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Biological Defense Research Lab ltr dtd 29Sep 1971

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TECHNICAL MANUSCRIPT 519* ... .....0

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INTERACTION OF116: VENEZUELAN EQUINE ENCEPHALOMYELITIS VIRUS

0*5000*OWITH NEUTRALIZING ANTIBODY:II. THE PERSISTENT VIRUS FRACTION

Nicholas Na0ho"

1H E LC O- C d) Lm

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MARCH 1969. t~L) u C

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Frederick, Maryland u- m

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DEPARTMENT OF THE ARHYFort Detrick

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TECHNICAL MANUSCRIPT 519

INTERACTION OF VENEZUELAN EQUINEENCEPHALOMYELITIS VIRUS WITH

NEUTRALIZING ANTIBODY:II. THE PERSISTENT VIRUS FRACTION

Nicholas Hahon

'..

* ~. b ;i* . . ;t

SExperimental Aerobioloty DivistonAUROBIOLO & EVALUATION LABORATORIES

Project 13562602AO59 March 1969

I

2

In conducting the research described in this report, theinvestigator adhered to the "Guide for Laboratory AnimalFacilities and Care," as promulgated by the Committee onthe Guide for Laboratory Animal Facilities and Care of theInstitute of Laboratory Animal Respurces, National Academyof Sciences-National Research Council.

ACKNOWLEDGMENT

The technical assistance of W. Douglas Zimmerman is gratefullyacknowledged.

ABSTRACT

The persistent virus fraction that results from the interaction ofVenezuelan equine encephalomyelitis (VEE) virus with antiviral serumis an infectious virus-antibody complex (sensitized virus) that canbe neutralized by anti-IgG serum. The quantities of virus sensitizedby VIE antiserum and neutralized by anti-IgG serum depend on theconcentration of these sera. In contrast to the marked temperatureand time dependence of VEE virus neutralization by antiviral serum,neutralization of sensitized virus by anti-IgG serum is more rapid(almost complete within I min at 35 C) and less sensitive to temperature.Evidence that virus sensitization preceded neutralization indicatesthat the persistent virus fraction is formed before virus neutralizationhas actively begun. Within certain limits, neutralization of sensitizedvirus by anti-Ig serum is species-specific. Differences in the abilityof anti-IgG, anti-IgA and anti-IgH sera to neutralize sensitized virusindicate that the reaction is also influenced by the specificity ofthe anti-imunglobulin.

Sensitized virus was partially neutralized by goat antiserum tomonovalent Feb fragments on human Igi and, to a lesser degree, by theVc fragment. Sensitized virus was neutralized by an in vitro mixtureof these fragments to almost the same degree as that neutralized bygoat antiserum to intact human IgG. Indirectly, these findings suggesta role for the Pc fragment in virus neutralization.

3

CONTENTS

Acknowledgment ............ . . . . . . . 2Abstract ..................... ........... .. . . 2

I. INTRODUCTION ........... o . .................. 5

II. MATERIALS AND METHODS . .... ... ... . . 5A. Virus L. . . ..... 5B. Virus Antiserum . . . . . . . . . . ....... 5C. Cell Lines .. oo......... o6

D. Kinetic Neutralization Procedure ........... . 6E. Immunological Reagents . . ... . . 6F. Virus Sensitization and Assay . . . . .. . . #. 6

III. RESULTS ..... . . o. ................. 7A. Multiplicity Curve . . . .. . . ......... . ... 7B. Factors Influencing Appearance and Magnitude of Persistent

Virus Fraction . . o o .. . .. .. .. . . ... ... .. . 7C. Neutralization of Persistent Virus Fraction by Anti-IgG

Serum .. . . . . . . . . . . . . .13

D. Parameters of Neutralization of Sensitized Virus by Anti-Ig *

IV. DISCUSSION . . . .............. . o . . . . . o . . 23

Literature Cited.. .. . . . . . . . .o 27Distribution List ...... . .. . .. . . . . . . .. . . 31DD Form 1473 o . . . . . . o . o o .. . . . .. .. 33

FIGURES

l. Multiplicity Curve of Neutralization of VES Virus . . . . . 82. Kinetic VER Virus Neutralization Reactions with Prior Filtration

or Sonic Treatment (20 kc/sec for 2 win) of Virus o . o . . . . o 103. Effect of Heated (56 C, 30 win) and Fresh Guinea Pig Serum on

the Kinetic VEE Virus Neutralization Reaction . . ...... 114. Levels of Persistent Virus Fractions in Kinetic VEZ Virus

Neutralization Reactions Assayed on Different Cell Lines ..... 125. Kinetic Neutralization of VEZ Parent, Progeny, and Attenuated

Viruses by Monkey Antiserum to Parent Virus . . . . . . . . . . . 146. Kinetic Neutralization of VEE Virus by Monkey Antiserum and the

Neutralization of the Persistent Virus Fraction by Goat Anti-Monkey IXC Serum . . . . . . ....... . . . . . . . . . . 15

4

TABLES

1. Effect of VEE Antiserum Concentrations on Virus Sensitization 17L2. Neutralization of Sensitized VEE Virus by Different Concentrations

of GatAnti-Monkey IgG Serum. . ........... . . 183. Effect of Time and Temperature on the Neutralization of*Sensitized

VEE Virus by Goat Anti-Monkey IgG Serum. .. ............ 194. Sensitization Preceding Neutralization of VEE Virus by Antibody 205. Neutralizatior of Sensitized VEE Virus by Goat Anti-IgG Serum

ot Lffcrz.nt Specie!!. ......... *.. ... .. 216. Sensitization of VEE Virus by Human or Monkey*Antiserum with

Cross-Neutralization by Homologous and Reterologous Goat Anti-

1. Neutralization of Sensitized (VEE Human Antiserum) Vius by ..... 2

Different Classes of Goat Anti-Human lmmunoglobulins . . . . . . . . 22S. Neutralization of Sensitized (VEE Humn Antiserum) Virus by

Goat Antiserum to Papain-Digested P'ragmentz ' IgG .. .. .. .. .23

5

I. INTRODUCTION*

Recent studies on the interaction of VEE virus with antibody showedthat neutralization reaciioas were initially first order and then followedcurvilinear kinetics.' This anomalous behavior, reported with differentanimal-virus and bacteriophage neutralization systems, 2-, has beenattributed to a small but definite fraction of a viral population thatresists neutralization even in the presence of excess antiserum afterprolonged treatment. Several hypotheses have been proposed to accountfor this phenomenon: (i) heterogeneity of ,iral population .f whicha distinct portion resists neutralization," (ii) aggregation of virusparticles by antibody,"0 (iii) nonavidity of antibody, 6 (iv) dissociationof the virus-antibody complex," (v) preaggregation of virus in suspension,'3

and (vi) formation of infectious virub-antiody complexes that stericallyhinder attachment of additional antibody.13 In view of supportiveexperimental evidence for each of these diverse hypotheses, the possibilityexists that the manifestation of a non-neutralizable virus fraction oftenobserved in kinetic neutralization tests may involve several factors,acting either individually or ;oncertedly: e.g., the state of virus, thequality of antibody, the host cell system, or the circumstances ofneutralization. At present, the phenomenon has not been adequately explainedor the problem completely resolved.

