http://repub.eur.nl/res/aut/2478/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/3681/ 1999-06-04T00:00:00Z Intro: More than 15 years after the discovery of HIV-1 as the causative agent of AIDS, and numerous attempts to develop a vaccine, it has become clear that the efficacy of the currently considered HIV-1 vaccine candidates will generally be limited. This is at least in part due to the relative resistance of so-called primary HIV strains to neutralization by HIV-1 envelope specific antibodies: even the most potent HIV-1 neutralizing antibodies failed to provide protection in in vivo models, at concentrations that can be maintained for longer periods in human vaccinees. http://repub.eur.nl/res/pub/3664/ 1999-01-01T00:00:00Z 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. http://repub.eur.nl/res/pub/8469/ 1999-01-01T00:00:00Z 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. http://repub.eur.nl/res/pub/3659/ 1998-10-23T00:00:00Z 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. http://repub.eur.nl/res/pub/3603/ 1997-01-01T00:00:00Z 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 infe http://repub.eur.nl/res/pub/3565/ 1996-01-01T00:00:00Z Specific pathogen free (SPF) domestic cats were inoculated with tissue homogenate obtained from a Chinese leopard (Panthera pardus japonensis) that had died in a North American zoo from a natural infection with canine distemper virus (CDV). The cats developed a transient cell-associated CDV viraemia along with pronounced lymphopenia but did not show any clinical symptoms. Plasma neutralizing-antibody titres against the homologous CDV (A92-27/4, isolated from the Chinese leopard) were consistently higher than against the CDV vaccine strain 'Bussell'. The Chinese leopard CDV isolate showed in vitro biological properties reminiscent of virulent, wild-type CDV strains. Sequence analysis of the H gene of two large felid CDV isolates from the USA (A92-27/4 and A92-6) revealed up to 10% amino acid changes including up to four additional potential N-linked glycosylation sites in the extra-cytoplasmic domain as compared to CDV vaccine strains. Phylogenetic analysis was performed using the entire coding region of the H gene and a 388 bp fragment of the P gene of several morbillivirus species. Evidence was obtained that recent CDV isolates from different species in the United States (including isolates from large felids), Europe and Africa are significantly distinct from CDV vaccine strains. All wild-type CDV isolates analysed clustered according to geographical distribution rather than to host species origin. By sequence analysis a CDV epizootic among large felids in a Californian safari park was linked to a virus which most likely originated from feral non-felid carnivores. http://repub.eur.nl/res/pub/3587/ 1996-01-01T00:00:00Z http://repub.eur.nl/res/pub/22018/ 1995-10-11T00:00:00Z Feline immunodeficiency virus (FIV) is classified as a member of the genus Lentivirus (subfamily Lentivirinae) of the Retroviridae family on basis of its morphology, biochemical characteristics, genomic organization, Mg'+ dependent reverse transcriptase, and nucleotide sequence homology with other members of this genus. Lentiviruses cause chronic, lifelong infections in their respective host species, which may be followed by a slowly progressive and degenerative disease. FIV was first isolated from a domestic cat suffering from an immunodeficiency syndrome reminiscent of AIDS in humans. Subsequently it was shown that upon FIV infection, domestic cats may develop an immunodeficiency syndrome, hallmarked by secondary and opportunistic infections. The clinical stages preceding the final stage of feline AIDS, are similar to those observed in humans with HIV infection: the acute stage is followed by the asymptomatic carrier (AC) stage, after which a persistent generalized lymphadenopathy (PGL) gradually leads to the stage of AIDS related complex (ARC). Then the animal develops full blown AIDS. The different stages of F1V infection of cats may often not be quite distinct and rapid transitions between the respective stages may be observed (see below). FIV infections occur virtually worldwide in domestic cats and to date three viral clades (A, B, and C) with partially overlapping geographical distributions have been identified on basis of env sequences. http://repub.eur.nl/res/pub/3535/ 1995-01-01T00:00:00Z Cats were immunized three times with different recombinant feline immunodeficiency virus (FIV) candidate vaccines. Recombinant vaccinia virus (rVV)-expressed envelope glycoprotein with (vGR657) or without (vGR657 x 15) the cleavage site and an FIV envelope bacterial fusion protein (beta-Galactosidase-Env) were incorporated into immune-stimulating complexes or adjuvanted with Quil A. Although all immunized cats developed antibodies against the envelope protein, only the cats vaccinated with the rVV-expressed envelope glycoproteins developed antibodies which neutralized FIV infection of Crandell feline kidney cells. These antibodies failed to neutralize infection of thymocytes with a molecularly cloned homologous FIV. After the third immunization the cats were challenged with homologous FIV. Two weeks after challenge the cell-associated viral load proved to be