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

Baldinotti, F

Bandecchi, P

Bendinelli, M

Matteucci, D

Osterhaus, A

Siebelink, K

Tozzini, F

PubMed

Tozzini, F.

Matteucci, D.

Bandecchi, P.

Baldinotti, F.

Siebelink, C.H.J. (Kees)

Osterhaus, A.D.M.E. (Albert)

Bendinelli, M.

Erasmus University Digital Repository

JOURNAL OF CLINICAL MICROBIOLOGY, June 1993, p. 1626-16290095-1137/93/061626-04$02.00/0Copyright © 1993, American Society for MicrobiologyNeutralizing Antibodies in Cats Infected with FelineImmunodeficiency VirusFRANCO TOZZINI,1 DONATELLA MAITEUCCI,2 PATRIZIA BANDECCHI,1 FULVIA BALDINOTTI 2KEES SIEBELINK,3 ALBERT OSTERHAUS,3 AND MAURO BENDINELLI2*Department ofAnimal Pathology' and Department ofBiomedicine,2 University ofPisa, I-56127 Pisa,Italy, and Laboratory ofImmunobiology, National Institute of Public Health andEnvironmental Protection, 3720 BA Bilthoven, The Netherlands3Received 31 August 1992/Accepted 23 February 1993Sera from cats experimentally infected with five isolates of feline immunodeficiency virus (FlY) from variousgeographical regions and from FIV enzyme-linked immunosorbent assay-seropositive field cats from fourEuropean countries neutralized the Petaluma strain of FIV (FlV-P), originally isolated in California, at hightiters. 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. deNoronha, J. Gen. Virol. 72:617-622, 1991), these findings indicate that most if not all FIV strains circulatingin Europe and the United States share important neutralization-inducing epitopes.Feline immunodeficiency virus (FIV), a widespread causeof disease in domestic cats (2, 9, 24), shares many propertieswith human immunodeficiency virus (HIV) and is thereforeconsidered a suitable model for studying aspects of AIDSpathogenesis and treatment which cannot be easily ad-dressed with humans (15, 19). Nonetheless, little is knownabout the ability of FIV to induce neutralizing antibodies(NA). Using a procedure based on infectivity inhibition,Fevereiro et al. (5) found an antibody that neutralizes theprototype Petaluma strain of FIV (FIV-P), originally isolatedin California (16), in a limited number of domestic cats fromthe United States as well as in cats experimentally infectedwith FIV-P. In the present study, using a recently developedsyncytium formation inhibition assay, we showed that FIVstrains which circulate in the United States and in Europeare extensively cross-neutralized.The neutralization assay used has been described else-where (22). Briefly, 0.1-ml volumes of twofold dilutions(from 1:8 to 1:16,096) of sera heated at 56°C for 30 min weremixed with 0.1 ml of FIV diluted to contain 1,000 syncytium-forming units/ml, incubated at room temperature for 1 h, andthen inoculated into 0.5 ml of medium in duplicate wells of24-well plates which were seeded with 104 Crandell felinekidney (CrFK-MESNA) cells adapted to grow in a mediumcontaining a low concentration of fetal bovine serum. Eachplate included control immune and negative sera. Six dayslater, the plates were stained with 0.2% crystal violet in 30%methanol and examined microscopically. Only syncytia con-taining seven or more nuclei were counted. The neutralizingtiter was expressed as the reciprocal of the highest dilutionof serum which prevented the formation of syncytia. Unlessotherwise stated, FIV-P was used in the neutralizationassay. In some experiments, the Dutch isolate Amsterdam-6(FIV-A6), which was obtained from a free-roaming cat in theNetherlands and adapted to replication in CrFK-MESNAcells, was also used. Both FIV-P and FIV-A6 consistentlyproduce syncytia on freshly infected CrFK-MESNA cells.The viral stocks used ranged in titer between 4 x 103 and 5x 104 syncytium-forming units/0.1 ml.* Corresponding author.We first examined the sera of specific-pathogen-free (SPF)cats infected with various FIV isolates. Viruses used forinfection included FIV-P, two Italian isolates (Pisa-Ml andPisa-M2 [FIV-M2]), one British isolate (Glasgow-8), oneDutch isolate (Amsterdam-19), and the viral progeny of aninfectious molecular clone (19kl) of the last virus (20). Thegenetic relatedness of these isolates was the subject of arecent report (18). All of the sera of infected cats neutralizedFIV with titers ranging from 512 to 8,192, whereas preinfec-tion sera diluted 1:8 did not. The presence of NA correlatedwith reactivity in an enzyme-linked immunosorbent assay(ELISA) for FIV antibody (CITE Combo FIV-FeLV; Ag-ritech Systems, Portland, Maine) (data not shown). Figure 1shows the kinetics of NA production in SPF cats inoculatedwith FIV-M2. Low levels of NA were already detectable insome cats at day 5 postinfection (Fig. 1, insert), but theseantibodies were most likely acquired passively with theplasma used to infect the animals, as the titers declinedsteadily in the following days. Actively produced NA be-came clearly