This report describes an attempt to elucidate the phenomenon of thenon-neutralizable virus fraction as it relates to the VEE virus-antibodysystem.

II. HATE IALS AND METHODS

A. VIRUS

The source and prer ration of the Trinidad strain of VEE virus wasdescribed previously.i The attenuated VEE virus strainis was obtainedfrom Dr. William A. Hankins, Fort Detrick, in the form of a susp. -sionderived from infected chick fibroblast cell cultures. Both virus strainswere assayed by an imunofluore-acent cell-counting procedure. 4

B. VIRUS ANTISKROM

VEE antiserum was obtained from rhesus monkeys that had been exposedI month earlier to an aerosol of approximately 1,000 cell-infecting units(CIU) of virus. Human antiviral serum Was obtahed from a convalescentpatient. Sets were inactivated at 56 C for 30 min.

* This report should not bc used as a literature citation in material to bepublished in the open literature. Readers interested in referencing theinformation contained herein should contact the author to as-rtalnwhen and where it may appear in citable form.

6

C. CELL LINES

The McCoy cell line was used routinely for the assay of virus; itscultivation and maintenance has been described previuasly.' Other establishedcell lines and their media for growth were hamster kidney and L-929 cells,medium 199 with 10% fetal calf serum (FCS); guinea pig lung cells, basalmedium Eagle (BME) with 10. FCS and 0.5. lactalbumin hydrolysate; Chang'shuman conjunctiva cells, 109 with 10% FCS. Hamster kidney a7d L-929 cellswere maintained with medium 199 and 5% FCS; guinea pig lung and humanconjunctiva celils with BIE and 5% FCS.

D. KINETIC NEUTRALIZATION PIOCEDURE

Although the reactants and experimental conditions of neutralizationvaried; In geniral, a suspension containing 1 x 108 CIU of virus per ml wasmixed with an equal volume of appropriteiy diluted monkey antiviral serum.Roth reactants were prewarmed to the temperature employed for incubation(35 C) before mixing. At intervals of incubation, 2 ml of the mixture waswithdrawn from the reaction tube and placed immediately into a vial heldin ice wter to arrest neutralization. Test samples were then diluted incold phosphate-buffered saline (PBS), pH 7.1, free of calcium and magnesiumions, and assayed for unneutralized virus. A control mixture, consistingof virus and normal monkey serum of toe same concentrations and volumesas the test mixtures, was employed with each kinetic neutralization deter-mination. The surviving fraction of virus was determined from the ratio ofunneutralized virus in the reaction tube to the virus titer in the controltube at the designated incubation time intervals.

I . IINJOLOGICAL RZAGENTS

Normal goat serum was obtained from Pentex Laboratories, Kankakee,Illinois. Coat anti-monkey games globulin (IgG) serum and goat antiserumto human, chicken, guinea pig, rabbit, bovine, and mouse lgC were obtainedfrom kicrobiological Associates, Inc., Bethesda, Maryland. Goat antiserum 4

to human albumin, immoglobulins, and Fab (1&11) and Fc fragments wereobtained from Hyland Laboratories, Loa Angeles, California. Fragments wereprepared by pepsin digestion of anti-IgC serum. All sera were heated at56 C for 30 min and diluted in PRS.

F. VIRUS SEBSITIZATIOI AN) ASSAY

The ters virus sensitization refers to the interaction of virus withantiviral serum to form an infectious virus-antibody complex."f! In thisreport, sensitised virus is used synonyuosly with persistent virus fraction.VIE virs vas sensitized by mixing 1 x 10 CIU/ml of virus with an equalv~lum of a 1:50 dilution of either monkey antiviral servm or of normalmam*Ay sertm (control) and incubating at 35 C for I hour. Sensitized

7 1virus, in contrast to unsensitized virus, was susceptible to neutralizationby goat anti-monkey IgG serum. To test for sen irb'ri, treated viruswas generally mixed with an equal volume of a 1:5 dilution of goat anti-monkey IgG serum and incubated at 4 C for 10 min. A control mixtureconsisted of treated virus and normal goat serum. Appropriate dilutionsof test samples were made in cold PBS and assayed for surviving virus.A reduction (0.3 log, units or greater) of the survivirg virus fraction,calculated by subtracting the titer (log,, remaining after incubationwith goat anti-monkey IgG serum from the titer (log,,) of the control, wasindicative of virus sensitization.

III. RESULTS

A. MULTIt'LICITf CURVE

Kinetic curves of VEE virus neutralization with different antiscrumconcentrations are linear initially and then undergo a decrease in slopethat may reach a horizontal line.1 4 The degree of this curvature appearsto be dependent on the ratio of serum to virus; it is more pronouncedwhen the ratio is increased. This dependency was investigated further bydetermining the neutralization curve at equilibrium of differentconcentrations of serum mixed with a constant quantity of virus (I x 10CIU/ml). After incubation at 35 C for 2 hours plus 4 C for I hour, reactionmixtures were appropriately diluted and Assayed for surviving virus.

A multiplicity curve resulted that resembled a kinetic curve when thelogarithm of surviving virus were plotted against antiserum dilutions(Fig. I). Fros the origin, the multiplicity curve was linear and thenbecame horizontal, even in the presence of high antiserum concentrations.The survival of virus being constant at this portion of the curve indicatedthat there is a fraction of virus, termed the persistent virus fraction.

that resists neutralization.

B. iACT(S INFLUENCING APEARANCE AND ,MAGNITUDE OF PERSISTENT VIRUSFRACTION

A report that the persistent fractio.i of virus populations was causedby aggregated virus1Z prompted an invesrigation to determine whetherthe virus preparation used in this study vis siailarly affected. Kineticneutralization tests were made with ".10 d.lutions ot monkey antiviralserun mixed in equal volumes vit. ui4'lttered uirus, sonic-treated (20 kc,'sec,2 sin) virus, or virus filtered thro jh 0.2-p or 0 .1 -. porosity membraes(Millipore Corp., bedford, Mass.) in accord with the described procedure.Appropriate tubes containing normal serus and virus served as controls.Reactions were carried out in the manner described earlier. Results inFigure 2 show that neutralization reactions proceeded initially at a linear

1.0

04-

w

16

5 10 15 20 25

Serum Dilution, 10-3

FIGURE 1. Multiplicity Curve of Neutralization of VEE Virus.

