Changes in the SU can modulate the susceptibility of feline immunodeficiency virus to TM-derived entry inhibitors.

Feline immunodeficiency virus (FIV) provides a valuable animal model by which criteria for the development of HIV-1 inhibitors can be explored. Previous studies had shown that a synthetic 8-mer peptide modeled on the tryptophan-rich motif of the ectodomain of the viral transmembrane glycoprotein (TM) is a potent inhibitor of FIV The observation that inhibition efficiency varied somewhat depending on FV strain prompted the present study in which we investigated whether changes in the surface (SU) glycoprotein can affect virus susceptibility to TM-derived peptide inhibitors. This was done by examining how effectively selected entry inhibitors blocked the infectivity of well characterized variants and molecular clones of the prototype isolate of FIV The results have shown that substitutions in the SU can indeed modulate virus susceptibility to TM-derived entry inhibitors. Interestingly, we also observed a parallelism between reduced susceptibility to entry inhibitors and broad resistance to antibody-mediated neutralization.

Feline immunodeficiency virus-infected cat sera associated with the development of broad neutralization resistance in vivo drive similar reversions in vitro.

We previously reported that, upon reinoculation into cats, a neutralization-sensitive, tissue culture-adapted strain of feline immunodeficiency virus constantly reverted to the broad neutralization resistance typical of primary virus isolates and identified residue 481 in the V4 region of the surface glycoprotein as a key determinant of the reversion. Here, we found that well-characterized immune sera, obtained from cats in which such reversion had occurred, selected in tissue culture in favor of virus variants that also had a neutralization-resistant phenotype and had amino acid 481 changed, thus indicating that the host's humoral immune response is capable of driving the reversion in the absence of other intervening factors. In contrast, a second group of immune sera, elicited by a virus variant that had already reverted to neutralization resistance in independent cats, induced the emergence of escape mutants lacking broad neutralization resistance and neutralized fewer virus variants. It is proposed that the viral variants used to produce the two sets of sera may have generated different antibody repertoires.

During readaptation in vivo, a tissue culture-adapted strain of feline immunodeficiency virus reverts to broad neutralization resistance at different times in individual hosts but through changes at the same position of the surface glycoprotein.

The broad resistance to antibody-mediated neutralization of lentiviruses recently isolated from infected hosts is a poorly understood feature which might contribute to the ability of these viruses to persist and to the failure of experimental vaccines to protect against virulent viruses. We studied the underlying molecular mechanisms by examining the evolution of a neutralization-sensitive, tissue culture-adapted strain of feline immunodeficiency virus upon reinoculation into specific-pathogen-free cats. Reversion to broad neutralization resistance was observed in seven of seven inoculated animals and, in individual hosts, started to develop between less than 4 and more than 15 months from infection. After comparison of the envelope sequences of the inoculum virus, of an additional 4 neutralization-sensitive in vitro variants, and of 14 ex vivo-derived variants (6 neutralization sensitive, 5 resistant, and 3 with intermediate phenotype), a Lys-->Asn or -->Glu change at position 481 in the V4 region of the surface glycoprotein appeared as a key player in the reversion. This conclusion was confirmed by mutagenesis of molecularly cloned virus. Analysis of viral quasispecies and biological clones showed that the intermediate phenotype was due to transient coexistence of neutralization-sensitive and -resistant variants. Since the amino acid position involved was the same in four of four recent revertants, it is suggested that the number of residues that control reversion to broad neutralization resistance in FIV might be very limited. Amino acid 481 was found to be changed only in one of three putative long-term revertants. These variants shared a Ser-->Asn change at position 557 in region V5, which probably collaborated with other mutations in long-term maintenance of neutralization resistance, as suggested by the study of mutagenized virus.

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: reevaluation of neutralizing antibody levels elicited by a protective and a nonprotective vaccine after removal of antisubstrate cell antibodies.

In the feline immunodeficiency virus system, immunization with a fixed-infected-cell vaccine conferred protection against virulent homologous challenge but the immune effectors involved remained elusive. In particular, few or no neutralizing antibodies were detected in sera from vaccinated cats. Here we show that, when preadsorbed with selected feline cells, the same sera revealed clearly evident virus-neutralizing activity. Because high titers of neutralizing antibody in cell-adsorbed sera from 23 cats immunized with fixed-infected-cell or whole-inactivated-virus vaccines correlated with protection, it is likely that they were more important for protection than formerly realized. In vitro, the fixed-cell vaccine efficiently removed neutralizing antibody from immune sera while the whole-inactivated-virus vaccine was much less effective.

