lmmunochemlstrv 1975 Vol 12 pp 597 601 PergamonPress Printedill Great Britain

NEUTRALIZATION KINETICS OF WESTERN EQUINE ENCEPHALITIS VIRUS BY ANTIBODY FRAGMENTS N A T A L I E E. C R E M E R , J O H N L R I G G S and E D W I N H. L E N N E T T E V~ral and Rlckettsml Disease Laboratory, Cahfornm State Department of Health, Berkeley, California 94704, U S A (Recewed 29 October 1974) Abstract--Antibody against western equine encephalms virus from the isolated and purified IgG fraction of hyperlmmune rabbit serum was dlgested with papaln or pepsin and the neutrahzmg abdlty of the resulting 5S {F(ab')2) and 3 5S (Fab I, II) fragments compared to that of the 7S molecule. By plaque reduction assay, the neutralizing capacity and neutralization kmeucs of the F(ab') 2 fragment was as effective as the 7S molecule even if the protein concentration of the F(ab')2 was lowered to approximate the mole concentration of the 7S molecule. The neutrahzmg abdlty of the Fab fragments was less effective, the kinetics of neutralization slower and the nonneutralizable fractmn larger even when the concentration was adjusted to two times that of the whole molecule An mltml shoulder m the neutrahzatlon kinetic curve was seen with all the preparations ff neutrahzaUon was carried out at 4°C but ~t was less prominent or absent at 37~C No slgmficant &ssociatlon of the 7S-wrus, F(ab')2 or Fab II-wrus complexes occurred on dilution or somcat~on at the protein concentrations stu&ed At the lowest concentratmn of Fab I, s~gnlficant dissocmtlon of the vlrus-Fab I complex d~d occur after 5 mm somcat~on, resulting in a 24-37 per cent nonneutrahzable fraction as compared to 3 per cent after the m~tml incubation period No significant protectmn of infant mice occurred with any of the fragments after an hour incubation of the wrus-fragment mixtures at 37°C

INTRODUCTION In previous reports (Cremer et al., 1964, 1966) it was shown that papain a n d pepsin fragments of g a m m a globulin prepared in rabbits against western equine encephalitis (WEE) virus, when tested m an m vitro neutralization test (metabolic inhibition test), were able to neutralize the virus to approximately the same degree as did the whole antibody molecule. Neutrahzation occurred regardless of the species (chick or mouse embryo) used for the cell cultures. By in t,lt~o assay (mouse protectton test) the fragments showed little or no neutralizing ability. The present study was initiated in order to study the v~rus neutrahzatlon by more quantitative methods (plaque assay), and to study the kinetics a n d some factors which may influence to different degrees m vitro neutralizations of the vtrus by the whole molecule and by its fragments.

METHODS AND MATERIALS

Virus Two passage lines of western equine encephalms (WEE) vn'us, strain A 42 prewously described (Cremer et al, 1964) were used. One passage hne was carried only m Swiss mouse brain and the other only m chick embryo cells Plaque assay Infectious varus was determined by the plaque assay method. Cback embryo cell suspenmons were prepared as previously described (Cremer et al., 1966). The cells were grown in 60ram plastic petrl dishes in medmm 199 in Earle's balanced salt solution fortified with 10~o fetal bovine serum. The cultures were incubated at 36°C in

