NEUTRALIZATION OF VISNA VIRUS BY HUMAN SERA

THE ENTEROVIRUS DEPARTMENT, STATENS SERUMINSTITUT, COPENHAGEN, DENMARK NEUTRALIZATION OF VISNA VIRUS BY HUMAN SERA By HALLD~R THORMAR~ and HERDIS VON MACNUS Received 28.ix.62 In a previous paper (12) the results of neutralization tests with human sera against visna virus were reported. These preliminary experiments included sera from healthy individuals as well as sera from patients suffering from multiple sclerosis (MS). The reason for testing the sera of these patients against visna virus was the apparent similarity between visna infection in sheep and the human demyelinating diseases as pointed out by Sigurdsson & Palsson (8). In the preliminary study, visna virus (about 100 TCIDoo) was found to be neutralized by MS sera in dilutions up to 1: 16. However, it was not ascertained whether there was a difference in the neutralizing activity of MS sera and of normal human sera, and the nature of the neutralizing substance was not studied. The present paper reports the results of neutralization tests carried out with human sera in a larger number than tested in the preliminary study. Both normal sera and sera from patients with MS were examined. MATERIAL AND METHODS Tissue culture.-serially propagated cell cultures derived from the choroid plexus of normal sheep brain were used for virus titrations and for inoculation of serumvirus mixtures in the neutralization tests (9, 10). The cultures were grown in medium 199 containing 10 per cent sheep serum and 10 per cent calf serum, and were maintained in medium 199 with 1 per cent sheep serum. Before changing the cultures to the maintenance medium they were washed with Hanks salt solution to remove neutralizing substances present in the calf serum (12). Virus.-Visna virus, strain K485, in its 40th TC passage was employed. The virus was titrated in tenfold serial dilutions in roller tubes. The batch of virus used in the present experiments had an infectivity titer of about 10-6.5 per 0.1 ml. Sera.-- Sera from patients with multiple sclerosis were kindly supplied by the Neurological Department, Municipal Hospital, Copenhagen, and by the Neurological Department of the National Hospital, Copenhagen. The sera derived from patients suffering from multiple sclerosis in various clinical phases of the disease. 1 Present address: Institute for Experimental Pathology, University of Iceland, Keldur, Reykjavik, Iceland. The authors are indebted to Overlaege dr. med. Torben Fog of the Neurological Department, Municipal Hospital, Copenhagen, and to Professor dr. med. Mogens Fog of the Neurological Department, the National Hospital, Copenhagen, for generous supply of sera from patients with multiple sclerosis. 261

