A determinant of feline immunodeficiency virus involved in Crandell feline kidney cell tropism.

Viral progeny of the molecular clone 19k1 of feline immunodeficiency virus (FIV) can infect feline T-cells but not Crandell feline kidney (CrFK) cells. In contrast, the biological isolate FIV-AM6c, which was CrFK adapted by co-cultivation of FIV-AM6 infected thymocytes with CrFK cells, can infect both thymocytes and CrFK cells. The envelope gene of FIV-AM6c was amplified by polymerase chain reaction using DNA from infected CrFK cells, and subsequently cloned and sequenced. To map viral determinants of CrFK cell tropism, chimeric viruses with a 19k1 background containing envelope gene fragments of FIV-AM6c were constructed. CrFK cells were transfected with DNA of these chimeric clones and co-cultivated with thymocytes. After 3 days the CrFK cells and the thymocytes were cultured separately. FIV antigen could be detected in most of the thymocyte cultures within 14 days and in one of the CrFK cultures after 52 days. The resulting virus from this CrFK culture can infect both CrFK cells and thymocytes. The results of this study indicate that the envelope region contains determinants of CrFK tropism. The delay in replication indicates that also determinants other than those identified here are involved in CrFK cell tropism. More chimeric clones are being studied at present to map these determinants.

Accelerated viraemia in cats vaccinated with fixed autologous FIV-infected cells.

We have vaccinated cats with fixed autologous FIV infected PBMC to determine whether autologous presentation of antigen is capable of inducing a protective immune response against homologous challenge. To this end autologous PBMC were infected with a FIV molecular clone (19k1). When infection was established, cells were inactivated by dialysis against paraformaldehyde. Upon vaccination, cats developed a virus specific immune response as measured by ELISA against the Gag protein of FIV. No antibodies against the envelope protein were detected with a peptide ELISA. Virus neutralizing antibodies however could be detected with a neutralization assay based on infection of CrFK cells, but not in an assay based on infection of primary T-cells. Although vaccination led to the induction of these virus-specific immune responses, vaccinated cats were not protected against homologous challenge but showed an accelerated viraemia upon infection. This was shown both by PCR and cell-associated viral load. The possible mechanisms underlying this observation are discussed in this paper.

IP-10 in chronic hepatitis C patients treated with high-dose interferon.

INTRODUCTION: Interferon-g-inducible protein-10 (IP-10) serum levels are associated with IL28B genotype and may predict response to interferon÷ribavirin-based therapy in chronic hepatitis C patients. Our aim was to relate IP-10 levels before and during treatment to treatment outcome, viral HCV-RNA kinetics and IL28B genotype. PATIENTS AND METHODS: A cohort of chronic hepatitis C patients was treated with high-dose interferon for six weeks, followed by standard peginterferon÷ ribavirin for 24 or 48 weeks. IP-10 and HCV-RNA levels were frequently determined before, during and after treatment. RESULTS: IP-10 levels increased from log2.56 pg÷ml at baseline to log3.48 pg÷ml at Day 1 and gradually diminished thereafter. IP-10 levels at any time point were not statistically different between patients with or without sustained viral response (SVR). Patients with IL28B CC genotype had significantly lower baseline IP-10 levels (p = 0.019) and a higher increase of IP-10 levels from baseline to Day 1 than patients with IL28B non-CC genotypes (p = 0.015). Patients with HCV-RNA decline ≥ 2.28log10 at Day 1 had significantly lower baseline IP-10 levels (p = 0.016) and a higher increase of IP-10 levels from baseline to Day1 (p = 0.047) than patients with HCV-RNA decline of < 2.28log10 at Day 1. CONCLUSIONS: In patients treated with high induction dose interferon, IP-10 levels at any time point were not predictive for SVR. Low baseline IP-10 levels and a higher increase of IP-10 levels from baseline to Day 1 were associated with IL28B CC genotype and HCV-RNA decline ≥ 2.28log10 at Day 1. This suggests that, in our cohort, for prediction of SVR the added value of IP-10 to IL28B genotype and early viral kinetics is limited.

Neutralization of feline immunodeficiency virus by polyclonal feline antibody: simultaneous involvement of hypervariable regions 4 and 5 of the surface glycoprotein.

Sites involved in antibody-mediated neutralization of feline immunodeficiency virus were mapped by reciprocal exchange of envelope fragments or amino acids between molecular clones of feline immunodeficiency virus with different susceptibilities to neutralization by a polyclonal cat serum. Combinations of mutations within HV-4 or within HV-4 and HV-5 changed the susceptibility of the viruses to neutralizing antibody.

Induction of feline immunodeficiency virus specific antibodies in cats with an attenuated Salmonella strain expressing the Gag protein.

Salmonella typhimurium aroA strains (SL3261), expressing high levels of the Gag protein of feline immunodeficiency virus (FIV) fused with maltose binding protein (SL3261-MFG), were constructed using an invertible promoter system that allows the stable expression of heterologous antigens at levels toxic for bacteria. A SL3261 strain expressing the B subunit of cholera toxin by a similar system (SL3261-CtxB) served as a control in FIV-immunization experiments. Cats immunized once orally or intraperitoneally with SL3261-MFG or SL3261-CtxB all developed serum antibodies to SL3261 lipopolysaccharide and against maltose binding protein or the B subunit of cholera toxin, respectively. Two intraperitoneal immunizations with SL3261-MFG also resulted in the development of Gag specific serum antibodies. Two oral immunizations with SL3261-MFG primed for a Gag specific response, which was demonstrated upon FIV challenge. All challenged cats became infected and no significant differences in viral loads were found between SL3261-MFG and SL3261-CtxB immunized cats.