a 5°0 CO2 mr-humldtfied atmosphere. For infectious virus assay wral samples m 0"2 ml amounts were allowed to adsorb onto the cell monolayers for 2 5 hr at 36°C The cultures were then overlaid with 1 5% agar in the growth medium with the serum reduced to 5% After a 48-hr incubation period, neutral red was added to a final concentratlon of 1 10,000 and the plaques were counted after a further lncubatxon period of 24 hr. Antlsera Hyperlmmune antlsera were prepared m rabb~ts using the mouse passage hne of virus as antigen and the same dosage as previously described (Cremer et al, 1964) A schedule of four weekly injections of the wrus m incomplete Freund's adjuvant followed by one month's rest and three more weekly injections was used. Preparatton and analyses of IgG and fragments Isolauon of ),-globuhn by ammomum sulfate precipitation and DEAE column chromatography, estlmat~on of protein, analyses of lgG and fragments by mlcrolmmunoelectrophoresls, Ouchterlony test and analytical centrffugatlon were done as previously described (Cremer et al., 1964). Papam and pepsin digestion of IgG and isolation of fragments by column chromatography were also done as previously described (Cremer et al, 1964, 1966) B~ologw acttt, zt) of fraoments, mouse protection Mouse protection tests were performed as previously described (Cremer er al, 1964) with the exception that the virus-antibody mixtures were incubated for 1 hr at 37°C before inoculation into the 2-3-day old rmce, rather than at 4:C as previously done The neutrahzatlon index (NI) was calculated by the Reed-Muench formula (Reed and Muench, 1938) The LDso of vtrus was determined by reaction of WEE virus with normal rabbit IgG isolated and purtfied in the same manner as the ant~-WEE IgG 597

598

NATALIE E CREMER, JOHN L. RIGGS and EDWIN H LENNETTE

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Fig 2 Neutrahzatxon of WEE virus. Equal volumes of varying concentrations of the whole antibody molecule and fragments Fab I and II were mixed with an equal volume of WEE virus (approximately 560 PFU/ml) and were held for 1 hr at 37°C The mixtures were then assayed for surviving virus by the plaque technique RESULTS

Plaque morphology

Fig. 1 A. Small plaque vtrus. B. Large plaque virus. Small plaque 1 x and large plaque 0'5 x magnification

Biological activity of fragments ,n vitro For deterrmnation of virus neutrahzlng activity of antibody fragments and of the whole molecule, equal volumes of the appropriate dilutions of the antibody samples (protein concentration diluted initially to 0'02%) and 200-1000 plaque formmg units (PFU) per ml of virus were thoroughly mixed and held in a water bath at either 37cC or 5°C. At the end of the incubation period, 0"2 ml samples were assayed for infectious virus by the plaque assay method All assays were done in triplicate Eighty per cent plaque reduction was taken as the end point. For kinetic studies (performed at 37°C and at 5:C) equal volumes of appropriately diluted antibody samples in medium 199 in Earle's BSS containing 2% FBS and virus diluted m the same medium to contain approximately 2 x 105 PFU/ml were mixed Antibody samples and virus preparatmns were equilibrated at 5°C or at 37°C prior to mlxmg. At intervals of incubations after mixing, 1 100 and 1' 1000 dilutions were made and immediately plated for plaque assay of infectious virus. A virus-medium mixture was similarly treated and assayed at appropriate dilutions for infectious virus

O n monolayers of chick embryo cells, the chick embryo or mouse passage lines of the A-42 strain formed two plaques of differing morphology, one perfectly r o u n d large plaque, 1 0 m m average dmmeter a n d one irregular small plaque, 1.7 m m average diameter (Fig. 1). Variation in plaque size of W E E has been reported by others (Ushijima et al., 1962; M a r shall et al., 1962). The small plaque, selected for study, was p r e d o m i n a n t in b o t h virus passage lines. The virus stock, chick embryo passage, was plaque puri-

I01

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Dlssocmuon studies Equal volumes of appropriately diluted antibody samples (20, 10, 5, 2"5/~g protem/ml) or medium, and a dilution of virus stock containing approximately 1 × 103 PFU/ml were combined and incubated at 37°C, for 1 hr. The mixtures were assayed for surviving virus lmmedmtely after incubation, then diluted 1:20 with cold medium and assayed again. The mixtures were then somcated for 2 and 5 rain (Bronwill disintegrator, 20KC/sec) and reassayed after each period of somcatlon

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~.g Prol~tn/~z Fig. 3 Neutrahzatlon of WEE vtrus. Equal volumes of varying concentranons of the whole antibody molecule and pepsin digestion fragment (F(ab')2) were mixed with an equal volume of WEE virus (approximately 570 PFU/ml) and were held for 1 hr at 37°C. The mixtures were then assayed for surviving virus by the plaque technique.

