Improving the safety of viral DNA vaccines: development of vectors containing both 5' and 3' homologous regulatory sequences from non-viral origin.

Although some DNA vaccines have proved to be very efficient in field trials, their authorisation still remains limited to a few countries. This is in part due to safety issues because most of them contain viral regulatory sequences to driving the expression of the encoded antigen. This is the case of the only DNA vaccine against a fish rhabdovirus (a negative ssRNA virus), authorised in Canada, despite the important economic losses that these viruses cause to aquaculture all over the world. In an attempt to solve this problem and using as a model a non-authorised, but efficient DNA vaccine against the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV), we developed a plasmid construction containing regulatory sequences exclusively from fish origin. The result was an "all-fish vector", named pJAC-G, containing 5' and 3' regulatory sequences of ß-acting genes from carp and zebrafish, respectively. In vitro and in vivo, pJAC-G drove a successful expression of the VHSV glycoprotein G (G), the only antigen of the virus conferring in vivo protection. Furthermore, and by means of in vitro fusion assays, it was confirmed that G protein expressed from pJAC-G was fully functional. Altogether, these results suggest that DNA vaccines containing host-homologous gene regulatory sequences might be useful for developing safer DNA vaccines, while they also might be useful for basic studies.

Replacement of the human cytomegalovirus promoter with fish enhancer and core elements to control the expression of the G gene of viral haemorrhagic septicemia virus (VHSV).

This work explores some of the possibilities to replace human cytomegalovirus (CMV) core and/or enhancer promoter control elements to create new expression vectors for use with fish. The work is relevant to fish vaccination, since DNA vaccines use eukaryotic expression plasmids controlled by the human cytomegalovirus (CMV) promoter to be effective against novirhabdoviruses, such as viral haemorrhagic septicemia virus (VHSV), one of the most devastating fish viral European diseases. To reduce possible homologous recombination with fish genome, core and enhancer sequences from fish origin, such as trout interferon-inducible myxovirus protein (Mx), zebrafish retrovirus long terminal repeat (LTR) and carp ß-actin (AE6), were combined with those of CMV to design alternative hybrid promoters. The substitution of CMV core and/or enhancer with the corresponding elements of Mx or the LTR core maintained a similar in vitro protein G expression level than that obtained by using the CMV promoter. Vectors using the dsRNA-inducible Mx enhancer followed either by the LTR or the AE6 cores showed the highest in vitro protein G expression levels. Furthermore, synthetic constructs using the Mx enhancer maintained their polyI:C induction capabilities despite the core used. Some of these hybrid promoters might contribute to the development of all-fish-vectors for DNA vaccines while others might be useful for more basic studies.

An ELISA for detection of trout antibodies to viral haemorrhagic septicemia virus using recombinant fragments of their viral G protein.

An enzyme linked immunosorbent assay (ELISA) method to study serum antibodies to viral haemorrhagic septicemia virus (VHSV) was designed by using recombinant fragments of their G protein. By using this fragment-ELISA, we describe the binding of antibodies against recombinant G fragments of 45-445 amino acids present in VHSV-hyperimmunized trout sera. Fragments were designed by taking into account their tridimensional pH-dependent structure and functional domains. Sera were obtained from hyperimmunized trout following 4-5 intraperitoneal injections of VHSV antigens by using Freund's or saponin adjuvants. Sera from different hyperimmunized trout differed quantitatively rather than qualitatively in their recognition of solid-phase frg11 (56-110), frg12 (65-109), frg13 (97-167), frg14 (141-214), frg15 (65-250), frg16 (252-450) and G (G21-465) by Western blot and ELISA. However, titres were higher when using frg11, frg15 or frg16, rather than G21-465, suggesting higher accessibility to G epitopes. Further knowledge of the antigenicity of the G protein of rhabdoviruses by using fragments might be used to improve current vaccines. On the other hand, they might be used to dissect the trout antibody response to VHSV infections, to complement in vitro neutralizing assays, and/or to quantitate anti-VHSV antibodies in VHSV-infected/vaccinated trout, other fish and/or other body fluids such as mucus.

Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments.

Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.

New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus.

Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene of the viral hemorrhagic septicemia virus (VHSV), a rhabdovirus affecting fish. Eight siRNA sequences were first designed, synthesized and screened for inhibition of in vitro VHSV infectivity. Small hairpin (sh) DNAs corresponding to three selected siRNAs were then cloned into pRNA-CMV3.1/puro plasmids, transfected into EPC cells and transformed clones were obtained by puromycin selection. Sequence-specific interference with VHSV could only be observed with EPC clones transformed with a mixture of the three shDNAs, rather than with those clones obtained with individual sh DNAs. However, interference was not specific for VHSV as infection with an heterologous fish rhabdovirus, was also reduced to a similar extent. It was shown that this reduction was not due to an Mx response in the transformed cell clones. Here, we discuss some of the possible reasons for such data and future work directions. EPC clones stably expressing rhabdoviral specific siRNA sequences could be a strategy to further investigate the use of RNA interference for targeting costly fish pathogenic viruses.

Transfection improvements of fish cell lines by using deacylated polyethylenimine of selected molecular weights.

A new tool for DNA transfer to fish cell lines such as epithelioma papulosum cyprini (EPC) and rainbow trout gonad (RTG2), has been optimized by testing commercially available polyethylenimine (PEI) polymers as transfectant reagents. Deacylated 25 kDa PEI polymers were selected amongst the most active and then low toxicity deacylated PEIs fractions around 15 kDa were obtained by gel filtration chromatography to increase 3-4-fold their initial in vitro transfection efficiency. The EPC and plasmids coding for reporter genes were first used to optimize variable values for best expression by transfection with deacylated low toxicity PEI while both EPC/RTG2 and a plasmid coding for the glycoprotein G gene of the fish pathogen, viral haemorrhagic septicemia virus (VHSV) were then used to demonstrate some of their practical applications. Due to its relatively low price, defined chemical composition and availability, low toxicity deacylated PEI might be used for numerous applications for all those studying fish cell immunology in vitro as well as in vivo.

Acute cellulitis: an unusual manifestation of meningococcal disease.

We describe 2 patients who both developed cellulitis due to Neisseria meningitidis and review 8 other cases reported since 1966. Female patients outnumbered male patients by 8 to 2, and there were 5 children and 5 adults. Four cases were caused by the serogroup C meningococcus, 2 cases by serogroup B and 2 others by serogroup Y (the nature of the meningococcal group was not available in 2 cases). Diverse medical underlying conditions were present in 4 of the adult patients. The periorbital region (in all 5 children), limb (in 3 adults), neck (in 1 adult) and face and neck (in 1 adult) were the locations of the meningococcal cellulitis. In all 10 patients, a favorable clinical response to the antibiotic therapy was documented and no relapses occurred. These cases indicate that N. meningitidis should be considered as a causative agent of cellulitis in the appropriate clinical setting, particularly in children with signs of periorbital infection or adults with underlying diseases.

Mucormycosis of the sphenoid sinus in an otherwise healthy patient. Case report and literature review.

Paranasal and rhinocerebral mucormycosis refers to uncommon opportunistic fungal infections, reported to occur especially in association with diabetic acidosis (the most common), immunosuppressive therapy, malignancy, or other chronic debilitating disorders. However, patients who have no underlying disease have occasionally been affected. According to the literature reviewed, only 13 well-documented cases without any predisposing factor have been previously reported. We describe a unique case of sphenoidal mucormycosis in an otherwise healthy individual, and the first patient to present with headache as the only symptom. We emphasize the importance of a high index of suspicion for early diagnosis and prompt management.

Concentración plasmática de hierro y ferritina en niños parasitados por Giardia lamblia / TIBC and transferrin saturation index in children, they have parasitic infection by Giardia lamblia

Se informa de los hallazgos clínicos y hematológicos (hemoglobina, hierro, ferritina, capacidad total de saturación de transferrina, CST, índice de saturación de transferrina) registrados en 100 niños parasitados por Giardia lamblia. Las manifestaciones relativas a la parasitosis no fueron diferentes a lo ya descrito, pero se observó que 20 por ciento de los niños exhibían signos clínicos relacionados con anemia. Los niños se separaron en dos grupos: grupo A, con 20 niños (hemoglobina menor de 11 g/dL) y grupo B, con 80 pacientes (hemoglobina mayor de 11g/dL). El coeficiente de correlación entre el hierro y la ferritina fue negativo para ambos grupos (r=-0.128) y (r=-0.000103). Lo cual sugiere que la concentración de ferritina no es buen indicador de depósitos de hierro en pacientes con giardiasis. Las medianas de los grupos, A y B, se compararon con la prueba de Mann-Withney no encontrando diferencias significativas con respecto a hierro y ferritina. Seis niños, del grupo B se encontraban en el estadio II de la hipoferremia, en ello no hubo cambios significativos en las mediciones tomadas tres meses después. En nueve niños (9 por ciento) se encontró ferritina mayor de 50 µg/dL que puediera sugerir comportamiento al igual que la proteína C reactiva(au)