Attenuated Venezuelan Equine

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1 APPLIED MICROBIOLOGY, Oct. 1972, p Copyright Americn Society for Microbiology Vol. 24, No. 4 Printed in U.S.A. Erly Protection in Hmsters Immunized with Attenuted Venezueln Equine Encephlomyelitis Vccine FRANCIS E. COLE, JR., CARL E. PEDERSEN, JR., AND DAVID M. ROBINSON U.S. Army Medicl Reserch Institute of Infectious Diseses, Frederick, Mrylnd Received for publiction 23 June 1972 The rpid onset nd persistence of homologous nd heterologous protection induced by ttenuted Venezueln equine encephlomyelitis (VEE) vccine (TC-83) were studied in the hmster, by using chllenge response s the index of protection. At 8 hr postvccintion with 103 medin immunizing doses of TC-83 vccine, 15 to 20% of nimls were protected ginst chllenge with VEE virus s well s Estern nd Western equine encephlomyelitis viruses. The percentge of protection incresed with time postvccintion until 80 to 90% homologous nd heterologous protection ws chieved by 18 hr postvccintion. Temporl studies indicted tht erly protection (dys 1 to 6) correlted with vccine viremi, nd tht the percentge of protection ginst heterologous chllenge decresed with the cesstion of viremi. Dt re presented to indicte tht the erly protection phenomenon is one of interference, since little or no repliction of chllenge virus occurred when it ws dministered during the vccine viremi stge. In previous publiction (5) we reported tht, in the hmster, ttenuted Venezueln equine encephlomyelitis (VEE) vccine (TC- 83) produced excellent homologous protection s well s 59 nd 37% protection ginst Estern nd Western equine encephlomyelitis (EEE, WEE) virus chllenge, respectively. However, in those studies ll chllenges were mde 21 to 30 dys postvccintion. Becuse of the extensive VEE epizootics in Centrl Americ (R. 0. Spertzel nd R. W. McKinney, in press) nd Mexico (R. 0. Spertzel nd R. W. McKinney, in press) in 1969 to 1971, nd the subsequent outbrek of the disese in the United Sttes (R. 0. Spertzel, in press) in the summer of 1971, the studies described herein were conducted to determine if the TC-83 vccine ws cpble of inducing rpid protection. The significnce of this is pprent when one considers: (i) the speed with which VEE spreds in n equine popultion, nd (ii) the fct tht immuniztion is often performed in res where ctive cses re occurring. Also, it ws deemed desirble to determine whether the vccine would provide significnt, rpid cross-protection ginst EEE nd WEE, the two other equine encephlomyelitides endemic in those res of the United Sttes in which VEE outbreks could be expected to occur. Finlly, the possible mechnism(s) of erly protection ws investigted. MATERIALS AND METHODS Chllenge viruses. Cliforni strin WEE virus (3), Cmbridge strin EEE virus (3), nd Trinidd strin VEE virus (12) were employed s chllenge viruses. These strins were lethl to high titers, i.e., 107 to 1010 medin lethl doses (LDO) per milliliter, when given intrperitonelly (ip) to the hmster. Virus titrtions. All chllenge viruses were titrted vi the ip route in Lkeview strin golden Syrin hmsters (85 to 95 g) obtined from the Lkeview Hmster Colony, Newfield, N.J. Groups of six 604 hmsters were inoculted with 0.5 ml of log10 dilutions of virus in cold phosphte-buffered sline, ph 7.2, contining 1% norml rbbit serum (PBS). For viremi determintions with ttenuted VEE (TC- 83) vccine, blood specimens were diluted in cold PBS. Groups of six 1- to 3-dy-old mice (CD-1 strin; Chrles River Mouse Frm, Wilmington, Mss.) were then inoculted intrcerebrlly (ic) with 0.03 ml of log,, dilutions. Titrtions end points in hmsters nd suckling mice were determined by the method of Reed nd Muench (13) nd re expressed s LD,, per milliliter. Vccintion nd chllenge. The ttenuted VEE (TC-83) vccine hs been described (2, 10). In ll experiments, hmsters were inoculted ip with 10' medin immunizing doses (ID..) of the vccine. Chllenges were mde vi the ip route t the indi-