Immunogenicity of an anti-clade B feline immunodeficiency fixed-cell virus vaccine in field cats.

Attempts at vaccine development for feline immunodeficiency virus (FIV) have been extensive, both because this is a significant health problem for cats and because FIV may be a useful vaccine model for human immunodeficiency virus. To date, only modest success, producing only short-term protection, has been achieved for vaccine trials in controlled laboratory settings. It is unclear how relevant such experiments are to prevention of natural infection. The current study used a vaccine that employs cell-associated FIV-M2 strain fixed with paraformaldehyde. Subject cats were in a private shelter where FIV was endemic, a prevalence of 29 to 58% over an 8-year observation period. Cats roamed freely from the shelter through the surrounding countryside but returned for food and shelter. After ensuring that cats were FIV negative, they were immunized using six doses of vaccine over a 16-month period and observed for 28 months after the initiation of immunization. Twenty-six cats (12 immunized and 14 nonimmunized controls) were monitored for a minimum of 22 months. Immunized cats did not experience significant adverse effects from immunization and developed both antibodies and cellular immunity to FIV, although individual responses varied greatly. At the conclusion of the study, 0 of 12 immunized cats had evidence of FIV infection, while 5 of 14 control cats were infected. Thus, the vaccine was safe and immunogenic and did not transmit infection. Furthermore, vaccinated cats did not develop FIV infection in a limited clinical trial over an extended time period. Thus, the data suggest that a fixed, FIV-infected cell vaccine has potential for preventing natural FIV infection in free-roaming cats.

AIDS vaccination studies using feline immunodeficiency virus as a model: immunisation with inactivated whole virus suppresses viraemia levels following intravaginal challenge with infected cells but not following intravenous challenge with cell-free virus.

The feline immunodeficiency virus (FIV) provides an excellent model system for AIDS vaccination studies. In the present experiments we investigated the immunogenicity and the protective activity of two inactivated vaccines prepared from a primary virus isolate. One vaccine was composed of whole virus inactivated with paraformaldehyde and then purified (WIV) and the other of viral proteins extracted with Tween-ether (TEV). Both vaccines elicited robust antiviral responses, but neither conferred appreciable levels of resistance against systemic challenge with the homologous virus. In addition, we tested whether the WIV vaccine, that had appeared more immunogenic, could protect against nontraumatic intravaginal exposure to FIV-infected cells. Although the proportions of control and vaccinated animals that became infected following mucosal challenge were similar, the vaccinees had significantly lower viral burdens than the controls, thus suggesting that immunisation with the WIV vaccine had limited FIV replication following intravaginal challenge.

Kinetics of replication of a partially attenuated virus and of the challenge virus during a three-year intersubtype feline immunodeficiency virus superinfection experiment in cats.

The effects of preinfecting cats with a partially attenuated feline immunodeficiency virus (FIV) on subsequent infection with a fully virulent FIV belonging to a different subtype were investigated. Eight specific-pathogen-free cats were preinfected with graded doses of a long-term in vitro-cultured cell-free preparation of FIV Petaluma (FIV-P, subtype A). FIV-P established a low-grade or a silent infection in the inoculated animals. Seven months later, the eight preinfected cats and two uninfected cats were challenged with in vivo-grown FIV-M2 (subtype B) and periodically monitored for immunological and virological status. FIV-P-preinfected cats were not protected from acute infection by FIV-M2, and the sustained replication of this virus was accompanied by a reduction of FIV-P viral loads in the peripheral blood mononuclear cells and plasma. However, from 2 years postchallenge (p.c.) until 3 years p.c., when the experiment was terminated, preinfected cats exhibited reduced total viral burdens, and some also exhibited a diminished decline of circulating CD4(+) T lymphocytes relative to control cats infected with FIV-M2 alone. Interestingly, most of the virus detected in challenged cats at late times p.c. was of FIV-P origin, indicating that the preinfecting, attenuated virus had become largely predominant. By the end of follow-up, two challenged cats had no FIV-M2 detectable in the tissues examined. The possible mechanisms underlying the interplay between the two viral populations are discussed.

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: detailed analysis of the humoral immune response to a protective vaccine.