Normal human sera were obtained by kind permission of blood donors at the Blood Bank of Statens Seruminstitut through the courtesy of Dr. P. Ejby Ponlsrn. The donors represented the same age groups, ranging from 18 to 65 years, as the patients from which serum was received. Sera from children were kindly supplied hy Dr. Inger Petersen of the Enterovirus Department, Statens Seruminstitut. Neufralization tests.---serial twofold dilutions of the human sera were prepared in medium 199, and 0.3 ml of each dilution were mixed with an equal volume of visna virus diluted in medium 199 to an estimated concentration of 50' TCID5o per 0.1 ml of serum-virus mixture. The ph of the mixtures was adjusted to 7.3-7.5 by gassing the tubes with 5 per cent COz in air. After incubation the serum-virus mixtures were inoculated in 0.1 ml amounts into roller tube cultures, using two tubes per serum dilution. The roller tubes were incubated at 37",1 first in a roller drum for two days and subsequently in stationary racks. They were observed for cytopathic changes at intervals, and were discarded after 14 days. Neutralizing titers were calculated by the method of Karber (5) and are expressed as the reciprocal of the highest initial serum dilution which completely suppressed cytopathic changes in 50 per cent of the inoculated tubes. In each experiment the virus dilution employed was mixed with a 1 : 8 dilution of normal sheep serum to serve as a virus control. The virus control was titrated at the end of the incubation period, simultaneously with the inoculation of the serumvirus mixtures. Visna antiserum no. 5383, with a known neutralizing titer (111, was included as a positive control in all experiments. This serum was kindly supplied by Dr. PdZZ A. Pdlsson of the Institute for Experimental Pathology, Keldur, Iceland. Prior to inoculation into tissue cultures, the serum-virus mixtures were incubated at 37" for 18 hours, except otherwise stated. These conditions of incubation had previously been found to result in a maximal neutralization of visna virus by antiserum, with only a slight spontaneous inactivation of virus at the serum concentrations employed (11). RESULTS Effect of heating the sera to 56" for 30 minutes.-in pilot experiments carried out with human sera incubated with visna virus at 22" for 24 hours prior to inoculation into roller tubes, neutralizing titers varying from less than 4 to at least 16 were observed in unheated sera. Upon heating the sera to 56" for 30 minutes before mixing with the virus, the titers decreased to below 4. However, in serum dilution 1 : 4 the viral activity was markedly reduced, indicating some neutralization of virus by heated sera. In an attempt to increase this neutralizing effect, scrumvirus mixtures were incubated at 37" for various periods of time before inoculation into roller tubes. Table 1 shows the results of one exyeriment comparing the neutralizing activity in unheated and in heated sera incubated with virus at 22" for 24 hours or at 37" for 18 hours. It can be seen that by raising the incubation temperature the titers were considerably increased, both in unheated and in heated sera. In the latter, about half of the sera neutralized the virus in dilution 1: 1 or higher. This experiment showed that in addition to the "heat-labile" substance the human sera contained a "heat-stable'' substance which neutralized visna virus during incubation at 37" for 18 hours. The term "heat-stable" will in the following sections be used to denote the ability to resist heating to 56" for 30 minutes. Comparison of heat-stable neutralizing substance in MS sera and in 1 All temperatures given in ccntigrade.

~ ~~~~ ~ ~ ~ ~ ~ ~ ~~ ~ ~ normal sera.-to compare the content of heat-stable neutralizing substance in MS sera and in normal human sera a total of 109 heated sera of each kind was tested against visna virus. The results of two experiments comprising all the sera are shown in Table 2. It can be seen that almost an equal number of positive sera was found in each group. The number was somewhat lower when a larger virus dose was employed. The distribution of neutralizing titers was almost identical in the two groups. TABLE 1 Effect of Heating to 56" for 30 Minutes on the Neutralizing Actiuity of Human Sera against Visna Virus. Eight Sera, Heated and Unheated, Were Incubated with Virus' a) at 22" for 24 Hours, or b) at 37" for 18 Hours, before Inoculation into Tissue Cultures. Number of sera with titer no. 5383 220, 24 hrs. Not heated... 4 3 1 0 4 Heated... 8 0 0 0 0 180 Not heated... 0 0 1 7 8 370 ' hrs' Heated... a 1 4 0 5 256 30 TCID5o per 0.1 ml of mixture. TABLE 2 Neutralizing Substance against Visna Virus. Comparison of Titers in Sera from Patients with Multiple Sclerosis (MS) and in Normal Human Sera. The Sera Were Tested a) agains,t 5 TCID5o and b) against 50 TCID5o of Virus per 0.1 ml of Serum-Virus Mixture. MS... 57 7 11 14 9 7 9 50 a) Normal... 57 6 11 7 15 14 4 51 MS... 52 16 11 14 8 1 2 36 ') Normal... 52 13 17 12 7 3... 39 From the above experiments it was concluded that small amounts of the heat-stable neutralizing substance against visna virus were found widely distributed in adult human sera, and that there was no significant difference between sera from patients with multiple sclerosis and normal human sera with respect to the occurrence of this substance. Neutralizing substance in sera from children.-in an attempt to characterize the heat-stable substance it was first studied whether there was an age-correlated difference in its occurrence in normal human sera. Table 3 shows the neutralizing titers found in heated sera from 79 children ranging in age from 2 months to 11 years, compared with the titers found in sera from adults. Positive sera were found in all of

