Haemophilus influenzae type b carriage and novel bacterial population structure among children in urban Kathmandu, Nepal.

Haemophilus influenzae type b (Hib) is a major cause of invasive bacterial infection in children that can be prevented by a vaccine, but there is still uncertainty about its relative importance in Asia. This study investigated the age-specific prevalence of Hib carriage and its molecular epidemiology in carriage and disease in Nepal. Oropharyngeal swabs were collected from children in Kathmandu, Nepal, from 3 different settings: a hospital outpatient department (OPD), schools, and children's homes. Hib was isolated using Hib antiserum agar plates, and serotyping was performed with latex agglutination. Hib isolates from children with invasive disease were obtained during active microbiological surveillance at Patan Hospital, Kathmandu, Nepal. Genotyping of disease and carriage isolates was undertaken using multilocus sequence typing (MLST). Swabs were taken from 2,195 children, including 1,311 children at an OPD, 647 children attending schools, and 237 children in homes. Overall, Hib was identified in 5.0% (110/2,195; 95% confidence interval [95% CI], 3.9% to 6.4%). MLST was performed on 108 Hib isolates from children carrying Hib isolates and 15 isolates from children with invasive disease. Thirty-one sequence types (STs) were identified, and 20 of these were novel STs. The most common ST isolates were sequence type 6 (ST6) and the novel ST722. There was marked heterogeneity among the STs from children with disease and children carrying Hib. STs identified from invasive infections were those commonly identified in carriage. This study provides evidence of Hib carriage among children in urban Nepal with genetically diverse strains prior to introduction of universal vaccination. The Hib carriage rate in Nepal was similar to the rates observed in other populations with documented high disease rates prior to vaccination, supporting implementation of Hib vaccine in Nepal in 2009.

Immunity under the skin: potential application for topical delivery of vaccines.

With the technological advances in biomedical sciences and the better understanding of how the immune system works, new immunisation strategies and vaccine delivery options, such sprays, patches, and edible formulations have been developed. This has opened up the possibility of administering vaccines without the use of needles and syringes. Already topical immunisation is a reality and it has the potential to make vaccine delivery more equitable, safer, and efficient. Furthermore, it would increase the rate of vaccine compliance and greatly facilitate the successful implementation of worldwide mass vaccination campaigns against infectious diseases. This review gives a brief account of the latest developments of application of candidate vaccine antigens onto bare skin and describes some of our recent observations using peptide and glycoconjugate vaccines as immunogens.

Effect of physico-chemical modification on the immunogenicity of Haemophilus influenzae type b oligosaccharide-CRM(197) conjugate vaccines.

Haemophilus influenzae type b (Hib) poly-ribosyl-ribityl phosphate (PRP) oligosaccharide-CRM(197) conjugate vaccines from two different manufacturers (Hib A and Hib B) were subjected to adverse storage conditions and used to establish correlates between physico-chemical characteristics and immunogenicity. There were manufacturer-specific differences in the effect of freezing or freeze-thawing on the carrier protein conformation and the anti-CRM(197) or anti-PRP IgG response in rabbits whereas both conjugates showed similar stability when stored at elevated temperatures. Both oligosaccharide-CRM(197) conjugate vaccines formed apparent 'aggregates' of non-specifically associated higher molecular weight material when subjected to elevated temperatures or repeated freeze-thawing. Following subcutaneous injection of samples into CBA mice and New Zealand White rabbits, the amount of IgG raised against CRM(197) was significantly lower for samples incubated at 37 or 55 degrees C compared with those kept at 4 degrees C, consistent with the less well-folded conformation of the carrier protein observed at elevated temperatures. Moreover, there was a parallel reduction in the amount of IgG raised against PRP and the level of bactericidal antibodies induced by vaccines A and B stored at 55 degrees C consistent with the observed depolymerisation of the oligosaccharide chains. Carrier protein conformational changes resulting from storage under adverse conditions did not affect the immunogenicity to Hib PRP in laboratory animals unless associated with loss of bound saccharide presumably because the carrier protein retains continuous T(H) cell epitopes which are unaffected by conformational changes.

