Effect of Complement and Viral Filtration on the

APPLIED MICROBIOLOGY, JUlY 1968, p. 1076-1080 Copyright @ 1968 American Society for Microbiology Vol. 16, No. 7 Printed in U.S.A. Effect of Complement and Viral Filtration on the Neutralization of Respiratory Syncytial Virus RICHARD H. BAUGHMAN, JAMES D. FENTERS, GEORGE S. MARQUIS, JR., AND JACOB C. HOLPER Infectious Disease Research Division, Abbott Laboratories, North Chicago, Illinois 60064 Received for publication 23 April 1968 The addition of 10 hemolytic units of guinea pig complement has been shown to enhance the neutralizing capacity of respiratory syncytial (RS) immune sera produced in guinea pigs and ferrets. This same immune sera, when tested without complement, had little or no neutralizing capacity. The addition of complement to RS immune horse serum did not significantly increase its neutralizing capacity. Immune horse serum effectively neutralized RS virus without complement. Other studies indicated that a 50% tissue culture infective dose of between 30 and 100 should be used in RS serum neutralization tests and that incubation should be for 90 to 105 min at room temperature. The neutralizing capacity of guinea pig immune serum was not increased by the use of filtered virus. The rate of virus neutralization, however, was increased with the addition of 10 hemolytic units of complement. The neutralizing capacity of RS immune horse serum was much greater for filtered than for unfiltered RS virus. The addition of complement increased the rate of virus neutralization but did not increase the neutralizing capacity of the horse immune serum. A standard serum neutralization test for the evaluation of immunogenicity of respiratory syncytial (RS) virus would be essential to the development of RS virus vaccines. Dulbecco et al. (2) reported the apparent increase in neutralization of western equine encephalitis virus by the addition of normal rabbit serum as a cofactor. The addition of unheated normal rabbit or guinea pig sera has been reported (Potash et al., Bacteriol. Proc., p. 122, 1966) to have an enhancing effect on the neutralization of RS virus by immune sera. Considerable variation was experienced in our laboratory when using the above procedure. Rawls et al. (4) found that the addition of guinea pig complement would enhance the neutralizing capacity of rubella-immune serum. Yoshino et al. (8) reported the early detection of herpes simplex antibody by the addition of 10 hemolytic units of guinea pig complement. In this report, we will further define the complement requirements of specific antisera from various animal species as well as the effect of viral aggregate removal (6) by filtration on RS virus neutralization. MATERIALS AND METHODS Virus strains. The Simon strain of RS virus, which was provided by Dr. Chanock of the National Institutes of Health, was used exclusively in these experiments. The virus had been passaged several times in either bovine kidney tissue culture or HEp-2 tissue culture in our laboratory. The virus pools prepared in either bovine kidney tissue culture or HEp-2 cells titered between 105.0 and 106.0 50% tissue culture infective doses (TcID5o) per ml when titered in HEp-2 roller tubes at 37 C. All virus pools used in tests were unfiltered unless specified. Either pressure or vaccum filtration through an RA (1.2 pz) filter membrane (Millipore Corp., Bedford, Mass.) was used. Tissue culture and media. The HEp-2 cells were grown in Eagle's Basal Medium plus 10% heatinactivated (56 C for 30 min) fetal calf serum. Bovine kidney cells were grown in 0.5% lactalbumin hydrolysate medium plus 5% heat-inactivated fetal calf serum. Maintenance medium for both cell systems consisted of 96 ml of medium 199, 1 ml of vitamins, 1 ml of L-glutamine, 1 ml of essential amino acids, 50 units of polymyxin per ml, 100,g of neomycin sulfate per ml, and 0.09% sodium bicarbonate to maintain the ph at approximately 6.8 to 7.2. Serum. Guinea pig immune serum was prepared by the pooling of several portions of postvaccination sera from guinea pigs which had been inoculated with Formalin-inactivated RS virus Simon vaccine. Animals had received two 0.5-ml injections, intraperitoneally, at 2-week intervals. Bleedings were made at 2, 4, and 6 weeks after the first injection. Normal or preimmune guinea pig serum was used 1076

