunogenicity profile observed in this study of healthy adults enabled studies in children to be initiated using 25 microg dosage.

In order to investigate the role of potential genotypic differences in three economically important barley cultivars, experiments were carried out to determine the influence of supplemental ultraviolet-B (UV-B, 280-320 nm) radiation on reactive oxygen species (ROS), antioxidant activity and photosynthesis. Greenhouse-grown barley (Hordeum vulgare L.) cultivars 'Cork', 'Prestige' and 'Golden Promise' showed different responses to supplemental 280-320 nm (UV-B) representing 100, 138 and 238% levels of ambient biologically active UV-B radiation, respectively. Among the three cultivars studied, cv. Golden Promise was the most tolerant to UV-B, cv. Prestige was slightly more sensitive than cv. Cork. A comparison with the other two cultivars showed that under supplemental UV-B, Golden Promise leaves (i) retained a higher quantum yield of photosynthesis under photosynthetically active radiation (PAR, 400-700 nm) corresponding to growth conditions; (ii) had the smallest decrease in both electron transport rate and non-photochemical quenching under high PAR; (iii) contained less oxidized ascorbate [measured as dehydroascorbate or electron paramagnetic resonance (EPR) detectable monodehydroascorbate radicals] than either Cork or Prestige. Under the highest UV-B level applied, Golden Promise leaves maintained the same activity of both monodehydroascorbate-reductase (MDAR) and ascorbate-peroxidase (APX) enzymes, as untreated controls, while MDAR markedly decreased in the other two cultivars and APX slightly increased in cv. Prestige. These features, together with the observation of directly EPR-trappable free radicals and the light-independent accumulation of monodehydroascorbate radicals in Cork and Prestige but not in Golden Promise leaves under high UV-B suggest that Golden Promise plants suffered less oxidative stress than the two other cultivars.

There is currently no standardized serum bactericidal antibody (SBA) assay for evaluating immune responses to meningococcal outer membrane vesicle or protein vaccines. Four laboratories, Manchester Health Protection Agency (MC HPA), New Zealand Institute of Environmental Science and Research Limited (NZ ESR), Norwegian Institute of Public Health (NIPH), and Chiron Vaccines (Chiron), measured SBA titers in the same panel of human sera (n=76) from laboratory staff (n=21) vaccinated with MenBvac. Blood samples were collected prevaccination, prior to each of the three doses of MenBvac given at 6-week intervals, and 6 weeks following the third dose. Initial results showed a number of discrepancies in results between the four participating laboratories. The greatest effect on titers appeared to be due to differences among laboratories in the maintenance of the meningococcal serogroup B test strain, 44/76-SL. A repeat study was conducted using the same frozen isolate (meningococcal serogroup B test strain 44/76-SL), freshly distributed to all four laboratories. Using SBA titers from the tilt method for all samples, and using MC HPA as the comparator, the results were as follows for NZ ESR, NIPH, and Chiron, respectively, using log(10) titers: correlation coefficients (r) were 0.966, 0.967, and 0.936; intercepts were 0.08, 0.15, and 0.17; and slopes were 0.930, 0.851, and 0.891. In both prevaccination and postvaccination samples from 15 subjects assayed by all four laboratories, similar increases in SBA (fourfold or greater) were observed (for 11, 11, 9, and 9 subjects for MC HPA, NZ ESR, NIPH, and Chiron, respectively), and similar percentages of subjects with SBA titers of>or=4 p revaccination and 6 weeks following each dose were found. The SBA assay has been harmonized between the four different laboratories with good agreement on seroconversion rates, n-fold changes in titers, and percentages of subjects with SBA titers of >or=4.