Construction and evaluation of an expression vector allowing the stable expression of foreign antigens in a Salmonella typhimurium vaccine strain.

Salmonella strains have great potential as live carriers of heterologous antigens to induce immunity against a variety of infectious diseases. However, the amount of heterologous antigen required to induce an adequate immune response may be toxic for the bacterium and result in cell death, overattenuation or loss of expression of the heterologous antigen. To solve this problem an expression vector was developed with a strong promoter located on a DNA fragment which is inverted at random. Antigen is only expressed in one particular orientation of the promoter. Thus a bacterial population harbouring the plasmid will consist of a subpopulation which does not produce heterologous antigen, and is therefore not affected in growth, persistence and dissemination within the host. Further, this non-producing population will continuously segregate antigen-producing bacteria. To evaluate the system, CtxB was used as a model antigen. Analysis of the plasmid DNA isolated from Salmonella revealed a selection against the promoter orientation that directs transcription of the ctxB gene. In spite of this, the vector was stably maintained in vivo and induced CtxB-specific IgA and IgG in mice. These results indicate that this kind of expression vector may offer a solution to the problem of unstable expression of foreign antigens in live bacterial vaccine strains.

Feline immunodeficiency virus subunit vaccines that induce virus neutralising antibodies but no protection against challenge infection.

Three experimental vaccines against feline immunodeficiency virus (FIV), all based on viral antigens presented via immune stimulating complexes (iscoms), were tested for their capacity to induce protection in cats from FIV infection. The respective vaccines consisted of FIV propagated in Crandell feline kidney (CrFK) cells (FIV-iscoms); FIV-iscoms spiked with recombinant vaccinia virus expressed FIV envelope glycoprotein incorporated into iscoms (FIV-iscoms + vGR657x15-iscoms) and vGR657x15-iscoms spiked with recombinant FIV Gag protein incorporated into iscoms (vGR657x15-iscoms + FIV-Gag-iscoms). Simian immunodeficiency virus envelope glycoprotein incorporated into iscoms, iscoms prepared with uninfected CrFK cells, and PBS served as controls. All cats vaccinated with vGR657x15-iscoms combined with FIV-iscoms or FIV-Gag-iscoms developed Env-specific plasma antibody responses. These antibodies neutralised FIV infection in CrFK cells, but failed to neutralise FIV infection in primary feline thymocytes. FIV-iscoms induced poor Env-specific responses and only one out of six cats developed antibodies that neutralised FIV in the CrFK cell based assay. Four weeks after challenge all cats proved to be infected, showing that none of the vaccine preparations provided protection. In contrast, 2 weeks after infection, virus infected peripheral blood mononuclear cells were only observed in cats vaccinated with FIV-iscoms + vGR657x15-iscoms or CrFK-iscoms and to a lesser extent in cats vaccinated with FIV-iscoms and vGR657x15-iscoms + FIV-Gag-iscoms, but not in cats vaccinated with SIV-iscoms or PBS. The differences found in cell associated virus loads amongst the respective groups are discussed in the light of antibody mediated enhancement of infectivity and protective effects provided by Gag-specific T cell responses.

Vaccination with experimental feline immunodeficiency virus vaccines, based on autologous infected cells, elicits enhancement of homologous challenge infection.

Cats were vaccinated with fixed autologous feline immunodeficiency virus (FIV)-infected cells in order to present viral proteins to the immune system of individual cats in an MHC-matched fashion. Upon vaccination, a humoral response against Gag was induced. Furthermore, virus-neutralizing antibodies were detected in a Crandell feline kidney cell-based neutralization assay, but not in a neutralization assay based on primary peripheral blood mononuclear cells. Despite the induction of these FIV-specific responses, vaccinated cats were not protected. Instead, accelerated virus replication was found, an observation similar to what previous experiments using other vaccine candidates have shown. Here, the results of the present study are discussed in the light of enhancement of lentivirus infections as a complicating factor in lentivirus vaccine development.

Salmonella typhimurium aroA recombinants and immune-stimulating complexes as vaccine candidates for feline immunodeficiency virus.

Two experimental feline immunodeficiency virus (FIV) vaccines were tested, either alone or in combination, in four groups of cats (A-D). One vaccine (SL3261-FIV) was composed of live attenuated Salmonella typhimurium aroA (SL3261) strains expressing the capsid (Gag) and part of the envelope (Env) proteins of FIV. The other was composed of FIV Gag and Env proteins incorporated into immune-stimulating complexes (iscom-FIV). Cats of group A were immunized four times with SL3261-FIV. Cats of group B were immunized twice with SL3261-FIV and then twice with iscom-FIV. Cats of group C were immunized twice with SL3261 expressing the B subunit of cholera toxin (SL3261-CtxB) and then twice with iscom-FIV. Cats of group D, which served as negative controls, were immunized twice with SL3261-CtxB and then twice with iscom into which the Gag and Env proteins of simian immunodeficiency virus (SIV) had been incorporated (iscom-SIV). Two weeks after the last immunization, all cats were challenged with FIV. At this time, cats immunized with iscom-FIV (groups B and C) showed strong plasma antibody responses to Gag and Env, whilst these responses were weak or undetectable in the cats immunized four times with SL3261-FIV (group A). Seven weeks after FIV challenge, Env-specific antibody responses had increased considerably in cats of all groups except group A. The mean virus loads in the cats of this group proved to be lower than those of the other groups at all time points, indicating partial protection.