2 VOL. 24, 1972 EARLY PROTECTION IN HAMSTER 605 cted times using 103 hmster LD,. of given chllenge virus. Norml hmsters used s chllenge controls for ll experiments consistently showed 98 to 100% deth rte with the dose of chllenge virus employed. Serology. Rndomly selected hmsters were bled t the specified periods postvccintion or postchllenge. Ser were stored t -20 C until tested for hemgglutintion-inhibiting (HI) ntibody, by the method of Clrke nd Csls (4), s modified by French nd McKinney (8). RESULTS Temporl studies. Initil studies were imed t determining: (i) the degree of homologous nd heterologous protection induced by the vccine during the first 10 dys postvcnintion, nd (ii) the presence of circulting HI ntibody or TC-83 virus, or both, during this period. Tble 1 is compiltion of dt from severl experiments in which hmsters were dministered TC-83 vccine on dy 0. On dys 1 through 10 postvccintion, representtive numbers of hmsters were exsnguinted for vccine viremi nd HI ntibody determintions, while other rndomly selected hmsters were chllenged with virulent VEE, EEE, or WEE viruses. Vccine viremi ppered s erly s dy 1 postvccintion nd persisted through dy 5 in the mjority of hmsters; however, viremi ws demonstrted in some nimls s lte s dy 7. Termintion of the viremic stge generlly coincided with the ppernce of HI ntibody to TC-83 virus. HI ntibody to TC-83 virus ws detected in individul nimls s erly s dy 5, wheres in TABLE 1. Response of hmsters vccinted with TC- 83 vccine nd chllenged with VEE, EEE, or WEE viruses Dy VEE Vc- Percent survivors (no./totl) post- HI cine fter ip chllenge with 103 LD[A) vcci- nti- virention body mi VEE EEE WEE 1 _ c +/_d 92 (23/25) 87 (26/30) 93 (37/40) (18/20) 90 (27/30) 77 (27/35) (46/50) 179 (123/155) 83 (132/160) (46/50) 77 (46/60) 77 (50/65) 5 +/- +/- 100 (20/20) 87 (26/30) 88 (30/34) 6 +/- +/- 100 (20/20) 77 (23/30) 97 (34/35) 7 +/- +/- 85 (17/20) 59 (17/29) 59 (20/34) (15/15) 72 (18/25) 63 (19/30) (14/15) 40 (10/25) 63 (19/30) (15/15) 48 (12/25) 57 (17/30) Dt compiled from results of severl experiments. Abbrevitions: VEE, EEE, WEE-Venezueln, Estern, nd Western equine encephlomyelitis, respectively; HL hemgglutintion-inhibiting. Nonvccinted (control) nimls exhibited 0 to 2% survivl fter chllenge. c A titer of > 1: 10 considered positive. d See Results. TABLE 2. Development of viremi nd HI ntibody in hmsters vccinted with TC-83 vccine Dy Vccine viremi VEE HI postvc- Percent ntibody cintion (no./totl) (rmen titerb (10/10) 2.2 < 10 ( ) (10/10) 3.7 < 10 ( ) (10/10) 5.7 < 10 ( ) (10/10) 2.9 <10 ( ) 5 90 (9/10) 1.4 < 10 ( ) 6 20 (2/10) ( ) 7 30 (3/10) ( ) 8 0 (0/10) (0/10) (0/10) 80 Ten different nimls bled ech dy; titers expressed s log,0 suckling mouse ic LD 1Jml. b Expressed s reciprocl of titer; pooled ser from two hmsters tested on ech dy postvccintion. For bbrevitions, see footnote to Tble 1. others ntibody did not pper until dy 8. A high degree of homologous nd heterologous protection ws fforded those nimls chllenged during the vccine viremi stge, viz. dys 1 through 6, fter which heterologous protection grdully decresed. Only 0 to 2% of nonvccinted, control nimls survived chllenge. A further study ws mde to quntify the viremi nd HI ntibody levels observed during the first 10 dys postvccintion. Hmsters were inoculted with TC-83 vccine nd were then rndomly selected nd exsnguinted for determintion of viremi (10 hmsters/dy) or HI ntibody (2 hmsters/dy). As shown in Tble 2, ll hmsters exmined on dys 1 through 4 were viremic, with pek men levels occurring on dy 3. All nimls tested for viremi fter dy 7 were negtive. HI ntibody ws not detected until dy 5, fter which there ws the expected increse in titer with time postvccintion. Mechnism of erly protection. In view of the high degree of homologous nd heterologous protection observed t 24 hr postvccintion, n dditionl study ws performed to determine the protection fforded t erlier periods postvccintion nd to relte this to vccine viremi. Subsequent to vccintion, hmsters were either bled for viremi determintions or chllenged with VEE, EEE, or