The feline immunodeficiency virus (FIV) cat model is extensively used to investigate possible vaccination approaches against AIDS in humans. Although consistent levels of protection have been achieved with FIV, as with other model systems, by immunizing with whole inactivated virus or fixed infected cells, the mechanisms responsible for protection are elusive. In previous studies we showed that cats immunized with a vaccine consisting of fixed infected cells were protected or unprotected against cell-free or cell-associated FIV challenge depending on the time interval between completion of vaccination and challenge. In an attempt to define possible humoral immune correlates of protection, selected sera harvested at the times of challenge from such cats were examined for anti-FIV-antibody titers and properties by using binding and functional immunological assays. Binding assays included quantitative Western blotting, enzyme-linked tests for antibodies to FIV glycoproteins and immunodominant linear epitopes, and tests for measuring conformation dependence and avidity of anti-viral-envelope antibodies. Functional assays included virus neutralization performed with two different cell substrates, complement- and antibody-dependent virolysis, blocking of reverse transcriptase, and an assay that measured the ability of sera to prevent FIV growth in cocultures of infected and uninfected cells. Despite the wide spectrum of parameters investigated, no correlation between vaccine-induced protection and the humoral parameters measured was noted.

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: homologous erythrocytes as a delivery system for preferential immunization with putative protective antigens.

Feline immunodeficiency virus (FIV) is a useful model for testing of criteria for AIDS vaccine development. In the protocol we adopted, we used a primary isolate of FIV as a source of antigen and, for challenge, plasma from cats infected with the homologous virus never passaged in vitro. Cat erythrocytes (RBC) were coated with the surface components of freshly harvested and purified FIV by means of biotin-avidin-biotin bridges and used to immunize specific-pathogen-free cats (four doses at monthly intervals; total amount of FIV antigen administered per cat, approximately 14 microg). Immunized cats developed moderate levels of antibodies directed mainly to surface components of the virion and clearly evident lymphoproliferative responses. Four months after the last dose of immunogen, FIV-immunized cats and control cats immunized with bovine serum albumin-coated RBC were challenged. Judged from the results of the subsequent 12-month follow-up, FIV-immunized cats exhibited at least some degree of protection. However, following rechallenge, most of the FIV-immunized animals became virus positive in spite of a booster immunogen dose given 2 months before the second challenge.

Effect of enzymatic deglycosylation on feline immunodeficiency virus sensitivity to antibody-mediated neutralization.

We have investigated the effect of deglycosylation with peptide-N-glycosidase F (PNGase F) on the sensitivity to inhibition by immune sera of two variants of the Petaluma strain of feline immunodeficiency virus (FIV-Pet), one sensitive to antibody-mediated neutralization because tissue culture adapted and the other, obtained by passaging the previous one in vivo, resistant to neutralization. The partial deglycosylation achieved did not appreciably affect FIV-Pet infectivity for T lymphoid cell cultures and did not increase the susceptibility to serum neutralization of the resistant variant but totally prevented neutralization of the sensitive variant. These finding suggest that the epitopes involved in neutralization of tissue culture-adapted FIV-Pet are effectively recognized by antibody only when the viral surface is properly glycosylated.

Autologous and heterologous neutralization analyses of primary feline immunodeficiency virus isolates.

Feline immunodeficiency virus (FIV) provides a model system with which the significance of neutralizing antibody (NA) in immunosuppressive lentivirus infections may be studied. To date, no detailed analysis of the neutralization properties of primary FIV isolates has been reported. In this study, we have conducted the first comprehensive study of the sensitivity to autologous and heterologous neutralization in a lymphoid cell-based assay of 15 primary FIV isolates and, for comparison, of one tissue culture-adapted strain. Primary isolates in general proved highly NA resistant, although there was considerable individual variation. Variation was also observed in the capacity of immune sera to neutralize heterologous FIV isolates. The ability of sera to neutralize isolates or for isolates to be neutralized by sera did not correlate with epidemiological and genetic relatedness or with the quasispecies complexity of the isolates. From the study of specific-pathogen-free cats experimentally infected with viral isolates associated with NA of different breadths, it appears that the development of FIV vaccines cannot rely on the existence of viral strains inherently capable of inducing especially broad NA responses.

Studies of AIDS vaccination using an ex vivo feline immunodeficiency virus model: protection conferred by a fixed-cell vaccine against cell-free and cell-associated challenge differs in duration and is not easily boosted.

Cats immunized with cells infected with a primary isolate of feline immunodeficiency virus (FIV) and fixed with paraformaldehyde were challenged with cell-free or cell-associated homologous virus obtained ex vivo. Complete protection was observed in animals challenged with cell-free virus 4 months after completion of vaccination (p.v.) or with cell-associated virus 12 months p.v. In contrast, no protection was observed in cats challenged with cell-free virus 12 or 28 months p.v. or with cell-associated virus 37.5 months p.v. Prior to the 28- and 37.5-month challenges, the animals had received a booster dose of vaccine that had elicited a robust anamnestic immune response. These results show that vaccine-induced protection against ex vivo FIV is achievable but is relatively short-lived and can be difficult to boost.