9

and similar rate with all virus preparations employed. Deviations fromlinearity first occurred with both unfiltered and sonic-treated virusesand resulted in equivalent levels of persistert virus fractions. Althoughthe linearity of the reactions continued slightly longer with filteredvirus preparations, the persistent virus fraction still appeared. Thelevel of these fractions was lower, however, than that of the unfilteredand sonic-treated virus preparations, These observations seem to indicatethat preaggregation of virus is not the dominant cause of the persistentVEE virus fraction, although aggregation may contribute to the levelof the persistent fraction.

In view of numerous reports attesting to the potentiation of virusneutralization by antiserum with the addition of heat-labile serumcomponents,' 1 - °20 these additives were investigated to determine whetherany change in the quantity of the persistent virus fraction resulted.Kinetic neutralization experiments were made with test mixtures consistingof 5 x 108 CIU/ml of virus, 1:10 dilution of monkey antiviral serum, andfresh or inactivated (56 C, 30 min) undiluted guinea pig serum. Controlmixtures consisted of virus, fresh or inactivated guinea pig serum, andnormal monkey serum. Reactants were prewarmed before mixing; the reactionswere carried out at 35 C. Both neutralization curves were linear for thefirst few minutes and then deviated from first-order kinetics within 10 minafter the onset of neutralization (Fig. 3). In the presence of freshguinea pig serum, the neutralization reaction velocity increased and thepersistent virus fraction was reduced but not eliminated.

It is generally recognized that the fraction of virus particlessurviving neutralization may depend on the cell type employed for virusassay.21 3 Different host cell systems were used to assay virus todetermine their effect on the quantity of the persistent virus fraction.A kinetic neutralization test was made in the usual manner using a 1-50dilution of monkey anti-viral serum; reaction samples taken at intervalsof incubation were then assayed in parallel on five cell lines: M4' oy,L-929, hamster kidney, Chang's human conjunctiva, and guinea pig lung.The human conjunctiva cell line was purposely included in the experimentbecause it was reported to be less capable of supporting VEE virusgrowth.3' Results in Figure 4 show that the kinetic neurralization curvesand the level of the persistent virus fractions were similar with allhost-cell systems. Among the cell systems tested, surviving virus fractionsdiffered from one another by less than 5% at that portion of theneutralization curve that reaches a conatant value. Dulbecco et al.3 alsofailed to demonstrate any difference in the shape and slope of multiplicitycurves with the limited cell types used for assay of surviving virus. Thesefindings, however, do not preclude the possibility that the choice of hostcells used in the assay of virus may influence the level of the persistentvirus fraction.

10

1.0

r

U..

CO

C~ ------ Sonic-treated virus

0.01-

Filtered (0.2 1A) virus

......................oFiltered (0.1 I)virus

000 110 is 20 25 30

Minutes

FIGURE 2. Kinetic VEZ Virus Neutralization Reactions with Prior

filtration or Sonic Treatment (20 kcluec for 2 min) of Virus.

1.01

Heated guinea pig serum

A:

0

C

Fresh guinea pig serum

0.,

000 10 20 30 40 50 60Minutes

FIGURE 3. Effect of Heated (56 C, 30 min) and Fresh Guinea pigSerum on the Kinetic VEIS Virus Neutralization Reaction.

12

1.0o-**--O- Guinea Pig Lung

o-.--o:Hamster Kidney

A: ~a ... a= MCOyI' -. : Chang's Human Conjunctiva

C .o

UM'

C '

0.1- Op -

5 10 15 20 25

Mi nutesFIIMz 4. Levels of Persistent Virus Fractions in Kinetic VEE VirusNeutralization Reactions Assayed on Different Celi Lines.j

13

To determine whether the persistent virus fraction constituted agepncically resistant population, surviving virus was passed twicein'racerebraliy in mice. Parallel kinetic neutralization tests wereperformed using a 1:50 dilution of monkey antiviral serum mixed withparent virus (original virus suspension) or progeny virus (persistentvirus fraction passed twice in mouse brains). A similar test was also

carried out with an attenuated VEE virus strain. Both the parent andprogeny viruses showed almost identical neutralization curves (Fig. 5);compared with both these viruses, the rate of neutralization of attenuatedvirus was greater. A persistent virus fraction was also manifested withthe attenuated virus; however it was lower in quantity than that notedwith the other two viruses- Kinetic neutralization tests performed undersimilar conditions with parent or attenuated viruses mixed with attenuatedmonkey antiviral serum resulted in neutralization cures comparable tothose shown for the corresponding viruses in Figure 5. These experimentsindicate that resistance of virus particles to neutralization, manifestedby a constant quantity of surviving virus, is not the result of a stablegenetiz mechanism. Experimental results obtained with different systemsof virus-antibody znd host cells support this conclusion.

4 'S'26

The persistent virus fraction incubated at 35 C for 30 min withadditional antiserum of high concentration was refractory to furtherneutralization. Similarly, it was unaffected after incubation withribonuclease (2 4g/ml). The latter, in agreement with findings withNewcastle disease virUS,4 indicates that the persistent virus fractionis not caused by the presence of infectious viral RNA.

C. NEUTRALIZATION OF PERSISTENT VIRUS FRACTION BY ANTI-IgG SERUM

Persistent virus fractions have been observed when lactic dehydrogenaseor herpes simplex viruses were incubated with their respective antisera.13 ''6

These fractions are in the form of infectious virus-antibody complexesthat may be neutralized by goat anti-mouse IgG serum. In a kineticneutralization experiment to determine whether these findings could beextended to the VEE virus-antibody system, dilutions (1:50) of monkeyantiviral or normal serum were mixed with equal volumes of I x 108 CIU/mlof virus suspension and then incubated at 35 C. Reaction mixtures Weresampled at different time intervals and assayed for unneutralized virus,Samples were also mixed with equal volumes of 1:5 dilutions of eithergoat anti-monkey IgG serum or normal goat serum to test for sensitizedvirus as described earlier. Results in Figure 6 show that the initialrate of neutralization in the mixture of virus and antiviral serum waslinear and then deviated as a result of a persistent virus fraction.The quantity of virus that was neutralized by VEE antiserum wasapproximately 1.0 log,, unit. Although the addition of normal goat serumto virus-antiserum mixtures did not alter the kinetic curve, goat anti-monkey IgG serum significantly reduced the Oersistent virus fraction by

- .I II

14

Progeny virus

o-o (Persistent parent virus fraction2X mouse brain passed)

1.0-~4Parent virus

\ \~ (-.-X Attenuated virus

'0

0.1

0.10

0 25105mAinutes

?103J . ti~iI C .tQ 1 & ot '(E iAreft, fo i *fl4 Lttelu*ted

ViusS y)WO4"ky AntS'T o nt V'dut

15

7.0. Normal Monkey

Serum -

Neutralized Virus

6.0.

