Genetic Diversity and Protective Efficacy of the RTS,S/AS01 Malaria Vaccine.

BACKGROUND: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

RTS,S malaria vaccine efficacy and immunogenicity during Plasmodium falciparum challenge is associated with HLA genotype.

Although RTS,S remains the most advanced malaria vaccine, the factors influencing differences in vaccine immunogenicity or efficacy between individuals or populations are still poorly characterised. The analyses of genetic determinants of immunogenicity have previously been restricted by relatively small sample sizes from individual trials. Here we combine data from six Phase II RTS,S trials and evaluate the relationship between HLA allele groups and RTS,S-mediated protection in controlled human malaria infections (CHMI), using multivariate logistic or linear regression. We observed significant associations between three allele groups (HLA-A∗01, HLA-B∗08, and HLA-DRB1∗15/∗16) and protection, while another three allele groups (HLA-A∗03, HLA-B∗53, and HLA-DRB1∗07) were significantly associated with lack of protection. It is noteworthy that these 'protective' allele groups are thought to be at a lower prevalence in sub-Saharan African populations than in the UK or USA where these Phase II trials occurred. Taken together, the analyses presented here give an indication that HLA genotype may influence RTS,S-mediated protective efficacy against malaria infection.

In vitro interaction of human HDL with human apolipoprotein A-II. Synthesis of apolipoprotein A-II-rich HDL.

The aim of this study was to define the specific affinity of human apolipoproteins A-I and A-II for HDL lipids and to investigate the possible transfer of apolipoproteins from the HDL molecule. For this purpose we incubated human HDL with increasing amounts of isolated apolipoprotein A-II. After incubation the reaction products were separated by gel chromatography and apolipoproteins A-I and A-II were quantified separately by immunonephelometry and HDL lipids by thin-layer chromatography. According to our results, apolipoprotein A-II progressively displaces apolipoprotein A-I to generate an HDL-like particle with identical lipid composition, hydrodynamic properties and lipid fluidity. These data indicate that apolipoprotein A-II is able to displace quantitatively apolipoprotein A-I from HDL in vitro, and that such a mechanism might contribute to the regulation of the HDL2 in equilibrium or formed from HDL3 distribution in plasma.

Reassembly of human apoproteins A-I and A-II with unilamellar phosphatidylcholine-cholesterol liposomes. Association kinetics and characterization of the complexes.

The kinetics of association between the human apoprotein A-I and apoprotein A-II and cholesterol dimyristoyl phosphatidylcholine (DMPC) vesicles are compared in this study and the lipid-apoprotein complexes are characterized. The association kinetics are followed by turbidity measurements monitoring the decrease of the vesicular size and by fluorescence polarization measurements monitoring the decrease in the mobility of the phospholipid acyl chains during complex formation. The influence of the incubation temperature and of the cholesterol/DMPC ratio has been studied by both techniques. Under all incubation conditions the apoprotein A-II associates more readily with cholesterol-DMPC vesicles than apoprotein A-I, as the kinetics are faster and the complex yield larger. With both apoproteins optimal complex formation takes place around the phospholipid transition temperature and around 10 mol% cholesterol. The apoprotein A-I/lipid association seems restricted to this narrow range for the temperature and the cholesterol/DMPC ratio, while the apoprotein A-II still associates with vesicles containing 20 mol% cholesterol and at temperatures up to 32 degrees C. The lipid-apoprotein complexes were isolated by gradient ultracentrifugation and by gel chromatography. According to these data the apoprotein A-II associates more readily than apoprotein A-I with cholesterol-DMPC vesicles to form protein-rich complexes, whilst the optimal apoprotein A-I-lipid association requires a more disordered lipid structure.

Towards standardization of specific protein assays.

