Studies on neutralization of japanese encephalitis virus (JEV)

34, 141-148 (1968)

VIROLOGY

Studies I. Further

on Neutralization Neutralization

Antivirus

of Japanese

of the Resistant Virus Fraction

IgG Antibody

and IgG Heterotype

TATSUO Institute

Encephalitis

IWASAKI

for Virus

AND

Research, Kyoto

Virus

by an Interaction

or Allotype

(JEV) between

Antibody

RYO OGURA University, Kyoto, Japan

Accepted September 25, 1967

When JE viruses interact with anti-JEV mouse IgG antibody (anti-JEV antibody), a resistant virus fraction remains which is not neutralized further by the antibody. This resistant virus fraction was neutralized by anti-mouse IgG rabbit IgG antibody (heterotype antibody) or anti-mouse IgG mouse IgG antibody (allotype antibody). The kinetics of this secondary neutralization was of the first order, as was that of the first neutralization by anti-JEV antibody. The rate of the secondary neutralization was proportional to the concentration of the heterotype or allotype antibody. The first neutralization could be reversed in part by dilution, and the susceptibility of the resistant virus fraction to anti-JEV antibody was increased upon dilution. On the contrary, the susceptibility of the resistant virus fraction to the heterotype or allotype antibody decreased with dilution, These findings suggested that the secondary neutralization of the resistant virus fraction was resulted in the interaction between reversibly combined anti-JEV antibody and the heterotype or allotype antibody. INTRODUCTION

A number of reports have appeared that are concerned with the persistence of a virus fraction resistant to neutralization following the reaction between the virus and the vira1 antibody. Although experiments on the nature of the resistant virus fraction have provided contradictory results, Jerne and Avegno (1956) and Mandel (1961) have demonstrated the occurrence of both reversibly and irreversibly neutralized virus. Moreover, Lafferty (1963a) demonstrated that the resistant fraction resulted from the presence in the antiserum of nonavid antibody which would combine reversibly with the virus surface but was unable to form a stable combination with the virus surface. Mandel reported (1958) that anti-rabbit r-globulin serum inhibited plaque formation of poliovirus in plate cultures that had been exposed to the virus-viral antibody (induced in rabbits) mixture. More recently, Notkins et al. (1966) have suggested from their

experimental findings with lactic dehydrogenase virus (LDV) that sensitization of the virus with antiviral antibody might play an important role in the resistance and susceptibility of a virus to neutralization by antiviral antibody, and an anti--y-globulin might prove useful in neutralizing the resistant fraction. In order to elucidate the nature of the resistant fraction, we demonstrated the further neutralization of the resistant virus fraction with anti-y-globulin and examined the kinetics of the reaction in our experimental system. This communication describes the results of these experiments. MATERIALS

AND METHODS

V&s strain. The Mukai strain of JEV used throughout these experiments has been described previously (Iwasaki and Inoue, 1961). Growth and assay of virus. The virus was grown on monolayer cell cultures of the 141

IWSSAKI

142

AND OGTT1:.4

PS(Y-15) ccl1 line (Inoue and >-amada, 1964). Infected cultures in which a cytopathic effect could be identified were subjected to t.hree rapid cycles of freezing and t.hnwing, and the cell debris was removed b!, centrifugation at 5000 ~1for 30 minutes. The supernutant, fluids were harvested and used as crude virus material. The virus was assayed for infectivit#y by the plaque assay method previously described (&to and 111011c, 196%, 1)). Cmcenfmt

imz and

put~$cafGn

of JKI’.