3 5.0-

2Sensitized VirusTo a Vi u

N outralizod

4.07

C-oat Anti-Monkeyq

2.0 -A

S 10 15 20 25 30 35 40 45 50 55 60

Minutes

FIGURE 6'. Kine~tic Neutralization of VEE Vir~s by Monkey Antiseruand the Neutra lization of the Ferslatent Virus Fra:tion by GoatAnti-Monkey IgG Serum.

more than 3.0 log,,, units. The total quantity of virus neutralized exceeded4.0 log,, units. These findings imply that the persistent VEE virus fractionexisted in the form of an infectious virus-antibody complex (sensitizedvirus) and that it could be neutralized by antibody not directed against thevirus.

D. PARAMETERS OF NEUTRALIZATION OF CENSITIZED VIRUS BY ANTI-IgG SERUM

To define the conditions of neutralization by anti-IgG serum, the effectof antibody' concentration on virus sensitization was first investigated bymixing different dilutions of monkey antiviral serum with 5 x 108 CIU/mlof virus in equal volumes and incubating the mixtures at 35 C for 1 hour.Portions of tue mixtures were assayed for unneutralized virus; other portionswere mi:,d with an equal volume of a 1:10 dilution of goat anti-monkey IgGserum, incubated Lt 35 C for 1 hour, and then assayed for surviving virus.Normal monkey or goat serR served as controls. The data in Tille I showthat 1:10 dilution of VEE antiserum neutralized the greatest amount of virus(2.0 logo); the quantity decreased in the presence of higher antiserumdilutions. The addition of goat anti-monkey IgG serum neutralized 3.7 log,,units more of virus with both 1:50 and 1:100 dilutions of VEE antiserum.In the presence of higher VEE antiserum dilutions (1:500 to 1:5,000), wherelittle virus neutr&lization occurred, more than 1.5 log 0 units of virus wereneutralized by anti-IgG serum. Virus sensitization, therefore, had alsoresulted with the high VEE antiserum dilutions. With the 1:10 dilutionof antiviral serum, anti-IgG serum was slightly inhibited in its neutraliza-tion of sensitized virus. That excess unbound IgG in the reaction mixturecan prevent anti-IgG from reacting with sensitized virus has been suggestedas the cause of the observed inhibition.13 In general, however, greaterconcentrations of antiviral serum sensitized more virus.

The effect of dirferent concentrations of goat anti-monkey IgC serum onthe neutralization of sensitized virus was determined by first mixing I x 108ClU/ml of virus with an equal volume of I:jO dilution of antiviral monkeyserum. Mixtures were incubated at 35 C for I hour to effect virus sensitiza-tion. In equal portions, sensitized virus suspension was mixed withdifferent dilutions of goat anti-monkey IgG serum or normal goat serum(control), incubated at 35 C for I hour, and then assayed for survivingvirus. Results show that neutralization of sensitIzed virus is dependenton the concentration of anti-IgG serum (Table 2). Greater amounts ofsensitized virus were neutralized with increased concentrations oi anti-IgC serum. .

The effects of time and temperature on the neutralization of sen'itizedVUZ virus by goat anti-monkey IgG serum were investigated. Virus wassensitized as described earlier and then mixed with anti-IgG serum. Mixturesvere incubated at 35 C or 0 C, sampled at prescribed time intervals, andthen as ayed for sjrviving virus. Results in Table 3 show that sensitizedvirus was neutralized by anti-IgG serum almost completely within I min at35 C br*. less rapidly at 0 C.

j 17

S0

4 A . U f -.4 () Cl -4 01 1

hi > -4

-. 0

044 -4 0

H 0- 'A mf ' P

f-4 04. (n 1n.4 4

U> c 0

> 140

1-. 4' 04 .4 0

vf 4Jj14U.

-'41

0 Ajo1 a

0 id

04~~~~. D-s. % N0

> .aa c U

w >k 4 (n ..

10 -AIkIC). V.

18

TABLE 2. NEUTRALIZATION OF SENSITIZED VEE VIRUS BY DIFFERENT CONCENTRATIONSOF G(XT ArTI-MONKE IgG SERUWe

Dilution of Surviving VirusGoat Anti-Monkey Virus Titer, Fraction,

IgO Serua logl0 CIU/ml loglo CIU/Ml

1:2.5 2.4 -4.3

1:5 3.1 -3.6

1:12.5 4.1 -2.6

1:25 4.6 -2.1

1:50 4.7 -2.0

1:250 5.3 -1.4

1:2.5 (normal goat serum) 6.7 0.0

a. Appromimately I x 108 CIU/ml of virus was mixed with an equal volume of1:50 dilution of VIE monkey antiserua and incubated at 35 C for 1 hourto effect virus sensitization. Anti-IgG was mixed with sensitized virusand incubated at 35 C for 1 hour.

75°. i. ':;L a

19

TABLE 3. EFFECT OF TIME AND TEMPERATURE ON THE NEUTRALIZATIONOF SENSITIZED VEE VIRUS BY GOAT ANTI-MONKEY IgG SERUfh/

35C OCTime, Surviving Virus Surviving Virusmin Virus Titer Fraction Virus Titer Fraction

1 4.4 -2.3 4.u -2.5

3 4.5 -2.2 4.2 -2.3

5 4.4 -2.3 4.3 -2.2

15 4.4 -2.3 4.3 -2.2

Control 6.7 0.0 6.5 0.0

a. Approximately 5 x 108 CIU/ml of virus was mixed with an equalvolume of 1:50 dilution of VEE monkey antiserum and incubated at35 C for I hour to effect virus sensitization. Portions ofsensitized virus were mixed with equal volumes of 1:10 dilutionsof goat anti-moniey IgG serum and incubated (35 C or 0 C) forprescribed time periods. Both reactants were at the desiredincubation temperature before mixing. Sensitized virus mixedwith 1:10 dilutions of normal goat serum and incubated (35 C or0 C) for 15 min served as controls. Samples were diluted inice-cold PBS and assayed for surviving virus. All data indicatelog'- CIU/ml.