evident at 5 to 6 weeks of infection (approxi-mately at the time of seroconversion, as detected by othermethods [reference 13 and data not shown]), reached aplateau 3 to 4 months later, and then remained stable for thesubsequent period of observation. As shown by periodicisolation of virus from the peripheral blood mononuclearcells by a standard procedure (2), all cats in this groupdeveloped a persistent FIV infection.We then examined sera obtained from naturally infecteddomestic cats from different European countries. The do-nors were of many breeds and ages and of both sexes andhad different living conditions. Infection was determinedserologically by ELISA and in most Italian cats was con-firmed by virus isolation. The sera of 43 out of 45 infectedcats neutralized FIV at titers ranging from 32 to 4,096 (theother two serum samples, diluted 1:8, reduced syncytiumformation by over 70%). In contrast, the sera of 17 out of 19noninfected cats were not reactive at a 1:8 dilution. Only twoserum samples from culture-negative, ELISA-seronegativecats showed titers of 8 and 16, respectively. In the infectedanimals, NA titers appeared unrelated to the country oforigin (Table 1) or to the clinical status of the cats (data notshown).1626Vol. 31, No. 6NOTES 162716,384>. 1,024020 6 10 141822+12832640) 8z10 20 30 4.0Days2 6 10 14. 18 22Months post infectionFIG. 1. NA production in six SPF cats inoculated intravenously with FIV-M2 at an age of 4 to 5 months. This virus is propagated in vivoby monthly passages in SPF cats and at the dose used (2 ml of freshly collected blood) leads to a positive FIV ELISA in 4 to 6 weeks andto a reduction in the number of circulating CD4+ lymphocytes by almost two-thirds in 1 year (10). During the period of observation, one catshowed progressive emaciation and was killed. The insert shows in more detail the NA titers detected during the first 40 days of infection (+and - indicate reactivity and nonreactivity, respectively, in the ELISA).TABLE 1. FIV NA in sera of FIV ELISA-negative and -positivefield cats from different European countriesNA titerELISA result and No. of serum No. of serumcountry of origin' samples samples with 95%examined NA Meanb confidencelimitsSeronegative, Italy 19 2 10SeropositiveItaly 21 21 495 213-1,150The Netherlands 6 6 287 70-1,179United Kingdomc 14 12d 1,124 53-2,488Switzerland' 4 4 512a All but two FIV-seronegative cats examined were clinically healthy. Incontrast, many infected animals presented clinical manifestations (gingivitis,diarrhea, dermatitis, upper respiratory tract infections, anorexia, emaciation,pyrexia, anemia, lymphopenia, lymphadenopathy, or neurological signs)compatible with FIV infection.b Geometric mean of titers Z8. The means of the infected groups are notsignificantly different.c Sera provided by Oswald Jarrett, University of Glasgow.d At the lowest dilution tested (1:8), the two serum samples that werescored as NA negative reduced FIV-induced syncytium formation by over70%.' Sera provided by Hans Lutz, University of Zurich.The previous data demonstrated the existence of a highdegree of serological cross-reactivity among FIV strainswhich circulate in Europe and the United States. To furtherinvestigate this aspect of FIV, we simultaneously titratedagainst FIV-P and FIV-A6 a panel of 15 serum samplesknown to contain NA. The sera neutralized the two isolateswith similar efficiencies, irrespective of the virus used toimmunize the animals or the geographical origin of thedonors (data not shown).The results presented above are at variance with thosereported for HIV, whose sensitivity to antibody-mediatedneutralization is generally markedly influenced by the sourceof sera (12). As prior studies with HIV had indicated that, invitro, certain antibodies may cause a dissociation betweensyncytium formation and virus replication and/or produceneutralization even if added to cultures after virus is boundto cells (14, 23), we investigated how seropositive cat serabring about the inhibition of syncytium formation by FIV.To address this point, we studied the effect of thoroughlywashing off the input virus-serum mixtures after absorption.The representative data presented in Table 2 show thatinhibition of syncytium formation is not dependent on thecontinuous presence of serum in the test cultures andcorrelates well with inhibition of viral antigen accumulationin the supernatants. In further studies, the addition ofhigh-titer neutralizing sera to cell cultures that had beeninfected 4 h earlier did not prevent the formation of theexpected numbers of syncytia, although the syncytia pro-VOL. 31, 1993J. CLIN. MICROBIOL.TABLE 2. Effect of removing virus-serum inocula on neutralization, as determined by syncytium formation and viral antigen productionResults with serum:M2-IV M2-VIIbSerumdilution With removal" With removalNo. of syncytiaa No. of