Miguel Ángel Ayala

Identification of a new protective antigen of Bordetella pertussis

Antigenic proteins whose expression is induced under iron starvation, an environmental condition that bacterial pathogens have to face during colonization, might be potential candidates for improved vaccine. By mean of immune proteomics we identified novel antigens of Bordetella pertussis maximally expressed under iron limitation. Among them, Bp1152 (named as IRP1-3) showed a particularly strong reaction with human IgG purified from pooled sera of pertussis-infected individuals. Computer analysis showed IRP1-3 as a dimeric membrane protein potentially involved in iron uptake. Experimental data revealed the surface-exposure of this protein and showed its increase under iron starvation to be independent of bacterial virulence phase. Immunization of mice with the recombinant IRP1-3 resulted in a strong antibody response. These antibodies not only recognized the native protein on bacterial surface but also promote effective bacterial phagocytosis by human PMN, a key protecting activity against this pathogen. Accordingly, IRP1-3 proved protective against B. pertussis infection in mouse model. Expression of IRP1-3 was found conserved among clinical isolates of B. pertussis and positively regulated by iron starvation in these strains. Taken together these results suggest that this protein might be an interesting novel vaccine candidate.

Bordetella pertussis iron regulated proteins as potential vaccine components

Bordetella pertussis is the etiologic agent of whooping cough, an illness whose incidence has been increasing over the last decades. Pertussis reemergence despite high vaccination coverage, together with the recent isolation of circulating strains deficient in some of the vaccine antigens, highlight the need for new vaccines. Proteins induced under physiological conditions, such as those required for nutrient acquisition during infection, might represent good targets for better preventive strategies. By mean of serological proteome analysis we identified two novel antigens of B. pertussis potentially involved in iron acquisition during host colonization. We had previously demonstrated that one of them, designated IRP1-3, is protective against pertussis infection in mice. In the present study, we show that the other antigen, named AfuA (BP1605), is a highly antigenic protein, exposed on the bacterial surface, conserved among clinical isolates and expressed during infection. Immunization of mice with the recombinant AfuA induced opsonophagocytic antibodies which could explain the protection against B. pertussis infection conferred by mice immunization with rAfuA. Importantly, we found that the addition of rAfuA and rIRP1-3 proteins to the commercial three pertussis components acellular vaccine significantly increased its protective activity. Taken together, our results point at these two antigens as potential components of a new generation of acellular vaccines.

Acute copper intoxication after a Cu–Ca EDTA injection in rats

It has been claimed that Cu-Ca EDTA releases the Cu slowly into the blood when injected as a therapeutic agent for Cu deficiency, but when overdosing occurs, animals may die in a few hours after the injection, suggesting that Cu release is faster than expected. A total of 30 rats were divided into six equal groups and injected with Cu-Ca EDTA at 4, 6, 13, 33 or 64 mg/kg body weight, the sixth group acted as control. All rats were sacrificed 24 h later, with the exception of rats from groups 33 and 64 that died 7 and 2 h postinjection respectively. The rate of Cu transference to the liver increased linearly with the dose, from (X +/- S.E.) 14.88 +/- 3.14 in group 4, to 134.47 +/- 21.86 microg/h in group 64. Injected animals developed liver and kidney degeneration, hemorrhage and necrosis, which increased in severity with the Cu dose. Liver Cu accumulation was highest for group 13 (866.8 +/- 84.23 ppm DM), while in group 64 reached only 167.0 +/- 14.99 ppm. It is concluded that during acute Cu poisoning, the rate of transfer is responsible for the liver and kidney damage, rather than the total amount of transferred Cu to those organs, as it is the case in chronic Cu poisoning. The high rate of Cu transfer possibly surpasses the intrinsic mechanisms of Cu deposition and free Cu is left into the cell with the development of necrosis.

Prevalence of antibodies against Kilham virus in experimental rat colonies of Argentina

The Kilham rat virus (KRV) is a parvovirus originally isolated from a rat sarcoma in the late 1950s. The clinical signs associated with a natural KRV infection include foetal resorption in dams, runting, ataxia, cerebellar hypoplasia and jaundice in suckling rats, and sudden death, scrotal cyanosis, abdominal swelling and dehydration in juvenile rats. The ability of this virus to produce persistent infections has resulted in a high frequency of contamination of cell cultures and transplantable-tumor system. In addition, the virus may interfere with research in other ways. The remarkable resistance to environmental conditions determines the importance of the detection and control of this agent, especially in the laboratory animal production. This study determines the seroprevalence of Kilham antibodies from sera of adult rats from conventional facilities, using the haemagglutination inhibition test. The seroprevalence varied between 27.8% and 75%. This result confirms that the virus is circulating in Argentinean conventional facilities and might be interfering with research. The recognized Kilham virus may be prevented from supply sources by implementing a health monitoring schedule including a regular serological surveillance, and by keeping the animals under barrier systems.

Non-aversive photographic measurement method for subcutaneous tumours in nude mice:

We have developed a new method for the measurement of subcutaneous tumour volume which consists in taking photographs of mice in their home cages, to refine the standard method of measurement with calipers. We consider this new method to be non-aversive, as it may be more compatible with mice behavioural preferences and, therefore, improve their welfare. Photographs are captured when mice voluntarily go into an acrylic tube containing graph paper that is later used as a scale. Tumour volumes measured with the caliper and the non-aversive photographic method were compared to those obtained by water displacement volume and weight. Behavioural and physiological changes were evaluated to assess animal welfare. Significant differences were found between measurements obtained with the caliper and the non-aversive photographic method, v. the reference volume acquired by water displacement (P < 0.001). Nevertheless, there was good consistency for these measurements when tumours were measured repeatedly, with all Intra-Class Correlation Coefficients above 0.95. Mice on which the non-aversive photographic method was employed were significantly less reluctant to establish contact with the experimenter (P < 0.001) and behaved less anxiously in a modified-Novelty Suppressed Feeding test. Particularly, statistically significant differences were found in connection with the latency to eat an almond piece (P < 0.05), the frequency of grooming (P < 0.001) and the frequency of defecation (P < 0.001). Corticosterone concentration in faeces and blood glucose were determined and no significant changes were found. Therefore, we propose the non-aversive photographic method to measure subcutaneous tumours as a way to refine methodologies in the field of experimental oncology.

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis. It was found that this homolog, named AfuABpp, is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O‐antigen, a molecule that has been found to shield surface antigens on B. parapertussis, showed no influence on antibody recognition of AfuABpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.