To determine t'e time relati-nship between neutralization andsensitization of VEE virus by antib0ody, a kinetic neutralization test wascarried out in the usual manner; the concentration of reactants was similarto that of the preceding experiment. Reaction mixtures were sampled atdifferent time intervals and assayed for unneutralized virus. A portionof each sample was treated with anti-IlG serum and then assayed forsensitized virus. Results reveal that virus sensitization preceded virusneutralization (Table 4). During the 0 5- to 4-rin incubation time intervalvirus neutralization continued but the quantity of sensitized virusremained constant. Cursory evidernce of these events was observed in theearly sample period of a preceding experiment (Fig. 6). These data suggestthat a major portion of the persistent virus fraction was an entity at theonset of virus neutralization by antiviral serum. I

I

j 20

AEZ4, SENSITIZATION PRECEDING NEUTRALIZATIONOF VEE ViRUS BY ANTIBODY.-a/

Tim, Surviving Virus Fractionmin Neutralization Sensitization

0.5 -0.3 -.

1 -0,4 -4.3

2 -0.6 -4.3

4 -0.7 -4.3

Control 0.0 0.0

a. Approximately I x 108 CIU/ml of virus was mixedwith an equal volume of 1:50 dilution of VEmonkey antiserum or normal monkey serum, incubatedat 35 C and sampled at designated time intervals.Samplesn were held at 0 C, diluted in ice-cold PBS,and assayed for surviving virus. Portions ofundiluted virus-antiserum mixtures were mixed withequal volumes of 1:5 dilutions of goat anti-monkeyIgG serum or normal goat serum, held at 0 C for

-, - 10 min, and assayed for surviving virus. All dataindicate 1oglo CIU/ml.

b. Normal monkey or goat serum.

The capacit) of gwt anti-IgG aerum of different animal species toneutralirea virus that had been sensitized by monikey antiviral serum westested. Sensitiz~ed virus was mixed with anti-T~gG serum of different species,I incubated at 35 C for 10 min, and then assayed -Lor surviving virus. Resultsin Table 5 show timt maximal neutralization of sensitized virus occurredwith goat aunti-monkay serum and, to a lesser degree, with goat anti-humanIgG serum. That goat anti-human IjG seruim reacting with virus sensitizedIby ao nkey antieerum was capable of cross-neutraliz~ation is suggested. by thedate, Ant1.-I&G serum of other animal species did not significantly neutralizesensitized virus,

To explore further the cross-neutralization phenomenon, a reciprocalneittralizatiort test was made by reacting virus that had been sensitized witheither monkey or human antiviral serum with goat anti-monkey or anti-humanTgG serum. Almost complete reciprocity of neutralizat' n of sensitized virusby homologous and heterologous anti-ISG sera resulted (Table 6). Theexception was virus that had been sensitized with monkey antiviral serumand reacted with nnti-human IgG serum. The quantity of sensitized viyus

neutralized by this combination was less than that with the other test

c,)mbiations

21

TABLE 5. NEUT7ALIZATION OF SENSITIZED YEE ViRUS BY GOATANTX-IgG SMRVT OF DI_7RF 'T SFECIESLi

Surviving Virus

Goat Serum Virus Titer Fraction

Anti-monkey Igt4 3,1 -3.6

Anti-human IgG 5.7 -1.0

Anti-chicken IgG 6.7 0.0

Anti-guinea pig IgG 6.6 -0.1

Anti-rabbit lgG 6.5 -0.2

Anti-bovine IgG 6.5 -02

Anti-mouse IgG 6.5 -0.2

Norma 1 6.7 0.0

a. Approximately I x 108 CIU/ml of virus was mixed with an equalvolume of 1:50 dilution of VEE monkey antiserum and incubatedat 35 C for I hour to effect virus Be itization. Portionsof sensitized virus were then mixed with equal volumes of1:2.5 dilutions of goat antiserum to IgG serum of -achdifferent species and incubated at 35 C for 10 min. Samplesof each mixture were appropriately diluted in PBS andassayed for surviving virus. All data indicate log,, CIU/ml.

TABLE 6. SENSITIZATION OF VEE VIRUS BY HUMAN tR MONKEY ANTISERUH WITHCROSS-NEUTRALIZATION BY HOMOLOGOUS AND HETEROLOWUS

GOAT ANTI- IgG SERUIe/

Antiserum for Neutralization SerumVirus Sensitixation Goat Anti-Human IgG Goat Anti-Monkey IgG

Human -3.6 -3.5

Monkey -1.0 -3.6

a. approximately 1 x 108 CIU/ml of virus was mixed with an equal volumeof 1:50 dilution of vgE unkey or human antiserum and incubated at 35 Cfor 1 hour to effect virus sensitization. Portions of sensitized viruswere then mixed with equal volumes of 1:2.5 dilutions of goat anti-humanor anti-monkey IgG serum or of normal goat serum (control) and incubatedat 35 C for 10 min. Samples were diluted in PBS and assayed forsurviv.kng virus.

b. All data indicate log 0 CIU/ml.

22

The neut~alization of sensitized virus by different classes of im uno-globulins was determined by first sensitizing virus with human antiviralserum and then reacting the virus with different goat anti-human immuno-globulins. Maximal neutralization of sensitized virus was observed withani-IgC serum and, to a lesser extent, with anti-IgA serum (Table 7). Viruswe* not neutralized by either anti-IgM or anti-albumin sera. These resultsare similar to those reported by Notkins et al.26 who additionally notedneutralization of lactic dehydrogenase virus by anti-mouse IgF serum.

TALI 7. NEUTRALIZATION OF SENSITIZED (VEE HUMAN ANTISERUM) VIRUS BYDIPFERENT CLASSES OF COAT ANTI-HUMAN IMMUNO BULINS./

Goat Antiserum to Surviving VirusHuman Immnoglobulin Virus Titer Fraction

IgO 3.3 -3.6

IgA 4.5 -2.4

IgM 6.9 0.0

Albumin 7.0 0.0

Normal goat serum 6.9 0.0

a. Approimtely 1 x 108 CIU/ml of virus was mixed with an equal volume of1:25 dilution of VEE human antiserum and incubated at 35 C for 30 minto effect virus sensitization. Portions of sensitized virus were thenmixed with equal volumes of undiluted goat antiserum to different humanimmunoglobulins, albumin, or normal goat serum and incubated at 35 Cfor 10 min. Samples were diluted in PBS and assayed for survivingvirus. All data indicate loglo CIU/ml.

