The immunology of Epstein-Barr virus infection

Epstein-Barr virus is a classic example of a persistent human virus that has caught the imagination of immunologists, virologists and oncologists because of the juxtaposition of a number of important properties. First, the ability of the virus to immortalize B lymphocytes in vitro has provided an antigen presenting cell in which all the latent antigens: of the virus are displayed and are available for systematic study. Second, the virus presents an ideal system for studying the immune parameters that maintain latency and the consequences of disturbing this cell-virus relationship. Third, this wealth of immunological background has provided a platform for elucidating the role of the immune system in protection from viral-associated malignancies of B cell and epithelial cell origin. Finally attention is now being directed towards the development of vaccine formulations which might have broad application in the control of human malignancies.

Orally administered Giardia duodenalis extracts enhance an antigen-specific antibody response

We have identified novel adjuvant activity in specific cytosol fractions from trophozoites of Giardia isolate BRIS/95/HEPU/2041 (J. A. Upcroft, P. A. McDonnell, and P. Upcroft, Parasitol. Today, 14:281-284, 1998). Adjuvant activity was demonstrated in the systemic and mucosal compartments when Giardia extract was coadministered orally with antigen to mice. Enhanced antigen-specific serum antibody responses were demonstrated by enzyme-linked immunosorbent. assay to be comparable to those generated by the gold standard, mucosal adjuvant cholera toxin. A source of adjuvant activity was localized to the cytosolic component of the parasite. Fractionation of the cytosol produced fraction pools, some of which, when coadministered with antigen, stimulated an enhanced antigen-specific serum response. The toxic component of conventional mucosal adjuvants is associated with adjuvant activity; therefore, in a similar way, the toxin-like attributes of BRIS/95/HEPU/2041 may be responsible for its adjuvanticity. Complete characterization of the adjuvant is under way.

Genotoxicity investigation of a cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN), a potent cyanobacterial hepatotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria, is regularly found in water supplies in many parts of the world, and has been associated with the intoxication of humans and livestock. In this study, Balb/c mice were injected via the intraperitoneal (IP) route with a single dose of 0.2 mg/kg CYN. Animals were sacrificed at 6, 12, 24, 48 and 72 It. DNA was isolated from the mouse livers, and examined for strand breakage by alkaline gel electrophoresis (pH 12). Significant DNA strand breakage was observed in the mouse liver exposed to CYN, suggesting that induction of DNA strand breakage is probably one of the key mechanisms for CYN genotoxicity. (C) 2002 Elsevier Science Ltd. All rights reserved.

Effects of intravenous injection of Clostridium perfringens type D epsilon toxin in calves

In cattle, a neurological lesion similar to that produced in sheep and goats by Clostridium perfringens type D enterotoxaemia has been reported. However, no causal relationship has been established between this disease and the lesion in cattle. The effects of single and multiple intravenous injections of epsilon toxin in three calves aged 6 months were studied. A further calf was inoculated intravenously with saline solution and used as a control. Epsilon toxin invariably produced neurological signs within 2-60 min of the end of the injection process. Clinical signs consisted of loss of consciousness, recumbency, convulsions, paddling, opisthotonus, hyperaesthesia and dyspnoea. Gross changes consisted of severe acute pulmonary oedema, which was particularly marked in the interlobular septa. The histological lesions consisted of intra-alveolar and interstitial oedema of the lung and variable degrees of perivascular proteinaceous oedema in the internal capsule, thalamus and cerebellar white matter. No clinical or post-mortem changes were observed in the control calf. These results show that calves are susceptible to the intravenous injection of epsilon toxin, and that they can show at least some of the histological lesions produced in sheep and goats by this toxin. (C) 2002 Harcourt Publishers Ltd.

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx(2c), either alone (16%) or in combination with stx(1) (74%) or stx(2) (3%) PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.

