Avidity of IgG antibodies against meningococcal serogroup a polysaccharide and correlations with bactericidal activity in sera from meningitis patients and controls from Ethiopia.

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.

An outer membrane vesicle vaccine for prevention of serogroup A and W-135 meningococcal disease in the African meningitis belt.

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.

A novel meningococcal outer membrane vesicle vaccine with constitutive expression of FetA: A phase I clinical trial.

OBJECTIVES: Outer membrane vesicle (OMV) vaccines are used against outbreaks of capsular group B Neisseria meningitidis (MenB) caused by strains expressing particular PorA outer membrane proteins (OMPs). Ferric enterobactin receptor (FetA) is another variable OMP that induces type-specific bactericidal antibodies, and the combination of judiciously chosen PorA and FetA variants in vaccine formulations is a potential approach to broaden protection of such vaccines. METHODS: The OMV vaccine MenPF-1 was generated by genetically modifying N. meningitidis strain 44/76 to constitutively express FetA. Three doses of 25 µg or 50 µg of MenPF-1 were delivered intra-muscularly to 52 healthy adults. RESULTS: MenPF-1 was safe and well tolerated. Immunogenicity was measured by serum bactericidal assay (SBA) against wild-type and isogenic mutant strains. After 3 doses, the proportion of volunteers with SBA titres ≥1:4 (the putative protective titre) was 98% for the wild-type strain, and 77% for the strain 44/76 FetA(on)PorA(off) compared to 51% in the strain 44/76 FetA(off)PorA(off), demonstrating that vaccination with MenPF-1 simultaneously induced FetA and PorA bactericidal antibodies. CONCLUSION: This study provides a proof-of-concept for generating bactericidal antibodies against FetA after OMV vaccination in humans. Prevalence-based choice of PorA and FetA types can be used to formulate a vaccine for broad protection against MenB disease.

The form of selenium determines the response to supplementation in a selenium replete population.

In an ongoing study of selenium bioavailability, effects of supplementation with organic and inorganic forms of selenium were investigated in healthy, Norwegian women, aged 23-50 years. In phase I of the study, 58 women received 200 micrograms selenium per day either as selenite or selenium-rich pea flour for 3 months. The selenium tablets were taken together with placebo or ascorbic acid in a double blind design. Initial blood and serum selenium concentrations were 153 +/- 15 micrograms/l and 117 +/- 12 micrograms/l, respectively. These are average values for Norwegians. Indications of increased blood levels were seen in all groups, but the rise reached significance only for the subgroup receiving selenite and ascorbic acid, 14 micrograms/l, P less than 0.05. On the other hand, selenium analysis of 72-h urine samples confirmed that at an average 50 per cent of the selenium supplements had been absorbed. In phase II of the study, 28 of the participants continued for another 5 weeks, still on 200 micrograms Se per day, but this time consuming commercially available preparations. Of four preparations that were tested, two consisted of yeast Se. Only one of these produced a significant rise in blood and serum selenium levels, 60 and 55 micrograms/l respectively. Blood glutathione peroxidase values were not affected by any supplementation. The study demonstrates that different forms of organic selenium elicit widely different responses when administered to a relatively selenium-replete population, and that the explanation for this must be sought at the metabolic level.

Levels and interactions of heavy metals in sea birds from Svalbard and the Antarctic.

Samples of liver and kidney from 92 seabirds of ten species collected on Spitsbergen and in the Antarctic, were analysed for their content of copper, zinc, cadmium, lead, mercury and selenium. Significantly higher levels of copper and zinc were observed in birds from Spitsbergen than in those from the Antarctic, while the opposite was true for selenium. The highest cadmium levels were found in fulmar Fulmarus glacialis and macaroni penguin Eudyptes chrysolophus. A possibility of kidney damage due to cadmium exists. The highest mercury levels were recorded in brown skua Catharacta lonnbergi collected at Bouvetøya. Lead was not detected in any of the birds. Significant correlations were observed between levels of several of the metals studied, especially between cadminum and zinc and between mercury and selenium. However, for all birds, the highest correlation coefficients were observed when the molar concentrations of cadmium plus mercury, and selenium plus zinc, were used in the calculations. Thus several protective mechanisms may operate to diminish effects of heavy metal contaminants.