Neisseria meningitidis bacteria of serogroup A are causing recurring meningitis epidemics on the African continent. An outer membrane vesicle (OMV) vaccine against serogroup A meningococci made from a subgroup III serogroup A meningococcal strain was previously shown to induce antibodies with serum bactericidal activity (SBA) in mice. We have here further investigated the properties of OMV vaccines made from five different subgroup III serogroup A meningococcal strains grown in a synthetic medium with low iron content. In addition to the major outer membrane proteins (PorA, PorB, RmpM, Opa and OpcA), small amounts of the NadA, TdfH, Omp85, FetA, FbpA and NspA outer membrane proteins, as well as lipooligosaccharides, were detected in the vaccines. The OMV vaccines were used to immunise mice. Anti-meningococcal IgG antibodies in the mouse sera were analysed by immunoblotting and by enzyme-linked immunosorbent assay against OMVs, and against live meningococcal cells in SBA and a flow-cytometric assay. The vaccines induced antibodies with high SBA and opsonophagocytic activity. The strongest IgG responses were directed against PorA. Significant SBA responses were also observed against a subgroup III strain, which did not express PorA, whereas no SBA was observed against a clone IV-1 serogroup A strain. An OMV vaccine from serogroup A meningococci may be an alternative to polysaccharide and conjugate polysaccharide vaccines for Africa.

Protein-based, outer membrane vesicle (OMV) vaccines have previously proven to be efficacious against serogroup B meningococcal disease in Norway and Cuba. Currently, a public health intervention is going on in order to control a serogroup B epidemic in New Zealand. The scale-up and standardization of vaccine production required for controlling the New Zealand epidemic has allowed the establishment of large-scale GMP manufacturing for OMV vaccines. The outcome of this will be licensing of the vaccine in New Zealand and possibly other countries. The availability of licensed OMV vaccines raises the question of whether such vaccines may provide the opportunity to control other outbreaks and epidemics. For instance, such a vaccine could control a localised outbreak of group B meningococci in Normandy, France. "Tailor-made" vaccines, focusing on the sub-capsular antigens may also be considered for use in sub-Saharan Africa for the prevention of the recurrent outbreaks by serogroups A and W135 meningococci. This assumption is based on the epidemiological observation that meningococcal outbreaks in Africa are clonal and are strikingly stable regarding their phenotypic characteristics.

Chlorophyll fluorescence has been routinely used for many years to monitor the photosynthetic performance of plants non-invasively. The relationships between chlorophyll fluorescence parameters and leaf photosynthetic performance are reviewed in the context of applications of fluorescence measurements to screening programmes which seek to identify improved plant performance. The potential role of chlorophyll fluorescence imaging in increasing both the sensitivity and throughput of plant screening programmes is examined. Finally, consideration is given to possible specific applications of chlorophyll fluorescence for screening of plants for tolerance to environmental stresses and for improvements in glasshouse production and post-harvest handling of crops.

Ash (Fraxinus excelsior L.) and beech (Fagus sylvatica L.) seedlings were grown in the field under three levels of natural light: (1) open, (2) gap and (3) shade. Light acclimation of photosynthesis was characterized by means of modulated chlorophyll a fluorescence of intact leaves and growth parameters were measured at the end of the growing season. Measurements of maximum photochemical efficiency (Fv/Fm) of dark-adapted leaves at intervals through the day showed that ash had a higher Fv/Fm than beech in open and gap plots but not in shade plots. This indicated a larger build-up of photoinhibition in beech under gap and open conditions. Steady-state light response curves of the operating efficiency of PSII (F'q/F'm), the electron transport rate (ETR) and the photochemical efficiency factor (F'q/F'v) showed greater variability across light treatments in ash than in beech. Both species exhibited similar responses of non-photochemical quenching (NPQ) to light. When the data were normalized to the mean maximum irradiance in the growth environment, all photochemical parameters showed a reduction in variation across treatments, indicating that light acclimation in the two species occurred primarily through adjustments in rates of photochemistry. Adjustments in thermal heat dissipation were small in both species. This pattern was stronger in ash, suggesting a greater degree of phenotypic plasticity in photosynthetic capacity in this earlier successional species. Contrary to our expectations, the build-up of photoinhibition in beech did not appear to have a negative effect on total biomass accumulation relative to ash.

Serogroup A Neisseria meningitidis organisms of the subgroup III have caused epidemics of meningitis in sub-Saharan Africa since their introduction into the continent in 1987. The population structure of these bacteria is basically clonal, and these meningococci are strikingly similar in their major outer membrane antigens PorA and PorB. Protein-based vaccines might be an alternative to prevent epidemics caused by these meningococci; thus, we developed an outer membrane vesicle (OMV) vaccine from a serogroup A meningococcal strain of subgroup III. The serogroup A OMV vaccine was highly immunogenic in mice and elicited significant bactericidal activity towards several other serogroup A meningococci of subgroup III. The IgG antibodies generated were in immunoblot shown to be mainly directed towards the PorA outer membrane protein. The results presented demonstrate the potential of an OMV vaccine as an optional strategy to protect against meningococcal disease caused by serogroup A in Africa.

