RD-Ai59 715 NONSPECIFIC RESISTANCE INDUCED BY AN /IIMUNOPHRNCOLDGIC AGENT DERIVED FR..(U) ALABAMA UNIVUNIVERSITY DEPT OF MICROBIOLOGY A L WINTERS ET AL. N

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_TITLE (and Subtitle) S. TYPE or REPORT a PCRmOO COVIRCOAnnual: 8/16/83-8/15/84

La) NONSPECIFIC RESISTANCE INDUCED BY ANIMM1JNOPHAL'-ACOLOGIC AGENT DERIVED FROM a u'seeOG EoTwwEBORDE2'ELLA PERTUSSIS * OTATO RN UU~e

AU THOR(*) B OTATO RN U1911e

0 Alvin L. Winters, ?h.D., Paul A. LeBlanc, Ph.D. N00014-83-K-0597It) and Gary S. Sloan, Ph.D.

U PERFORMING ORGANIZATION NAME AND ADDRESS AN. 4A:QGRAM9 EMWPRO 1CT. TAMO

E~Department of Microbiology, Box BMThe University of Alabama

I University, AL 35486CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE

Jeanine A. Majde, Ph.D., Scientific Officer 1131/85Itmmunology Code 441, Cellular Biosystems; Group 13. NUMBER OF PAGES,

Dept. of the Navy, ONR, Arlington, VA 22217 14MONITORING AGENCY N AME &ADDRESS(If different from' Controlling Office) IS. SECURITY CLASS. (of Of* repen)

K UnclassifiedIse. *EC~ ASICATIONIDOWN GRADING

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IS. DISTRIBUTION STATEMENT (of this Report)

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I7. DISTRIBUTION STATEMENT (of the abstract eto,.Eln Stock 20. ilIf fiamt trust nap") L .

10. SUPPLEMENTARY NOTES

19. KEY WORDS (Continue on rovers* side It necessary and Idenify by block inumbef)

antiviral agent, Bordetela pertussi8, mutants, virulence factors,~ii pertussigen, imunomodulation, adenovirus, lipopolysaccharide, polysaccharide,

0... vesicular stomatitis virus, endotoxin, endotoxin protein, peritoneal exudate

4 E3 cells, virus migration, splenomegaly, hypothermia, neutralizing antibodies20. A 9STRPACT (Con tnue an revwre side It necessary and fdontitlP Ip 5149k IeebffJ

LJ..JTreatment of mice with Bordetella pertueeis vaccine resulted in* resistance to mouse adenovirus infection. Antiviral activity was associated

with surface components of B. pertussie. Acellular fractions with antiviralactivity were obtained by relatively gentle extraction methods, i.e., 1.0 MNaCl extraction or Waring blender treatment. B. pertussis lipopolysaccharide

#=mom extracted by the Westphal procedure possessed antiviral activity. Lipopoly-saccharide complexed with protein appeared to have more activity thanpurified lipopolysaccharide. B. pex'tusei8 vaccine did not induce-Li 1BOLT FC." 1473PIATO COIIO OFI IAS MUm 95IS0ee

85 02 04 161

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cross-reacting antibodies with either neutralizing or protective activitytoward mouse adenovirus. Peritoneal exudate cells obtained from micetreated with B. pertuseis vaccine inhibited vesicular stomatitis virusmultiplication in L-929 cells. Removal of adherent cells from theperitoneal exudate cell population increased the antiviral activitytoward vesicular stomatitis virus.

Mouse adenovirus migrates from the peritoneum to the blood withinminutes after intraperitoneal inoculation. Infection with sublethaldoses of mouse adenovirus resulted in a transient splenomegaly. Micedeveloped maximal neutralizing antibody titers after infection with aslittle as 10 plaque-forming units. Infection with lethal doses of mouseadenovirus resulted in a fever response (hypothermia) before death.

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Table 1. Characteristics of Brdoetlala 2otuiUsi Vaccines

Strain Dry Weight Agglut.1 Protein2 "Glucose"3 Mortality4(uQ) (ua) (uG) (deaths/total)

18323 166 + 35 2.8 0/5Tohana I 161 + 40 11.6 0/5BP347 169 + 41 9.7 0/5BP359 164 + 44 9.1 0/5Tohama III 250 + 150 --- 0/511615 250 - 143 --- 0/10

Controls:Connaught #1 625 --- 100 32.8 0/5Connaught 02 563 106 30.0 0/5Connaught 03 438 --- 97 32.8 0/5

jAgglutination with a 1:5 dilution of rabbit antiserum raised against

Connaught BPV.

