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.).

Inducing humoral and cellular responses to multiple sporozoite and liver-stage malaria antigens using exogenous plasmid DNA.

A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4(+) and CD8(+) T cell compartments. Furthermore, hepatic CD8(+) lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8(+) granzyme B(+) T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.

Cloning, expression, purification, and characterization of the major core protein (p26) from equine infectious anemia virus.

The gene coding for the major core protein (p26) of the lentivirus equine infectious anemia virus (EIAV) was cloned from EIAV infected serum, expressed in E. coli, and the resultant protein purified to electrophoretic homogeneity. The protein was expressed in a soluble form and was purified by conventional protein separation methods. When analyzed by SDS-PAGE, under both reducing and non-reducing conditions, the purified protein migrated as a 26 kDa monomer. Recombinant p26 (rp26), therefore, does not contain any intermolecular disulfide bond. Gel filtration chromatography also indicated that the protein occurs as a monomer in solution. Labeling of free sulphydryl groups with [1-14C]iodoacetamide suggests that none of the three cysteine residues of rp26 is involved in intramolecular disulfide bonds. The circular dichroism spectrum of rp26 was consistent with the following assignment of secondary structure elements: 51% a-helix, 15% beta-turn, and 34% aperiodic. Fluorescencespectroscopy revealed that the three tryptophan residues in rp26 occupy two different environments. These data support the conclusion that the recombinant protein is folded into an ordered and probably native conformation. Immunoblotting and enzyme immunoassay with EIAV infected sera demonstrated that recombinant p26 protein may be useful for diagnostic purposes.

Resistant starch lowers fecal concentrations of ammonia and phenols in humans.

We investigated the effect of resistant starch (RS) on markers of colonic protein metabolism. Eleven subjects participated in a randomized crossover study in which they consumed either high-RS (39 +/- 3 g/d, -chi +/- SEM) or low-RS (5 +/- 0.4 g/d) diets for 3 wk. All other macronutrients were kept constant. During the high-RS diet daily excretion of fecal nitrogen increased from 1.84 +/- 0.15 to 2.86 +/- 0.42 g/d (P < 0.01) and excretion of fecal phenols fell from 9.2 +/- 1.4 to 5.3 +/- 0.8 mg/d (P < 0.01). Fecal concentrations of ammonia decreased from 397 +/- 33 to 278 +/- 49 microgram/g (P < 0.01) and phenols decreased from 69 +/- 8 to 39 +/- 10 microgram/g (P < 0.001). Daily output of urinary ammonia, urea, phenols, and total nitrogen did not change significantly, but pH decreased from 6.4 +/- 0.1 to 6.2 +/- 0.1 (P < 0.05) during the high-RS period. These results suggest that RS significantly attenuates the accumulation of potentially harmful byproducts of protein fermentation in the human colon.

Food processing and maize variety affects amounts of starch escaping digestion in the small intestine.

Two meals which differed greatly in resistant starch (RS) concentration, but otherwise had similar macronutrient composition (including nonstarch polysaccharides), were fed for breakfast to five subjects with ileostomies. The high-RS meal included bread made from high-amylose maize, uncooked green banana flour, and coarsely ground uncooked wheat. The low-RS meal contained bread made from low-amylose maize, cooked green banana flour, and cooked wheat. The effluent produced over 14 h was analyzed for the total amount of starch escaping digestion. In the low-RS meal 51.8 +/- 6.2 g (mean +/- SD) starch was consumed and 2.4 +/- 0.6 g recovered in the effluent, while for the high-RS meal a total of 52.7 +/- 8.8 g starch was fed and 19.9 +/- 5.2 g recovered in the effluent. The ileostomy results provided additional validation of an in vitro resistant starch assay. Scanning electron micrographs of effluent from one subject who consumed the high-amylose bread revealed that many intact starch granules escaped digestion in the small intestine.

Effect of resistant starch on fecal bulk and fermentation-dependent events in humans.