Some heavy metals, essential elements, and chlorinated hydrocarbons in polar bear (Ursus maritimus) at Svalbard.

During the period 1978-1989, samples of liver, kidney and subcutaneous fat from 24 polar bears, Ursus maritimus, from Svalbard were analysed for mercury, cadmium, lead, copper, zinc, selenium, arsenic, HCB, DDE, PCBs (as Aroclor 1260 or Phenoclor DP6). In a selected number of liver (seven) and fat (three) samples, the composition of individual PCB congeners was studied by comparison with 23 individual PCB congeners (IUPAC nos 28, 52, 74, 101, 99, 110, 149, 118, 114, 105, 153, 141, 138, 187, 128, 183, 156, 157, 180, 170, 194, 206 and 209). In the seven liver samples, the concentrations of o,p'- and p,p'-isomers of DDT, TDE, DDE, alpha-, beta- and gamma-HCH, oxychlordane, heptachlor, heptachlorepoxide, aldrin and dieldrin were also determined. The hepatic concentrations of mercury, cadmium and lead in animals of all ages were 0.4-6.0, <0.1-1.2, and <0.5-1.6 microg g(-1), respectively. This indicates a moderate exposure. Concentrations of mercury and selenium were correlated (r=0.80). The levels of copper and zinc represented normal physiological concentrations. The concentrations of HCB, DDE and PCBs in fat were <0.05-1.5, <0.1-3.4 and 2.9-90 microg g(-1), respectively. The corresponding results for liver were <0.01-0.11, <0.1-0.5 and 0.1-78 microg g(-1), respectively. Six PCB congeners, PCB-99, -153, -138, -180, -170, and, -194 accounted for about 99 and 87% of the total PCB content (sum of the 12 congeners, nos. 28, 99, 153, 138, 128 + 187, 156, 157, 180, 170, 194, 206 and 209) in liver and fat, respectively. PCB-153 represented 37+/-3 30+/-16% of the sum PCB (sum of 12 congeners) in liver and fat, respectively. The range of the hepatic concentration of oxychlordane was 5-19 microg g(-1). Quantifiable concentrations of heptachlor, heptachlorepoxide, beta-HCH and dieldrin were also found in all the liver samples analysed. Low concentrations of p,p'- and o,p'-DDT were found in two of seven liver and two of two fat samples. Comparisons are made with investigations from Canada and Greenland. Possible effects of PCBs, especially on reproduction, cannot be excluded. Ringed seal, Foca hispida, and to some extent bearded seal, Erignathus barbatus, are the main food of the polar bear. It is therefore likely that the exposure to environmental pollutants occurs via the consumption of these two species.

Levels of polychlorinated biphenyls, organochlorine pesticides, mercury, cadmium, copper, selenium, arsenic, and zinc in the harbour seal, Phoca vitulina, in Norwegian waters.

Residue levels of the chlorinated hydrocarbons polychlorinated biphenyls (PCBs), total DDT, alpha-, beta- and gamma-hexachlorocyclohexane (HCH), hexachlorobenzene (HCB), and oxychlordane in blubber, and the elements mercury, cadmium, copper, selenium, arsenic, and zinc in liver, of 82 harbour seals, Phoca vitulina, were determined. The seals were found dead or dying in Norwegian waters during the disease outbreak caused by a morbilli virus in 1988. Of the chlorinated hydrocarbons, the highest concentrations were found of PCBs, which were 2-4 times higher than the total DDT concentrations. P,p'-DDE was the main contributor to the total DDT, and constituted about 80%. The PCB and total DDT concentrations ranged from 0.4-38 and 0.1-8.8 mg kg(-1), respectively. The mercury concentrations ranged from 0.1-89 mg kg(-1). Significantly higher mean levels of PCBs (13 mg kg(-1) and mercury (16 mg kg(-1)) were found in blubber and liver, respectively, of seals from the Southern coast of Norway, as compared to the corresponding mean levels in seals from the Oslofjord (8.8 and 4.1 mg kg(-1)), and at the Northwestern coast (5.8 and 7.9 mg kg(-1)), respectively. A significant positive correlation was found between the concentrations of selenium and mercury. When the seals were grouped according to sex and age, females of ageclass > 1 and pups of both sexes had significantly lower PCB and total DDT levels than males ageclass > 1. Significantly higher hepatic mercury levels were found in seals ageclass > 1 as compared to pups. Only low levels of the other organochlorines, cadmium and arsenic, were found. Copper and zinc were considered to be present at normal physiological levels. The present organochlorine and heavy metal concentrations gave no support to suggestions that organochlorines and heavy metal pollution may be directly involved in the observed seal deaths.