syncytiawithout removal No. of Optical without removal No. of Opticalsyncytia densityc syncytia density1:256 0 0 0.211 0 0 NDd1:512 0 0 0.203 0 0 ND1:1,024 0 0 0.219 0 0 0.2161:2,048 0 0 0.263 0 0 0.2131:4,096 0 0 0.271 0 0 0.2101:8,192 4 ± 2 0 0.242 0 16 ± 5 0.4731:16,384 18 ± 4 14 ± 4 0.846 8 22 ± 6 0.477FIV alone 61 ± 8 59 ± 10 2.708No FIV 0 0 0.227a Mean ± standard deviation.b Four hours after inoculation, that is, at a time when most FIV has become absorbed to CrFK-MESNA cells but no syncytia have formed (22), the FIV-seruminocula were washed off and replaced with fresh medium.c At the end of the 6-day incubation period, the cultures were examined by ELISA for FIV p24 antigen content as well as for syncytia. The p24 assay wasperformed on pooled supematants of the cultures as described elsewhere (21), with minor modifications.d ND, not determined.duced were smaller (data not shown). These results showthat inhibition of syncytium formation is due to a trueneutralization of FIV infectivity prior to its entry into cellsand not to down-regulation of syncytium formation or othermechanisms.This study confirms recent findings (5) showing that catsdevelop high-titer NA early after FIV infection. NA maycontribute to the long incubation period between infectionand illness observed in FIV-infected cats as well as to thefact that FIV transmission in utero is a rare event whichpossibly occurs only during primary infection (4, 15, 24, 25).However, FIV was readily isolated from the peripheralblood mononuclear cells of animals with high levels of NA intheir sera, indicating that NA are unable to terminate anestablished FIV infection. This is in keeping with previousresults (5) and with our present understanding of the role ofNA in HIV type infection (11, 12). It has been suggested thatNA mediate the reduction of high-titer viremia which occursin human plasma soon after infection (1). Nevertheless, FIVcould be isolated from plasma regardless of the NA titers(10).The neutralizing activity found at low titers in few appar-ently noninfected cats deserves further investigation. Sinceit was observed in cats who were also negative by Westernblot (immunoblot) assay (data not shown), the result doesnot seem to stem from the low sensitivity of the ELISAused. Since it was detected in field cats but not in SPF cats,explanations to be considered include cross-reactivity ofFIV with other cat pathogens (cats singly infected with felineleukemia virus or feline syncytium-forming virus, however,tested negative), the possibility that the neutralization testdetects FIV infections not detected by other currently usedserological methods (6, 8), the possibility that NA persistlonger than other antibodies and represent footprints of aneradicated FIV infection, and the possible existence ofnonspecific inhibitors.However, the most important finding of the present studyis the large degree of cross-neutralization exhibited by FIVisolates from different geographical areas, indicating thatFIV contains one or more highly conserved neutralizationepitopes. This information may be useful in the preparationof effective vaccines. HIV, which can be considered thehuman counterpart of FIV, also contains regions which elicitbroadly reactive neutralizing responses (3, 7, 11, 12), thoughits env gene variability appears to be higher than that of FIV(17). We are currently trying to identify the FIV epitope(s)involved in neutralization.This work was supported by grants from the Ministero dellaSanita-Istituto Superiore di Sanita "Progetto Allestimento ModelliAnimali per l'AIDS" and "Progetto FATHA," CNR, Rome, Italy.We are grateful to Marian C. Horzinek, University of Utrecht;Oswald Jarrett, University of Glasgow; Hans Lutz, University ofZurich; and Niels C. Pedersen, University of California, Davis, forsuggestions and reagents.REFERENCES1. Albert, J., H. Gaines, A. Sonnerborg, G. Nystrom, P. 0.Pehrson, F. Chiodi M. von Sydow, L. Moberg, K. Lidman, B.Christensson, B. Asji, and E. M. Fenyo. 1987. Isolation ofhuman immunodeficiency virus from plasma during HIV infec-tion. J. Med. Virol. 23:67-73.2. Bandecchi, P., D. 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Pedersen. 1989.Epidemiologic and clinical aspects of feline immunodeficiencyvirus infection in cats from the continental United States andCanada and possible mode of transmission. J. Am. Vet. Med.Assoc. 194:213-220.25. Yamamoto, J. K., E. Sparger, E. W. Ho, P. R. Andersen, T. P.O'Connor, C. P. Mandell, L. Lowenstine, R. Munn, and N. C.Pedersen. 1988. Pathogenesis of experimentally induced felineimmunodeficiency virus infection in cats. Am. J. Vet. Res.49:1246-1258.VOL. 31, 1993

Neutralizing antibodies in cats infected with feline immunodeficiency virus.

Neutralizing antibodies in cats infected with feline immunodeficiency virus.

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