This study investigated the effect of two diets, which differed in resistant starch (RS) concentration, on fecal bulk and fermentation-dependent events in 11 humans. Amounts of RS consumed were 5.0 +/- 0.4 and 39.0 +/- 3.0 g/d (mean +/- SEM) for the low- and high-RS diets, respectively. The two diets were fed for 3 wk each in a randomized crossover design. Fecal collections were made in the third week of each study period. The high-RS diet produced an increase (P < 0.01) in total fecal output (from 138 +/- 22 to 197 +/- 37 g/d) and lowered fecal pH (6.9 +/- 0.1 to 6.3 +/- 0.1). There were significant increases (P < 0.05) in the fecal concentrations and daily excretion of butyrate (+38% and +100%, respectively) and acetate (+26% and +72%, respectively) during the high-RS period. The fecal excretion (g/d) of nonstarch polysaccharides (NSP) also rose by 50% during the high-RS diet, suggesting that the presence of starch in the colon may affect the fermentation of NSP. Subjects reported an increase in flatulence and easier defecation. These results demonstrate that RS has a significant impact on putative markers of colonic health in humans.

A "universal" human influenza A vaccine.

We have previously reported on a universal human influenza A vaccine, based on the external domain of the transmembrane viral M2-protein (M2e) [Nature Medicine 5 (1999) 1119]. M2-protein is scarcely present on the virus but is abundantly expressed on virus-infected cells. The external domain, M2e, is 23-amino acids long and as such weakly immunogenic. But when presented on an appropriate carrier, such as hepatitis B virus core (HBc) particles, it induces a high titer antibody response that in mice effectively protects against a potentially lethal influenza infection. The advantage of M2e as an antigen is the conservation of its sequence that has hardly changed since the first influenza virus was isolated in 1933, despite numerous epidemics and several pandemics. Various constructs, e.g. M2e fused at the N-terminus of the HBc subunit or inserted in the immuno-dominant loop, were evaluated as a vaccine. They conferred full protection when administered together with an adjuvant. Several adjuvants were tested in conjunction with intraperitoneal vaccine administration, while the non-toxic enterotoxin mutant LT(R192G) was used for intranasal vaccination. Appropriate combinations of vaccine construct and adjuvant allowed to obtain anti-M2e IgG2a serum titers above 10,000, and this provided complete protection.

Dietary intake and faecal excretion of carbohydrate by Australians: importance of achieving stool weights greater than 150 g to improve faecal markers relevant to colon cancer risk.

OBJECTIVES: This study investigated, on 53 Australians consuming a typical Western diet, the relationship between dietary intake, faecal excretion of carbohydrate and changes in faecal markers believed to be relevant to colon cancer risk, for example faecal output, transit time and concentrations of phenols, ammonia and butyrate. DESIGN: Fifty-three subjects consuming their usual diet were asked to record and weigh all food consumed for a seven day period, and to collect faeces for three days during this period. SETTING: Geelong, Victoria, Australia. SUBJECTS: All volunteers were either staff and students of the university, or associates of the authors. INTERVENTIONS: None. RESULTS: Volunteers had the following dietary intakes of carbohydrate (g/d; mean +/- s.d.); starch 131 +/- 41 (including resistant starch (RS), 5 +/- 2), sugar 108 +/- 37 and non-starch polysaccharides (NSP) 14 +/- 7. Daily faecal output was 127 +/- 70 g and transit time 47 +/- 19 h. Analysis of faecal samples found 0.8 +/- 1.2 g RS and significant relationship with the concentration (mmol/L) of butyrate excreted n faeces (r = 0.34, P < 0.05). Dietary intake of RS was associated with higher concentrations of faecal ammonia (r = 0.34, P < 0.05), but this association was reversed when RS was combined with NSP in the diet (r = 0.07, NS). In contrast to dietary intake, the faecal excretion of RS was negatively related to faecal ammonia concentration (r = -0.40, P < 0.01) and positively related to faecal output (r = 0.64, P < 0.01). Individuals who consumed more NSP in their diet (19 +/- 7 g/d) excreted more than 150 g faeces per day and had higher quantities of faecal-RS and -NSP; faster transit times; higher concentrations of short chain fatty acids and lower concentrations of potentially harmful ammonia and phenols. CONCLUSIONS: The combination of RS and NSP in the colon may be required to achieve an optimal luminal environment conducive to 'colonic health'. The results also support the suggestion that faecal output (< or > 150 g/d) may provide a useful index of colon cancer risk. High faecal outputs are achieved through higher intakes of NSP (the major component of dietary fibre).

Online motion-gated dynamic three-dimensional echocardiography in the fetus--preliminary results.