2Protein determined by the method of Lowry and coworkers.

3Glucose equivalents measured by the anthrone reaction.

4Challenged with 4 LD50 of MAd1y4 seven days after treatment.

2.2 BIOCHEMICAL EXTRACTION OF BPV. Dr. Robert Lemon, ConnaughtLaboratories, Inc., Swiftwater, PA, developed several acellular fractionsof 1. pertusjsi for possible use as an alternative to the currently usedwhole cell vaccine with all of its associated side-effects. We haveanalyzed these fractions for their antiviral activity and have examined apolysaccharide and a lipopolysaccharide fraction of BPV also.

Acellular fraction 15A-1B was obtained from 4 day growth of phase I*1. pJtus (Connaught Laboratories, Inc., vaccine strain) onCohen-Wheeler agar. The cells were extracted with 1.0 N NaCl containing0.05 N sodium phosphate buffer (pH 7.2) and 0.02% thimerosol for 4 daysat 40C. The cells were sedimented end the supernatant decanted. Afractional precipitate (20 to 40% ammonium sulfate saturation) was made,

- dissolved in buffered saline, dialyzed, and designated fraction 15A-lB.A portion of 15A-1B was treated with 1.0% (v/v) Emulphogene BC 720(General Aniline & Film Corp., New York, NY). The mixture was incubatedfor 60 min at 40C and the precipitate was sedimented at 100,000 x S. for60 mn. The pellet was resuspended in phosphate buffered saline and thesuspension was adsorbed to 1.0% aluminum hydroxide gel. Thisalum-stabilized preparation was designated a& fraction 15A-1O6A.

Purified LPS was provided by Dr. Thomas W. Klein, University ofSouth Florida, Tampa, FL. The LPS was extracted from 1. perusa t.,strain 3779 BL2S4 by a modified Weetphal phenol-water procedure. LPS

-2-

1.0.0 BACKGROUND. Bordaele pertueeis vaccine (BPV) is administered toa large portion of the population in the United States. Our previousobservations indicated that BPV has immunomodulatory activity and inducesa resistant state to mouse adenovirus, a natural pathogen of mice.Infection with our strain of mouse adenovirus, designated NAdl2&4,results in a systemic infection that culminates in interstitial pneumoniaand death with hemorrhagic lungs observed on necropsy. Kirchner andcoworkers have reported a similar BPV-induced resistance against herpessimplex virus infection. Acellular extracts of B. portussis produced bygentle extraction of whole cells with 1.0 K NaCl induced a resistantstate to NAdl_,4 also. Thus, the isolation of an immunomodulatory,antiviral agent from 1- Dertus for possible human use appears to be afeasible project.

The efforts of the first year of the contract have been focused onidentifying the BPV component that is responsible for the antiviralactivity of the vaccine. Our preliminary findings indicate the activityresides in lipopolysaccharide (LPS) or a lipopolysaccharide-proteaincomplex.

2.0 IDENTIFICATION OF THE BPV COMPONENT WITH ANTIVIRAL ACTIVITY.Identification of the antiviral component of P. DertuseAs has beenapproached by both genetic analysis and biochemical extraction.Antiviral activity was assayed by administering BPV or an acellularcomponent of BPV to mice by the intraperitoneal route and thenchallenging the animal with a lethal dose of mouse adenovirus seven dayslater.

2.1 GENETIC ASPECTS OF BPV . Vaccines were developed from severalstrains of D. pertusJai. The organisas were aLntained on BG agar base(Difco Laboratories, Detroit, MI) supplemented with 17% defibrinatedsheep blood. Vaccines were prepared by harvesting 4-day growth fromCohen-Wheeler agar in phosphate buffered saline (pH7.2) and inactivatingthe cells by heating (560C for 30 min) in the presence of 0.02%thimerosel. The vaccines were adjusted to an estimated 4.0 mg (dryweight) per ml in saline-thimerosal diluent and stored at 40C.Uninoculated Cohen-Wheeler medium did not have antiviral activity.