There is still no general vaccine for prevention of disease caused by group-B meningococcal strains. Meningococcal lipopolysaccharides (LPSs) have received attention as potential vaccine candidates, but concerns regarding their safety have been raised. Peptide mimics of LPS epitopes may represent safe alternatives to immunization with LPS. The monoclonal antibody (MoAb) 9-2-L3,7,9 specific for Neisseria meningitidis LPS immunotype L3,7,9 is bactericidal and does not cross-react with human tissue. To explore the possibility of isolating peptide mimics of the epitope recognized by MoAb 9-2-L3,7,9, we have constructed two phage display libraries of six and nine random amino acids flanked by cysteines. Furthermore, we developed a system for the easy exchange of peptide-encoding sequences from the phage-display system to a hepatitis B core (HBc) expression system. Cyclic peptides that specifically bound MoAb 9-2-L3,7,9 at a site overlapping with the LPS-binding site were selected from both libraries. Three out of four tested peptides which reacted with MoAb 9-2-L3,7,9 were successfully presented as fusions to the immunodominant loop of HBc particles expressed in Escherichia coli. However, both peptide conjugates to keyhole limpet haemocyanin and HBc particle fusions failed to give an anti-LPS response in mice.

For evaluation of serum bactericidal activity (SBA) as surrogate for the efficacy of outer membrane vesicle (OMV) vaccines against Neisseria meningitidis serogroup B disease, we have reanalyzed data from a randomized double blind placebo-controlled efficacy trial involving 172000 secondary school students (aged 13-14 years) in Norway (1988-1991). A cohort of the efficacy trial consisting of 880 individuals was selected for immunogenicity studies. An efficacy of 87% was calculated for a 10-month observation period. However, after an observation period of 29 months, the estimated efficacy against group B disease induced by vaccination was 57%. The immunogenicity study showed that the SBA geometric mean titer (GMT) for the vaccinees was 2.4 before vaccination and 19.0 six weeks after the second vaccine dose. One year after vaccination the GMT was reduced to 2.8. A separate three-dose study with 304 adolescents showed that with a third dose at 10 months after the second dose (i.e. when cases of disease started to appear) a strong booster response was induced. Ten months after the second dose the SBA was reduced to near pre-immunization level. Following the third dose the SBA geometric mean titer of 2.7 increased to 62.3. One year after the third dose, the GMT was markedly higher than 6 weeks after the second dose (12.6 versus 8.8). Thus, protection after vaccination corresponds with the level of SBA. In order to reach lasting protective levels of SBA in a population, three vaccine doses are probably required. Measurements of SBA are likely to be useful for evaluating various upcoming formulations and improvements of immunization regimens for OMV vaccines.

Complement activation is closely associated with plasma endotoxin levels in patients with meningococcal infections. This study assessed complement activation induced by purified Neisseria meningitidis lipopolysaccharide (Nm-LPS), native outer membrane vesicles (nOMVs), LPS-depleted outer membrane vesicles (dOMVs), wild-type meningococci, and an LPS-free mutant (lpxA(-)) from the same strain (44/76) in whole blood anticoagulated with the recombinant hirudin analogue. Complement activation products (C1rs-C1 inhibitor complexes, C4d, C3bBbP, and terminal SC5b-9 complex) were measured by double-antibody EIAs. Nm-LPS was a weak complement activator. Complement activation increased with preparations containing nOMVs, dOMVs, and wild-type bacteria at constant LPS concentrations. With the same protein concentration, complement activation induced by nOMVs, dOMVs, and the LPS-free mutant was equal. The massive complement activation observed in patients with fulminant meningococcal septicemia is, presumably, an indirect effect of the massive endotoxemia. Outer membrane proteins may be more potent complement activators than meningococcal LPSs.