HPA-1a-mediated platelet interaction with monocytes in vitro: involvement of Fcgamma receptor (FcgammaR) classes and inhibition by humanised monoclonal anti-FcgammaRI H22.

To gain insight into mechanisms of platelet destruction and its possible inhibition during fetal/neonatal alloimmune thrombocytopenia (FAIT/NAIT) the binding to monocytes (Mo) of anti-HPA-1a-sensitised platelets, the initial step in IgG-mediated destruction by effector cells, was evaluated in an in vitro assay. Neonatal Mo were compared with adult Mo as effectors in the assay. Moreover, the potential involvement of Fcgamma receptor (FcgammaR) classes during platelet destruction in the disease was tested by using FcgammaR class-specific reagents as inhibitors of the binding reaction. Neonatal Mo were 37% less active than adult Mo in their interaction with anti-HPA-1a-sensitised platelets (p < 0.05). The FcgammaRI-specific reagents human monomeric IgG and humanised anti-FcgammaRI monoclonal H22 caused virtually complete inhibition of platelet binding to Mo. When compared to an intravenous immunoglobulin preparation the inhibitory activity of H22 was 10-100 x greater than that of the latter compound. Monoclonal anti-FcgammaRII IV.3 and anti-FcgammaRIII 3G8 decreased platelet binding by 70% and 64%, respectively, but only the anti-FcgammaRII inhibition was statistically significant (p < 0.001). Finally, anti-HPA-1a-sensitised platelets bound to 131H- but not to 131R- FcgammaRIIa transfected 3T6 mouse fibroblasts (p < 0.01), in an anti-HPA-1a-concentration-dependent manner. The results suggest that FcgammaRI and FcgammaRIIa may be involved in anti-HPA-1a-mediated platelet destruction by mononuclear phagocytes during FAIT/NAIT. Moreover, the much greater potency ofmonoclonal H22 than of intravenous immunoglobulin as an inhibitor of anti-HPA-1a-mediated Mo-platelet interaction, might render it superior to the latter agent in the maternal therapy of the disorder.

Serotype of Streptococcus pneumoniae capsular polysaccharide can modify the Th1/Th2 cytokine profile and IgG subclass response to pneumococal-CRM(197) conjugate vaccines in a murine model.

The cellular and antibody responses to type 14 and type 19F Streptococcus pneumoniae capsular polysaccharides (PS) conjugated to CRM(197) were investigated in a mouse model developed for pre-clinical evaluation and quality control of pneumococcal conjugate vaccines. Total IgG antibody and IgG subclasses against PS and the carrier protein for both conjugates were measured in addition to the T cell proliferation and cytokine profiles induced by these conjugates. While unconjugated PS 14 and 19F were at best only weakly immunogenic, both types of conjugate induced strong primary and secondary IgG responses to PS. The responses induced by the two conjugates to the carrier protein were very different; a high level of anti-CRM(197) IgG was induced only by the PS19F conjugate whereas a very weak response was induced by the PS14 conjugate. Interestingly, the IgG subclass distribution was different for the two conjugates; for PS19F conjugate, the IgG response was almost completely of IgG1 subclass with low levels of IgG3 and IgG2a while the response to PS14 conjugate was mainly of the IgG1 and IgG2a subclasses with a low level of IgG3. The anti-CRM(197) IgG subclass distribution was identical with that to the corresponding conjugated PS. Both types of conjugate induced strong T cell proliferation to recall antigens but induced different patterns of cytokine response in immune spleen cells which were indicative of a Th0 response or a mixture of Th1 and Th2 responses with a bias towards Th2 response in PS19F-CRM(197) immunised mice. In conclusion, PS14- and PS19F-CRM(197) conjugates induced different IgG subclass patterns as a result of inducing different patterns of cytokine response to the carrier protein. This indicates that the serotype of PS can modify the Th1/Th2 response to the carrier protein, which has a direct effect and can predict the IgG subclass of the PS response. Finally, we conclude that this model appears suitable for studying the immunogenicity and immune interaction of different components of multivalent pneumococcal conjugate vaccines and may be applicable to their pre-clinical evaluation and quality control.