VOL. 16, 1968 EFFECT OF COMPLEMENT ON RS VIRUS 1077 as control serum. Serum from a horse immunized with RS Long antigen was obtained from Flow Laboratories (Rockville, Md.). Preimmune horse serum was used as a control. Ferret immune sera were prepared by the injection of either live RS Long or RS Bernett antigen. Preimmune ferret serum was used as a control. All sera tested were heat-inactivated for 30 min at 56 C prior to use. Diluent. All virus and serum dilutions were made in magnesium saline containing 0.85% NaCl. Complement. Unheated normal guinea pig serum was used as the source of complement. Freshly processed serum was frozen in 2-ml portions at -20 C until use. A complement titration was done to determine the required dilution of normal serum to give 10 hemolytic units of complement per ml. Assay of virus. Serial 10-fold dilutions of RS virus were prepared and assayed in HEp-2 roller tubes at 37 C. Two or four tubes were inoculated with 0.1 ml per dilution. Titers were calculated according to the method of Reed and Muench (5). All titers mentioned in the text are expressed as log10 TCID5 per ml. Serum neutralization test. Serum to be tested was diluted in an appropriate diluent and heat-inactivated at 56 C for 30 min before testing. Each serum was diluted in two parallel series in 0.2-ml amounts in twofold increments. One series received 0.1 ml of complement diluted to give 10 hemolytic units per ml; the other series received 0.1 ml of diluent. All tubes then received 0.1 ml of virus, diluted to give approximately 100 TCID50 per ml of infectious virus. The normal serum which was the source of the complement was diluted in the same manner as above and tested for the presence of neutralizing antibody. Neutralization time for the above mixtures was 1 hr at room temperature. Neutralization slope-method A. Guinea pig immune serum used in the method A (dilutions of virusdilutions of sera) neutralization slopes was prepared by immunization with RS virus Simon-inactivated vaccine. Serum was heat-inactivated at 56 C for 30 min and then diluted in twofold increments in 0.2-ml amounts. Six sets of serum dilutions were prepared; half of them received 0.1 ml of complement and the other half received 0.1 ml of diluent. To all sets, an appropriate virus dilution was added. Neutralization time was 1 hr at room temperature. Test animal Neutralization slope-method B. Both guinea pig and horse immune sera were used for the method B (constant amount of virus-constant amount of sera) neutralization slopes. The guinea pig sera were used undiluted. The horse sera were diluted 1:10 in magnesium saline to avoid cytotoxicity seen with undiluted sera. Equal volumes of sera and virus preparations were mixed and samples were taken at appropriate intervals for infectivity tests to determine the unneutralized virus titers. Incubation was at room temperature. In the tests using 10 hemolytic units of complement, equal volumes of virus and complement were combined. This combination was then added to an equal volume of the appropriate serum sample. RESuLTs Effect of complement on RS virus-neutralizing antibody titers. To determine the effect of the addition of 10 hemolytic units of guinea pig complement on neutralizing antibody titers, sera from guinea pigs and ferrets were tested in a serum neutralization test with and without the addition of complement (Table 1). When tested against 100 TCID5o of RS virus Simon bovine kidney-grown antigen, the addition of complement increased the serum-neutralizing antibody titers fourfold or greater. For example, the neutralizing antibody titer of immune serum from guinea pig 5022, which had received live RS Simon vaccine, was increased sixfold by the addition of complement. The increase in antibody titer of the other sera tested was 4- to 48-fold. Neutralization slope-method A. The neutralization slopes done by method A (dilutions of both sera and virus) were observed for cytopathic effects for 3 consecutive days beginning 4 days postinoculation. Reciprocal serum-neutralizing antibody titers were calculated each day for each virus dilution. The addition of complement appeared to increase the neutralizing antibody titers of the immune serum (Fig. 1). An increase in neutralization titer was seen with the 10-1 and 10-2 dilutions of virus with the addition of complement when read on days 5 and 6 post- TABLE 1. Effect of complement on the neutralization of RS virus,a Vaccine Reciprocal serum neutralization titersb Immune serum Immune'serum Fold increase without complement with complement Guinea pig 5022 Live RS Simon 16 96 6 Guinea pig 1425 Inactive RS Simon <8 96 48 Ferret 149 Live RS Long 32.128 4 Ferret 230 Live RS Burnett 8 48 6 a Respiratory syncytial (RS) virus Simon (100 TCID50 per ml) used in test. Done in HEp-2 roller tubes at 37 C. b Preimmune serum titers were all <4, with and without complement.