The parent 8. 2_rtJish strain used to develop the ConnaughtLaboratories' BPV was ATCC 9797, a strain derived from 18323. Antiviralactivity was observed with both strains of the same lineage (Table 1).BPV made with strain Tohame 1, which was isolated independently of Strain18323 was protective also (Table 1). In addition, the "virulencefactors" of 1. Dertuoai1 did not play a role in the antiviral activity.Strains Tohama III and 11615 that do not express the virulence factorshad protective activity (Table 1). It was interesting to note thatstrain 11615 did not agglutinate with rabbit antiserum. This strain isthought to be a deep rough strain with a truncated lipopolyseccheridemolecule In the outer membrane of L. Dmr eai (Dr. Mark Peppler,University of Alberta; personal communication). In addition, we haveobtained mutants of strain Tohana I (BP347 and BP349) from Dr. AllisonWeiss and Stanley Falkow (Stanford University, Stanford, CA). Thesestrains were created by single site insertion of a transposon thatabrogated the "virulence factors" ; however SPV made from these strainsexhibited antiviral activity (Table 1).

4-1- -

extracted by the Westphal procedure from microorganisms other than 1.'i pertUasi&~j were obtained from Sigma Chemical Company, Inc., St. Louis, NO.

Coll surface polyseaccharide was harvested and partially purified bythe method of Conrad. Four day growth of phase I 6. RarituiAJL, strain18323, was harvested from Cohen-Wheeler agar medium using 0.01 Npotassium phosphate buffer (pH 7.0). The capsule (&lime) polysaccharidewas removed by brief shearing treatment in a Waring blender set at topspeed for 45 sac. The cells were removed by centrifugation, the pH ofthe supernatant was adjuated (pH 2.0) with 6.0 N H2S04 and thepolyaaccharide was precipitated with acetone. The precipitate wascollected by centrifugation, resuspended in water (pH 9.0), andre-precipitated at pH 3.0. The supernatant was dialyzed and concentratedin a flash evaporator.

Antiviral activity was detected in several acellular fractionsderived from D. oertussis (Table 2). The standard dose of BPV (250 ugdry weight) contained approximately tenfold the concentration necessaryto protect 50% of the test population. Fraction 15A-1B revealed at least13 proteins ranging in size from 16.5K to 139K upon polyacrylamide gelelectrophoresis. The gel pattern was similar to the pattern of outermembrane proteins isolated by Dobrogoaz and coworkers. Fraction 15A-1O8Atreated to remove LPS was markedly less active in stimulatinglymphocytosia, histamine sensitization, induction of peritoneal exudatecalls, and splenomagaly; however, the fraction retained antiviralactivity. Partially purified polysaccharide obtained by relativelygentle shearing of the cells possessed antiviral activity. Purified LPSexhibited antiviral activity also. Our preliminary data indicated thatthe 20 ug (dry weight) dose of LPS was near the minimum protective dose.

The LPS of 1. DgrtuasLS was unique in its antiviral activity (Table3). Purified LPS of four different gram-negative species did not haveantiviral activity. An antiviral activity of D. port uia&-derived LPSand derivatives has been reported previously by LeDur, Chaby, Szabo and

* coworkers. The protective activity of LPS observed by LeDur andcoworkers developed within 24 h. We may be examining the same activity;however, the duration of our antiviral effect (5 to 35 days) wouldsuggest that we are observing a different component of immunomodulation.

Additional evidence for a polysaccharide moiety being involved inthe antiviral activity is the observation that the vaccine can beinactivated by periodate treatment. Treatment of BPV with 100 mMperiodate for 48 h destroyed the protective activity (Table 4). Milderperiodate treatment as described by Tuomanen and Handley to inactivate 1.o RartiamAL glycocaylx adherence to bronchial ciliated epithelial cells didnot abrogate the protective activity.

Taken together, the date indicate that LPS is probably involved inthe BPV-induced immunomodulation that results in resistance to NAdlpt4.However, when one examines the relative specific activity of 1. pLPS in the various acellular fractions, one observes that LPS inconjunction with native protein appears to be more effective, e.g., BPV(25 ug dry weight) and 15A-1OSA versus purified LPS (20 ug dry weight),which was near the minimum effective dose. Sultzer, Craig, end coworkers,have observed that the endotoxin-associated proteins of D. agzjjuJUe. areunique in their adjuvant activity. Their findings also suggest that LPScomplexed with endotoxin-associated proteins might have increasedantiviral activity. We are currently focusing our efforts on theisolation of an LPS-protein complex from 1. ELaJ&.