Horizontal transmission of Shiga toxin-producing Escherichia coli within groups of dairy calves

To examine the dissemination of Shiga-toxigenic Escherichia coli (STEC) within cattle groups, dairy calves on two farms utilizing different calf-rearing practices were exposed to a traceable STEC strain. Test strain dissemination differed significantly between farms, with a higher prevalence being associated with group penning. Pen floors and calf hides may be the main environmental mechanisms of transmission. Dairy calf husbandry represents a control point for reducing on-farm STEC prevalence.

Protection of mice from group A streptococcal infection by intranasal immunisation with a peptide vaccine that contains a conserved M protein B cell epitope and lacks a T cell autoepitope

Infection with group A streptococci (GAS) can lead to rheumatic fever (RF) and rheumatic heart disease (RHD) which are a major health concern particularly in indigenous populations worldwide, and especially in Australian Aboriginals. A primary route of GAS infection is via the upper respiratory tract, and therefore, a major goal of research is the development of a mucosal-based GAS vaccine, The majority of the research to date has focused on the GAS M protein since immunity to GAS is mediated by M protein type-specific opsonic antibodies. There are two major impediments to the development of a vaccine-the variability in M proteins and the potential for the induction of an autoimmune response. To develop a safe and broad-based vaccine, we have therefore focused on the GAS M protein conserved C-region, and have identified peptides, J8 and the closely related J8 peptide (J14), which may be important in protective immunity to GAS infection. Using a mucosal animal model system, our data have shown a high degree of throat GAS colonisation in B10.BR mice 24 h following intranasal immunisation with the mucosal adjuvant, cholera toxin B subunit (CTB), and/or diptheria toxoid (dT) carrier, or PBS alone, and challenge with the M1 GAS strain. However, GAS colonisation of the throat was significantly reduced following intranasal immunisation of mice with the vaccine candidate J8 conjugated to dT or J14-dT when administered with CTB. Moreover, J8-dT/CTB and J14-dT/CTB-immunised mice had a significantly higher survival when compared to CTB and PBS-immunised control mice. These data indicate that immunity to GAS infection can be evoked by intranasal immunisation with a GAS M protein C-region peptide vaccine that contains a protective B cell epitope and lacks a T cell autoepitope. (C) 2002 Published by Elsevier Science Ltd.

Parenteral and mucosal delivery of a novel multi-epitope M protein-based group A streptococcal vaccine construct: investigation of immunogenicity in mice

Primary vaccine strategies against group A streptococci (GAS) have focused on the M protein-the target of opsonic antibodies important for protective immunity. We have previously reported protection of mice against GAS infection following parenteral delivery of a multi-epitope vaccine construct, referred to as a heteropolymer. This current report has assessed mucosal (intranasal (i.n.) and oral) delivery of the heteropolymer in mice with regard to the induction and specificity of mucosal and systemic antibody responses, and compared this to parenteral delivery. GAS-specific IgA responses were detected in saliva and gut upon i.n. and oral delivery of the heteropolymer co-administered with cholera toxin B subunit, respectively. High titre serum IgG responses were elicited to the heteropolymer following all routes of delivery when administered with adjuvant. Moreover, as with parenteral delivery, serum IgG antibodies were detected to the individual heteropolymer peptides following i.n. but not oral delivery. These data support the potential of the i.n. route in the mucosal delivery of a GAS vaccine. (C) 2002 Elsevier Science Ltd. All rights reserved.

Analysis of toxin profiles in three different fish species causing ciguatera fish poisoning in Guadeloupe, French West Indies

A grey snapper (Lutjanus griseus), a grouper (Serranidae) and a blackjack (Caranx lugubris) were implicated in three different ciguatera poisonings in Guadeloupe, French West Indies. A mouse bioassay indicated toxicity for each specimens: 0.5-1, greater than or equal to 1 and > 1 M Ug g(-1), respectively. After purification by gel filtration chromatography, the samples were analysed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). The toxin profiles differ from one fish to another. C-CTX-1 was detected at 0.24, 0.90 and 13.8 ng g(-1) flesh in the snapper, grouper and jack, respectively. It contributed only to part of the whole toxicity determined by the mouse bioassay. Other toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or -2, and five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the flesh of the grouper with [M+ + H](+) ions at m/z 851.51, 857.50, 875.51, 875.49 and 895.54 Da. Some of these compounds have the same mass range as some known dinoflagellate toxins. In conclusion, this study confirms the usefulness of LC-MS analysis to determine the ciguatoxins levels and the toxin profile in fish flesh hazardous to humans.