A new international reference preparation for proteins in human serum has been recently proposed in Europe from the Community Bureau of Reference (Brussels) under the name CRM 470 (Certified Reference Material) and in the United States from the College of American Pathologists under the name RPPHS (Reference Preparation for Proteins in Human Serum). This serum based material offers the opportunity to the laboratories to standardize their serum protein concentrations, and to improve dramatically the transferability of the results from studies to the local working conditions. The International Federation of Clinical Chemistry strongly recommends the use of this preparation by all laboratories. The Belgian Institute of Hygiene and Epidemiology expects an improvement in the agreement of the results of the three proteins (IgA, IgG, CRP) included in its external quality control of the clinical laboratories. These proteins were omitted from the controls of the second semester of 1994, but would reappear in early 1995, after the conversion of the laboratories to the new protein calibrators. Of course, this implies the assignment of new reference ranges for most of the serum proteins, some of them being significantly modified (alpha 1-antitrypsin, haptoglobin, transferrin, C3, IgM). The clinical chemists must inform the physicians of these changes.

Medical and technical usefulness of measurement of kappa and lambda immunoglobulin light chains in serum with an M-component.

Kappa and lambda-immunoglobulin light chains were measured to evaluate their usefulness for identifying monoclonal components in serum. Reference concentrations of the Ig light chains were evaluated by the analysis of the serum of 50 blood donors. Two hundred and fifty patients were selected for the presence of an M-component in their serum, which was detected by serum electrophoresis on agarose. The heavy and light chains of the M-component were identified by immunofixation. The concentrations of the three major immunoglobulin classes and of the two light chain types were measured by immunonephelometry. On the basis of the Ig kappa/lambda ratio alone, the Ig light chain type was correctly identified in 76% of the M-components, and there were no misidentifications. By comparing the calculated concentrations of M-component and residual polyclonal Ig with the measured concentrations of the 3 major Ig classes, it was possible to identify the heavy chain type of 58% of the M-components. These observations suggest that M-components can often be identified by the measurement of Ig light chains, a procedure involving less work and time than immunofixation. Moreover, the follow up of gammapathies could benefit from quantitative parameters evaluated from the Ig light chain concentrations: the concentration of the M-component, the ratio of M-component vs total Ig of the same class, and the ratio of M-component vs the sum of the 3 major Ig classes.

Accidental ingestion of a zinc and copper sulfate preparation.

CASE REPORT: An 86-year-old woman accidentally ingested a preparation containing zinc and copper sulfate. At ninety minutes after ingestion, the peak plasma concentration was 1979 micrograms/dL for zinc and 209 micrograms/dL for copper, suggesting preferential absorption of zinc. The major complications were gastric and bronchial inflammation due to the corrosive properties of these compounds. Systemic manifestations also developed with cardiovascular failure and renal insufficiency, but the patient made a complete recovery. In addition to symptomatic treatment, chelation therapy with dimercaprol and D-penicillamine was given for 48 h. CONCLUSION: The available clinical and toxicokinetic data do not support the benefits of chelation in addition to supportive therapy.

Pleural fluid lactate in pleural effusion.

Pleural fluid lactate (PFL) and blood lactate (BL) concentrations were simultaneously measured in samples from 46 patients with pleural effusion. PFL exceeded 6 mmol/l in all 15 patients with pyogenic bacterial pleurisy but in only 5 of the other 31 patients. We have found that a PFL-BL difference greater than or equal to 6 mmol/l has a sensitivity of 100% and a specificity of 93.8% in detecting pyogenic pleural effusions. Results are available within 1 h of sample collection, so that PFL-BL difference may become a useful aid in the early assessment of pleural effusions.

Immunoassay of low concentrations of albumin in urine by latex particle counting.

We describe here a nonisotopic immunoassay, based on particle-counting technology, for the determination of urinary albumin. The assay takes only 35 min and has been fully automated on the IMPACT (Acade Diagnostic Systems, Brussels, Belgium) machine. The system measures albumin within a linear range between 6.25 and 50 mg/L and has a detection limit of 0.4 mg/L. Analytical recoveries at three concentrations ranged between 96% and 102%. Within-run precision ranged from 1.6% to 9.5%. The method was compared with a commercial nephelometric immunoassay system and a correlation coefficient of 0.996 was found for 216 urine samples. No antigen excess affects the shape of the curve in our system, whereas in nephelometry a 3 g/L solution of albumin starts to decrease the dose-response curve.