significantly higher in the cats immunized with vGR657 and vGR657 x 15 than in the other cats. The cats immunized with vGR657 and vGR657 x 15 also developed antibodies against the Gag proteins more rapidly than the cats immunized with beta-Galactosidase-Env or the control cats. This suggested that immunization with rVV-expressed glycoprotein of FIV results in enhanced infectivity of FIV. It was shown that the observed enhancement could be transferred to naive cats with plasma collected at the day of challenge. http://repub.eur.nl/res/pub/3536/ 1995-01-01T00:00:00Z 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. http://repub.eur.nl/res/pub/3537/ 1995-01-01T00:00:00Z Viral progeny of two molecular clones of feline immunodeficiency virus (FIV), 19k1 and 19k32, were tested in a virus neutralization assay. In this assay the infection of thymocytes with FIV19k1 was neutralized by serum S1422, derived from an SPF cat 22 weeks after infection with FIV19k1. We previously reported that a point mutation at position 560 in hypervariable region-5 (HV-5) of 19k1 confers resistance to virus neutralization (Siebelink et al., 1993, J. Virol. 67:2202-2208). Viral progeny of the other molecular clone, FIV19k32, which differs in the envelope protein in only six amino acids from 19k1, was not neutralized. In order to map sites involved in virus neutralization we constructed chimeric clones by reciprocal exchange of 19k1 and 19k32 envelope gene fragments. Reciprocal exchange of a 1662 bp fragment, encoding almost the whole surface protein, which differs in five amino acids between these two clones, resulted in exchange of the phenotype. Amino acids of the envelope protein of 19k1 and 19k32, in which these clones differ, were substituted by point mutation. We demonstrated that one of these mutations, a substitution of leucine to serine at position 483 in HV-4, also conferred resistance of 19k1 to neutralization by serum S1422. http://repub.eur.nl/res/pub/3545/ 1995-01-01T00:00:00Z 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. http://repub.eur.nl/res/pub/3552/ 1995-01-01T00:00:00Z To gain further insight into the ability of subunit vaccines to protect monkeys from experimental infection with simian immunodeficiency virus (SIV), two groups of cynomolgus macaques were immunized with either recombinant SIVmac32H-derived envelope glycoproteins (Env) incorporated into immune-stimulating complexes (iscoms) (group A) or with these SIV Env iscoms in combination with p27gag iscoms and three Nef lipopeptides (group B). Four monkeys immunized with recombinant feline immunodeficiency virus Env iscoms served as controls (group C). Animals were immunized intramuscularly at weeks 0, 4, 10, and 16. Two weeks after the last immunization, monkeys were challenged intravenously with 50 monkey 50% infectious doses of virus derived from the J5 molecular clone of SIVmac32H propagated in monkey peripheral blood mononuclear cells. High titers of SIV-neutralizing antibodies were induced in the monkeys of groups A and B. In addition, p27gag-specific antibodies were detected in the monkeys of group B. Vaccine-induced cytotoxic-T-lymphocyte precursors against Env, Gag, and Nef were detected on the day of challenge in the monkeys of group B. Env-specific cytotoxic-T-lymphocyte precursors were detected in one monkey from group A. In spite of the observed antibody and T-cell responses, none of the monkeys was protected from experimental infection. In addition, longitudinal determination of cell-associated virus loads at weeks 2, 4, 6, 9, and 12 postchallenge revealed no significant differences between vaccinated and control monkeys. These findings illustrate the need to clarify the roles of the different arms of the immune system in conferring protection against primate lentivirus infections. http://repub.eur.nl/res/pub/3486/ 1994-04-05T00:00:00Z In order to monitor the antibody response to feline immunodeficiency virus (FIV) in cats, following experimental and natural infection, enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant env and gag proteins and p24-specific monoclonal antibodies. It was shown that in experimentally infected cats an env protein-specific antibody response was directly followed by a gag protein-specific response. Furthermore, an ELISA for the detection of env protein-specific serum antibodies proved more sensitive in identifying experimentally and naturally infected cats than ELISAs demonstrating gag protein-specific antibodies. It was concluded that, like in HIV infection of humans, the detection of env protein-specific serum antibodies in addition to gag protein-specific antibodies is not only an important tool in the diagnosis of the infection but also in studies concerning the pathogenesis of the disease. http://repub.eur.nl/res/pub/3460/ 1993-01-01T00:00:00Z We infected a specific-pathogen-free cat (cat 14) with molecularly cloned feline immunodeficiency virus clone 19k1 (FIV19k1 [K. H. J. Siebelink, I. Chu, G. F. Rimmelzwaan, K. Weijer, A. D. M. E. Osterhaus, and M. L. Bosch, J. Virol. 