Peter M. Smooker

Evaluation of antigens of Fasciola gigantica as vaccines against tropical fasciolosis in cattle

Vaccine trials were conducted in Brahman cross cattle evaluating the efficacy of 4 native antigens purified from adult Fasciola gigantica flukes, and 1 recombinant F. gigantica antigen, as vaccines against tropical fasciolosis. The antigens tested were native glutathione S-transferase, cathepsin L, paramyosin, fatty acid binding protein (FABP), and a recombinant FABP expressed in E. coli, and were formulated in 1 or more of several adjuvants (Quil A, Squalene Montanide 80, MF59-100, Auspharm, NAGO, polylactoglycolide microspheres, Algammulin, DEAE, Freunds). Vaccination induced low, moderate or high antibody titres to the various antigens which were dependent on the adjuvant. Low but significant reductions in fluke burdens (31%, P < 0.026) and fluke wet weight (36%, P < 0.041) were only observed in cattle vaccinated with the native FABP in Freunds adjuvant. There was no correlation between total antibody titres to FABP and protection. The protection observed in cattle vaccinated with native FABP of F. gigantica supports the notion that this class of proteins is a useful target for protection of animals against Fasciola and extends the efficacy of FABPs to the tropical liver fluke. This is the first report of vaccination of cattle against F. gigantica with a purified protein.

THE SENSITIVITY AND SPECIFICITY OF TWO METHODS FOR DETECTING FASCIOLA INFECTIONS IN CATTLE

Counts of Fasciola spp. eggs in faeces and measurements of antibody concentration to the excretory/secretory antigens of Fasciola spp. by ELISA were related to the numbers of flukes in the livers of 92 cattle killed in the abattoirs of Hanoi City, Vietnam. In this population, about 22% of the cattle had no flukes, another 22% had between 1 and 10 flukes, 44% between 11 and 100 flukes and 12% had more than 100 flukes in their livers. Of the 14 animals less than 2 years of age, only three were infected. At 2 years of age the mean number of flukes per liver was 10 whereas at 3 years and older, the mean varied between 60 and 80 flukes. Prevalence of infection was 78.3%. No eggs of Fasciola spp. were detected in the faeces of one third of infected cattle and 60% of the counts were less than 100 eggs per gram. The sensitivity of the egg counting method was 66.7% and specificity 100%, overall accuracy was 73.9%. Corresponding values for the ELISA method were 86.1, 70 and 82.6%, respectively. The positive and negative predictive values for the egg counting method were 100 and 45.5% and for the ELISA method were 91.2 and 58.3%, respectively.

Immunological approaches for the control of fasciolosis

The immunological relationship between liver flukes and their mammalian hosts is being unravelled by in vivo and in vitro studies. Vaccine studies in cattle and sheep with purified antigens (fatty acid binding protein, FABP; glutathione S-transferase, GST; cathepsin L, CatL; hemoglobin) have shown that high reductions in worm burdens (31-72%) and egg production (69-98%) can be achieved, raising the realistic possibility that immunological control of Fasciola infection is a commercially achievable goal. Combination vaccines may also be feasible since a cocktail of CatL and hemoglobin elicits a significant 72% protection in cattle. Analysis of immune responses to Fasciola during infection in ruminants suggests that chronic infection correlates with a type 2 helper T cell response, implying that type 1 helper T cell responses are down-regulated in fasciolosis. Recent results studying the resistance of Indonesian Thin Tail (ITT) sheep to F. gigantica have shown that this breed exhibits high innate (or rapidly acquired) resistance to infection and acquires a higher level of resistance after a primary challenge. Initial studies suggest that the resistance of ITT sheep to F. gigantica may be determined by a major gene. Merino sheep also acquire resistance to F. gigantica. In contrast, ITT and Merino sheep do not exhibit resistance to F. hepatica. These results suggest that there are fundamental differences between these two species of Fasciola in the biology of their interaction with the sheep immune system. In vitro studies on immune mechanisms of killing of juvenile fluke have shown that juvenile larvae of F. hepatica are susceptible to antibody-dependent killing by activated rat macrophages in vitro which is mediated by nitric oxide. Future studies on the immune effector mechanisms expressed by resistant sheep which control infection by F. gigantica will lead to new knowledge which may allow the design of more effective vaccines for fasciolosis.