-3-

Table 2. Activities of BdstlJla aertuasiJ Vaccine and AcellularExtracts

Treatment Dry Weight Protein LPS 1 ,2 Biologic Activity ortality3(£.0.) (ua) (UG) (uQ) (deaths/total)

BPV, 250 51 2.34 LPF, HSF, PEC- 0/21Connaught stimulation,

splenomegaly

BPV, 25 5 0.23 19/47Connaught

15A-1B, --- 46 147 LPF, HSF, decreased 3/16Connaught splenomegaly

15-108A, --- 12 .039 decreased LPF,HSF, 4/20Connaught and PEC stimulation,

splenomegaly notdetected

Purified 20 <0.2 40 --- 0/5LPS, 3779

Polsacch, 415 11.5 100 --- 0/518323

Diluent ........... 21/21

1 The efficiency of detection was assumed to be similar for free andbound LPS as reported by Jorgesen and Smith.

2 Amebocyte lysate assay, Sigma Chemical Company, St. Louis, NO.

Challenged with 2-10 x 107 PFU MAdljL4 seven days after treatment.

3.0 MECHANISM OF BPV-INDUCED ANTIVIRAL ACTIVITY. In anticipation ofisolation of a purified component with antiviral activity, preliminarystudies of possible mechanisms of immunomodulation were carried out.

* 3.1 INDUCTION OF A HUNORAL RESPONSE.The serum of BPV-treated mice did not contain crosareacting antibody

that would neutralize NAdl14 I& vitro. However, several publishedreports indicated that certain monoclonal antibodies do not neutralizeviruses, but will protect a test animal against a challenging virusinfection. Serum was obtained from mice treated with BPV for 21 days.Passive immunization against NAd1aj 4 infection was attempted byinoculating 0.5 ml of the serum into each animal 12 h prior to viruschallenge. No protection was observed.

-4-

'* 4 . *.o .- . .... ......-... 4.... . -44 * • .o . . - - * * p ,. * 4 " . 4

Table 3. Activity of Purified Lipopolysaccharidesl

Treatment, Nortality2

(20 uo) (deaths/total)

Lecherichia gqJj, Serotype 055:B5 5/5

SVLbrLo cholerae, Serotype Inaba 5699 5/5

SlmILD.lla 3isurium 5/5

I2oU*aeJl. peLtussLs, strain 37793 0/5

Purified by the Westphal phenol-water extraction method.

Challenged with 2 x 107 PFU NAdl2L4 seven days after treatment.

3 Provided by Dr. Thomas W. Klein, University of South Florida,Tampa, FL.

Table 4. Modification of PV-

Reaction Conditions Prior to Animal Injection Nortalityl(deaths/total)

BPV (no treatment) 0/5

BPV treated with 0.1 N M104 for 4S h at 250C. 5/5

BPV treated with 0.5 mH NaI04 for 30 win at 250C. 0/5

SChallenged with 4 LD50 of NAdlmk4 seven days after treatment.

3.2 INDUCTION OF A CELLULAR RESPONSE. Peritoneal exudate calls (PECa)were chosen for study because of their direct exposure to BPV duringtreatment, ease of harvest, and the prior report by Norahan and coworkersof antiviral activity of PECa following lnunomodulation. Vesicularstomatitis virus (VSV) infection was chosen for the Initial developmentof en La viltro assay of antiviral activity due to the ease and rapidityof the VSV plaque assay; one day as compared to 12-14 days for NAdl1L 4.Nice were Inoculated I.p. with BPV or vaccine diluent

-5-

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(saline-merthiolate;SM) and seven days later peritoneal lavage wasperformed to harvest PECe. Cells were cultured in microtiter plates or35 a culture dishes. The following cell concentrations were added toeach microtiter wall, singularly or in combination:

L929 - 4.0 x 103 cellsSK-PECs - 1.0 x 105 cellsBPV-PECs - 1.0 x 105 cells

The cell culture systems were infected by adsorbing 20 PFU of VSV,aerotype Indiana, to L-929 cells for 2.0 h. This strain of VSV did notproduce a lethal infection in mice with 108 PFU or leas. Twelvemicrotiter cultures were pooled for each point assayed. The growth cycleof the virus appeared to be essentially complete within 60 h of infectionin L-929 cell cultures (Figure 1). Combination cultures using the aboveeffector cell (PEC) concentration to obtain an effector to target cellratios of 25:1 were constructed also. Virus was adsorbed to L-929 cellcultures (calculated NO0 of 1.0 PFU per 200 cells) for 2.0 h to allowentry of the virus into the permissive cell and then effector cells(either SN-PECs or BPV-PECa) were added. As before, 12 microtiter wellswere pooled for each assay of infectivity. Effector cella harvested fromSN-treated mice had no observable effect when cocultured withVSV-infected L-929 cells, whereas effector calls from BPV-treated micedecreased the ability of L-929 cell cultures to synthesize virus byapproximately 90% (Figure 1). This observation was confirmed bystatistical analysis of titers obtained from ten individual pools ofmicrotiter well cultures 60 h after infection (Table 5).

I | I I I

60

5.0

~40-

30

* 2.0-

1.0

* 0 12 24 36 48 60 72TIME AFTER INFECTION (IIx

Figure 1. Growth Curve of Vesicular Stomatitis Virus in L-929Cell and Peritoneal Exudate Cell Cocultures. Vesicularstomatitis virus was adsorbed to cultures of L-929 cells.After virus adsorption either no cells (0l), SN-induced PICa(0), or IPV-induced PICa (0) were added and virus Infectivitywas assayed on L-929 cells thereafter.

>,> -... ".. . . . .." ". ..". " . - ." " " "" . .. -. ..... " .' , .. "-. - i" '

Table 5. Activity of BPV-induced Peritoneal Exudate Cell onVesicular Stomatitia Virus Synthesis in L-929 Celia

Treatment of Infected Cultures Virus Synthesized 60 h After Infection(PFU x 10-6 )

meoan Standard Deviation

Control (no PEC addition) 18.4tc 7.8

SX-PEC coculture 37.1t-, 28.8

BPV-PEC coculture 2.&t' 0.9

a Student's t-test, d.f. a 18, P a .059.b Student's t-test, d.f. a 18, P m .001Student's t-test, d.f. a 18, P < .001

Although there were many more cells in the PEC population, ascompared to the L-929 cell population in the cultures, the PECa werenotably less permissive (Figure 2). In addition, the BPV-induced PECawere less permissive then the SN-induced PEC cultures. Thus, the PECfraction in a combination culture of PECs and L-929 cells wouldcontribute less than one percent of the progeny virus.

A portion of the inhibition of virus synthesis was due toInteraction of the BPV-induced PECa and the cells infected initially.Virus was adsorbed to L-929 cell cultures (calculated NOI of 1.0 PFU per200 cells) for 1.0 h to allow entry of the virus into the permissivecil. The infection was carried out in 35 ma culture dishes with thesame density of L-929 cells per unit area as was used in the microtiterwells. Effector cells (either SN-PECa or BPV-PECa) were added and thecombination culture was Incubated an additional 2.0 h. Excess L-929 cellsat a concentration that would forma monolayer within 24 h were thenadded and 1.0 h later an agar-based overlay medium was added to localizevirus cytopathology for visualization of infectious centers or plaques.Approximately half of the Infectious centers were inactivated bySPV-induced PICa (Table G). Virus Infection cycles in L-929 cellosubsequent to the initial round of multiplication were probably Inhibitedalso.

The activity of BPV-induced PECa was specific and was directedtoward virus-infected L-929 target cells and not toward both uninfectedand infected cells. L-929 cells were planted at a concentration of 40

-; cells per 30 m dish and cultured 24 h. SPV-Lnduced or SN-Lnduced PECswere added to the L-929 cell cultures at the previous effector cell totarget cell ratio. The cells were then maintained in coculture for 6days and the colonies of L-929 cells counted.

71

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-J

2.0

p" I I a -I -- J -

-. ".0 12 24 36 W8 70 5 4

TIME AFTER PFECTKON (Oh&V

Figure 2. Growth Curve of Vesicular Stoatitle Virus In L-929Cell end Peritoneal Exudate CeLs. VeaIcular stometitia virus weadsorbed to cultures of L-2 cells (0), SK-induced PKCs (0), or

SBPV-induced PECS (O) and virus Infectivity was e&syed on L-92cells thereafter.