Neutralmauon Kinetics by Antibody Fragments IC

599

F(ab')2 as effective as the whole molecule m neutrahzmg a given number of infecuous virus particles. Figures 2 and 3 give representative neutralization curves performed at 37°C.

Kmencs of neutralizatton of WEE wrus by antibody and Its fragments

I

20 40 go go 16o ~2o MInu?es

Fig 4 Kmetms of neutrahzatlon at 4°C of WEE virus

by the whole anubody molecule and pepsin dlgestmn fragment, F(ab')2 at approximately equal mole concentrations Equal volumes of the antibody samples (7S at 5 #g/ml, F(abh at 3'3/~g/ml)and of WEE virus (2'2 x l0 5 PFU/ml) were m~xed assayed at indicated intervals for surviving virus

fled by picking isolated small plaques at limiting dilutions and passing the isolates two additional times. Stock virus was then prepared and assayed for mfecuous virus and plaque morphology. Plaques of both types were ewdent in the stock virus prepared in this manner, therefore three further plaque purificatmn passages were carried out. Again evidence was obtamed that both types of plaques appeared m stock virus prepared from the sixth plaque passage with the small plaque the predommant type. Since the frequency of reversion of small plaque type to large plaque type was very small ( < 15o), and the small plaque type could be counted with greater ease, it was used in the experiments described below

Neutralization of WEE virus by the whole antibody molecule and its fragments On the bas~s of protein concentration the Fab fragments (FI and FII were less effectwe and the fragment

The kinetics of neutralizatmn at 37°C and 5°C with F(ab')2 were the same as those with the whole antibody molecules using either the plaque purified chick embryo virus (Figs. 4 and 5) or the nonplaqued mouse passage virus. The protein concentration of the antibody preparatmns was adjusted on a mole basis. There was an mmal lag in neutrahzat~on at 5°C which in different experiments extended from 15-35min. After this mmal lag, a strmght hne was obtained by plotting the surviving virus fraction vs time on semMogarithmlc paper. At increased antibody concentratlons the duration of the initial lag was reduced. Neutralization of virus at 37°C occurred more slowly and was less effective with the fragments relative to the 7S molecule despite the use of twice the protein concentration. (Fab = 10 #g protem/ml, 7S molecule = 5 #g protein/ml) (Fig. 6).

Effects of dllutton and somcation on stability of WEE virus wtth the whole antibody molecule and its digestion fragments No significant &ssocmtmn was observed at the end of the 5 mm sonicauon period between the virus and the whole molecule, the F(ab')2 or Fab II fragments at the higher protein concentrations. There was a trend toward a greater nonneutralgable fractmn after 1:20 dfluuon and somcatmn at the lowest protein concentration tested The increase in nonneutrahzed wrus however was shght. At reduced protein concentration the Fab I-virus complex showed slgmficant &ssocmtlon In two experiments after the 5 mln period of somcatlon 24-37 per cent of the wrus mocu-

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tel

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Fig 5 Kinetics of neutrahzaUon at 37°C of WEE virus by the whole antibody molecule and pepsin digestion frag- Fig 6 Kinetics of neutrahzatlon at 37°C of WEE virus ment, F(ab'h at approximately equal mole concentrations. by the whole antibody molecule and the Fab papam fragEqual volumes of the antibody samples (7S at 5 #g/ml, ments Equal volumes of anUbody sample (7S at 5 #g/ml F(ab'h at 3 3 #g/ml) and of WEE virus (1.5 x l0 s PFU/ml) and Fab at 10 #g/ml) and of WEE virus (2.8 x 10s PFU/ were rmxed and assayed at m&cated intervals for surv]vmg ml) were mixed and ~2 ml assayed at ln&cated intervals virus. for survavmg virus [MM 12

6 7

k

600

NATALIE E CREMER. JOHN L RIGGS and EDWIN H LENNETTE Table 1 Dissociation studies Percent nonneutralized virus b~ ~la~ue assay E)~.

Antibody

Protein concentration ~g/ml

Incubation, I he

~8 none~ 7S

86oOC ~] -1