Humoral responses in mice following vaccination with DNA encoding glutathione S‐transferase of Fasciola hepatica: effects of mode of vaccination and the cellular compartment of antigen expression

The humoral responses in mice following vaccination with DNA constructs encoding Fasciola hepatica glutathione S‐transferase (GST) have been evaluated. GST47 cDNA was subcloned into two DNA vaccine vectors, VR1012 and VR1020, which direct expression to the cytoplasmic and extracellular compartments, respectively. Expression was confirmed by transfection into COS 7 cells. Groups of mice were vaccinated with these constructs, by either intramuscular injection with the VR1012‐or VR1020‐based constructs, or intradermal vaccination (with a gene gun) with the VR1020‐based construct. Vaccination with the construct designed for secretion resulted in an increased humoral response compared to vaccination with the nonsecretory construct. The level of the total humoral response after vaccination with the secretion construct was not dependent on the route of vaccination. However, the isotype profile of the response differed between the groups; intramuscular vaccination with the construct directing cytoplasmic expression yielded an immuoglobulin (Ig)G2a dominant (Th1‐type) response, intradermal vaccination with the secretory construct a IgG1/IgE dominant (Th2‐type) response, and intramuscular vaccination with the secretory construct a mixed isotype response. These results demonstrate that the immunogenicity of a DNA vaccine based on Fasciola GST, as well as the isotype of the response against GST, is determined by the mode of vaccine administration.

Expression library immunization protects mice against a challenge with virulent rodent malaria.

Although several candidate vaccine antigens have been developed for malaria, there is as yet no effective single vaccine available. There is a growing consensus that the ultimate malaria vaccine will be multivalent, requiring the identification of a suitable cocktail of antigens. However, evaluation of the multitude of potential malaria vaccine antigens in suitable combinations is a daunting task. Here we describe the validation of expression library immunization (ELI) as a tool for the discovery of sequences protective against malaria infection. A genomic Plasmodium chabaudi expression library was constructed comprising ten separate pools of 3000 plasmids. Over three vaccine trials using biolistic delivery of pools composed of 616 to 30,000 plasmids we report up to 63% protection of mice from a challenge with P. chabaudi adami DS, a highly virulent strain. Overall, ELI protected 36% of vaccinated mice against virulent challenge compared with only 3.2% survival of control mice. These results demonstrate that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.

Atypical (mild) forms of dihydropteridine reductase deficiency : neurochemical evaluation and mutation detection

ABSTRACT: We investigated two patients with an atypical (mild) form of dihydropteridine reductase (DHPR) deficiency. Both responded to the loading test with tetrahydrobiopterin; their plasma phenylalanine levels were lowered from 278 μmol/L to 85 and 48 μmol/L and from 460 μmol/L to 97 and 36 μmol/L after 4 and 8 h, respectively. In one of the patients, a combined loading test with phenylalanine followed by tetrahydrobiopterin was also carried out and showed a profile typical for DHPR deficiency. The phenylalanine hydroxylation rate was calculated to be 43 and 87%, 4 and 8 h after cofactor administration, respectively. Diagnosis was confirmed by the absence of DHPR activity in the patients erythrocytes. In cultured fibroblasts, residual activity of 4 and 10%, respectively, was found. Excretion of urinary pterins was essentially normal, and the biopterin to neopterin ratio in cerebrospinal fluid was increased. Although in both patients cerebrospinal fluid homovanillic acid was found to be normal, and 5-hydroxyindoleacetic acid was substantially reduced, there was no sign of neurologic alterations until the age of 2 y. However, one of the patients recently developed deceleration of head growth, whereas psychomotor development continued to be normal for age. Using the chemical cleavage method on the amplified cDNA, mismatches of T to G at nucleotide 659 and of G to A at nucleotide 475, respectively, were identified. These results also demonstrate that screening for tetrahydrobiopterin deficiency by urinary pterin analysis alone can miss some newborns with mild DHPR deficiency and that all children with tetrahydrobiopterin defects need full neurochemical evaluation together with analysis of the enzyme activity.