To determine whether antiserum to monovalent fragments of IgG has thecapacity to neutralize sensitized virus, the virus was sensitized with humanantiviral serum and then incubated with goat antiserum to either Fab or Fcfragments of human IgG. A mixture of antisera to Fab and Fc fragments wasalso reacted with sensitized virus. Results in Table 8 reveal that antibodyto monovalent Fab fragments was capable of neutralizing sensitized virus.Antibody to the 7c fragment also neutralized virus that had been sensitized,but the quantity was 1.0 log,, unit less than that neutralized by antibodyto Fab fragments. The mixture of antisera to both Fab and Fc fragmentsneutralixd virus to almost the same degree as intact anti-IgG serum.I irectly, the evidence suggests that the Fc fragment, devoid of combiningsites but containing structural features necessary for other biologicalfunctions,0 7 plays a role in virus neutralization. These findings, inrelation to available evidence on the neutralization of viruses by IgGfragments, are discussed subsequently.

23

TABLE 8. NEUTRALIZATION OF SENSITIZED (VEE HUMAN ANTISERUM) VIRUS BYGQ4T ANTISERUM TO PAPAIN-DIGESTED FRAGMENTS OF IgO-/

Goat Antiserum to Surviving VirusHuman IgG Fragments Virus Titer FrActionb/

Fc 6.1 -0.6

Fab 5.1 -1.6

Fc + Fab 3.9 -2.8

IgG 3.7 -3.0

Normal goat serum 6.7 0.0

a. Approximately 1 x 108 CIU/ml of virus was mixed with an equal volume of1:25 dilution of VEE human antiserum and incubated at 35 C for 30 winto effect virus sensitization. One part undiluted goat antiserum toIgG fragments of normal goat serum were mixed with two parts sensitizedvirus and one part PBS diluent. Mixtures were incubated at 35 C for10 mi , appropriately diluted in PBS, and assayed for surviving virus.All data indicate logl0 CIU/ml.

IV. DISCUSSION

The studies reported here indicate that the persistent virus fractionmanifested when VEE virus interacted with neutralizing antibody was not thedirect result of aggregation of virus particles, heterogeneity of viruspopulation, infectious viral RNA, or the host cell system employed for virusassay. Nor was dissociation of the virus-antibody complex a factor,because previous studies with VEE virus demonstrated that neutralized viruswas not appreciably reactivated under physiologic conditions.1 The factthat prolonged incubation of virus-serum mixtures in the presence of excessvirus antiserum did not result fn fu,:ther virus neutralization is additionalevidence against a dissociation phenomenon. The level of the persistentvirus fraction was only slightly lowered when heat-labile serum factorsfrom fresh guinea pig serum were added to virus-antibody mixtures. Thepersistent fraction of V39 virus was in the form of an infectious virus-antibody complex (sensitized virus) that could be neutralized by anti-IgGseruw, ua antibody not directed and inactive against the virus itself.

24

Investigatiou of determinants that may affect the formation and neutrali-zatioa of the infectious complex showed that the quantities of virus sensitizedby antiviral serum and neutralized by anti-IgG serum increased with higherconcentzations of these respective sera. With the highest antiviral serumconcentration employed for virus sensitization, however, neutralization ofsensitized virus by anti-IgG serum was slightly inhibited. This phenomenonhas been attributed to unbound IgG that prevents anti-IgG antibodies fromreacting with sensitized virus.13 In this study, the reverse situationwas also noted, with high concentrations of anti-IgG serum inhibitingneutralization of virus by VER antiserum. When virus, monkey antiviralserum, and goat antiserum to monkey IgG or norml goat serum were mixedsimultaneously and incubated, virus neutralization by antiviral serum wasmrkedly inhibited in reaction tubes containing anti-IgG serum but not intubes containing normal goat serum.

Virto sensitized with VEN monkey antiserum was not neutralized by anti-IgG sera of different animal species. An exception to these findings was thepartial neutralization of sensitized virus by goat anti-human IgO serum.,Reciprocity of neutralization of sensitized virus (formed with monkey or humanantiviral serum) was almost complete by homologous and heterologous anti-I4 sera (Table 6). The cross-reactivity is probably the consequenceof a comon globulin antigenic determinant shared by species of the Primateorder. In general, however, neutralization of sensitized virus by anti-IgCserum appears to be species-specific.

The specificity of the anti-imunoglobulin also af!fected the neutralizationof sensitized VII virus. axiiml neutralization was noted with anti-ISCserum and to a lesser extent with anti-IgA serum; anti-IgM serum had noneutralizing effect. Although this suggests that VRE virus was not sensitizedwith ISM antibody, additional tests are needed to determine whether viruscan be sensitized with IgM antiviral antibody and then neutralized by anti-ISM serum before this assumption is warranted. a s

In contrast to the marked temperature and time dependence of V59 virusroutralisation by antiserum,1 neutrulization of sensitized virus by anti-IgG serum was rapid and less sensitive to temperature. Sensitized VIE viruswas neutralized almost completely within 1 min at 35 C and within 3 to 4 minat 0 C. Previous studies showed that sensitized lactic dehydrogenase 2 s andherpes samplex viruses13 were neutralized by anti-ISG serum at 4 C within1 sin and 15 sec respectively. Differences iii temperature did not signi-ficantly affect the reaction. Evidence that sensitization of VM virusprecedes neutralization (Table 4) is highly -ertinent to the problem of thepersistent virus fraction. Apparently, a ma.ior portion of the persistent

I virus fraction is forme before virus neutralization by antiviral serumhas actively begun. The foiation of virus-antibody complexes, which arenot necessarily neutral, prior to viral inactivation has been reported inother virus neatralisatiou system.* 6 °2 s These findings suggest that virussensitization in some way prevents additional ant'viral antibody, which is

777,

25

necessary for neutralization, from attaching to virus particles. Thataggregation of sensitized VEE virus particles my be a factor in theinhibition of virus neutralization is not supported by this study orby findings with other viruses. "

1 29*2u Antibodies to both monovalentfragments of IgG and intact IgG were capable of reaching and netitralizingsensitized VEE virus rapidly and effectively. It is also relevant thatmonovalent Fab fragments of papain-digested antit'era to influenza orherpes simplex viruses (not prone to produce aggregates) sensitizedand reduced the neutralization of these viruses by undigested antiviralsere. 5s '3 0 The neutralization of low concentrations of sen~itized VUEvirus by anti-IgG serum precludes lattice formation as a possible causeof inhibition of virus neutralization.