Clostridium difficile-associated diarrhea after norfloxacin.

Although theoretically the spectrum of antimicrobial quinolone antibiotics are at low risk of producing Clostridium difficile overgrowth and diarrhea, a patient developed this clinical problem while receiving norfloxacin. We review the antimicrobial activity of the quinolone antibiotics, with respect to their predisposition for producing C. difficile-induced diarrhea.

Measurement of beta 2-microglobulin in serum by a particle-enhanced nephelometric immunoassay.

A particle-enhanced immunoassay of beta 2-microglobulin in serum is described. It is based on the agglutination of complexes formed between the serum beta 2-microglobulin and latex particles coated with F(ab')2 fragments of polyclonal anti-beta 2-microglobulin antibodies. The analytical range of the method is 0.50 to 16 mg/l; it can be extended by appropriate dilution to 0.12 to 80 mg/l with good precision (CV less than 5% over the whole range). The accuracy and the precision are confirmed by a good correlation with radioimmunoassay (n = 123, r = 0.993). No error due to antigen excess was observed, even up to 292 mg/l. The main advantages of the method are its simplicity, its low cost per test and its high sensitivity (final dilution of the sample at 1/1200) with no known interference. The calibration curve is stable for at least 2 weeks.

Displacement of the human apoprotein A-I by the human apoprotein A-II from complexes of (apoprotein A-I)-phosphatidylcholine-cholesterol.

Reassembly experiments, involving isolated human apoproteins A-I and A-II and (dimyristoylglycerophosphocholine)-cholesterol vesicles were performed with apoprotein mixtures at apoprotein A-I/A-II molar ratios varying between 0 and 3. The apoproteins were incubated at 24 degrees C. 28 degrees C and 32 degrees C with either pure dimyristoyl-glycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 2, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after labeling the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A-I and A-II were quantified separately by immunonephelometry. The content of apoprotein A-I was also monitored by measuring the intrinsic tryptophan fluorescence. The results suggest that apoprotein A-II has a greater affinity than apoprotein A-I for the phospholipid-cholesterol vesicles and that apoprotein A-II is able to quantitatively displace apoprotein A-I from the lipid-protein complexes. The content of apoprotein A-II in the complexes increases proportionally to the concentration of apoprotein A-II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A-II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A-I is displaced by 2 mol human apoprotein A-II.

Dissociation of apolipoprotein AI from apoprotein-lipid complexes and from high-density lipoproteins. A fluorescence study.

The dissociation of the apoprotein AI (apoAI) from the apoAI-dimyristoylglycerophosphocholine complex and from human high-density lipoproteins (HDL), was induced by incubation with guanidinium hydrochloride at concentrations between 0 M and 7 M. The kinetics and extent of denaturation were followed by monitoring both the fluidity of the lipid phase by fluorescence polarization measurements and the protein conformation by measuring the tryptophanyl fluorescence emission. The association with lipids protects apoAI against denaturation both in HDL and in the apoAI-phospholipid complex. The results of the kinetic and end-point measurements suggest that the denaturing effect of guanidine hydrochloride on the apoAI-lipid complex and on HDL is a two-step process. It involves the dissociation of the apoAI-phospholipid bond, as evidenced by fluidity measurements: this effect is maximal between 3 M and 4 M guanidine hydrochloride. The conformational change of the apoAI protein into a randomly coiled structure with the tryptophanyl residues exposed to the solvent is maximal between 4 M and 6 M guanidine hydrochloride. HDL and the apoAI-phospholipid complex have a closely similar behaviour towards denaturation by guanidine hydrochloride indicating that the phospholipid-apoAI association in HDL is primarily responsible for the stability of the lipoprotein molecule.

Displacement of apo A-I by A-II in lipid-apoprotein complexes and human HDL.