Characterization of a second nuclear gene, AEP1, required for expression of the mitochondrial OLI1 gene in Saccharomyces cerevisiae

Due to mutation in a single nuclear locus, AEP1, the temperature-conditional pet mutant ts1860 of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature of 36°C. The presence at this temperature of near-normal levels of the cognate oli1 mRNA in mutant ts1860 indicates that, as previously shown, the product of the AEP1 gene is required for translation of the mitochondrial oli1 transcript. In this study the AEP1 gene has been cloned from a wild-type yeast genomic library by genetic complementation of a temperature-conditional aep1 strain at the restrictive temperature. A 2,330-bp genomic fragment which restores subunit 9 synthesis in aep1 mutant strains was characterized. This fragment encoded five open reading frames: the longest of these, at 1,554 nucleotides, was identified as the AEP1 gene, since disruption of this reading frame generated a non-conditional pet strain unable to synthesize subunit 9. The predicted product of AEP1 is a basic, hydrophilic protein of 59,571 Da which possesses a putative mitochondrial address sequence. Hybridization studies with AEP1-specific probes indicate that the gene is located on chromosome XIII and produces several poly(A)+ transcripts ranging in size from 0.9 to 2.7 kb. None of the identified reading frames share significant homologies with entries of several data bases.

Induction of specific T-cell responses, opsonizing antibodies, and protection against Plasmodium chabaudi adami infection in mice vaccinated with genomic expression libraries expressed in targeted and secretory DNA vectors.

ABSTRACT It has been proposed that a multivalent malaria vaccine is necessary to mimic the naturally acquired resistance to this disease observed in humans. A major experimental challenge is to identify the optimal components to be used in such a multivalent vaccine. Expression library immunization (ELI) is a method for screening genomes of a pathogen to identify novel combinations of vaccine sequences. Here we describe immune responses associated with, and the protective efficacy of, genomic Plasmodium chabaudi adami DS expression libraries constructed in VR1020 (secretory), monocyte chemotactic protein-3 (chemoattractant), and cytotoxic T lymphocyte antigen 4 (lymph node-targeting) DNA vaccine vectors. With splenocytes from vaccinated mice, specific T-cell responses, as well as gamma interferon and interleukin-4 production, were observed after stimulation with P. chabaudi adami-infected erythrocytes, demonstrating the specificity of genomic library vaccination for two of the three libraries constructed. Sera obtained from mice vaccinated with genomic libraries promoted the opsonization of P. chabaudi adami-infected erythrocytes by murine macrophages in vitro, further demonstrating the induction of malaria-specific immune responses following ELI. Over three vaccine trials using biolistic delivery of the three libraries, protection after lethal challenge with P. chabaudi adami DS ranged from 33 to 50%. These results show that protective epitopes or antigens are expressed within the libraries and that ELI induces responses specific to P. chabaudi adami malaria. This study further demonstrates that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.

A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria.

ABSTRACT The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethal Plasmodium chabaudi adami DS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.

Molecular analysis of dihydropteridine reductase deficiency: identification of two novel mutations in Japanese patients

Abstract Mutations in the dihydropteridine reductase (DHPR) gene result in hyperphenylalaninaemia and deficiency of various neurotransmitters in the central nervous system, causing severe neurological symptoms. We studied two Japanese patients with DHPR deficiency and identified a missense and a splicing error mutation, respectively. A homozygous missense mutation (tryptophan36-to-arginine) was detected in patient 1. The mutation abolished DHPR activity according to in vitro expression studies. The DHPR mRNA in patient 2 was markedly decreased. Reverse transcription-polymerase chain reaction of the mRNA generated a cDNA fragment with a 152-bp insertion. The inserted sequence contained a termination codon, which was likely to affect the stability of the mRNA. Analysis of genomic DNA showed that the insertion was derived from putative intron 3 of the DHPR gene, and an intronic A-to-G substitution was present adjacent to the 3′-end of the inserted sequence. The nucleotide change generated a sequence similar to an RNA splice donor site and probably activated an upstream cryptic acceptor site, thus producing an abnormal extra exon.