Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component

Background

A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge.

Methodology/Principal Findings

NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected.

Significance

The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00392015","term_id":"NCT00392015"}}NCT00392015     

Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm

Background

Rice is the primary source of food for billions of people in developing countries, yet the commonly consumed polished grain contains insufficient levels of the key micronutrients iron (Fe), zinc (Zn) and Vitamin A to meet daily dietary requirements. Experts estimate that a rice-based diet should contain 14.5 µg g⁻¹ Fe in endosperm, the main constituent of polished grain, but breeding programs have failed to achieve even half of that value. Transgenic efforts to increase the Fe concentration of rice endosperm include expression of ferritin genes, nicotianamine synthase genes (NAS) or ferritin in conjunction with NAS genes, with results ranging from two-fold increases via single-gene approaches to six-fold increases via multi-gene approaches, yet no approach has reported 14.5 µg g⁻¹ Fe in endosperm.

Methodology/Principal Findings

Three populations of rice were generated to constitutively overexpress OsNAS1, OsNAS2 or OsNAS3, respectively. Nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grain of all three of the overexpression populations, relative to controls, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations. Selected lines from each population had at least 10 µg g⁻¹ Fe in polished grain and two OsNAS2 overexpression lines had 14 and 19 µg g⁻¹ Fe in polished grain, representing up to four-fold increases in Fe concentration. Two-fold increases of Zn concentration were also observed in the OsNAS2 population. Synchrotron X-ray fluorescence spectroscopy demonstrated that OsNAS2 overexpression leads to significant enrichment of Fe and Zn in phosphorus-free regions of rice endosperm.

Conclusions

The OsNAS genes, particularly OsNAS2, show enormous potential for Fe and Zn biofortification of rice endosperm. The results demonstrate that rice cultivars overexpressing single rice OsNAS genes could provide a sustainable and genetically simple solution to Fe and Zn deficiency disorders affecting billions of people throughout the world.     

Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.     

The spatial dynamics of pollen beetles in relation to inflorescence growth stage of oilseed rape: implications for trap crop strategies

Semi‐field‐scale arrays of oilseed rape (Brassica napus L.) (Brassicaceae) plants were used to observe the development of distributions of pollen beetles (Meligethes aeneus Fabricius) (Coleoptera: Nitidulidae) in a simulated trap crop system where inflorescence growth stage alone was used to manipulate the pest. Over two successive years, pairs of 1 m spaced square arrays of 100 glasshouse‐grown plants were placed 40 m apart in the field in May, and were subject to natural infestation by pollen beetles. The test plot of each pair had a simulated trap crop, with an outer row of plants at early flowering stage intended to protect more susceptible inner plants at late bud stage, and the control plot had all plants at the late‐bud stage, simulating a standard crop situation. Pollen beetles were counted daily on each plant for 10–13 days. The spatio‐temporal development of plot infestation was analysed in relation to the distribution of racemes in bud and raceme in flowers using Spatial Analysis by Distance IndicEs (SADIE), and tests of edge and centre distribution. Inflorescence growth stage characteristics were shown to be important in determining the spatial distributions of pollen beetles. In control plots, the numbers of racemes in bud and in flower were never edge or centre distributed. In test plots, racemes in flower were always edge distributed, and racemes in bud began edge distributed and became centre distributed. Pollen beetle numbers were usually spatially associated with the abundance of racemes in bud and/or in flower. In control plots, pollen beetles were neither edge nor centre distributed, but in test plots they maintained a significant edge distribution for 7–10 days. At the end of the experiments, females were more centre distributed in the test plots than males, and were more closely associated with racemes with buds, whereas males were more associated with racemes with flowers. In early flowering stage plants, the number of racemes in flowers were a good indicator of the abundance of racemes in buds, but this relationship was lost as flowering progressed. Although flowering racemes provide strong cues for immigrating pollen beetles, the abundance of buds may be a more important determinant of residence time, particularly for females, and is therefore a critical determinant of trap crop effectiveness.     

Assessing the impact of episodic pollution

The increased tightening of controls on industrial and municipal wastewater discharges has resulted in steady improvements in the quality of many important rivers over recent years. However, episodic pollution, particularly from farm wastes and combined sewer overflows continues to pose a major problem, and is one of the main causes of poor quality rivers today. Despite our acknowledgement of this continuing problem, very little is known of the mechanistic basis of responses and recovery of aquatic organisms and communities exposed to intermittent pulses of common pollutants. The majority of ecotoxicological studies to date have been concerned with the effects of continuous exposure. Although such studies may provide a means of predicting the impact of episodic pollution events, a more appropriate test design would be to assess toxicity under pulsed and fluctuating exposure. Studies should also include a post-exposure observation period and should consider recovery of individuals and communities. This paper reviews the results of reported studies relevant to the investigation of episodic pollution and pays particular attention to the effects of magnitude, duration and frequency of exposure. Results of field investigations using an in situ bioassay technique are also presented to emphasize the importance of field validation of proposed water quality criteria for intermittent pollution events.     

Novel inhibitors of enkephalin-degrading enzymes. II: N5'-substituted-4-thioxohydantoic acids as aminopeptidase inhibitors

Some 2-substituted-(2'-aminophenyl)-4-thioxohydantoic acids (o-amino PTC-amino acids) have antinociceptive activity when administered (icv) alone (IC50 = 0.04-0.87 microM/animal) and show a striking prolongation of the antinociceptive action of (D-Ala-2 D-Leu5)-enkephalin (DADL) in combination. The effects are thought to be mediated via opioid receptors since they are naloxone-reversible. Although inhibitors of the enkephalin degrading puromycin-insensitive, bestatin-sensitive aminopeptidase (possibly aminopeptidase M) their action is weak (IC50 = 32 microM leucine, 536 microM, glycine) and they might be considered to have a direct antinociceptive effect on opioid receptors. The titled compounds constitute novel 'lead' compounds for the development of potent aminopeptidase M inhibitors.