The aim of this study was to visualise the fetal heart in dynamic three dimensions (4-D) during an ultrasound (US) scan (online), rather than after (offline). With special pairing and sequential setting to minimise interference between two scanners, umbilical arterial Doppler waveforms (UADWs) from one scanner were used as an online motion gating source to trigger simultaneous 3-D cardiac structural data acquisition by another. Of 25 data sets from 10 fetuses, 18 were acquired in 15 to 30 s per set with > or = 50% Doppler waveforms efficiently converted to triggering signals. Of 15 valid 4-D data sets, 10 were reconstructed in 2 to 20 min, compared to over 2 h previously reported (mainly for offline gating). Fine structures (including chordae tendinae and trabecular muscles) were depicted in six sets. The main problems in degrading 4-D images were extensive shadowing (6) from bony structures during rigid mechanical scanning, and random motion artefacts (6) from prolonged setting-up time with a complex combination of several systems. Integration of these systems is, therefore, recommended.

Conversion of umbilical arterial Doppler waveforms to cardiac cycle triggering signals: a preparatory study for online motion-gated three-dimensional fetal echocardiography.

To remove motion artefacts, a device was built to convert "noisy" umbilical arterial Doppler waveforms (UADWs) from an ultrasound (US) system into sharp ECG R-wave-like cardiac cycle triggering signals (CCTSs). These CCTSs were then used to gate a simultaneous (online) 3-D acquisition of sectional fetal echocardiograms from another US system. To test the conversion performance, a study was carried out in sheep fetal twins. Pulmonary arterial flow waveforms (PAFWs) from implanted probes were traced, in the meantime, to determine the reference cardiac cycle. Interference caused by running the two nonsynchronised US systems was controlled to three degrees (not-noticeable, moderate, and severe), together with high (> or = 40 cm/s) and low (< 40) flow velocities on UADWs. The conversion efficiency, assessed by the percentage of UADWs converted into CCTSs, was in the range of 83% to 100% for not-noticeable and moderate interference, and 0% to 71% for severe interference. The triggering accuracy, assessed by [(time lag mean between the onsets of PAFWs and corresponding CCTSs) -- (its 99% confidence level)] / the mean, was 90% to 96% for the not-noticeable interference high- and low-flow groups and for the moderate interference high-flow group; 19% to 93% for the moderate interference low-flow group; and from not obtainable up to 90% for the severe interference groups. The results show that UADWs can be used as a satisfactory online motion-gating source even in the presence of moderate interference. The major problems are from severe interference or moderate interference with low-flow velocity, which can be minimised/eliminated by the integration of the individual systems involved.

Effect of feeding quandong (Santalum acuminatum) oil to rats on tissue lipids, hepatic cytochrome P-450 and tissue histology.

Quandong kernels are a traditional Aboriginal food item; they are rich in oil and contain large amounts of an unusual fatty acid, trans-11-octadecen-9-ynoic acid (santalbic acid), but it is not known whether this acid is absorbed and/or metabolized. The oil was fed at 12.6% of total energy content in semi-synthetic diets to groups of male Sprague-Dawley rats for 10 and 20 days. Santalbic acid was found in the lipids of plasma, adipose tissue, skeletal muscle, kidney, heart and liver but not in brain. Hepatic microsomal cytochrome P-450 activity in animals fed for 20 days was significantly higher (P < 0.05) than in controls. Histopathological examination did not reveal any lesions in the tissues of any animal fed quandong oil. The fact that santalbic acid was readily absorbed, widely distributed in tissues and was associated with an elevated level of hepatic cytochrome P-450 indicates that further studies are required to investigate whether or not there is a hazard associated with the human practice of consuming quandong kernels.

A system for logging incontinence events using a simple disposable sensor.

Many elderly people entering residential or nursing care are already incontinent to some degree, relying on incontinence pads to deal with the consequences. A proportion of these people have been shown to exhibit a regular pattern in their incontinence, which opens up the possibility of mitigating the problem by instituting an individual toileting regime for the person. This can reduce their reliance on incontinence pads, both improving their quality of life, and reducing the cost of care. This paper covers the development and evaluation of a sensor for detecting incontinence events, suitable for use in this setting, and describes the design of an associated electronic logger. The devices form part of an assessment system intended to identify a pattern in incontinence where it exists, and to help with the design of the toilet regime for an individual. The requirement is that the system must reliably record incontinence events, and present the information describing them in a manner appropriate to the users of the devices, who are likely to be non-technical and non-specialist.

Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity.

Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

Evidence for the in vivo deamidation and isomerization of an asparaginyl residue in cytosolic serine hydroxymethyltransferase.