The aim of this study was to define the specific affinity of human apo A-I and apo A-II for HDL lipids and to investigate the possible transfer of apoproteins from the HDL molecule. For this purpose we incubated apo A-I -- lipid complexes prepared "in vitro", as well as human HDL with increasing amounts of isolated apo A-II. After incubation the reaction products were separated by gradient ultracentrifugation and gel chromatography. The apoproteins were quantitated separately by immunonephelometry and the apo A-I content was monitored by measuring the intrinsic tryptophan fluorescence. These results suggest that apo A-II has a higher affinity than apo A-I for the lecithin-cholesterol vesicle and that 2 mol apo A-II are able to displace 1 mol apo A-I from the apo A-I lipid complexes. Analogous results were obtained with HDL where two mol apo A-II substitute to 1 mol apo A-I to yield en apo A-II - rich HDL with identical lipid composition, hydrodynamic properties and fluidity. Such a mechanism might contribute to the regulation of the HDL2 in equilibrium with HDL3 distribution in plasma.

Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 1. Calorimetric and potentiometric titration of the native apoA-I protein and of the apoA-I protein-dimyristoyl lecithin complex.

The ionization behaviour of native apoA-I protein is compare to that of its complex with synthetic dimyristoyl lecithin in studies using calorimetric, potentiometric and spectrophotometric titration. In the presence of phospholipids, 10 out of 21 lysines together with 22 acidic residues are masked in the complex. All tyrosines remain accessible to titration below pH 13. The apparent ionization enthalpy of the 11 lysine residues is not affected by the presence of phospholipids. These data are consistent with discrete binding sites located in the apoprotein helical segments as suggested by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. A tentative localisation of lysine, arginine, aspartic acid and glutamic acid residues directly involved in phospholipid binding is suggested, assuming that such helical regions are involved in apoprotein-phospholipid association.

Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 2. Potentiometric titration of the native apo-A-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin.

A comparison of the ionization behaviour of the human apoA-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin is based on potentiometric titration of the basic and acidic residues and spectrophotometric titration of the phenolic groups. Experimental data suggest that a number of lysine, arginine, aspartic acid and glutamic acid residues are masked in the complexes. For each of these amino acids and in all three proteins the number of masked residues is consistent with the content of those regions predicted to be involved in lipid binding by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. These data taken together with the results of calorimetric and titration experiments with the apoA-I protein reported in the accompanying article [Rosseneu et al. (1977) Eur. J. Biochem. 79, 251-257] strongly support the general nature of the proposed model and further suggest that ionic interactions have some role in the formation of the dimyristoyl lecithin/apolipoprotein complexes.

Fluorescence depolarization studies and phase transition in human apoprotein . phospholipid complexes.

The microviscosity of unilamellar vesicles of dimyristoyl-3-sn-phosphatidylcholine and that of phosphatidylcholine . apoprotein complexes was followed by fluorescence depolarization after labeling with 1,6-diphenyl-1,3,5-hexatriene. The transition temperature from gel-crystalline to liquid-crystalline phase in 24 degrees C for the dimyristoyl-phosphatidylcholine vesicles and is shifted to around 30 degrees C in the complexes between phosphatidylcholine and apoA-I, apoA-II, apoC-I, apoC-III proteins while the cooperativity of the transition is decreased. At temperatures below the transition of the phospholipid, the microviscosity of the complexes of phosphatidylcholine with apoA-I, apoA-II and apoC-I proteins is lower than that of the phosphatidylcholine, while the opposite effect is observed above 30 degrees C. The phosphatidylcholine . apoprotein complexes isolated on a Sepharose 6B column have a molecular weight around 100 000 and a phosphatidylcholine/apoprotein ratio of 2--2.6 (w/w). The microviscosity measurments at 35 degrees C performed after elution of the column enable the complex to be detected. The size and microviscosity of the apoprotein . phosphatidylcholine complex is compatible with a model where the vesicular structure has disappeared and the amino acid side chains present hydrophobic interaction with the phosphatidylcholine acyl chains.