Although the quality of antibody participating in VEt virus sensitizationand neutralization was not investigated, it has been postulated that thepersistent virus fraction arises, at least in part, from the presenceof nonavid antibody in antiserum.o*31'a3 This type of antibody cancombine reversibly with the virus surface but does not form a stableunion. Results of a recent study on the properties of both early and late7S and 19S neutralizing antibodies to herpes simplex virus indicate thatboth common and distinctive properties exist in the ability of theseantibodies to sensitize virus for subsequent neutralization by anti-IGserum or complement. 3 It appears that the quality of aratibody prtici-pating in virus neutralization reactions could be a factor contributingto the phenomenon of the persistent virus fraction.

The assessment of individual antibody subunits in the interactionbetween virus and antibody has been facilitated within recent years withthe demonstration that antibody molecules my be degrac.ted ensymtically. 3 4

Monovelent Fab fragments of goat anti-mouse serum or goat anti-rabbitIgG serum have been shown to neutralize, in part, lactic dehydrogenaseviruses and poliovirus.26 respectivftly. The former virus was notneutralized by the Fc fragment. Although the Fc fragment does not containantigen-binding sites and has not been specifically related to antigen-binding activities,3 limited virus neutralization activity of thisfragment has been reported. 3 s It has been assumed that contaminationwith undigested IG or other active fragments is responsible for theactivity exhibited by the Fc fragment. 35, 3 In this study on VIS virusneutralization, sensitized virus was partially neutralized by goatantiserum to monovalent fragments of human IUG and, to a lesser degree,by the Fc fragment. It was also noted that VII virus sensitized withantiviral human serum was neutralized by a mizxture of goat antisera toab and Pc fragments of human WgG (Table 8). Sensitised VIE virus was

neutralized to almost the same degree as that neutralized by goatantiserum to intact human 1g. Indirectly, thesr i aug .st a rolefor the Fc fragment in virus neutralization. Observations by othcrs onthe inability of Fab fragments to establish and mintuin neutralizationof virus3 6 - 3 favor the concept that the Fc fragment may complete the

26

structural configuration necessary for antibody to exert its most efficientit.....ralising power. Effective virus neutralization, therefore, is not onlya function of the antigen-binding site# of the antibody but may dependon the entire structure of the IgG molecule.3 6

Although the experiments reported here do not clearly define themechanism by which neutralization of sensitized virus by anti-IgG serumoccurs, the evidence adds support to hypotheses proposed by others. 1 6 5 2

A secondary reaction is involved in the interaction of anti-IgG withsensitised virus. Neutralization of the infectious virus-antiserum complexby anti-IgG is effected by an Increase in size of the complex or the formationof bridges that cover or block critical Infective site# on the virus particle.The inefficiency of monovalent Fab antibody fragments to block completelythe reactive site* on the infectious virus-antibody complex may be relatedto their reduced size or the need for the ic antibody fragment to provide acamplete and effective configuration on the complex. Electron microscopystudies have revealed changes in the size of virus-antibody complexes thatmy be related to the valency of Fab fragments.'0 Fractionation techniques

of antibody molecules, the use of anti-IgG serum to assay sensitized virus,and electron microscopy examinations of virus-antibody coaplexes offer t.hemeans for elucidating further the stages and mechanism Involved in virusneutralization.

27

LITERATURE CITED

1. Hahon, N. 1969. Interaction of Venezuelan equine encephalonyelitisvirus with neutralizing antibody: I. Neutralization kinetics andreaction reversibility. J. Gen. Virol. 4:77-88.

2. Delbruck, M. 1945. Effects of specific antisera on the growth ofbacterial viruses (bacteriophages). J. Bacteriol. 50:137-149.

3. Dulbecco, R.; Vogt, H.; Strickland, A.G.R. 1956. A study of thebasic aspects of neutralization of two animal viruses, western equineencephalitis and poliomyelitis virus. Virology 2:162-205.

4. Granoff, A. 1965. The interaction of Newcastle disease virus andneutralizing antibody. Virology 25:38-47.

5. Hashimoto, N.; Prince, A.M. 1963. Kinetic studies on the neutrali-zation reaction between Japanese encephalitis virus and antiserum.Virology 19:261-272.

6. Lafferty, K.J. 1963. The interaction between virus and antibody:I. Kinetic studies. Virology 21:61-75.

7. Lehane, D.E., Jr.; Clark, H.F.; Karson, D.T. 1968. Antigenicrelationships among frog viruses demonstrated by the plaque reductionand neutrali-ation kinetics tests. Virology 34:590-595.

8. Overby, L.R. 1967. Bacteriophages as model system to studyantigenic properties of viruses. BioScience 17:807-812.

9. Thormr, H. 1963. Neutralization of Visna virus by antisera fromsheep. J. I.unol. 90:185-192.

10. Bradish, C.J.; Farley, J.O.; Ferrier, H.E.N. 1962. Studies on thenature of the neutralization reaction and the competition forneutralizing antibody between components of the virus system offoot-and-mouth disease. Virology 18:378-400.

11. Puackas de St. Groth, S.; Watson, C.S.; Reid, A.F. 1958. Theneutralization of animal viruses: 1. A model of virus-antibodyinteraction. J. Immunol. 80:215-224.

12. Wallis, C.; ?elnick, J.L. 1967. Virus aggregation as the cause ofthe non-neutralizable persistent fraction. J. Virol. 1:478-4U.

13. Ashe, W.K.; Notkins, A.L. 1966. Neutralization of an infectiousherpes simplex virus-antibody complex by ati-y-globulin. Proc. gat.Acd. Sol. U.S. 56:4,7-451.

28

14. Hahen, H.; Cooke, K.O. 1967. Primary virus-cell interactions inthe imunofluorescent assay of Venezuelan equine encephalomyelitisvirus. J. Virol. 1:317-326.

15. Berge, T.O.; Banks, I.e.; Tigertt, W.D. 1961. Attenuation ofVenezuelan equine encephalomyelitis virus by in vitro cultivation inguimea-pig heart cells. Amer. J. Hyg. 73:209-218.

16. Notkins, A.L.; Mahar, S.; Scheele, C.; Goffman, J. 1966. Infectiousvirus-antibody complex in the blood of chronically infected mice.J. Up. Med. 124:81-97.

17. Baughman, R.H.; Fenters, J.D.; Marquis, G.S., Jr.; Halper, J.C.1968. Effect of complement and viral filtration on the neutralizationof respiratory syncytial virus. Appl. Microbiol. 16:1076-1080.

18. Iwasaki, T.; Ogura, R. 1968. Studies on complement-potentiatedneutralizing antibodies (C'-PHAb) induced in rabbits inoculated withJapanese encephalitis virus (JEV): I. The nature of C'-PNAb.Virology 34:46-59.