Incubation time for feline immunodeficiency virus cultures.

In a recent report, Fiscus et al. (S. A. Fiscus, S. L. Welles, S. A. Spector, and J. L. Lathey, J. Clin. Microbiol. 33:246-247, 1995) have shown that qualitative human immunodeficiency virus cultures can be terminated at day 21 with minimal false-negative results. We have evaluated a large number of qualitative and quantitative feline immunodeficiency virus (FIV) isolations to determine how long FIV cultures should be incubated to obtain reasonably certain results. The rate at which FIV cultures became positive was influenced by whether the cats under study were naturally or experimentally infected, the duration of in vivo infection, and the number of infected peripheral blood mononuclear cells seeded. The results show that cultures for FIV isolation should be kept for 5 to 6 weeks.

Vaccination protects against in vivo-grown feline immunodeficiency virus even in the absence of detectable neutralizing antibodies.

So far, vaccination experiments against feline immunodeficiency virus have used in vitro-grown virus to challenge the vaccinated hosts. In this study, cats were vaccinated with fixed feline immunodeficiency virus-infected cells and challenged with plasma obtained from cats infected with the homologous virus diluted to contain 10 cat 50% infectious doses. As judged by virus culture, PCRs, and serological analyses performed over an 18-month period after the challenge, all of the vaccinated cats were clearly protected. Interestingly, prior to challenge most vaccines lacked antibodies capable of neutralizing a fresh isolate of the homologous virus.

[Clinical usefulness of a computerized tomography study of morphology of the femoral canal in patients candidates for hip prosthesis]. / Utilità clinica dello studio con tomographia computerizzata della morfologia del canale femorale nei pazienti candidati a protesizzazione dell'anca.

In the last few years hip arthroplasty has been increasingly used and both metaphysis and diaphysis of the proximal femur are studied preoperatively for best compliance between prosthesis and bone. Indeed, the best results can be obtained by reducing the risk of stem end mobilization, which means to choose the prosthesis fitting the femoral canal best and to limit the use of cement prostheses, which are at high risk of mobilization in time, to advanced osteoporosis patients. We used a simple and repeatable CT technique to study femoral canal structure and size. After accurately positioning the patients inside the gantry, we acquired some axial scans at scheduled levels referring to the horizontal midline of the lesser trochanter. The axial scans were acquired 2 cm above and 5 and 10 cm below the horizontal midline in 105 patients. Measurements were bilateral in 13 patients. Finally, the results were compared with the surgical outcome. Femoral canal cross-diameters and cortical bone width varied greatly, which variations were confirmed in bilateral measurements and in the same patient between the two femurs. Femoral canal structure, which can be remodelled, influences the choice of the stem and thus the amount of cement. We believe this method to be fundamental for correct surgical planning and for best treatment outcome.

[Computer-assisted report generation and image transmission in bedside chest x-rays in intensive therapy units]. / Refertazione assistita e trasmissione delle immagini mediante computer nei toraci a letto dei reparti di terapia intensiva.

Since a few years ago, in our department the bedside chest X-rays of intensive care patients have been reported by means of a computer program which has also storing function. This computer program is a guideline for the radiologist and is organized in pages having a logical sequence. The program has proved very useful in learning the correct reporting of bedside chest X-rays. The nosographic data of the patients, the ventilatory and the technical data are stored for a better clinico-radiological correlation. The last four reports are displayed on the monitor to better understand the patient's history. The other reports become part of a "historical" archive. Most important is the cooperation with the referring physician: to make the most of it, a system has been implemented which sends the images from the Radiology Department to Intensive Care. The images are filmed with a camera and then digitalized on 1024 x 768 matrix with 16 million colors and 256 gray levels. Each workstation is composed of: AT286 computer with 60-MB hard disk, hardware or the digitalization and compression of images, a high-resolution monitor, an intercommunication system, and a modem. It is possible to zoom on the images, but a close-up on the image with the camera is better for improved spatial resolution. The images are stored on the hard disk: each image requires 3M bytes, but it can be compressed down to 25:1 with no detail loss. The images are transmitted via modem in at least 20 seconds/image. More images can be sent out-line. During transmission, it is possible to talk by the intercommunication system, pointing out structures on the monitor or drawing objects on both sides of the system. In our experience, image quality is good. We are therefore considering extending the network to other Departments and making the transmission of images of pathologic specimens possible. The natural evolution of this system is the teleconsult.