PLGA-dendron nanoparticles enhance immunogenicity but not lethal antibody production of a DNA vaccine against anthrax in mice.

Dendriplexes, complexes of dendrons and condensed plasmids containing the gene for protective antigen (PA) of Bacillus anthracis, were encapsulated in poly-lactide-co-glycolide (PLGA) particles using the double emulsion method. The two dendrons employed are a dendron with three C(18) chains (C(18) dendron) and one with no attached hydrocarbon chains (the C(0) dendron). Three types of particles were examined, namely PLGA-C(18) dendriplexes, PLGA-C(0) dendriplexes and the control PLGA-naked DNA system. These were characterised by standard biophysical methods such as photon correlation spectroscopy (PCS) and scanning electron microscopy to select the complexes for in vivo testing. Three intramuscular immunizations were carried out using 14 microg of DNA per dose at weekly intervals in BALB/c mice. Antibodies against rPA were measured using ELISA. Results indicate that the PLGA-C(18) dendriplex particles produced superior levels of anti-PA IgG antibodies in comparison to animals immunized with the PLGA-C(0) dendriplex particles. The level of antibody production was dependent on the number of immunizations, higher antibody levels being measured after two booster vaccinations. However toxin neutralizing antibodies were absent in all treatment groups, and it is likely that the mice lack protection against lethal toxin and anthrax infection. Further studies are needed to optimize the formulation of DNA vaccines and increase the level of anti-lethal toxin antibodies and enhance their functionality.

Transcriptional and mutational analysis of the Helicobacter pylori urease promoter.

Urease is an essential virulence factor of the human gastric pathogen Helicobacter pylori, and is expressed to very high levels. The promoter of the urease operon contains sequences resembling the canonical -10 and extended -10 motifs, but no discernible -35 motif. To establish the role of different motifs and regions in the urease promoter, we fused the urease promoter to a genomic lacZ reporter gene in H. pylori, made substitutions in the aforementioned promoter motifs, and also made deletions in the upstream sequences removing regulatory sequences. Substitutions in the -10, extended -10 and predicted -35 motifs all significantly altered expression of the lacZ reporter gene, demonstrating their importance in transcription of the H. pylori urease operon. In contrast, sequential deletions upstream of the -35 region did not affect expression of the lacZ reporter gene. This demonstrates the modular structure of the H. pylori urease promoter, where basal levels of transcription are initiated from a typical sigma(70) promoter, which requires -10 and extended -10 motifs, and also its -35 motif for efficient transcription. Upstream sequences are not involved in basal levels of urease transcription, but play an important role in responses to environmental stimuli like nickel.

Assessing clonality of Vibrio cholerae Inaba isolates by characterization of nonsense mutations in wbeT.

The transferase gene wbeT of six clinical isolates of Vibrio cholerae O1 biotype El Tor was analysed. Two unique mutations were identified in the wbeT gene of three Inaba isolates. Due to their random nature, mutations in wbeT can be used to determine the clonal origin of clinical Inaba isolates.

Role of the Helicobacter pylori outer-membrane proteins AlpA and AlpB in colonization of the guinea pig stomach.

The human gastric pathogen Helicobacter pylori expresses several putative outer-membrane proteins (OMPs), but the role of individual OMPs in colonization of the stomach by H. pylori is still poorly understood. The role of four such OMPs (AlpA, AlpB, OipA and HopZ) in a guinea pig model of H. pylori infection has been investigated. Single alpA, alpB, hopZ and oipA isogenic mutants were constructed in the guinea pig-adapted, wild-type H. pylori strain GP15. Guinea pigs were inoculated intragastrically with the wild-type strain, single mutants or a mixture of the wild-type and a single mutant in a 1: 1 ratio. Three weeks after infection, H. pylori could be isolated from stomach sections of all animals that were infected with the wild-type, the hopZ mutant or the oipA mutant, but from only five of nine (P = 0.18) and one of seven (P = 0.02) animals that were infected with the alpA or alpB mutants, respectively. The hopZ and oipA mutants colonized the majority of animals that were inoculated with the strain mixture, whereas alpA and alpB mutants could not be isolated from animals that were infected with the strain mixture (P < 0.01). Specific IgG antibody responses were observed in all animals that were infected with either the wild-type or a mutant, but IgG levels were lower in animals that were infected with either the alpA or the alpB mutants, compared to the wild-type strain (P < 0.05). In conclusion, absence of AlpA or AlpB is a serious disadvantage for colonization of the stomach by H. pylori.

Differences in immunogenicity and protection in mice and guinea pigs following intranasal immunization with Helicobacter pylori outer membrane antigens.

Mice and guinea pigs were intranasally immunized with either recombinant lipoprotein 20 or Helicobacter pylori outer membrane vesicles (OMV). Cholera toxin was used as mucosal adjuvant. In mice, both vaccines elicited systemic and local IgG responses, which correlated with significantly lower levels of H. pylori colonization. In contrast, only OMV proved immunogenic in guinea pigs, with the development of both systemic and local immune responses. These antibodies did not, however, correlate with protection in these animals, which suggests that vaccine formulation is as important as choice of antigen in the development of an H. pylori vaccine.

Investigation in a model system of the effects of combinations of anthrax and pertussis vaccines administered to service personnel in the 1991 Gulf War.

The toxicity and immunogenicity of the anthrax and pertussis vaccine combinations used in the 1991 Gulf War was assessed in NIH, A/J and Balb/c mice. Inoculation of pertussis vaccines, vaccine combinations, or aluminium salt caused illness, splenomegaly and significant weight loss. Although some animals recovered eventually, a lethal form of ascites developed in some NIH mice and body weights of A/J and Balb/c mice remained below normal levels. Inoculation of anthrax vaccine produced little effect. Exposure to diluted vaccine combinations produced less serious side effects of shorter duration. Single vaccinations induced specific IgG1 antibodies whereas a mixture of IgG1 and IgG2a was produced after multiple injections. Antigen stimulation of spleen cells from mice exposed to pertussis vaccines induced high levels of NO and IL-6, whereas stimulated spleen cells from mice exposed to anthrax vaccine produced only low levels of IL-6. In mice, pertussis vaccines act as an adjuvant for anthrax vaccine, but these vaccines are also the major cause of toxicity of the vaccine combination. The relatively high vaccine dose used, together with the low sensitivity of mice to anthrax toxin, emphasises that caution should be exercised in applying these results to human recipients of these vaccines.

Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii and group VS116 by PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans.

OBJECTIVE: To evaluate the diagnostic performance of two polymerase chain reaction (PCR) procedures using skin biopsies of 20 erythema migrans (EM) and 24 acrodermatitis chronica atrophicans (ACA) patients. METHODS: One assay amplified a fragment of the outer surface protein (Osp) A gene. The second method amplified the spacer region between the 5S and 23S rRNA genes; hybridization of this fragment allowed identification of Borrelia burgdorferi sensu lato species. RESULTS: Among EM patients, both assays detected Borrelia DNA in 15 samples. Among ACA patients, the ospA PCR detected 15 positives and 10 samples were positive by 5S-23S PCR. In 19 samples one species was detected, 15 skin biopsies contained Borrelia afzelii, and Borrelia garinii was found in two patients. Group VS116 was detected in two EM patients, and therefore this group has pathogenic potential. Mixed infections of B. afzelii and B. garinii, group VS116 or B. burgdorferi sensu stricto were found in three EM and three ACA patients. CONCLUSIONS: Diagnosis of EM and ACA by PCR is useful and knowledge of the presence of species may be used to predict the course of disease or the need for further antibiotics.