The crude virus nlaterial was concent.ratcd and purified by a procedure involving conwntrat,ion by :L t(wo-phase polymer system and purifcation by Sephadex G-100 gel filtration, as described previously (Iwasaki and Ogura, 1964; Nakai, 1965): Sodium d&ran sulfat#e fiS and polyet,h;vlcne glycol 6000 \\-ere :tdded to the crude material of th(, virus 10 give a final composition of 0.2 (\\-1\\,) and 6.45 (2 (\v/\\-), respectively. The mixture was allowed to stand overnight $11; 4” for phase separat,ion. The viruses I\-erc c~uccntr:~ted into a small hott’om phase :md an int c~rphwse. To get, a new phase syst,em wit,h the bott,om phase and the interphase, NaCl \v:w added up to :t concentration of nbout 1 mole. All the virus act’ivity t\-as found in t#he t,op phase. The virus makrial in the top phase was further purified b> gel filtration on :1 Sephadex G-100 column. The eluted viruses in a single peak were concent rated by polycthylenc glycol 6000, and the final virus m:krial was used. Inacfizlatim of’ airus by ulfraciolef (1-L’) li&. .JEV was ‘inact,ivat,ed by 17 light in a petri di& agitat)ed 15 cm alvay from a l;,-\vat t8 germicidal lamp. This t’reatment resuked in :I loss of 7.G log,, in virus infect.ivity in 12 minut,es. ‘IJV irradiation under this conditit n1 did not affect t#he antigenicity of .JE;V. P,~eparafion (?f’anpi-JET’ Sew?n. Anti-,JEV sern lucre prepared in S-week-old mice, strain BALB/C. Mice were injected intraperit,one:dly 3 times at. intervals of 3 days ivith 0.5 ml of highly purified, 13~-irrndiatZed JEV antigen containing lo9 5 PFU before T:V-irracii:ltion, and 3 lveelrs after the first . . . mJectwn, m addit,ional injection was given. Iking n-eel; 4, the mice were bled. The

blood was allo\\-cd t,o clot and the serum W:U collected and inactivated by heating at, 56” for 30 minutes, and t,hen stored at -70” . FracfimLation of I& antibodies. As &scribed by Fahey et al. (1958)) for chromatography on IYEAE-cellulose, antisera wre dialyzed against 0,015 31 in a pH 8.0 potassium phosphak buffer and applied to :I 2 x 20 cm column of DIUE-cellulose equilibrated I\-ith the same hoffw. lr :k similar procediw lvit II irljections of norm:d guinea pig Ig(;. :\ntimouse IgG mouse serum was prep:wcl b:, injecting A !,J:W mice irltr:lprritoIre:rllv with TgG obt,uincd from norm:d IMLH ;i’ tnicc, three times a. keel< for :i ~~wl;s. Stwutl ~-as obtained 7 days :&cr tho lwt injection. All IgG fractiolw obtained From o:wh :ultiserum gave strong precipitation b:r.rktls in tests with 11~ C’,jrIllicro-Ouchterlon!. responding Tg( ;. RESULTS

I1pproxin~at’ely 10” PYl./ml of JF3’ \Y:I~ mixed with an equal volunw of a 1: 10 dilution of anti-JEV mouse I& antibod?.; IgG obt,ained from normal mouse sera served as the control. The mixtures \vere incubated at 37” in a lvater bath, and ;it wrious time int#ervaln samples \vere removed from t,he reaction mixture. The reaction mixtures were serially diluted and the virus titers determilled. The virus survival is plotted in Fig. I. The virus was r:lpitlly neutralizcrl to 10WO of tlw initial titw for 1.5 minut,es, and thcrenfter much more slo~4y. as has been described earlier (Lklhccco e/ al., 1956). This resistant frwtion was desigwited as the persistwt fraction (IMbwco

NEUTRALIZATION

OF JEV

143

TABLE 1 EFFECT OF THE HETEROTYPE OR ALLOTYPE IgG ANTIBODY ON INFECTIVITY OF NONRESISTANT AND RESISTANT VIRUS FRACTIONS

\

-

W

Nonresistant Resistant fractiona fraction* incubated at incubated 37” for at 37” for 0 min

0

5

IO 15 20 TIME IN MINUTES

25

30

FIG. 1. Kinetics of neutralization of JEV. Equal volumes of JEV (8 X 106 PFU/ml) and anti-JEV antibody (1:lO) were mixed. The mixture was incubated at 37”. At various intervals samples were removed and assayed. et al., 1956) or the protect.ed ferty, 1963a).

