Publications

In a recent article, Oscherwitz et al. endeavor to enhance the immunogenicity of a multiple antigenic peptide (MAP) vaccine that targets a loop-neutralizing determinant (LND) of Bacillus anthracis protective antigen (PA) (1). They showed in a previous study (2) that this MAP, consisting of four copies of amino acids 305 to 319 of PA (PA 305-319) extending from a lysine core, can elicit humoral immunity in rabbits that is specific and strongly neutralizing for the 2?2-2?3 loop in domain 2 of PA. However, LND-specific antibodies were not detected in rabbits immunized with whole PA, and it was concluded that PA 305-319 may be a cryptic epitope. T cell assays in PA 305-319-immunized mice showed that this peptide appeared to lack activity as a T helper cell epitope. We have good evidence that humans exposed to PA, either following cutaneous anthrax infection or through vaccination, generate long-term, T cell memory to both whole PA and PA 305-319. In a study including samples from naturally exposed patients recovered from cutaneous anthrax, from vaccine-hyperimmunized AVP (anthrax vaccine precipitated) donors, and from recombinant PA-vaccinated donors, positive T cell gamma interferon (IFN-?) enzyme-linked immunospot (ELISPOT) assay responses were detected across all groups on restimulation of peripheral blood mononuclear cells (PBMC) with both whole PA and PA 305-319 (Table 1; unpublished data). This shows that humans exposed to B. anthracis and whole PA recognize and generate memory to PA 305-319, suggesting that it is immunodominant rather than cryptic. Donors expressed a range of HLA haplotypes, with a majority being HLA-DR4 positive. While many important discoveries on the immunology of infection are made in experimental models, the human T cell response to PA in this respect differs from that of both rabbits and mice. In this light, the epitope used by Oscherwitz and colleagues may indeed be more relevant to eliciting appropriate human immunity than previously envisaged.

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of African swine fever virus (ASFV). This assay targets the topoisomerase II gene of ASFV and its specificity was confirmed by restriction enzyme digestion of the reaction products. The analytical sensitivity of this ASFV LAMP assay was at least 330 genome copies, and the test was able to detect representative isolates of ASFV (n = 38) without cross-reacting with classical swine fever virus. The performance of the LAMP assay was compared with other laboratory tests used for ASF diagnosis. Using blood and tissue samples collected from pigs experimentally infected with ASFV (Malawi isolate), there was good concordance between the LAMP assay and real-time PCR. In addition to detecting the reaction products using either agarose gels or real-time PCR machines, it was possible to visualise dual-labelled biotin and fluorescein ASFV LAMP amplicons using novel lateral flow devices. This assay and detection format represents the first step towards developing a practical, simple-to-use and inexpensive molecular assay format for ASF diagnosis in the field which is especially relevant to Africa where the disease is endemic in many countries.

The positive-stranded RNA genome of classical swine fever virus (CSFV) encodes 12 known proteins. The first protein to be translated is the N-terminal protease (Npro). Npro helps evade the innate interferon response by targeting interferon regulatory factor-3 for proteasomal degradation and also participates in the evasion of dsRNA-induced apoptosis. To elucidate the mechanisms by which Npro functions, we performed a yeast two-hybrid screen in which the anti-apoptotic protein HAX-1 was identified. The Npro–HAX-1 interaction was confirmed using co-precipitation assays. A dramatic redistribution of both Npro and HAX-1 was observed in co-transfected cells, as well as in transfected cells infected with wild-type CSFV, but not in cells infected with an Npro-deleted CSFV strain.