The RmpM protein has been reported to be present only in pathogenic Neisseria species. In the present study we demonstrate that this protein is also present at least in N. lactamica and N. sicca strains. The N. lactamica protein reacts with a RmpM-specific monoclonal antibody (185,H-8), having a molecular mass ( approximately 31 kDa) slightly lower than that of the meningococcal RmpM, and mouse antibodies from sera against outer membrane vesicles from both N. lactamica and N. sicca strains cross-react with the meningococcal RmpM. PCR and hybridization experiments with a complete rmpM probe agree with the immunodetection experiments. Our results strongly suggest that the meningococcal RmpM should not be considered a virulence marker, and the presence of this protein in the commensal species agrees with its role as a structural protein, proposed for the RmpM, which should be considerably conserved in the Neisseria species.

It is reported here that the PorB3 porin proteins of serotype 4 and 15 are poorly accessible for antibody binding on live Neisseria meningitidis bacteria, whereas the allelic PorB2 and the PorA outer membrane protein appear to be highly accessible. This was revealed by flow cytometry analysis using several mouse monoclonal antibodies (mAbs) as well as PorB3 specific antibodies isolated from post vaccination and patient sera. However, strong antibody binding to the PorB3 protein was observed after killing the bacteria with ethanol. The reason for the lack of epitope exposure could be a shielding effect of the carbohydrate chains of lipopolysaccharides (LPS) possibly combined with short extra-cellular loops in the PorB3 protein. The findings indicate that the PorB3 protein is not an optimal target for protective antibodies induced by vaccination.

Alternative strategies exist for prevention of group B Neisseria meningitidis (meningococcal) disease through vaccination (see Chapters 5 , 8 , 13 , 14 in this volume). However, the most promising approach to date has been the use of outer-membrane vesicle (OMV) vaccines for induction of bactericidal antibodies against cell-surface outer-membrane proteins (OMPs).

Enzyme-linked immunosorbent assay (ELISA, EIA) is a highly versatile and sensitive technique that can be used for quantitative as well as qualitative determination of almost any antigen or antibody. Reagents are stable, non-radioactive and, in most cases, commercially available. Owing to the simplicity and versatility of the method, ELISA represents probably one of the most used methods for studying antibody responses and antibody levels. Since Engvall and Perlman's first paper describing the ELISA in 1971 (1), almost all laboratories working in serology or immunology have designed their own assays with different protocols for coating with antigens, incubation conditions, detecting systems, and ways of reporting of the results. In most cases, there is no need for strict interlaboratory standardization of ELISAs and each laboratory will develop a system that suits their needs. However, for some ELISAs, e.g., used in diagnostic laboratories and in vaccine trials, standardization is important, and this is considered in "Meningococcal Disease" Edited by AJ Pollard and MCJ Maiden, (1a).

Chrysanthemum inducum-hybrid 'Coral Charm', Hibiscus rosa-sinensis L. 'Cairo Red' and Spathiphyllum wallisii Regel 'Petit' were grown in natural light in a greenhouse at three levels of irradiance using permanent shade screens. Light acclimation of photosynthesis was characterized using modulated chlorophyll a fluorescence of intact leaves. A close correlation was found between the degree of reduction of the primary electron acceptor Q(A) of Photosystem II (PS II) approximated as the fluorescence parameter 1-q(P), and light acclimation. The action range of 1-q(P) was 0-0.4 from darkness to full irradiance around noon, within the respective light treatments in the greenhouse, indicating that most PS II reaction centres were kept open. In general, the index for electron transport (ETR) measured by chlorophyll fluorescence was higher for high-light (HL) than intermediate-(IL) and low-light (LL) grown plants. However, HL Chrysanthemum showed 40% higher ETR than HL Hibiscus at light saturation, despite identical redox states of Q(A). The light acclimation of the non-radiative dissipation of excess energy in the antenna, NPQ, varied considerably between the species. However, when normalized against q(P), a strong negative correlation was found between thermal dissipation and ETR measured by chlorophyll fluorescence. To be able to accommodate a high flux of electrons through PS II, the plants with the highest light-saturated ETR had the lowest NPQ/q(P). The possibility of using chlorophyll fluorescence for quantification of the energy balance between energy input and utilization in PS II in intact leaves is discussed.