Rabbit liver cytosolic serine hydroxymethyltransferase exists in several subforms which have different isoelectric points. Incubation of the purified enzyme with chymotrypsin cleaves the enzyme at Trp14. The released amino-terminal 14-mer peptide was shown to exist in three forms of equal concentration. The peptides differ in structure only at the asparaginyl residue at position 5. In addition to asparagine at this position we found both aspartyl and isoaspartyl residues. The deamidation of Asn5 does not appear to occur during the purification of the enzyme. The in vitro rate of deamidation of Asn5 in the enzyme is more than 5-fold slower than the rate of deamidation of this residue in the free 14-mer peptide. The isoaspartyl residue at position 5 serves as a substrate for protein carboxyl methyltransferase both in the free 14-mer peptide and the native enzyme. The enzyme which has had the amino-terminal 14 residues removed by digestion with chymotrypsin still exists in several forms with different isoelectric points. Reaction of peptides from this enzyme with carboxyl methyltransferase suggests that there is at least one more asparaginyl residue in this enzyme other than Asn5 which has undergone deamidation with the formation of isoaspartyl bonds.

Simple high-performance liquid chromatographic analysis of phenol and p-cresol in urine and feces.

Previous reports which present methods of analysis of phenol and p-cresol by HPLC are usually designed for the detection of these compounds in urine, can be complicated by the use of uncommon equipment or additional techniques such as steam distillation or derivatisation, or concentrate on the detection of phenol rather than p-cresol. In this paper we report a simple method suitable for the analysis of phenol and p-cresol in both urine and feces, based upon extraction into ether following acid hydrolysis and UV detection.

Use of alpha-N,N-bis[carboxymethyl]lysine-modified peroxidase in immunoassays.

Horseradish peroxidase was activated by periodate oxidation of the carbohydrate moiety and then modified by the covalent attachment of alpha-N,N-bis[carboxyethyl]lysine (CM-Lys) by reductive alkylation using sodium cyanoborohydride. The resultant CM-Lys peroxidase was charged with nickel ions and then used as a specific labeling reagent for histidine-tagged recombinant proteins. This labeling method was effective for proteins that are soluble or insoluble in the absence of chaotropic agents. The labeled proteins were very effective in direct sandwich enzyme-linked immunosorbent assay for detecting antibodies against the protein in sera as demonstrated by assays for antibodies to such diverse viral proteins as hepatitis B surface and core proteins, hepatitis C core and helicase protein (NS3), and retroviral core proteins.

Antibody production against hepatitis C virus core and nonstructural 3 proteins is highly sensitive to deficits in T-cell function.

The influence of suppression of CD4+ and CD8+ T cells on the humoral responses to hepatitis C virus (HCV) core and nonstructural 3 proteins was studied. An increasing viral burden cannot substitute for the lack of functional T cells in maintaining humoral HCV-specific responses.

Antibodies against thrombospondin-related anonymous protein do not inhibit Plasmodium sporozoite infectivity in vivo.

Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required for Plasmodium sporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain of Plasmodium yoelii and located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve.

Immune responses to the hepatitis C virus NS4A protein are profoundly influenced by the combination of the viral genotype and the host major histocompatibility complex.

The interaction between the host major histocompatibility complex (MHC) and the genotype of the hepatitis C virus (HCV) was analysed using synthetic full-length non-structural (NS) 4A proteins, residues 1658-1712, of genotypes 1b, 2b, 3a, 4a and 5a. Human and murine antibodies specific for the five NS4A genotypes analysed focused on residues 1688-1707. In immunized B10 H-2 congenic mice, the H-2d, H-2f and H-2s haplotypes were good responders to NS4A, irrespective of the viral genotype. In contrast, the H-2k haplotype was a low or non-responder to all NS4A genotypes, except for genotype 2b. Also, H-2f- and H-2s-restricted NS4A genotype 1b-specific T-cells focused on residues 1670-1679 and 1683-1692, respectively, whereas H-2k-restricted NS4A genotype 2b-specific T-cells focused on the carboxy terminus. Interestingly, H-2f-restricted genotype 1b-specific T-cells did not cross-react with T-cell site analogues of seven other genotypes, whereas the H-2s-restricted, genotype 1b-specific T-cells cross-reacted with genotypes 1a, 4a and 5a. Thus the combination of viral genotype and host MHC profoundly influences the ability to mount an HCV NS4A-specific immune response.