3 606 COLE, PEDERSEN, AND ROBINSON APPL. MICROBIOL. WEE viruses. Tble 3 is summry of the results of such study performed t 8, 12, nd 18 hr postvccintion. The importnce of vccine viremi in the erly protection stge is reflected in the responses to the chllenge viruses. As the percentge of viremic nimls incresed with time postvccintion, so lso did protection ginst homologous nd heterologous chllenge. The results observed t 18 hr postvccintion re comprble to those seen previously t 24 hr postvccintion (cf. Tble 1). To gin further insight into the mechnism of erly protection, we studied those hmsters tht survived chllenge dministered during the period of highest vccine virem, i.e., dys 1 through 4 postvccintion. In seprte experiments, groups of hmsters were vccinted on dy 0 nd then chllenged with VEE, EEE, or WEE viruses on dys 1, 2, 3, or 4. Control nimls received only TC-83 vccine on dy 0. On dy 17 postvccintion, representtive numbers of hmsters tht survived chllenge with virulent VEE, EEE, or WEE viruses s well s hmsters tht hd received only TC-83 vccine on dy 0 were exsnguinted, nd HI ntibody titers were determined. The remining nimls in ech survivl group were rechllenged with the sme virus tht hd been dministered on dys 1, 2, 3, or 4. As controls, hmsters tht hd received only TC- 83 vccine on dy 0 nd norml hmsters of the sme ge were lso chllenged. On dy 34, rndomly selected surviving nimls from ll groups were bled for HI ntibody determintions. Results of these studies re summrized in Tble 4. As indicted, there ws virtully no difference in dy 17 survivl rtes between those nimls tht hd received previous homologous chllenge nd those tht received only TC-83 vccine on dy 0. Since significnt repliction of the originl (dy 1 to 4) chllenge virus should hve incresed the bility of TABLE 3. Erly response of hmsters vccinted with TC-83 vccine nd chllenged with VEE, EEE, or WEE viruses Percent survivors Percent (no./totl) Hr Percien fter ip chllenge with postvc- 103 cintion viremi LDO' (no./totl) VEE EEE WEE 8 20 (2/10) 15 (3/20) 15 (3/20) 20 (4/20) (5/10) 30 (6/20) 25 (5/20) 25 (5/20) (5/5) 90 (18/20) 80 (16/20) 90 (18/20) All chllenge control hmsters died t ech time period with ech chllenge virus. For bbrevitions, see footnote to Tble 1. TABLE 4. Response to homologous rechllenge in TC-83 vccinted hmsters tht survived primry chllenge with VEE, EEE, or WEE viruses Percent Primry (no./totl) Homologous men HI chllenge surviving titerc (rnge) on dy 1 to 4 rechllenge postvc- on dy 17 cintionb with Dy 17 Dy LD.,, VEE 98 (49/50) ( ) (80-320) Controld 100 (27/27) ( ) ( ) EEE 39 (47/120) (10-20) ( ) Controld 35 (15/43) (< 10-40) ( ) WEE 42 (52/125) (< 10-20) ( ) Controld 44 (19/43) (< 10-40) ( ) Dt compiled from results of severl experiments. For bbrevitions, see footnote to Tble 1. "Received TC-83 vccine on dy 0, nd chllenge virus indicted on dy 1, 2, 3, or 4 postvccintion. creciprocl of geometric men nd rnge determined using pooled ser of two hmsters ech; minimum of five pools tested ginst pproprite ntigen (VEE, EEE, or WEE). d Received TC-83 vccine on dy 0, but were not chllenged until dy 17. these nimls to withstnd rechllenge, one cn ssume tht little or no repliction occurred. This pprent lck of repliction by the originl chllenge virus is verified by the HI titers of nimls t the time of rechllenge on dy 17. Comprble titers were seen in the chllenged (primry) nd control groups for ech of the three viruses studied. These dt re suggestive of n interference phenomenon medited directly or indirectly by the TC-83 vccine. Supporting this belief is the indirect evidence of the bility of EEE nd WEE viruses to replicte in the bsence of interfering virus, even in nimls previously protected by inocultion with TC-83 vccine. As shown in the results of HI tests performed with ser obtined from hmsters on dy 34 (i.e., 17 dys fter rechllenge), mrked increses of similr mgnitude in HI titers ginst EEE nd WEE viruses occurred with both the previously chllenged nd control groups. Effects of vccintion fter or t time of chllenge. As result of our demonstrtion of the rpid onset of viremi in nimls dministered TC-83 vccine (Tble 3), it ws of prcticl significnce to determine whether vcci-