Neisseria meningitidis is one of the most common causes of purulent meningitis all over the world. Large epidemics caused by meningococci have spread during the last decade throughout vast areas of Africa, also outside the region referred to as the classic "Meningitis Belt". Globally, this organism each year causes about 300,000 cases and 30,000 deaths; most of these are children. Meningococci of serogroup A cause a major part of these epidemics, and a remarkable feature of the epidemical situation is that the bacteria differ very little in antigenic properties as they belong to the same clonal group of meningococci. Immunization with safe and effective vaccines is the most efficient way of combatting these epidemics. The currently available polysaccharide vaccines against serogroup. A meningococcal disease do not induce long-term immunological memory and do not provide adequate protection of children below two years of age. There is an urgent need for a vaccine that induces long-term immunological memory in all age groups, so it can be included in the routine vaccination program. In contrast to serogroup C, the development of conjugate vaccines against serogroup A meningococcal disease has not yielded the positive results hoped for. The development of alternative protein-based vaccines therefore needs to be intensified.

The meningococcal PorA protein is considered a promising vaccine candidate. Although much is understood regarding the structure of PorA proteins, little is known about the structure-function relationships of PorA antibodies. The aim of this study was to compare the functional and molecular characteristics of a human monoclonal antibody (MAb) and three murine MAbs specific for the PorA P1.7 serosubtype. Murine MAbs 207,B-4 (immunoglobulin G2a [IgG2a]) and MN14C11.6 (IgG2a) were both bactericidal and opsonophagocytic for P1.7-expressing meningococci, whereas human MAb SS269 (IgG3) and murine MAb 208,D-5 (IgA) initiated neither effector function. Epitope mapping with synthetic peptides revealed that MAbs 207,B-4 and 208,D-5 recognized the sequence ASGQ, which is the same specificity motif that a previous study had established for SS269 and MN14C11.6. Nucleotide and amino acid sequence analyses of the variable regions of the four MAbs showed that the SS269 V(H) region belonged to the VH3 family and was approximately 70% homologous to those of the murine MAbs which were all from the 7183 family, whereas the SS269 V(L) region belonged to the Vlambda1-b family and was less than 40% homologous to those of the murine MAbs which were all members of the Vkappa1 family. The Fab fragment of SS269 was cloned and expressed in Escherichia coli and was shown by enzyme-linked immunosorbent assay analyses to bind as well as intact SS269 MAb to P1.7,16 serosubtype group B strain 44/76. We conclude that distinct differences exist in the effector function activities and variable region gene sequences of human and murine P1.7-specific MAbs despite their recognition of similar epitopes.

Neisseria meningitidis, the cause of epidemic meningitis and acute lethal sepsis, synthesizes surplus lipopolysaccharides (LPSs) during growth, which are released as outer membrane vesicles (OMV) or "blebs". Meningococcal disease severity is related to plasma LPS levels. We have compared the biological activities of native outer membrane vesicles (nOMV) to those of purified Nm-LPS (Nm-LPS) and LPS-depleted OMV (dOMV) prepared from N. meningitidis. The LPS content of nOMV was determined spectrophotometrically by quantifying KDO and by silver-stained SDS-PAGE gels. The morphology of the preparations was studied by transmission electron microscopy. The Limulus amoebocyte lysate (LAL) assay was used to quantify LPS in the plasma solutions. The preparations were diluted in endotoxin-free heparin plasma to equal amounts of LPS (w/w) in the range 50-5000 pg/ml. The biological reactivity was tested by: (i) a monocyte target-assay (monocyte purity > or =96%); and (ii) a whole blood model, measuring the secretion of TNF-alpha and IL-6 induction of procoagulant activity in monocytes (PCA). In both models, nOMV induced dose-dependent cell responses (TNF-alpha, IL-6, PCA) similar to purified Nm-LPS, whereas dOMV induced minimal responses. However, LAL activity was significantly higher for nOMV than for purified Nm-LPS and dOMV. The cellular responses of purified Nm-LPS and nOMV were reduced (>95%) by a specific anti-CD14-antibody.