Serum levels of tumor necrosis factor-alpha, interleukin-1, and interferon-gamma in beta(o)-thalassemia/HbE and their clinical significance.

Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and interferon-gamma (IFN-gamma) were estimated by conventional ELISA kits in 60, 42, and 58 Thai patients, respectively, with beta(o)-thalassemia HbE and found to be above the normal range in 13%, 21%, and 33% of the patients, respectively. Using high-sensitivity ELISA systems, an additional 10 beta(o)-thal/HbE patients were compared with 9 controls for concentrations of circulating TNF-alpha and IL-1beta, and 9 and 5 patients, respectively, but only 1 and none of the controls, respectively, showed values above the normal ranges. In patients with abnormally high IFN-gamma levels, basal hemoglobin values were significantly lower than in those with normal levels of the cytokine (mean +/- SEM: 6.03+/-0.24 vs. 7.08+/-0.18, p < 0.05), although circulating concentrations of soluble transferrin receptors (sTrF) and absolute reticulocyte counts were similar in the two groups. Patients with raised or normal levels of TNF-alpha, IL-1alpha, or IL-1beta had similar basal hemoglobin values. In a phagocytosis assay, monocytes of patients with raised serum levels of IFN-gamma showed significantly more attached or ingested IgG-coated red cells than those of patients with normal concentrations of the cytokine (mean +/- SEM: 192+/-22 vs. 140+/-14 per 100 monocytes, p < 0.05). Moreover, in 3 of 4 of the former patients, the number of attached or ingested IgG-coated red cells per 100 monocytes was above the 95% reference limit for the latter patients. The results suggest that IFN-gamma aggravates the anemia of beta(o)-thal/HbE by activating mononuclear phagocytes for destruction of red cells but not by inhibiting erythropoiesis. The elevated serum levels of TNF-alpha and IL-1 could contribute to complications of the disease, such as cachexia and thromboembolic phenomena.

Immunoglobulin G subclasses of anti-human platelet antigen 1a in maternal sera: relation to the severity of neonatal alloimmune thrombocytopenia.

The monoclonal antibody immobilization of platelet antigen (MAIPA) technique was employed to detect and semiquantitatively assess total IgG anti-HPA-1a and its subclasses in sera of mothers who gave birth to severely thrombocytopenic (< 50 x 10(9) platelets/L)(n = 14) or mildly thrombocytopenic/unaffected (> 50 x 10(9) platelets/L)(n = 13) neonates. There was no statistically significant difference between the IgG anti-HPA-1a subclass composition of the 2 groups of sera. The majority of sera (26/27, 96%) showed IgG1 + IgG3 while 17/27 (63%) had all 4 subclasses of the antibody. No significant differences between the severely thrombocytopenic and mildly thrombocytopenic/unaffected groups were detected in the levels of IgG, IgG1, IgG2 or IgG4 of the antibody. However, the values of IgG3 anti-HPA-1a were significantly higher in the severely thrombocytopenic than in the mildly thrombocytopenic/unaffected group of sera with only little overlap (median 2.94 vs. 1.68, range 1.36-9.71 vs. 1.50-2.84, respectively; p < 0.01). The results suggest that maternal IgG3 anti-HPA-1a has predictive value for severe thrombocytopenia of the neonate. However, a prospective study of IgG HPA-1a subclasses in a greater number of maternal sera at different times of pregnancy is needed to test if IgG3 anti-HPA-1a is predictive of the degree of fetal/neonatal thrombocytopenia.

Increased serum levels of macrophage colony-stimulating factor (M-CSF) in alpha- and beta-thalassaemia syndromes: correlation with anaemia and monocyte activation.