1078 BAUGHMAN ET AL. APPL. MICROBIOL. inoculation. Undiluted virus appeared to over- 5.75 whelm the antibody present in the immune serum. 55 Neutralization slopes-method B. Neutraliza- 525 tion slopes by method B (constant amount of q virus-constant amount of sera) were conducted q5 i- in an effort to determine the effect of the addition 4.25 of 10 hemolytic units of complement on the, 40 amount of surviving infectious virus after various '5 35 X m 3.25 - z> 128-3.0 - E L 4 DAY READINGS 2.75 - : 128 - U 4 DAY READINGS WITH COMPLEMENT 2.5 _ 0 5 DAY READINGS 2.25 Z 96_*0 5 DAY READINGS WITH COMPLEMENT 20 E 5A 6 DAY READINGS 1 30 45 60 75 90 105 120 135 150 165 180 2 64 A 6 DAY READINGS WITH COMPLEMENT G NEUTRALIZATION TIME IN MINUTES 48 FIG. 3. Neutralization of RS virus Simon by im- D mune guinea pig serum with the addition of comple- O a32met-oto - ment. Symbols: control serum (X), virus control + 0c A@U C' (0), immunized serum (A), virus control (0). ui 24 Z 16 0 5 750 4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~47 z 50 z 5 N ~ VIUDIUIOX 46 A O0A _jtcd5' A El 4 5 \ / 12 DY4 0 003 0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. 1.1 D50'S4O DAY 5 3 ONDAYUNDIUTE DILUTION Ai- TCID5'S ONDAY 4 300 300 30 TCID50'S ON DAY 5 3000 1000 100 325 TCISO' O DA 6 3000 1000 100 325' I I I FIG. rig. 1. 1lveutrallzation Neutralization f of RS virus Simon with 0 15 30 45 60 75 90 105 120 135 150 160 180 195 210 ~~~~~~~~~NEUTRALIZATION TIME IN guinea pig immune serum by use of different virus MINUTES dilutions both with and without complement. FIG. 4. Neutralization of RS virus Simon by immune horse serum without the addition of complement. Symbols: control serum (X), immunized serum (A), 6.0- virus control ( ). D 5.75- - ~~ x x ~ periods of incubation. Without complement, X maximal neutralization occurred after incubation 5.25iX X for 135 min at room temperature (Fig. 2). When 50 complement was added to the virus-immune > 4.75A serum mixture (Fig. 3), the time required for CD \ o \ / maximal neutralization was decreased to 105 > 4.5 min of incubation. In addition, a much greater 4.25 4.25 \ / amount of virus was neutralized by guinea pig immune serum with complement than was neu- 4.0 - tralized by guinea pig immune serum alone. 3.75 RS virus-immune horse sera were also tested 35 - I I I I I I I by method B neutralization slopes to determine 15 30 45 60 75 90 105 120 135 150 165 180 the effect of guinea pig complement on its neu- NEUTRALIZATION TIME IN MINUTES tralizing capacity. From the data obtained (Fig. FIG. 2. Neutralization of RS virus Simon by im- 4 and 5), it appears that the addition of 10 mune guinea pig serum without the addition of com- hemolytic units of guinea pig complement does plement. Symbols: control serum serum (A), virus control (*). I (X), immunized not enhance the neutralizing capacity of the RS virus-immune horse serum. -~~~~

VOL. 16, 1968 EFFECT OF COMPLEMENT ON RS VIRUS 1079 Effect of filtration on viral neutralization. The 5.25 recent report by Wallis and Melnick (6) pointed 50E out the effect of viral aggregation on the neutrali- CD zation of various viruses by specific antibody. W 425 X X The following studies were carried out to de- Ix termine the effect of filtration of RS virus on 35 - subsequent neutralization by immune serum with > 3.25 - and without complement. RS virus-immune 2-- horse serum was tested by using filtered virus 2.5 - both with and without complement. Without 2.25 the addition of complement (Fig. 6), the virus 2.01.75 titer had decreased by 2.75 log1o to 1020 TCID5o 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 per ml after incubation for 165 min. With the NEUTRALIZATION TIME IN MINUTES addition of 10 hemolytic units of complement FIG. 7. Neutralization of filtered RS virus Simon (Fig. 7), the total neutralizing capacity of the by immune horse serum with the addition of complehorse immune serum was not increased, as the ment. Symbols: control serum (X), virus control () maximal decrease in titer after incubation for immunized serum (A), virus control + C' (0). 164 min was also 2.75 log1o of virus. However, the virus titer decreased more rapidly (a 1.75-6.0 -,Q log1o decrease in titer after 30 min of incubation) when complement was added (Fig. 7). Without 5.75 S-the addition of complement, no significant de- X x x crease x in virus titer was observed after incubation 5.25 t \ os for at least 30 min. so- f/ldentical I tests were done with guinea pig 4.5_Xtimmune serum and filtered virus with and with- _ \ out complement. The filtration of the virus did not appear to affect the neutralizing capacity VS4.25 Y --s of the guinea pig immune serum. However, the 4.0 addition of complement did increase the rate of virus neutralization (Fig. 3) with unfiltered 3.75 ~I- I virus. 3.5 15 30 45 60 75 MU 105 120 135 150 165 180 195 210 D iscussion NEUTRALIZATION TIME IN MINUTES FiG. 5. Neutralization of RS virus Simon by im- Morgan (3), working with western equine mune horse serum with the addition of complement. encephalitis virus, reported that the apparent Symbols: control serum (X), virus control + C' (0), loss in neutralization titer of rabbit immune sera immunized serum (A), virus control (0). stored at 4 C could be reactivated by the addition of guinea pig complement. Whitman (7) 6.25 also found an enhanced neutralizing effect of 6.0 unheated normal human, guinea pig, or monkey 5.75 -sera on human immune sera to western equine 525 1{--Xencephalitis X @ virus. Dozois et al. (1) found rabbit 5.25 - o 5. II -x-x complement to enhance the neutralizing activity cc 4.754( \of rabbit immune sera to western equine en- '425 ; cephalitis virus. Rawls et al. (4) also found that 4.0 - the addition of fresh guinea pig complement Z, 3.75 - \, enhanced rubella antibody titers in sera obtained 3.25 >both from individuals with recent rubella in- 3.0 - fections and from individuals with remote 2.72 - \ histories of rubella. Yoshino and Taniguchi (8) 2.52s L employed \ 10 hemolytic units of guinea pig com- 2.0 15 30 45 60 15 0 go 105 120 135 150 165 180 195 210 plement for the detection of early complement- NEUTRALIZATION TIME IN MINUTES requiringherpes simplex virus antibody in rabbits. FIG. 6. Neutralization of filtered RS virus Simon Our results indicate that the neutralizing caby immune horse serum without the addition of com- pacity of RS virus-immune sera was enhanced by plement. Symbols: control serum (X), immunized the addition of 10 hemolytic units of guinea pig serum (A), virus control (0). complement.