4 VOL. 24, 1972 EARLY PROTECTION IN HAMSTER 607 ntion would lso be efficcious if crried out simultneously, or fter chllenge with VEE, EEE, or WEE viruses. In limited study, hmsters were dministered TC-83 vccine 48 or 24 hr fter chllenge s well s simultneously with the chllenge viruses. As shown in Tble 5, little or no protection ws fforded nimls vccinted fter chllenge. The degree of protection seen ginst WEE virus chllenge is perhps due to the slower invsiveness of this virus in the hmster, lthough no studies to confirm this were conducted. DISCUSSION Although extensive immuniztion progrms nd some studies hve been conducted with TC-83 vccine in mn (1, 7, 11; P. J. Brtelloni, personl communiction) nd nimls (5, 6, 14), no informtion hs been vilble regrding the bility of the vccine to induce rpid homologous nd heterologous protection. In the present study we hve demonstrted tht protection in the hmster strts s erly s 8 hr postvccintion, nd tht erly protection closely prllels the viremi resulting from infection with the vccine virus. Homologous nd heterologous protection ws gretest during the period of mximum vccine viremi, i.e. dys 1 to 6. The persistence of heterologous protection fter this period ws not unexpected in view of the results of n erlier study in which we demonstrted the bility of TC-83 vccine to elicit 59 nd 37% protection ginst EEE nd WEE virus chllenges, respectively, 21 to 30 dys postvccintion (5). Our dt on erly protection strongly suggest tht n interference phenomenon is responsible for the degree of protection observed. In this cse the presence of replicting, or t lest, circulting virus (i.e., TC-83 vccine) prevents superinfection of host niml with other viruses. The filure of the chllenge viruses to replicte to ny pprecible extent when dministered during the vccine viremi stge ws indirectly demonstrted in two wys: (i) hmsters which survived such chllenge did not show incresed resistnce to rechllenge dministered fter the vccine viremi stge, nd (ii) the HI ntibody responses of hmsters tht were vccinted nd chllenged were similr to those of hmsters tht were only vccinted. Evidence of the nonspecific nture of the erly protection is provided by results of experiments in which it ws shown tht EEE nd WEE viruses do, indeed, replicte in the bsence of interfering virus, nmely in the sme nimls tht hd been TABLE 5. Effect of vccintion with TC-83 vccine following inocultion of hmsters with virulent VEE, EEE, or WEE viruses Virulent virus Survivors (no./totl) by hr of chllenge preimmuniztion:b 0 48 hr 24 hr (simul- tneous) VEE 0/5 0/5 0/5 EEE 0/5 0/5 0/5 WEE 0/5 1/5 3/5 Chllenge ws 103 LD50, ip t 48 hr or 24 hr prior to or simultneously with vccine. For bbrevitions, see footnote to Tble 1. "All hmsters received 103 ID,0 of TC-83 vccine, ip t time 0. previously protected by inocultion with TC- 83 vccine. Here, deth or mrked increses in HI ntibody titers to EEE nd WEE viruses were similr in both test nd vccine control nimls. It should be noted tht the filure of lte chllenge with virulent VEE virus to increse the homologous ntibody titer is in greement with the results of our erlier study (5). Our findings, in generl, re in greement with those of Hern nd Riney (9), who showed tht vriety of lbortory nimls were protected ginst homologous nd heterologous chllenge s erly s 24 hr postvccintion with nother ttenuted strin of VEE virus. In our study, no ttempt ws mde to determine whether the observed interference ws interferon-medited. In terms of field use of the ttenuted VEE vccine in equines, the results of these erly protection studies re of mjor importnce. For exmple, Spertzel nd McKinney reported tht during the 1969 Centrl Americn epizootic of VEE, ll equine cses of VEE stopped 7 to 10 dys fter vccintion, even in res experiencing ctive cses of VEE in nonimmunized nimls (R. 0. Spertzel nd R. W. McKinney, in press). Similr results were reported by Eddy et l. (6). If one ssumes tht the mjority of those equines which died or exhibited clinicl illness during the 7- to 10- dy period were infected prior to or shortly fter vccintion, then one cn lso ssume tht protection occurs in equines erlier thn 7 dys postvccintion. These ssumptions re supported by Wlton's nlysis of the VEE outbreks in Nicrgu nd Cost Ric, which indictes tht the vccine will protect equines within 3 dys even in the presence of concurrent equine VEE infections in given re (T. E. Wlton, in press).