fraction

(Laf-

E$ect of the Heterotype or Allotype Antibody m Further Neutralization of the Resistant Virus Fraction Many investigators have reported on the nature of the persistent fraction. Jerne and Avegno (1956), Mandel (1958, 1961), and Lafferty (1963a) have indicated that in the resist’ant virus fraction there are reversibly and irreversibly neutralized viruses. Jerne and Avegno (1956) and Lafferty (1963a) suggested that reversibly neutralized viruses were caused by nonavid antibody in the serum. Moreover, Lafferty (1963b) demonstrated that the monovalent antibody produced by the digestion of immune y-globulin wit’h papain could protect influenza virus from irreversible neutralization by divalent antibody, and suggested the nature of nonavid antibody from these findings. On the other hand, Mandel (1958) and Notkins et al. (1966) showed from their findings that an anti-r-globulin was capable of neutralizing the resistant virus fraction.

Anti-JEV mouse IgG Anti-mouse IgG rabbit W Anti-mouse IgG mouse I@ Anti-guinea pig IgG rabbit IgG Anti-guinea pig IgG mouse IgG Normal rabbit IgG Norma1 mouSe ‘gG

- -I

60 0 60 min minI min ____

7.2c 7.2

5.2 7.0

5.3 5.3

5.1 3.8

7.3

7.1

5.3

3.6

7.2

7.0

5.3

7.2

7.0

5.3

7.2 7.2

7.0 7.0

5.3 5.3

a Nontreated JEV were referred to as nonresistant fraction. b JEV which were incubated at 37” for 30 minutes with anti-JEV antibodies were referred to as resistant fraction. c Virus titers for infectivity were expressed in PFU per milliliter (loglo).

Therefore, in order to see whether the heterotype or allotype IgG antibody neutralizes the resistant fraction of JEV, approximately 10’ PFU/ml of JEV was incubated with an equal volume of a 1: 10 dilution of anti-JEV antibodies at 37” for 30 minutes when the reaction had reached a stationary phase, and then the mixture was further incubated with an equal volume of a 1: 10 dilution of the various IgG preparations at, 37” for 1 hour. The reaction mixtures were serially diluted and the virus titer was determined. As shown in Table 1, anti-mouse IgG rabbit IgG antibody and anti-mouse IgG mouse IgG antibody neutralized the resistant virus fraction while other IgG preparations had no effect. Kinetics of Neutralization of the Resistant Virus Fraction by the Heterotype or Allotype Antibody In order to elucidate the kinetics of neutralization of the resistant virus fract,ion

144

IWASAKI

AND

OGURA

E$ect of Concentration of the Heterotype 01 Allotype Antibody on the Rate of Neutralization qf the Resistant Virus Fracticm Attempts were made t’o determine the effects of t’he concentratjion of heterotype or allotype on the rate of neutralization. The virus, in a concentration of 5.5 X 106 PE’U/ml, n-as incubated with an equal volume of a 1: 10 dilution of anti-.JIW mouse IgG a&bodies at’ 37” for 30 minutes

c

FIG. 2. Kinetics of neutralization of the resistant virus fraction by heterotype or allotype antibody. Equal volumes of virus (5 X lo6 PFU/ml) and anti-JE\antibody (1:lO) were mixed. The mixtures were incubated at 37”. After 30 minutes’ incubation, heterotype antibody (1:lO) or allotype antibody (1: 10) was added to each mixture. After various intervals samples were removed and assayed. 0: Titer of the mixture consisted of JEY and anti-JE\. antibody; 0: Tit.er of the mixture consisted of the resistant virus fraction and heterotype a&body; A: Titer of the mixture consisted of the resistant virus fraction and allotype ant,ibody.

by the heterot’ype lo7 approximately incubated with an

or

&type

FIG. 3a. Effect of concentration of heterotype antibody on the rate of neutralizat,ion of the resist,ant JEV fraction. The kinet,ic curves were determined by using the resistant JEV fraction at the concentration of 7 X lo4 PFU/ml. The concentrations of the antibody were I:1 (0), I:3 (a), and 1:lO (A). 0.5 0.5‘i f