1080 BAUGHMAN ET AL. APPL. MICROBIOL. Neutralization slopes, done by using dilutions of both virus and serum, proved the importance of not overwhelming the antibody present in immune sera with a high multiplicity of virus. These tests indicate that a TCID5o of 30 to 100 per ml of test virus should be used in neutralization tests when readings for cytopathic effects are made 5 to 6 days postinoculation. An enhancing effect of complement was observed on the serum titers when readings were made 5 to 6 days postinoculation. Neutralization slopes, done with a constant amount of both virus and serum, further showed the effect of complement on the neutralizing capacity of guinea pig immune serum. With the addition of complement, only 105 min were required for maximal neutralization, as compared to 135 min without complement. Yoshino and Taniguchi (9) observed that an increase in incubation time would also increase the neutralization of herpes simplex immune sera without the addition of complement. Wallis and Melnick (6) reported that aggregated non-neutralizable fractions of several viruses could be removed by filtration. Poliovirus, vaccinia virus, herpes virus, reovirus, and influenza virus could not be completely neutralized unless passed through a proper-sized filter membrane. They found, however, that adenovirus could be completely neutralized without prior filtration of the virus. The neutralizing capacity of RS virus-immune horse serum was greatly increased with the use of filtered virus over that observed with unfiltered RS virus. The addition of complement did not appear to increase the maximal neutralizing capacity of the RS virus-immune horse serum to filtered RS virus, but did increase the rate of neutralization. The maximal neutralizing capacity of guinea pig immune serum for RS virus was not increased with the use of filtered virus. Complement did, however, appear to increase the rate of virus neutralization. ACKNOWLEDGMENTS We gratefully acknowledge the excellent technical assistance of Alan J. Emerick throughout these experiments. We are also grateful to Marcella A. Zinzilieta for helpful advice while this work was in progress. This work was supported by the Vaccine Development Board of the National Institutes of Health under contract PH43-62-487. LITERATURE CITED 1. Dozios, T., and J. Wagner. 1949. The influence of certain serum factors on the neutralization of equine encephalomyelitis virus. J. Immunol. 62:319-331. 2. Dulbecco, R., M. Vogt, and A. G. R. Strickland. 1956. A study of the basic aspects of neutralization of two animal viruses, western equine encephalitis virus and poliomyelitis virus. Virology 2:162-205. 3. Morgan, I. 1945. Quantitative study of the neutralization of western equine encephalomyelitis virus by its antiserum and the effect of complement. J. Immunol. 50:359-371. 4. Rawls, W., J. Desmyter, and J. L. Melnick. 1967. Rubella virus neutralization by plaque reduction. Proc. Soc. Exptl. Biol. Med. 124:167-172. 5. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty per cent end points. Am. J. Hyg. 27:493-497. 6. Wallis, C., and J. L. Melnick. 1967. Virus aggregation as the cause of the non-neutralizable persistent fraction. J. Virol. 1:478-488. 7. Whitman, L. 1947. The neutralization of western equine encephalomyelitis virus by human convalescent serum. The influence of heat labile substances in serum on the neutralization index. J. Immunol. 56:97-108. 8. Yoshino, K., and S. Taniguchi. 1966. Evaluation of the demonstration of complement-requiring neutralizing antibody as a means for early diagnosis of herpes virus infections. J. Immunol. 96:196-203. 9. Yoshino, K., and S. Taniguchi. 1965. Studies on the neutralization of herpes simplex virus. Virology 26:44-53.