19. Ralis, W.; Desmyter, J.; Melnick, J.L. 1967. Rubella virus neutrali-zation by plaque reduction. Proc. Soc. Exp. Biol. Med. 124:167-172.

20. Yoshino, K.; Taniguchi, S. 1966. Evaluation of the demonstrationand compleimnt-requiring neutralizing antibody as a means for earlydiagnosis of herpes virus infections. J. Itasunol. 96:196-203.

21. FreIdman, M. 1954. The effect of the host on the properties and sensi-tivity of a bacterial virus. J. Bacteriol. 68:274-278.

22. Kalmanson, G.M.; Bronfenbrenner, J. 1942. Evidence of serologicalheterogeneity of polyvalent "pure line" bacteriophage. J. Immnol.45:13-19.

23. Kjell4, L.N.; Schlesinger, R.W. 1959. Influence of host cell onresidual infectivity of neutralized vesi:ular stomatitis virus.Virology 7:2)6239.

24. Flardy, F.M. 1959. The growth of Vnezutlan equine encoophalomyelitisvirus in various tissue cultures. Amer. J. HyS. 70:21-27.

25. Keller, 1. 1968. Studies on the mechanism of the enzymatic reactivationof antibody-neutralised poliovirus. J. Lvaunol. 100:1071-1079.

26. Notkins, A.L.; Mage, K.; Ashe, W.K.; Mahar, S. 1968. Neutralizationof sensitized lactic dehydrogenase virus by anti-y-globulin. J.1uiol. 100:314-320.

27. Pub", J.L. 1962. et. 3*eneity of y-globulins. Advances Imiunol.2:41-109,

7* 7A.------ ______________- -

t

29

28. Yoshino, K.; Taniguchi, S. 1965. Studies on the neutralization ofherpes simplex virus: III. Mechanism of the antibody-potentiatingaction of complement. Virology 26:61-72.

29. Westaway, E.G. 1968. Antibody responses in rabbits to the group Barbovirus Kunjin: Serologic activity of the fractionatedimmunoglobulins in homAlogov. and heterologous reactions. J. Iuuunol.100:j69-580.

30. Lafferty, K.J. 1963. The interaction between virus and antiboly:II. Mechanism of the reaction. Virology 21:76-90.

31. Jerne, N.K.; Avegno, P. 1956. The development of the -aage-inactivating properties of serum during the course of sjeciftcimmunization of an animal: Reversible and irreversi1le inactivation.J. Immunol. 76:200-208.

32. Kjellen, L. 1962. Studies on the interactions of adenovirus,antibody, and host cells in vitro. Virology 18:448-456.

33. Hamper, B.; Nvtkias, A.L.; Mage, M.; K:ehn, N.A. 1968. heterogeneityin the properties of 7S and 19S rabbit-neutralizing antibodies toherpes simplex virus. J. Immunol. 100:586-593.

34. Porter, R.R. 1959. The hydrolysis of rabbiL gaina-jlobulin andantibodies with crystalline papain. B.ochem. J. 73:119-126.

35. Keller, R. 1966. The stability of neutraliza'_ion of poliovirus bynative antibody and enzymatically derived fragments. J. Immunol.96:96-106.

36. Cremer, N.E.; Riggs, J.L.; Fujimoto, F.Y.; agene, S.J.; Ota, 1.I.;Lennette, E.H. 1964. Neutralizing activity of fragments obtainedby papain digestion of viral antibody. J. Immunol. 93:283-292.

37. Kjell4n, L. 1964. Reactions between adenovirut antigens and papaindigested rabbit immune globulin. Arch. Gen. Virumforach. 14:185-200.

38. Kjellen, L. 1965. On the capacity of pepsin-digested antibody toneutralize Adenovirus infectivity. Iounology 8:557-565.

39. Vogt, A.; Kopp, R.; Maas, G.; Reich, L. 1964. Poliovirus type 1:Neutralization by papuin-digested antibodies. Science 145:1447-1448.

40. Almeida, J.D.; Cinader, P.; Naylor, D. 1965. Univalent fragmentsof antibody: A study by electron ricroscopy. Is.snocheaitry 2:169-176.

33

D0CtKEHT CONTROL DATA. - R D(3wfys""tm ofM~ .1 .0. be* of sbashe, OWd AW.64m AN001000 now he wGE awr ean~ ,sPM. i. CI...US

I, 0*t*NAIIO ATIVIYY (C5iam vw) A*5 1119901T 96CUMTY CLASOVICATIOl.

Department of the Army UnlasiieFort Detrick, Frederick, Maryland, 21701 *0x

'4TKIACTION OF V&NEUElAX ZIQUIME ENCEFHALO)MYELITIS VIRUS WITH HEtTllALIZIPLANrTluOW: 11. THE PERSIZET VIRUS FRACTION

IL neseMPIva more* (1rp. eI u"00 mod hwmelImtd.C

Nicholas (NMI) Hahon

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Department of the ArmyFort Detrick, Frederick, Maryland, 21701

The persistent virus fraction that results from the interaction of Venezuelanfequine ancephalomyiin (VII) virus vith antiviral serum io an 'fectious virus-antibod) compla (sensitized virus) that can be neutralized by enti-IgG serum. Thequantities of virus ..nsitixed by VII antiserum and neutralixed by anti- Wg loorilsdepend on the coceatration of theme *or&. In contrast to the marked teperatureend timis dependence of VIE virus neutralization by antiviral serusm, neutraliaticaof sensitized virus by anti-IgC serum is more rapid (almost complete within I mm Iat 3$ C) and lars, sensitive to temperature. Kvidonce that virus sensitizationpreceded neutralization Indicates that the persistent virus fraction is forotudW~ore virus neutrallatioa hee actively begun. Within Icertain limits, neutrali-

sation of sendItfzed virus by anti-UGC serum is species-specific. DifferencesIs the ability of anti-IW, ati-I&A, and anti-ISgM sera to neutralize sensitizedvirus indicat, that the reaction is also influenced by the specificity of the anti-

Sensitized virus was pertially neutralized by Soot antiserum to mofrove lentFeb fragpients on humn ISO and, to a Lesor degree, by the Vc fragment. Sensitized4virvo woo neutralized by an in vitro mixture of these fragments to almost the samedegr~ee so that neutrolized by goat antisrum t, intact human WG. Indirectly,there findiags sougeat a role for the Fc fragment in virus neutralization.

IA. KAY VC'4dS Neutralizer* Sensi tvity*V*eeuelan equine eicepbs loftylitis virus Antiserum Hoot virus relations

IttUdi tuiaglling Viruses Cell lines