~ ~ the age groups, and a characteristic age pattern could not be demonstrated in the distribution of the neutralizing substance. Sera from infants seemed to contain the substance in high titers. TABLE 3 Nrutralizing Substance against Visna Virus. Distribution in Sera from Indiuitlunls of Various Age Groups. Number of sera with titer No. positite Age group _. ~ ~ - ~. <4 i 4 I 8 ' 216 No. tc5trd 0-6 mos.... 0 1 8 3 12/12 6-12 mob.... 4 1 7 1 9/13 12-18 mos.... 0 5 3 2 1 O/l 0 18-24 mos.... 2 2 3 a 517 2-4 years... 7 5 2 0 7/14 4-7 years... 4 5 3 1 9/13 7-11 years... 3 3 2 2 7/10 20-35 years... 10 12 3 0 15/25 Absence of neutralizing activity in human gamma globulin. --In ordtr to study whether the neutralizing substance was found in the gamma globulin fraction of human sera, seven preparations of human gainrna globulin were tested against visna virus together with a few randomly selected donor sera which served as controls. The gamma globulin was prepared at Statens Seruminstitut from sera of blood donors and was kindly supplied by Dr. Albert Hansen of the Department of Biochemistry. No neutralizing activity against visna virus was detected in any of the gamma globulin preparations employed in a concentration of 5 per cent, while all the donor sera were positive in a dilution of 1 : 4 to 1 : 8. As a further control the gamma globulin preparations were tested in a neutralization test against 50 TCID5o of poliovirus, type 11, kindly carried out by Dr. fnger Petersen of the Enterovirus Department of Statens Seruminstitut. This virus dose was found to be neutralized by the gamma globulin in a concentration as low as 0.0025 per cent. Stability of the neutralizing substance to heating at 80".-To study further the heat stability of the neutralizing substance which resisted inactivation at 56" for 30 minutes, five MS Sera and five normal sera which had been found to contain heat-stable neutralizing substance in a titer of 8 to 16, were diluted 1: 2 in Hanks' salt solution and heated to 80" for 30 minutes. Another aliquot of each serum was heated to 60" for 30 minutes. Visna antiserum No. 5383 was treated in the same manner to serve as a control. The heat treated sera were then tested against visna virus together with an unheated aliquot of each serum. The results are presented in Table 4 which shows that the neutralizing substance which resisted heating to 60" for 30 minutes also resisted exposure to 80" for 30 minutes. On the other hand, the titer of antiserum No. 5383 was reduced about &fold by this treatment. Stability of the neutralizing substance to treatment with trgltsin,

potassium periodate, and ethyl ether.-in an attempt to obtain sonic information about the nature of the heat-stable substance, the effect of a few chemical reagents on the neutralizing activity was studied. Firstly, a number of human sera were mixed with trypsin (Difco 1 : 250) in a final concentration of 0.1 per cent and incubated at 37" for 30 minutes. Treatment with trypsin was found not to reduce the neutralizing activity of the sera, as compared with control sera incubated in the same manner either with inactivated trypsin or with phosphate buffer. TABLE 4 Effect of Heating on the Neutralizing Actiuity of Human Sera. Heated and Unheatcd Aliquots of 10 Sera Were Tested against Visna Virus (40 TClDjo per 0.1 ml). ~~~ - ~~~~ Number of sera positive in titer Titer of no. 5383 Unheated... 0 0 1 4 5 256 60" / 30 min.... 0 1 5 3 1 256 80" / 30 min.... 0 3 2 2 3 45 Similarly, human sera were mixed with potassium periodate in a final concentration of 11200 M and incubated for 30 minutes either at room temperature or at 37". Before mixing the sera with virus in the neutralization test, glycerol was added to inactivate the remaining periodate (2). The treatment with periodate did not influence the titer of the neutralizing substance, as compared with controls incubatcd in the samc manner either with saline or with a mixture of periodate and glycerol. Incubation with an equal amount of ethyl ether at 4" for 24 hours did not affect the neutralizing activity of human sera. DISCUSSION It was found in the present study that the majority of the human sera tested contained a neutralizing substance against visna virus. In addition to a heat-labile substance, which was inactivated by heating to 56" for 30 minutes, a heat-stable activity was demonstrated in a large part of the sera. This activity was found as often in sera from healthy individuals as in sera from patients with multiple sclerosis, a finding which does not not support the assumption that there is an immunological relationship between visna virus and a hypothetical agent of the human disease. As to the nature of the heat-stable substance which was found to neutralize visna virus, two possibilities may be considered. Firstly, this substance might be a true antibody against a human virus antigenically related to visna virus, or secondly, we might be dealing with some kind of a heat-stable non-specific substance capable of inactivating visna virus.