Table 6. Activity of SPV-Lnduced Perltoneal Exudate Cells on VesicularStomatitis Virus Infectious Centers

Treatment of Infected Cells Infectious Centers in L-92 CultureNn Standard Deviatlion

Control (no PEC addition) *2oSSDC 6.6

S5-PEC coculture 77.40_vt 11.0

BPV-PEC coculture SS.0t'c 6.4

• Student's k-test. d.f. a 1, P * .004.Student's &-test, d.f. n 10, P ( .001.Student's L-test, d.f. a If. P * .009.

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Li.BPV-induced PECs exhibited some toxicity toward uninfected L-929

calls; however, the amount of colony formation indicated that nonspecifictoxicity did not play a ma3or role in the antiviral activity ofSPV-induced PECa (Table 7). Coculturing of SN-induced PECs with L-929cells markedly decreased the number of microcolonies that developed.This decrease did not appear to be due to toxicity. The coculture ofSN-induced PECa and L-929 cells resulted in a marked increase of L-929cell migration or spreading and enumeration of discrete colonies wasdifficult.

An initial characterization of the PEC population with antiviralactivity was carried out. Infected L-929 cells were cocultured witheither PECa or PEC& minus the adherent cell population. SN-induced andBPV-induced PECa were harvest as described previously. A portion of eachcell suspension was planted in 60 a cell culture petri dishes andincubated 2.0 h. Nonadherent cells were removed by washing and ed3ustedto the same concentration as the total PEC population. Approximately 50xof the total population was adherent cells, consequently the nonadherentcell population was enriched approximately twofold.

Table 7. Colony Formation in Cocultures of L-929 Cells and PeritonealExudate Cells

Treatment Colonies after 6 Days of Culture

Mean Standard DeviStion

Control (no PECa added) 30.3'C 4.9

SM-PEC coculture 9.30-b 2.8

BPV-PEC coculture 23.60 b 4.1

a Student's t-test, d.f. a 18, p < .001.

Student's t,-teat, d.f. a 18, p < .001.Student's t-tost, d.f. w 18, p - .003.

Removal of the adherent cell population increased the antiviralactivity of BPV-induced PECa (Table 8). Either the cell populationresponsible for the antiviral activity was enriched by the procedure orthe adherent cell population in the original PEC suspension wasinhibiting the antiviral activity of that suspension.

Taken together, our data indicate that BPV-induced PECs exhibit anantiviral activity toward VSV infected L-929 cell; however, the systemdoes not provide an easily assayed expression of the antiviral activity,i.e., a relative large number of cultures and statistical analysis wouldbe necessary to evaluate a given acellular fraction of I. partu .1.U . Ourfuture efforts will be directed toward using herpes simplex virusinfected cells as target cells.

4.0 PATHOGENESIS OF NAdlLI4. Studies of MAd1M&4 pathogenesis wereextended to aid in the Interpretation of the model used for screeningantiviral activity of 1. h.

* -9

4.1 MIGRATION OF THE INOCULUN. The kinetics of MAdl 4 migration fromthe peritoneel cavity after inoculation was examined. Nice were injectedi.p. with 10 4 PFU (sublethal dose) and then a pocket bleed and peritoneallavage were performed. Infectious virus was assayed in each system atvarious times after infection. Unexpectedly the virus appeared in theplese very soon after in3ection into the peritoneum (Table 9). most ofthe virus had migrated out of the peritoneum by 3.0 h. The virusexhibited very low titers in the circulating blood by 24 h and thenincreased after 3 to 4 days, probably due to active replication.Significant changes in the number of cells or cell types in theperitoneum during this period were not observed.

:- 4.2 SYSTEMIC INFLAMMATION. Infection with a sublethal dose of MAdliQ4resulted in a significant, transient splenomegaly after infection (Figure3). The increased spleen size was due In part to an increaad number ofcalls in the spleen. Infection with a sublethal dose of MAd1g&4 did notinduce a fever response of hypothermia characteristic of mice (Figure 4).In contrast, lethal dose infection induced a fever response at least twodays before death (Figure 5).