The infant rat infection model previously developed to evaluate protective ability of passively administered murine antibodies to group B meningococcal (MenB) surface antigens was adapted for human sera. Several challenge doses were tested, aiming at sensitive detection of protection with little interassay variability. Doses of 10(5) and 10(6) colony forming units of strain IH5341 (MenB:15:P1.7,16) injected intraperitoneally gave consistently high levels of bacteremia and meningitis developed in 6 h in 50-100% of the pups. A monoclonal antibody mAb735 to the MenB capsule, injected 1-2 h before bacterial challenge, gave full protection at a dose of 2 microg/pup. Sera from adult volunteers immunized with a MenB outer membrane vesicle vaccine reproducibly reduced bacterial counts in the blood and cerebrospinal fluid, whereas a normal human serum, lacking bactericidal and opsonophagocidal activity, was unprotective.

Context: Meningococcal disease occurs worldwide, and serogroup B disease accounts for a large proportion of cases. Although persons younger than 4 years are at greatest risk for serogroup B meningococcal disease, vaccine efficacy has not been demonstrated in this age group., Objective: To evaluate serum bactericidal activity (SBA) against homologous vaccine type strains and a heterologous Chilean epidemic strain of Neisseria meningitidis as a potential correlate for vaccine efficacy., Design: Double-blind, randomized controlled trial conducted between March 14 and July 20, 1994. All blood samples were taken by December 1994., Setting: Santiago, Chile, where a clonal serogroup B meningococcal disease epidemic began in 1993., Participants: Infants younger than 1 year (n = 187), children aged 2 to 4 years (n = 183), and adults aged 17 to 30 years (n = 173)., Intervention: Participants received 3 doses of outer-membrane protein (OMP) meningococcal vaccine developed in either Cuba or Norway or a control vaccine, with each dose given 2 months apart. Blood samples were obtained at baseline, prior to dose 3, and at 4 to 6 weeks after dose 3., Main Outcome Measure: Immune response, defined as a 4-fold or greater rise in SBA titer 4 to 6 weeks after dose 3 compared with prevaccination titer., Results: Children and adult recipients of either meningococcal vaccine were more likely than controls to develop an immune response to the heterologous epidemic strain. After 3 doses of vaccine, 31% to 35% of children responded to the vaccine vs 5% to placebo; 37% to 60% of adults responded to vaccine vs 4% to placebo (P<.05 vs control for all). Infants, however, did not respond. In contrast, against homologous vaccine type strains, the response rate was 67% or higher among children and adults and 90% or higher among infants (P<.001 vs control for all). Subsequent SBA against 7 isogenic homologous target strains identified class 1 OMP as the immunodominant antigen., Conclusions: These data suggest that neither serogroup B OMP meningococcal vaccine would confer protection during a heterologous epidemic. However, epidemic strain-specific vaccines homologous for class 1 OMP are promising candidates for the control of epidemic serogroup B meningococcal disease.

Antibodies against the class 4 outer membrane protein (OMP) from Neisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard beta-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.

Sera from vaccinees and controls who contracted serogroup B meningococcal disease during the blinded and open parts of a two-dose protection trial in Norway were compared for antigen-specific and bactericidal antibodies against vaccine strain 44/76 (B:15:P1.7,16). From 16 of 20 (80%) vaccinees and 26 of 35 (74%) controls, one or more serum samples (n = 104) were collected during the acute phase (1 to 4 days), early convalescent phase (5 to 79 days), and late convalescent phase (8 to 31 months) after onset of disease. Binding of immunoglobulin G (IgG) to the major outer membrane antigens (80- and 70-kDa proteins, class 1, 3, and 5 proteins, and lipopolysaccharide [LPS]) on immunoblots was measured by digital image analysis. Specific IgG levels in vaccinees increased from acute to early convalescent phases, followed by a decline, while controls showed a small increase over time. Vaccinees had significantly higher levels than controls against class 1 and 3 porins and LPS in acute sera, against all antigens during early convalescence, and against class 1 and 3 porins in the later sera. Vaccinees who were infected with strains expressing subtype P1.7,16 proteins demonstrated a level of IgG binding to protein P1.7,16 with early-convalescent-phase sera that was fourfold higher than that of those infected with other strains. Bactericidal titers in serum against the vaccine strain were 192-fold higher for vaccinees than those for controls during early convalescence, but similarly low levels were found during late convalescence. A vaccine-induced anamnestic response of specific and functional antibody activities was thus shown, but the decrease in protection over time after vaccination indicated that two vaccine doses did not induce sufficient levels of long-term protective antibodies.