Serum levels of M-CSF were determined by an ELISA method in 29 and 34 patients with HbH disease (alpha 1/alpha 2 or alpha 2/HbCS) or beta zero-thal/HbE, respectively, in 28 haematologically normal subjects and in five patients with anaemia due to iron deficiency or myelodysplasia. In HbH disease and beta zero-thal/HbE, M-CSF concentrations were significantly higher than those in the normal subjects [986 +/- 138 and 1385 +/- 133, respectively, vs. 500 +/- 33 pg/ml (mean +/- SEM); p < 0.01, and p < 0.001, respectively]. By contrast, in patients with anaemia due to iron deficiency, M-CSF levels were within the normal range. In HbH disease and in beta zero-thal/HbE, M-CSF levels correlated inversely with mean basal Hb values (r = -0.39, p = 0.05 and r = -0.60, p < 0.001, respectively). In addition, in some of the HbH and beta zero-thal/HbE patients, monocyte ADCC activities towards red cells were tested and found to be approximately twice as high as those in normal controls [38.3 +/- 5.7 and 30.7 +/- 4.6 vs. 17.8 +/- 1.8% specific lysis (mean +/- SEM), respectively; p < 0.01 and p < 0.02, respectively]. When thalassaemic patients and normal controls were considered together there was a significant correlation between M-CSF levels and monocyte ADCC activities (r = 0.51, p < 0.02). The results suggest that in HbH disease and in beta zero-thal/HbE, raised serum M-CSF contributes to the anaemia by enhancing the effector function of mononuclear phagocytes towards red cells.

Role of Fc gamma RIIa (CD32) in IgG anti-RhD-mediated red cell phagocytosis in vitro.

To test the role of Fc gamma RIIa in IgG anti-RhD-mediated phagocytosis three IgG1 and two IgG3 human monoclonal anti-D antibodies were tested for ability to mediate binding/phagocytosis of cDE/cde and -D-/-D- red cells by Fc gamma RIIa-R131 and Fc gamma RIIa-H131 cDNA-transfected 3T6 fibroblasts. Both IgG3 monoclonal antibodies brought about -D-/-D- cell interaction with IIa-transfected fibroblasts, while only one of them, Og3, mediated binding of cDE/cde targets. Although Fc gamma RIIa expression was three times greater on IIa-R131 than on IIa-H131 fibroblasts, the latter bound significantly more Og3-coated cDE/cde- and IgG3 anti-D-sensitized -D-/-D- cells, respectively, than the former effectors and showed some phagocytosis of the -D-/-D- targets. IgG1 anti-D antibodies were inactive in mediating red cell interaction with the fibroblasts. Moreover, monoclonal anti-Fc gamma RII IV.3 partially inhibited the phagocytosis by adult or fetal monocytes of Og3-sensitized cDE/cde cells. Fc gamma RIIa-H/H131 monocytes exhibited higher phagocytic indices towards these targets than monocytes of other IIa allotypes. The results indicate that Fc gamma RIIa can participate in the phagocytosis of red cells coated with IgG3 anti-D; in this case the allotype of the receptor will modify the extent of red cell destruction.

A major role of class I Fc gamma receptors in immunoglobulin G anti-D-mediated red blood cell destruction by fetal mononuclear phagocytes.

OBJECTIVE: To examine Fc gamma receptor (Fc gamma R) classes that direct immunoglobulin (Ig) G anti-D-mediated red blood cell interaction with fetal mononuclear phagocytes. METHODS: Mononuclear phagocytes isolated from fetal blood and spleen at 20-35 and 12-15 weeks' gestation, respectively, were tested in a modified mononuclear phagocytosis assay against IgG anti-D-coated red blood cells in the absence and presence of Fc gamma R class-specific monoclonal antibodies as inhibitors. Monocytes from cord and adult blood served as controls. RESULTS: In the absence of any inhibitor, attachment and phagocytosis indices of fetal monocytes were similar to those of their newborn and adult counterparts but markedly less than those of mononuclear phagocytes from fetal spleen. Blockade of the high-affinity Fc gamma RI caused a profound (more than 93%) reduction in red blood cell attachment and phagocytosis indices of fetal as well as newborn and adult monocytes. It also brought about a marked decrease in the attachment and phagocytosis indices of mononuclear phagocytes from fetal spleens (64 and 81%, respectively). With fetal spleen mononuclear phagocytes, anti-Fc gamma RII lacked any significant effect on their interaction with red blood cells, whereas anti-Fc gamma RIII caused a significant (43%) inhibition of their phagocytosis. CONCLUSION: Immunoglobulin G anti-D-mediated attachment to and phagocytosis by fetal mononuclear phagocytes of red blood cells is well developed early during the second trimester. High-affinity Fc gamma RI plays a major role in the effector function of circulating monocytes and splenic mononuclear phagocytes, whereas Fc gamma RIII, expressed strongly on the latter effectors, participates in target ingestion.