Inhibitors of COP-mediated Transport and Cholera Toxin Action Inhibit Simian Virus 40 Infection

Simian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway. We now show that the initial entry step is blocked by brefeldin A and by incubation at 20degreesC. Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway. This intracellular trafficking pathway is also brefeldin A sensitive. Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to betaCOP. In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action. Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection.

Solution Structures of the cis- and trans-Pro30 Isomers of a Novel 38-Residue Toxin from the Venom of Hadronyche Infensa sp. that Contains a Cystine-Knot Motif within Its Four Disulfide Bonds

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-HI:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-HI:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing, the presence of a triple-stranded antiparallel sheet consistent with the inhibitor cystine-knot (ICK) motif.

Solution structure of CnErg1 (Ergtoxin), a HERG specific scorpion toxin

The three-dimensional structure of chemically synthesized CnErg1 (Ergtoxin), which specifically blocks HERG (human ether-a-go-go-related gene) K+ channels, was determined by nuclear magnetic resonance spectroscopy. CnErg1 consists of a triple-stranded beta-sheet and an a-helix, as is typical of K+ channel scorpion toxins. The peptide structure differs from the canonical structures in that the first beta-strand is shorter and is nearer to the second beta-strand rather than to the third beta-strand on the C-terminus. There is also a large hydrophobic patch on the surface of the toxin, surrounding a central lysine residue, Lys13. We postulate that this hydrophobic patch is likely to form part of the binding surface of the toxin. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Pesquisa de antigenos aglutinantes "major" 1, 2 e 3 em cepas de Bordetella pertussis, isoladas de crianças com coqueluche atendidas no Hospital de Isolamento Emílio Ribas de São Paulo, Brasil

Em 30 cepas de Bordetella pertussis isoladas de crianças com coqueluche, atendidas no Hospital de Isolamento Emílio Ribas de São Paulo, foram pesquisados os antígenos aglutinantes ''major" 1, 2 e 3. Levando-se em conta a presença combinada dos três antígenos, as provas de soro-aglutinação rápida em lamina revelaram que 25 (83,3%) cepas possuiam os fatores 1, 2 e 3, enquanto que 3 (10,0%) e 2 (6,7%) foram positivas, somente, para 1, 2 e 1, 3, respectivamente. Os resultados foram discutidos, considerando-se a importância deste antígeno no preparo de vacinas.

Bordetella pertussis en estudiantes adolescentes de la Ciudad de México

OBJETIVO: Estimar la seroprevalencia a Bordetella pertussis en escolares y sus contactos escolares y familiares. MÉTODOS: Un total de 12.273 estudiantes de 12 a 15 años de edad, de 14 escuelas secundarias públicas de la Ciudad de México fueron estudiados durante los meses de Septiembre 2002 a Marzo 2003. Se tomó muestra de exudado nasofaríngeo en adolescentes con tos de más de 14 días de evolución. La infección fue confirmada por la técnica de reacción en cadena de polimerasa. Se realizó estudio de contactos escolares y familiares. RESULTADOS: La incidencia de tos fue de 5 para 1.000 estudiantes. De los 61 estudiantes con tos incluidos en la muestra, 20 (32,8%) fueron positivos para Bordetella. De los 152 contactos escolares, 16 (10,6%) resultaron positivos, y ocho tenían tos. Uno de esos contactos fue el director de una de las escuelas responsable de más del 60% de los casos positivos (12/20), quien también dio lecciones a diez de los estudiantes infectados. De los 29 familiares, ocho (27,6%) fueron positivos, pertenecientes a tres familias. CONCLUSIONES: Los resultados muestran que la frecuencia de la enfermedad fue similar al comunicado en la población adolescente de otros países. Sin embargo, este trastorno no tiene necesariamente signos clínicos de la tos persistente y está sujeto a la existencia de infectados asintomáticos con Bordetella.