Table 8. Activity of Nonadherent BPV-induced Peritoneal Exudate Cells onVesicular Stomatitia Virus Synthesis in L-929 Cells

Treatment Virus Titer after 60 h(PFU x 10-6)

Mean Standard Deviation

Control (no PEC addition) 10.6 3.8

SM-PEC coculture 3.5& 1.5

SK-PEC (minus adherent cells) coculture 1.80 1.3

BPV-PEC coculture 2.5b 0.6

BPV-PEC (minus adherent cells) coculture 1.3b 0.4

a Student's &-test, d.6. - 8, p - .083b Student's 1-test, d.f. a 8, p s .004

- 10 -

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Table 9. Virus Migration from the Peritoneal Cavity to the Bloodstroaml

Time After Infection Peritoneal Cavity plasms2K(h) (PFU) .(PFU)

0.*08 19,800 3080.*25 7,300 3960.42 9,500 4841.0 5,000 1,1003.0 280 3086.0 920 2,44212 20 11024 10 4448 120 4472 60 96896 140 550268 90 220

1 Nice were injected i.p. with 104 PFU.

2Based on a total plasma volume of 1.10 al as reported byFriedman.

* 4.3 HUNORAL RESPONSE. Nice respond to sublethal dose infection withdetectable neutralizing antibodies within 14 days after infection (Table

* 10). As little as 10 PFU will cause seroconversion of the mouse (Table11). These data reinforce the extreme caution one must use in working

* with NAdlpt4 and thereby prevent possible communication between the viruslaboratory and the animal facility.The role of interferon in mouse adenovirus infection Is questionable atthis time. We have repeatedly fail to demonstrate significant increasesin levels of interferon in the blood or peritoneal lavage after virusinfection. In addition, reproducible increases of interferon after BPV

* treatment were not observed. Numerous attempts to induce interferon inL-929 cells by NAd1q4 infection have failed. Finally, the sensitivity

* of KAdlg&4 to the effects of interferon was fourfold less than VSV.

* 5.0 PUBLICATIONS FRON THIS CONTRACT.

Winters, A. L., D. W. Daggett, W. R. Benjamin, H. K. Brown, and T. W.Klein. 1985. Resistance to adenovirus Infection after administration ofSordstshi* RSZAA vaccine In mice. Infect. Immun. 47: (in press).

Winters, A. Lop Do W. Daggett, J. Do Loa, Go L. Sloan# R. Do Lesson, andR. S. Stinson. 1985. Immunomodulation by Bgrdsalla mpgM..eLas: Antiviraleffects. 1A, C. R. Kanclark and W. Hennessen (ads). Proceedings of theFourth International Symposium on Pertusais. S. Karger, N.Y., (in proe).

1.2

1.0

o 0.80X

~02

ow

""0.4-

* 0.2

011 3 5 7 10 14 21

TIME AFTER INFECTION (days)

Figure 3. Splenomegaly Following KAd1p.t4 Infection. Nice were

inoculated i.p. with 104 PFU (0) or L-929 cell extract control(13). Unin3ected mice were used as control also (0). Pointsrepresent the average splenic index of 3 to 15 mice.

ri

%

S- 12-

38.0

360

21.0-

Figure .Temprature f MiceFollown Mtd1~neto

23.0-rooma temrp.

34.0

2hreftr rahpon epeetstema tempertreo

3.0.

Dose.~~~~~~~~~~~~~rca tiewrencuaem~.wih2ax17p.U*Rca tmeaue

vea baieda 1 hitevasthrefer Ec smblre4eetsa

indviualmose

MICL%

.d :. * - * * . 6. 0- . .-

Table 10. Development of Neutralizing Antibodies After NAdIML4Infectioni

Time of Serum Sample Titer2 ,3

(days)

7 < 10

14 47

21 160

28 213

35 267

1ice were infected by i.p. inoculation with 1.0 x 104 PFU.

2 One unit was defined as the antibody concentration necessary toneutralize 16 TCID5o in an L929 culture in a microtiter well.

3 Represents the average titer in a group of three mice.

Table 11. Dose-response of HAd1L4 and Neutralizing Antibodies

Virus Dose Neutralizing Antibody

(PFU) (Titer)1, 2

0 (L-929 cell extract) ( 10

101 213

102 133

104 160

Represents the average titer in a group of three mice.

I Serum obtained 21 days after virus inoculation i.p.

I -14

w' m

ToI

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DTICS0,5