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Summary. Clostridium sordeiiii produces two toxins, designated HT (haemorrhagic toxin) and LT (lethal toxin), that are similar to toxins A and B of C . dzBciZe. The physicochemical properties of toxins HT and A were remarkably similar. The specific biological activities of toxin HT were almost the same as those of toxin A, and their NH,-terminal sequences shared close homology. The properties of toxins LT and B were similar, as were their NH2-terminal sequences, but toxin B was much more cytotoxic than toxin LT. Immunodiffusion analysis with specific antibodies showed that although toxins B and LT shared major antigenic determinants, each had unique epitopes. The results suggest that toxins B and LT have diverged more than toxins A and HT. Immunoblotting with antibodies to the toxins of C. dzficile showed that toxins HT and LT had common antigenic determinants. In trodue tionSeveral studies have implicated Clostridium sordefiii as a cause of diarrhoea and enterotoxaemia in domestic and. more recently, as an agent of toxic shock-like syndrome in man.*? This species was once suspected to be the cause of pseudomembranous colitis (PMC) in man because the cytotoxicity of faecal filtrates from PMC patients was neutralised by C. sordelfiiHowever, C . sordeiiii could not be isolated from the faeces of patients with PMC. This discrepancy was clarified when C. dificile was isolated from faecal samples of patients with PMC,9-' and it was shown that the toxins produced by this organism are neutralised by C. sordellii 2* l 3 Two toxins, A and B, have been purified from culture supernate of toxigenic strains of C. dzficife. ''-I6 Both are large proteins that are lethal to animals and cytotoxic. Toxin A is a potent enterotoxin that produces a haemorrhagic fluid response in the rabbit ileal loop assay.C . sordeifii produces two toxins that are similar to toxins A and B, which explains why C . sordeffii antitoxin neutralises the toxins of C . drficife. The production of two distinct toxins by C. sordefiii was first described by Arseculeratne er aI.,*' who extracted a haemorrhagic toxin from sporulating cells and an oedema-producing toxin from vegetative cells. The oedema-producing toxin was more lethal than the haemorrhagic toxin and the toxins are now referred to as LT (lethal toxin) and HT (haemorrhagic toxin) respectively. We have already described the purification of toxin HT by ultrafiltration and immuno-affinity chromatography with a monoclonal antibody (MAb) to toxin A, and have shown that toxin HT has biological activities and immunological properties similar to those of toxin A. * Popoff has purified toxin LT and shown that it is immunologically related to toxin B.22 The toxins produced by C. dzflcile and by C. sordeiiii have similar physicochemical as well as immunological and biological properties but they are not identical. Therefore, it was of interest to compare the properties of these toxins in more detail. Materials and methods Protein determinationProtein concentration was estimated by the method of Bradford23 w...

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The goal of this study was to compare the immune response and the protection capacity induced by the dengue virus 2 (DENV-2) American and Asian genotypes in Macaca fascicularis monkeys. Animals were infected with American or Asian DENV-2 strains and challenged 1 year later with a DENV-2 Asian genotype strain. The viremia and monkey antibody levels were similar for the different strains after primary and secondary infection; however, the functionality of the antibody response was different. A limited viral replication was demonstrated after the secondary infection in all the monkeys. No virus was isolated in tissue culture, while reverse transcription-PCR showed a late positive reaction in four of five challenged monkeys. The immunoglobulin M response pattern and the detection of antibodies to specific proteins by Western blotting supported the protection data. Despite the demonstration of the protective effect after homologous challenge, a strong anamnestic antibody response was observed.Dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are caused by any of four closely related but antigenically distinct dengue virus (DENV) serotypes (DENV-1, DENV-2, DENV-3, and DENV-4).

The increasingly limited availability and high cost of the hitherto most commonly used monkey species in dengue vaccine research has augmented the importance of identifying alternative suitable models for these studies. In this study we examined the capacity of green monkeys (Chlorocebus aethiops sabaeus) to develop dengue viremia, and thus provide a potential model for dengue vaccine testing. Monkeys were inoculated with two different doses of dengue virus type 2. All animals in both groups became viremic after inoculation of the virus. In the lower dose group, mean viremia duration of 5.66 days was detected, whereas in the group that received the 10 6 PFU dose, viremia had a mean duration of only 1.66 days. Antibody titers were similar to those obtained in previous experiments with rhesus and cynomolgus macaques. We conclude that green monkeys develop viremia and antibody responses and therefore provide a potential model for the preclinical evaluation of novel candidates for dengue vaccines.

The suitability of dengue 2 envelope domain III recombinant fusion proteins [(fusion (PD5) and insertion (PD3) variants)] for inducing functional antibodies and a protective immune response in nonhuman primates has been reported. However, the evaluation of the antibody response after immunization did not correlate with the protection data as measured by viremia detection. Here, we characterized the anamnestic immune response after viral challenge in monkeys immunized with the dengue 2 recombinant proteins in an attempt to define correlates of protection useful for vaccine studies. Monkeys immunized with PD5 (most protected group) exhibited an earlier increase in the anti-DENV-2 IgM response after challenge compared to control animals. Hemagglutination-inhibiting (HAI) antibodies were increased significantly earlier in PD5-immunized animals compared to those immunized with PD3. The fully protected monkeys showed the earliest HAI antibody response. These results underline the usefulness of the anamnestic antibody response for supporting protection data. The induction of an early HAI and IgM antibody response after challenge suggest a protective role against dengue virus (DENV) infection in monkeys, supporting their use as correlates of protection in vaccine studies.

A recombinant vaccine that expresses the envelope (E) gene of dengue virus type 4 was tested for immunogenicity and protection in Macaca fascicularis. One hundred micrograms of semipurified recombinant E protein (E4rec) expressed in Pichia pastoris was used to immunize three animals. Neutralizing antibodies to dengue 4 virus with a titer of 1:30 were detected in all immunized monkeys prior to challenge. Animals were challenged with 10 5 plaqueforming units of dengue 4 virus. One vaccine-immunized monkey was protected from viremia, while the other two were partially protected. Monkeys immunized with E4rec elicited the highest neutralizing antibody titers (P < 0.05) ranging from 1:85 to 1:640 at day 30. In both immunized and control animals, the longest duration of viremia correlated with earliest and highest level of IgM antibody to dengue virus. The vaccinated animals showed anamnestic antibody responses upon virus challenge, indicating successful priming by the recombinant vaccine. Our results suggest that E4rec expressed in P. pastoris can provide partial protection against viremia. However, the results were not effective enough to use it as a vaccine candidate. Further work is required to improve the quality of the immunogen.