5 608 COLE, PEDERSEN, AND ROBINSON APPL. MICROBIOL. Bsed on the studies reported here, one might lso expect rpid, solid protection of equines ginst nturlly occurring WEE nd EEE during the erlier periods postvccintion. Indeed, decrese in WEE cses concurrent with the VEE vccintion cmpign during the summer of 1971 in southern Cliforni ws noted (W. C. Reeves, personl communiction). Thus, the ttenuted VEE vccine cn be expected to elicit rpid protection not only ginst VEE virus, but lso ginst the other two mjor equine encephlitides. Our previous studies indicte tht such heterologous protection cnnot be expected from n inctivted vccine prepred from VEE virus (5). LITERATURE CITED 1. Aleviztos, A. C., R. W. McKinney, nd R. D. Feigin Live, ttenuted Venezueln equine encephlomyelitis virus vccine. I. Clinicl effects in mn. Amer. J. Trop. Med. Hyg. 16: Berge, T. O., I. S. Bnks, nd W. D. Tigertt Attenution of Venezueln equine encephlomyelitis virus by in vitro cultivtion in guine pig hert cells. Amer. J. Hyg. 73: Byrne, R. J., G. R. French, F. S. Yncey, W. S. Gochenour, P. K. Russell, H. H. Rmsburg, 0. A. Brnd, F. G. Scheider, nd E. L. Buescher Clinicl nd immunologic interreltionship mong Venezueln, Estern nd Western equine encephlomyelitis viruses in burros. Amer. J. Vet. Res. 25: Clrke, D. H., nd J. Csls Techniques for hemgglutintion nd hemgglutintion-inhibition with rthropod-borne viruses. Amer. J. Trop. Med. Hyg. 7: Cole, F. E. Jr., nd R. W. McKinney Cross-protection in hmsters immunized with group A rbovirus vccines. Infect. Immunity 4: Eddy, G. A., D. H. Mrtin, W. C. Reeves, nd K. M. Johnson Field studies of n ttenuted Venezueln equine encephlomyelitis vccine (strin TC- 83). Infect. Immunity 5: Feigin, R. D., R. F. Jegr, R. W. McKinney, nd A. C. Aleviztos Live, ttenuted Venezueln equine encephlomyelitis virus vccine. II. Whole-blood mino cid nd fluorescent-ntibody studies following immuniztion. Amer. J. Trop. Med. Hyg. 16: French, G. R., nd R. W. McKinney Use of O- propiolctone in preprtion of inctivted rbovirus serologic test ntigens. J. Immunol. 92: Hern, H. J., nd C. T. Riney Cross-protection in nimls infected with group A rboviruses. J. Immunol. 90: McKinney, R. W., T. 0. Berge, W. D. Swyer, W. D. Tigertt, nd D. Crozier Use of n ttenuted strin of Venezueln equine encephlomyelitis virus for immuniztion in mn. Amer. J. Trop. Med. Hyg. 12: Pekrek, R. S., G. A. Burghen, P. J. Brtelloni, F. M. Cli, K. A. Bostin, nd W. R. Beisel The effect of live ttenuted Venezueln equine encephlomyelitis virus vccine on serum iron, zinc, nd copper concentrtions in mn. J. Lb. Clin. Med. 76: Rndll, R., nd J. W. Mills Ftl encephlitis in mn due to the Venezueln virus of equine encephlomyelitis in Trinidd. Science 99: Reed, L. J., nd H. Muench A simple method of estimting fifty percent endpoints. Amer. J. Hyg. 27: Spertzel, R. O., nd D. E. Khn Sfety nd efficcy of n ttenuted Venezueln equine encephlomyelitis vccine for use in Equide. J. Amer. Vet. Med. Ass. 159: Downloded from on December 25, 2018 by guest