Immunization of mice with Plasmodium TCTP delays establishment of Plasmodium infection.

Translationally controlled tumour protein (TCTP) may play an important role in the establishment or maintenance of parasitemia in a malarial infection. In this study, the potential of TCTP as a malaria vaccine was investigated in two trials. In the initial vaccine trial, Plasmodium falciparum TCTP (PfTCTP) was expressed in Saccharomyces cerevisiae and used to immunize BALB/c mice. Following challenge with Plasmodium yoelii YM, parasitemia was significantly reduced during the early stages of infection. In the second vaccine trial, the TCTP from P. yoelii and P. berghei was expressed in Escherichia coli and used in several mouse malaria models. A significant reduction in parasitemia in the early stages of infection was observed in BALB/c mice challenged with P. yoelii YM. A significantly reduced parasitemia at each day leading up to a delayed and reduced peak parasitemia was also observed in BALB/c mice challenged with the nonlethal Plasmodium chabaudi (P.c.) chabaudi AS. These results suggest that TCTP has an important role for parasite establishment and may be important for pathogenesis.

Molecular typing of Salmonella enterica serovar Sofia in Australia by pulsed-field gel electrophoresis and repetitive element PCR typing.

AIMS: In this study, we used two molecular fingerprinting methods to investigate the genetic and clonal relationship shared by Australian Salmonella Sofia isolates. METHODS AND RESULTS: A total of 84 Australian Salm. Sofia isolates from various states in Australia were typed using pulsed-field gel electrophoresis (PFGE) (XbaI and SpeI) and repetitive element PCR (REP1R-I primer). The previous problem of DNA degradation of Salm. Sofia strains was solved by modifying the lysis solution used to treat the bacterial plugs, allowing Salm. Sofia to be subtyped using PFGE. Molecular typing of isolates resulted in the generation of eight XbaI, six SpeI and five REP1 pattern profiles. Individual typing methods showed low discrimination index values (<0·5), indicating the poor discriminatory ability of the methods. However, the combination of the typing methods was able to improve the discrimination of isolates, further dividing them into 16 subtypes and raising the index value to 0·721. CONCLUSIONS: The combination of typing methods was shown to be the best approach to fingerprint Salm. Sofia. The Australian Salm. Sofia isolates only showed limited genetic diversity and probably share a clonal relationship. A majority of the Salm. Sofia isolates were not geographically restricted with the predominant pattern subtype observed amongst the isolates from various states. SIGNIFICANCE AND IMPACT OF THE STUDY: We have successfully devised a PFGE protocol that counteracts DNase activity of Salm. Sofia, enabling typing of this serovar.

Construction and immunogenicity of Salmonella vaccine vector expressing HIV-1 antigen and MCP3.

UNLABELLED: This study aims to determine the efficacy of Salmonella enterica serovar Typhimurium STM-1 bearing MCP-3 gene as a delivery vehicle for the HIV gag gene (in particular p24 gene) and HIV env gene. The STM1 delivery HIV-p24 vaccination was carried out in the form of a recombinant or a DNA vaccine whereas only a DNA vaccine was used for HIV env . Naked DNA vaccination was also tested and immune responses were evaluated following immunisation in mouse model. RESULTS: vaccination cellular immune responses induced by recombinant p24 STM1 (STM1/pHly-p24) were greater than those elicited by the p24 DNA vaccine in STM1 (STM1/VR-p24), (but statistically not significant) than those induced by oral vaccination. However, IgA responses induced by oral vaccination with either a recombinant or DNA vaccine of p24 in STM1 are higher than those induced by IP vaccination. In addition, the numbers of cells secreting IL4 are reduced after oral vaccination with STM1/VR-p24/MCP3. However, for the HIV p24 antigen, STM1/MCP3 preferentially induces IFNgamma-secreting splenocytes. CONCLUSIONS: This result confirms other studies that Salmonella was able to deliver HIV antigens to the immune system and induced specific immune responses to the HIV antigen and for the HIV p24 antigen, STM1/MCP3 induces secretion of IFNgamma.

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.

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.

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.

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.

The protective efficacy of MSP4/5 against lethal Plasmodium chabaudi adami challenge is dependent on the type of DNA vaccine vector and vaccination protocol.

The enhancement of immunogenicity of malarial DNA vaccines is important if they are to have practical application in protecting against blood-stage malaria. Here we describe three different DNA vaccine vector types used in conjunction with the blood-stage merozoite surface protein 4/5 (MSP4/5), the murine homologue of Plasmodium falciparum MSP4 and MSP5, in an attempt to enhance survival against lethal Plasmodium chabaudi adami DS blood-stage challenge. MSP4/5 was inserted into VR1020 (secretory), monocyte-chemotactic protein-3 (MCP-3) (chemoattractant), and cytotoxic T-lymphocyte antigen 4 (CTLA4) (lymph node targeting) vectors. Mice were immunized intradermally via gene-gun, IM injection, or boosting with recombinant MSP4/5 protein. Antibody responses after boosting were predominantly of the IgG1 and IgE isotypes, with low avidity antibodies produced in DNA primed groups. Despite antibody responses comparable to recombinant protein immunization, boosting mice primed with antigens encoded by MCP-3 and CTLA4 vectors did not enhance survival compared to vector control groups. Gene-gun vaccination using VR1020/MSP4/5 followed by recombinant MSP4/5 boosting, or gene-gun DNA vaccination alone using MCP-3/MSP4/5, resulted in enhanced survival compared to empty vector control mice. The results suggest that the enhancement of survival against lethal blood-stage malaria challenge after utilizing MSP4/5 DNA vaccination is therefore highly dependent on the route and type of vaccine vector employed.