66:1091-1097, 1992]). Serum of this cat obtained 22 weeks postinfection (serum 1422) neutralized FIV19k1 but not FIV19k32, which is 99.3% identical to FIV19k1 in the envelope gene. Serum 1422 also neutralized virus isolated from cat 14 at weeks 2 and 32 postinfection. We then cultured FIV19k1 in the continuous presence of serum 1422, which resulted in a delay in virus replication of 6 weeks. The resulting virus population appeared to be resistant to virus neutralization by serum 1422. Nucleotide sequencing of the env open reading frame of this presumed escape mutant revealed the presence of one silent and two substitution mutations, both of the latter in hypervariable region 5. Through the construction of chimeric viruses and site-directed mutagenesis, we demonstrated that one of these mutations, the substitution of lysine to glutamine at amino acid position 560 in hypervariable region 5, was sufficient to allow the escape of FIV19k1 from neutralization by serum 1422. http://repub.eur.nl/res/pub/3471/ 1993-01-01T00:00:00Z Sera from cats experimentally infected with five isolates of feline immunodeficiency virus (FIV) from various geographical regions and from FIV enzyme-linked immunosorbent assay-seropositive field cats from four European countries neutralized the Petaluma strain of FIV (FIV-P), originally isolated in California, at high titers. In addition, FIV-P and a European isolate proved equally susceptible to neutralization by all sera tested. Coupled with observations by Fevereiro et al. (M. Fevereiro, C. Roneker, A. Laufs, L. Tavares, and F. de Noronha, J. Gen. Virol. 72:617-622, 1991), these findings indicate that most if not all FIV strains circulating in Europe and the United States share important neutralization-inducing epitopes. http://repub.eur.nl/res/pub/3434/ 1992-01-01T00:00:00Z Replication-competent molecular clones of feline immunodeficiency virus (FIV) were isolated directly from the DNA of bone marrow cells of a naturally FIV-infected cat. After transfection in a feline kidney cell line (CrFK) and subsequent cocultivation with peripheral blood mononuclear cells (PBMC), the viral progeny of the clones was infectious for PBMC but not for CrFK cells. PBMC infected with these clones showed syncytium formation, a decrease in cell viability, and gradual loss of CD4+ cells. The restriction maps of these clones differed from those obtained for previously described molecular clones of FIV derived from cats in the United States. The predicted amino acid sequence similarity of the envelope genes of the two clones was 99.3%, whereas the similarities of the sequences of the clones to those of two molecular clones from the United States, Petaluma and PPR, were 86 and 88%, respectively. Most of the differences between the amino acid sequences of the two clones and those of the clones from the United States were found in five different hypervariable (HV) regions, HV-1 through HV-5. The viral progeny of one of these clones was inoculated into two specific-pathogen-free cats. The animals seroconverted, and the virus could be reisolated from their PBMC. http://repub.eur.nl/res/pub/3411/ 1990-01-01T00:00:00Z To assess the value of feline immunodeficiency virus (FIV) infection as a model for human immunodeficiency virus (HIV) infection in man, we studied the impairment of certain immunological functions following natural or experimental FIV infection. Proliferative responses of peripheral blood mononuclear cells (PBMC) from symptomatic and asymptomatic cats after naturally or experimentally acquired FIV infection, induced by activation with the mitogens concanavalin A, pokeweed mitogen, or lipopolysaccharide or by stimulation with human interleukin-2 (IL-2), were significantly lower than the proliferative responses found with PBMC from noninfected control cats. Also IL-2 production levels of mitogen-activated PBMC from naturally infected symptomatic cats were significantly reduced. These data confirm that the pathogenesis of FIV infection in the cat, like HIV infection in man, is characterized by a serious malfunction of the immune system. http://repub.eur.nl/res/pub/3325/ 1987-01-01T00:00:00Z Five asymptomatic human immunodeficiency virus (HIV)-seropositive ; male homosexuals were immunized with the recall antigens tetanus toxoid (TT) and the three types of poliovirus present in diphtheria, tetanus, and polio vaccine. Four weeks after immunization, the in vivo response to booster immunization, the in vitro pokeweed mitogen (PWM)-induced IgG secretion, and the in vitro T cell-dependent and T cell-independent antigen-induced antibody response were assayed. Increase in serum antibody titer to TT and poliovirus was low and normal, respectively. In all five subjects studied, a high rate of spontaneous IgG production, including antibodies directed toward HIV was observed. Addition of PWM to the cultures induced suppression of the spontaneous IgG secretion. Only one donor showed a slightly increased IgG production after stimulation with PWM. Peripheral blood mononuclear cells of four of the five HIV-seropositive individuals did not produce TT, or poliovirus-specific antibodies when stimulated with the respective T cell-dependent antigens. However, stimulation of these peripheral blood mononuclear cells with TT coupled to agarose beads, which was shown to be T cell-independent, resulted in the generation of IgG anti-TT antibody-forming cells. http://repub.eur.nl/res/pub/3289/ 1984-01-01T00:00:00Z