Macrophages are a major source of acetaldehyde in circulating acetaldehyde-albumin complexes formed after exposure of mice to ethanol.

C57BL mice were depleted of macrophages by an intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (DCMDP), and control mice were uninjected or injected with empty liposomes. One day after injection, a proportion of the DCMDP-treated and control mice was continuously exposed to ethanol vapor for 4 days. Albumin fractions were separated from the sera of both ethanol-unexposed and ethanol-exposed animals and tested for cytotoxicity against a monolayer of A9 cells using two indicators of cytotoxicity: detachment of adherent cells and a decrease in the ability of cells to reduce tetrazolium. The results show that, in mice exposed to ethanol, macrophages are a major source of the acetaldehyde in circulating cytotoxic acetaldehyde-albumin complexes and presumably also of free acetaldehyde.

The expression of IgG Fc receptors on circulating leucocytes in the fetus and new-born.

The expression of Fc-gamma-R (FcR) classes on circulating leucocyte lineages (lymphocytes, monocytes, granulocytes) was determined by flow cytometry in 46 fetuses at 18-35 weeks of gestation and in 11 full-term neonates, and compared to that in 20 adults. Classes of FcR present on adult leucocytes could be detected on the corresponding fetal cells as early as 18 weeks of pregnancy. Generally, in the fetus, FcR expression was lower than in the adult while in the neonate it approached values found later in life. However, percentages of Fc-gamma-RIII-positive fetal/new-born monocytes, and those of FcR-positive new-born granulocytes were considerably raised above adult levels. The modified pattern of FcR expression on fetal/new-born leucocytes is likely to influence their IgG-mediated effector activities towards targets such as red cells and platelets.

Activation of monocytes for the immune clearance of red cells in beta zero-thalassaemia/HbE.

We have recently provided evidence that IgG antibodies play a role in the destruction of red cells in thalassaemia syndromes. In order further to delineate factors involved in the clearance of thalassaemic cells, monocytes of 30 Thai patients with beta zero-thal/HbE (17 non-splenectomized and 13 splenectomized) and 16 normal controls were examined for their ability to bind and phagocytose normal red cells coated with IgG anti-Rh(D). In beta zero-thal/HbE, the mean number of red cells attached to the monocytes was approximately 3-fold greater than in normal controls and the number ingested 30% higher. Among the non-splenectomized patients, the number of red cells attached to and ingested by the monocytes, correlated inversely with mean basal Hb levels, suggesting that activation of mononuclear phagocytes for the immune clearance of red cells is a factor in determining the severity of the anaemia. As Fc-gamma-RI is of primary importance in the recognition of IgG-coated red cells by monocytes, leucocytes from 10 beta zero-thal/HbE patients (four non-splenectomized and six splenectomized) and five normal controls were investigated for their expression of Fc-gamma-RI by flow cytometry. In beta zero-thal/HbE there was an approximately 3-fold increase in the percentage of leucocytes expressing this receptor; the receptor was up-regulated on monocytes and induced on granulocytes. The up-regulation of Fc-gamma-RI in beta zero-thal/HbE is likely to be an important component in the activation of monocytes and in mediating their enhanced effector function towards antibody-coated cells.