0.4 0.4 1

5 ‘$ 0.3 ” 2 ,o ‘; 0.2-

antibody,

PFU/ml of virus was equal volume of a 1: 10 dilut,ion of antiserum at 37”, samples were removed at, various intervals of time, and the virus titer was determined. The results with ant,i-mouse IgG rabbit IgG antibody and anti-mouse IgG mouse IgG antibody are shown in l;ig. 2. It) n-as demonstrakd that t,he heterotype or allotype antibody could neukalize the resistant virus fra&on by a fir&order reaction.

r

z”

73 bo.l0.1 ” ‘a yy -,I0 0 t I

clI// Relative

Concentration

Heterctype

FIG.

ization body.

I IO

5

3b. Relationship and concentration

of

Antibody

between rat,e of neutralof the heterotype anti-

NEUTRALIZATION

when the reaction phase, and then were carried out fraction, in which

had reached a stationary the kinetics experiments using the resistant virus the virus titer was 11.2 X

145

OF JEV TABLE EFFECT

OF DILUTION RESISTANT

2

ON REACTIVATION VIRUS FRACTION

-

Virus-antibody

Titer of virus sur-

mixture

Titer of virus Dilusurvival before tion dilution factor Virus PFU AntiIO.2 ml body

A

F,\

21 x 103

1:lO

112 f

10.8”

30 x 10s

1:lO

152 f

12.2

\ L-

OF THE

5 10 50 10 100

vival after dilution

-. 25 15 8 22 6

f f f f f

4.1 3.5 3.0 3.9 2.9

0'

a Standard deviation of plaque numbers of 4 bottles. 1

5

IO Time

I5

20

in

Minutes

25

30

FIG. 4a. Effect of concentration of allotype antibody on the rate of neutralization of the resistant JEV fraction. The kinetic curves were determined by using the resistant JEV fraction at the concentration of 7 X lo* PFU/ml. The concentration of the antibody was 1:l (01, 1:3 (a),

and 1:lO (A).

E$ect of Dilution 012 Reactivation Resistant Virus Fraction

0.6 -

i,

lo3 PFU/ml. The relative concentrations of heterotype antibody used were 1:3: 10, and those of the allotype antibody were 1: 2: 10. The results of experiments with heterotype or allotype antibody are shown in Figs. 3a, b and 4a, b. The rate of neutralization by the heterotype or allotype antibody was found to be proportional to the concentration of the antibody. of the

To understand the mechanism of the secondary neutralization of the resistant virus fraction by heterotype or allotype antibody, it is necessary to clarify the nature of the resistant virus fraction, although many investigators have described it. First, tests were conducted to determine whether or not the virus titer of the re-

OS-

z ):

sistant

Relative

Concentration

Allotypc

Antibody

of

FIG. 4b. Relationship between rate of neutralizat.ion and concentration of allotype antibody.

virus

fraction

was increased

upon

dilution. Mixtures of JEV and anti-JEV antibody were incubated at 37” for 30 minutes, when the reaction had reached a stationary phase. Some mixtures were assayed without dilution, and others were diluted. The diluted mixtures were incubated again at 37” for 1 hour and assayed. The results are shown in Table 2. Partial reactivation of the resistant virus fraction upon dilution could be observed in these experiments.