Chemical and morphological studies of liver from eider (Somateria mollissima) in Svalbard with special reference to the distribution of copper.

Specimens of male and brooding female eider (Somateria mollissima) were collected in Svalbard. Chemical analyses revealed hepatic copper concentrations ranging from 20 to 1050 micrograms per g wet weight. This is in agreement with previous results. The selenium, zinc and cadmium values were equal to or slightly higher than previously recorded. It is suggested that the wide variation in copper concentration is a result of differences in intake of copper-containing food among the birds. High selenium intake may enhance copper accumulation. Starvation influences the concentration of zinc and also copper. Zinc concentrations were significantly higher in females. This may be secondary to starvation. The percentage of copper recovered among the soluble proteins was inversely related to the copper content. The distribution of the soluble proteins reflects a normal copper metabolism. Microscopic studies showed prominent dark granules, positive with the rubeanic acid test for copper, confined to hepatocytes. By electron microscopy, the granules appeared as large irregular, electron-dense bodies that, by X-ray microanalysis, were found to contain copper. There were no signs of liver injuries such as necrosis and fibrosis. Apparently, the eider has evolved a high capacity for copper storage.

Seasonal siderosis in female eider nesting in Svalbard.

Specimens of female and male eider (Somateria mollissima) were collected in Svalbard. Atomic absorption analyses revealed mean hepatic iron concentrations of 280 micrograms per g wet weight 2 to 3 weeks before egg laying, 2620 micrograms per g after 2 to 3 weeks brooding and 800 micrograms per g 2 weeks after hatching. At the highest concentration, there was massive siderosis with the iron located in both parenchymal and non-parenchymal cells. No signs of liver injury were seen. The female eider fasts completely from the start of egg laying to the end of hatching. The non-parenchymal iron deposits are probably the result of catabolism of blood and lean tissue, i.e., translocation of body iron. The parenchymal siderosis can hardly be explained by liver weight loss alone. An increased iron absorption preceding the egg laying is suggested.

Influence of season and diet on liver and kidney content of essential elements and heavy metals in Svalbard reindeer.

Samples of liver (n = 78) and kidney (n = 60) from Svalbard reindeer (Rangifer tarandus platyrhynchus Vrolik) collected at four different seasons in Svalbard were analysed for their content of Cd, Pb, Cu, Zn, Mn, and Se. The study shows that when animals are exposed to large seasonal variations in both the quality and quantity of food, it is crucial to relate element concentrations to the physiological condition of the animal, e.g., to look at seasonal fluctuations in the total element content of the different organs.

Selenium concentrations in the human thyroid gland.

Recently, we found that prediagnostic serum selenium concentration was significantly lower for cases developing thyroid cancer (n = 43) than for controls. We assumed that redistribution of serum selenium into the affected tissue took place in the prediagnostic period. The present study was carried out to determine the physiological concentration of selenium in the thyroid, since very few data are available in the literature. The concentrations of selenium in the thyroid (n = 45) and liver samples from Norwegians who had died because of acute illness or accidents were determined by hydride generation atomic absorption spectrometry. The mean selenium concentration was found to be 0.72 +/- 0.44 microgram/g in the thyroid and 0.45 +/- 0.11 microgram/g in the liver tissue. The surprisingly high concentration of selenium in apparently normal thyroids indicates that selenium has important functions in this organ. The remarkably broad range, together with the observation that no significant correlation exists between thyroid and liver concentrations, suggest that factors other than the selenium status are important determinants for the selenium concentration in the thyroid gland. This observation is consistent with our hypothesis that in carcinogenesis, prediagnostic processes influence the serum-/thyroid-ratio of selenium.

Preclinical immunogenicity study of trivalent meningococcal AWX-OMV vaccines for the African meningitis belt.

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.

Preclinical immunogenicity and functional activity studies of an A+W meningococcal outer membrane vesicle (OMV) vaccine and comparisons with existing meningococcal conjugate- and polysaccharide vaccines.

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.