Fasciola: kinetics and quality of humoral responses to fatty acid binding protein and cathepsin l following delivery as DNA vaccines in mice.

The humoral response to DNA vaccination of mice with two important Fasciola antigens has been investigated. Both F. gigantica fatty acid binding protein (FABP) and F. hepatica cathepsin L5 (FhCatL5) were shown to be expressed in COS 7 cells and induced a humoral response when delivered as secretory constructs in mice. FABP induced an IgGl dominant response, with significant IgE, IgG2a, and IgG2b responses also present, indicating a mixed Th1/Th2 response. The total Ig response peaked at 1:24,500 and antibody titers were sustained for at least 32 weeks. In contrast, the delivery of FABP as a nonsecreted construct did not result in the induction of a measurable humoral response. FhCatL5 was delivered as a secretory construct, with secretion mediated by the native F. hepatica signal sequence, which was shown to operate in COS 7 cells. The humoral response peaked at 1:2000 at week 8 and was sustained for at least 20 weeks. Antibody isotype analysis demonstrated a Th2-like response, which was qualitatively different from that obtained for FABP with an IgE dominant response, and lower titers to IgG1 and IgG3. The results demonstrate that Fasciola antigens can be delivered as DNA vaccines, but that the quality of the response varies between antigens and is influenced by the method of vaccine delivery.

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.

A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica.

The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.

A series of mutations in the dihydropteridine reductase gene resulting in either abnormal RNA splicing or DHPR protein defects. Mutations in brief no. 244. Online.

Five novel mutations are described which result in the rare hyperphenylalaninemia DHPR-deficiency. Three of these are located at different intron/exon boundaries within the DHPR gene, and disrupt the maturation of the DHPR transcript such that little full-length mRNA can be detected by RT-PCR. Each mutation alters a conserved nucleotide within the splice site consensus sequence, and results in the skipping of an exon and, in one case, the activation of an inappropriate splicing signal. Two further mutations are missense mutations resulting in a non-conservative amino acid change within the DHPR protein (L14P and G17V) and are associated with a severe phenotype.

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.

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.

Dihydropteridine reductase deficiency: physical structure of the QDPR gene, identification of two new mutations and genotype-phenotype correlations.

Dihydropteridine reductase (DHPR) is an enzyme involved in recycling of tetrahydrobiopterin (BH4), the cofactor of the aromatic amino acid hydroxylases. Its deficiency is characterized by hyperphenylalaninemia due to the secondary defect of phenylalanine hydroxylase and depletion of the neurotransmitters dopamine and serotonin, whose syntheses are controlled by tryptophan and tyrosine hydroxylases. The DHPR cDNA has been cloned and mapped on 4p15.3. In the present study we report the genomic structure of the DHPR gene (QDPR). This gene includes seven exons within a range of 84-564 bp; the corresponding introns are flanked by canonic splice junctions. We also present a panel of PCR primers complementary to intronic sequences that greatly facilitates amplification of the gene and provides a genomic DNA approach for mutation detection. We have used this approach to study six patients with DHPR deficiency. Four known mutations (G23D, H158Y, IVS5G+ 1A, R221X) and two new mutations (Y150C and G218ins9bp) were found. The Y150C mutation was found in compound heterozygosity with G23D, a mutation always associated with a severe phenotype in homozygous patients. This patient has an intermediate phenotype (good response to monotherapy with BH4). The mutant enzyme for Y150C was expressed in an E. coli system. Comparison of its kinetic parameters with those of the G23D mutant enzyme showed that it is not as effective as the wild-type enzyme, but is more active than the G23D mutant. This patient's intermediate phenotype is thus due to the mild DHPR mutation Y150C. Correlations between genotypes and phenotypes were also found for the other mutations.

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.

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

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.

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, Freund's). 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 Freund's 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.

A mutation causing DHPR deficiency results in a frameshift and a secondary splicing defect.

In our analysis of mutations causing DHPR deficiency we identified a patient in whom there was an aberrant transcription pattern detected by PCR of DHPR cDNA. However, unlike the pattern observed as a result of most splicing mutations, there is some full length transcript. The mutation was located and is a single nucleotide deletion at position 570/571 of the DHPR cDNA sequence and results in a frameshift and premature termination after the addition of six amino acids. The mutation is present in a homozygous state in the patient and in a heterozygous state in both parents. The exon which is deleted at high frequency in the patient is the putative exon 4, which is remote from the mutation, and confirms our observation that exon 4 skipping is a relatively common event.

Molecular basis of dihydropteridine reductase deficiency.

The spectrum of mutations causing dihydropteridine reductase is reviewed. A total of 12 point mutations have been described that map in the DHPR cDNA, resulting in amino acid substitutions, insertions and premature terminations. A further two mutations are described which result in aberrant splicing of DHPR transcripts. The application of the mutation identification to diagnostics and clinical treatment is discussed.