146

IWASAKI

AND TABLE

EFFECT OF DILUTION

OGUKA 3

OF THE RESISTANT VIRUS FR.LCTION ON SUSCEPTIHILIW ~~~~~~~~ IgG ANTIBODIES Dilution

kG

1 Beforea

a Titers b Titers

(log,, PFU/ml) of the diluted

TO

factor of the resistant virus fraction

5 ..-. __&ifter” Before After

20 -.---~_.--. Before .ifter

100 Before

After

of the diluted specimens before incubation at 37” with various Ig(+ antibodies. specimens after 1 hour of inclthation at 37” with various Ig( + ant ibodirs.

h’flect of Dilution of the Resistant Vivus Fraction ma Susceptibility to Neutralixation by anti-JEV, or Heterotype 0) Allotype Antibody A study was made of the alterations of suscept.ibility of the virus fraction resistant to antiJEV, or heterotype or allotype antibodies upon dilution. After 30 minutes’ incubation at 37”, mixtures of JEV and anti-JEV mouse IgG antibody were assayed. Then t’he mixtures were diluted and incubated with an equal volume of a 1: 10 dilution of anti-JEV, or of heterotype or allotype antibody at 37” for 1 hour and assayed again. As seen in Table 3, the virus in the resistant fraction showed increased suscept,ibility to anti-JEV antibody, but decreased susceptibility to heterotype or allotype ant,ibody upon dilution. DISCUSSION

The experiments described in this paper show that .JEV in the resistant fraction upon neutralization by anti-JEV antibody could be further neutralized by heterotype or allotype antibody, and that the susceptibility of t,he resistant virus fraction to heterotype or allotype antibodies decreased upon dilution, while that to anti-.JEV antibody was in part restored upon dilution. These findings suggest that a reversible combination between the virus and the viral antibody exists in the resistant. virus fraction, and that het,erotype or allotype

antibodies interact wi-ith t’he anti-J P:V :Lnt.ibody which is reversibly combined t,o t,he virion, with resulting loss of infectivity. The existence of a reversible ncutralizntion in the resistant fraction has been demonskated by many investigators (,Jernc and Avegno, 1956; I’azekas de St. Grot.h et al., 1958; E’azekas de St. Groth and Webst,er, 1963; Mandel, 1961; KjelIBn, 1962; Lafferty, 1963a, b; Westaway, 1965), ahhough contradictory reports have been preswted (Dulbecco et al., 1956). hloreover, Lafferty (196&j attributed, at least. in part the persistence of the resistant fraction, the so-called protected fraction (1963a), to the presence of nonavid antibody that would combine reversibly with t’he virus surface but was unable to form a stable combinat.ion \vith it. He indicated that the reaction between virus and antibody occurred in tM.0 stages-- the first a reversible combination, :wd the second stabilization of the combinut,io~l-~ulltl suggest’ed that the primary comkin:&n represent’ed the interaction of one active sit,e 011 t,he ant#ibody molecule n-ith a11 antigenic site 011 the surface of the virus part’icle. md the second stabilization represented the combination (Jf the free site of the antibody with another sit,e on the same virus particle. Therefore, the secondary neutr:tlization of t’he resistant fraction by the heterot-ype or allotPype ant’ibody could be :ktribut.ed t 1)the secondary stabilization rcsponw by t,he heterotype or allotype antibt dy. I’urt.hcr-

NEUTRALIZATION

more, t,o consider the mechanism of the secondary neutralization by the allotype antibody, it may be convenient to know the localization of allotypic specificity on the mouse IgG molecule. Recently, the localization of allotypic specificity was found to be carried on the Fe- fragment of the IgG molecule (Lieberman et al., 1965; Dray et al., 1965; Potter et al., 1966). Accordingly, the allotype antibody molecule would combine wit’h the PC locus of the anti-JEV mouse IgG molecule, which combines with t’he surface of the virus. If some of the viral antibody molecules combine reversibly with sites on the virus particle in close proximity, they might form a bridge between two viral antibodies. This bridge formation would result in the same effect as the two-site attachment of the viral antibody to the surface of the virion. The mechanism of the secondary neutralization by the heterotype antibody can be understood as analogous to the former case, although the fraction of the heterotype antibody may contain various molecules corresponding to the various sites of the viral ant,ibody molecules. However, we do not yet have any information on the occurrence of the resistant virus fraction in the secondary neutralization by the heterotype or allotype antibody. ACKNOWLEDGMENTS We wish to thank Mrs. E. Ochiai technical assistance.

for her skillful

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147

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14s

IWASAKI

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AND

OGURA

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