In an attempt to distinguish between these two possibilities, the neutralizing substance was studied with regard to a few characteristics considered to be typical of true antibodies (4,6). The results, showing a) absence of neutralizing activity in the gamma globulin preparation tested, b) an apparent absence of an age pattern indicativc of ininiunc response, and c) resistance of the substance to heating at 80", seein to indicate that the neutralizing substance described in the present study was not a true antibody. Apart from the above evidence against an antibody status of thr neutralizing substance, its nature remains unknown. The resistance to treatment with periodate indicates that it is not a mucopolysaccharidc (3, 7). Furthermore, preliminary experiments carried out to study whether we were dealing with viral inactivating lipids (1, 13, 14) wcrc negative. S U hf M A R Y The majority of human sera studied in neutralization tests against visna virus were found to contain substances which neutralized the virus. These seemed to be of two kinds, one which was inactivated by heating to 56" for 30 minutes, and another which was not affected by this heating. The latter substance was most easily detected after incubation with virus at 37" for 18 hours. It was found as often in sera from normal blood donors as in sera from patients with multiple sclerosis. It was found in sera from children ranging in age from 2 months to 11 years and no typical age pattern could be demonstrated. The heatstable factor was not found in preparations of human gamma globulin and aparently it was not destroyed either by heating to 80" for 30 minutes, or by treatment with trypsin, periodate or ethyl ether. It is concluded that the heat-stable substance most likely is a non-specific inhibitor of visna virus. REFERENCES 1. Cusals, J. & Olitsky, P. K.: Inactivation of certain neurotropic viruses in vitro by serum lipids. Science 106: 267-268,1947. 2. Fuzekas de St. Groth,S. & Grahan1,D.M.: Modification of virus receptors by metaperiodate. 11. Infection through modified receptors. Aust. J. Exp. Biol. Med. Sci. 27: 83-98,1949. 3. Hirst, G. K.: The nature of the virus receptors of red cells. 1. Evidence on the chemical nature of the virus receptors of red eells and of the existence of a closely analogous substance in normal serum. J. Exp. Mcd. 87: 301-314, 1948. 4. Karzon, I). T.: Studies on a neutralizing antibody against canine distemper virus found in man. Pediatrics 16: 809-818,1955. 5. Karber, G.: Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Arch. f. exp. Path. u. Pharmakol. 162: 480-514,1931. 6. Klein, M.: The significance of human antiviral neutralizing substances in animal sera. Ann. N. Y. Acad. Sci. 70: 362-368,1958. 7. Mandel, B. & Racker, E.: Inhibition of Tbeiler's encephalomyelitis virus (G D VII strain) of mice by an intestinal mucopolysaccharide. 11. Purification and properties of the mucopolysaccharide. J. Exp. Med. 98 : 417-426,1953.

267 8. Siyurdsson, B. & Palsson, P. A.: Visna of Sheep. A slow demyelinating infection. Brit. J. Exp. Path. 39: 519-528,1958. 9. Siyurdsson, B., Thormar, H. & Paisson, P. A.: Cultivation of visna virus in tissue culture. Arch. ges. Virusforsch. 10 : 368-381,1960. 10. Thormar, H.: Stability of visna virus in infectious tissue culture fluid. Arch. ges. Virusforsch. 10: 501-509,1960. 11. Thormar, H.: Neutralization of visna virus by antisera from sheep. J. Immunol. 1963. 12. Thormar, H. & Siyurdarddttir, B.: Growth of visna virus in primary tissue cultures from various animal species. Acta path. et microbiol. scandinav. 55 : 180-186,1962. 13. Utz,J.: Effect in uitro of specific lipid fractions of animal sera on psittacosis virus. Proc. Soc. Exp. Biol. Med. 69: 186-189,1948. 14. Ufz,J.: Studies on the inactivation of influenza and Newcastle disease viruses by a specific lipid fraction of normal animal sera. J. Immunol. 63: 273-279, 1949.