OBJECTIVE : To investigate the immunological efficacy and mosquito infectivity of a candidate dengue 2 virus vaccine.

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Biological and mmunological Studies of a Dengue 2 Candidate Virus Vaccine Principal nvestigators : Robert Mcair Scott, LTC, MC Ananda isalak, M.. Douglas J. Gould, Ph.D. Associate nvestigators : Frank E.

1 Biological and mmunological Studies of a Dengue 2 Candidate Virus Vaccine Principal nvestigators : Robert Mcair Scott, LTC, MC Ananda isalak, M.. Douglas J. Gould, Ph.D. Associate nvestigators : Frank E. Chapple, MAJ, VC Phillip -K. Russell, COL, MC Walter E. Brandt, Ph.D. John L. Bwn, MAJ, YC OBJECTVE : To investigate the immunological efficacy and mosquito infectivity of a candidate dengue 2 virus vaccine. BACKGROUD : Dengue emorrhagic Fever (DF) is a severe public health pblem in Southeast Asia and represents a potential threat to military tops operating in areas where the dengue viruses are prevalent. For these reasons dengue virus strains are being sought for the preparation of vaccines. Over the past several years, a dengue 2 virus strain has been isolated under conditions suitable for vaccine development (1). The candidate vaccine virus (Sl) is a small plaque variant isolated by plaque selection fm dengue 2 PR-159, a strain obtained during the 1969 dengue 2 epidemic in Puerto Rico. Compared to the parent strain, (PR-159 PGMK-6), the small plaque variant (PR-159 S1-PGMK-19) was temperature sensitive and appeared to have decreased virulence in suckling mice. While the parent strain pduced relatively consistent viremia in ndian Rhesus monkeys on days three to seven following inoculation, the S1 strain only occasionally pduced viremia. When viremia did occur, it was as late as the tenth day following inoculation. Despite the inconsistency of viremia, the Sl strain appeared to be % stngly immunogenic. Following initial testing in the rhesus monkeys the S1 strain was further passed four cimes in fetal rhesus lung cells. A candidate vaccine (PR-159 (S1) Lot l,pgmk-19 FRL-4) was prepared. The purpose of this investigation was to study the clinical and immunological affect of this candidate vaccine strain on ndian Rhesus monkeys and to determine its infectivity for mosquitoes biting these monkeys. METODS : Twc strains of dengue 2 virus were received fm Dr. Walter E. Brandt, Department of Virus Diseases, Walter Reed Army nstitute of Research. At the time of preparation t e fzen parent strain (PR-159 PGMK-6) was reported to 5 have a titer 1.5 x 1 PFU/ml at 35OC and the lyophilized candidate vaccine stgain (PR-159 (Sl) PGMK-19 FRL-4, Lot 1) was reported to have a titer of 1.9 x 1 PFU/ml at 35'C. The viruses were shipped on dry ice and held at -7 C. The virus preparations were rapidly thawed and reconstituted just prior to use. ndian Rhesus monkeys (Macaca mulatta) were screened for hemagglutination inhibition () antibody against dengue 1-4, Japanese encephalitid, Vesselsbn and Tangat antigen and for neutralizing () antibody against dengue 2. Fifteen

monkeys wer"e selected which were negative for these antibodies. Monkeys received either the candidate vaccine or. the parent strain. Each monkey was inoculated subcutaneously in the left arm wish O.

General,condition, appetite, temperature organomegaly, lymphadenopathy and presence or absence of petechia were noted.

- eparinized blood was separated and plasma and leukocytes were submitted for virus assay.

Virus isolations were attempted by the'direct and delayed plaque assay technique.

On days one thugh ten followig inoculation; 3, 3-5 day old femal mosquitoes pvided by the Department of Medical Entomology were allowed to feed on each monkey.

2 monkeys wer"e selected which were negative for these antibodies. Monkeys received either the candidate vaccine or. the parent strain. Each monkey was inoculated subcutaneously in the left arm wish O.S ml of- the appointed virus strain containing an estimated-o.s -1 x 1 PFU. The monkeys were examined before inoculation and daily thughout the course of the experiment. General,condition, appetite, temperature organomegaly, lymphadenopathy and presence or absence of petechia were noted. Bloowas obtained prior to inoculation and on days one thugh 1, 15, 3 and 6 following inoculation. Blood was submitted for hemogram and platelet count. - eparinized blood was separated and plasma and leukocytes were submitted for virus assay. Methods for separation of leukocytes are described elsewhere in this annual report (The solation of Dengue Virses fm Leukocytes). Virus isolations were attempted by the'direct and delayed plaque assay technique. Serum was submitted for selogy by tests and plaque reduction neutralizations tests (PRT) against ptotype dengue 1-4 antigens. On days one thugh ten followig inoculation; 3, 3-5 day old femal mosquitoes pvided by the Department of Medical Entomology were allowed to feed on each monkey. Engorged mosquitoes were separated, incubated for 14 days at 32 C, sacrificed by quick freezing and stored at -7 C. Ten mosquitoes were pooled, (one pool/monkey/day) sonicated in 1. m1 of Roswell Park Memorial nstitute. (RPM) 164 media supplemented with glutamine, sodium bicarbonate and 1% fetal calf serum, and centrifuged at 1 x g for 3 minutes. The supernatant was assayed for virus by direct/and delayed plaque techniques. Six mosquitoes/day/ monkey were ecapitated; had squashed preparations were examined for dengue. antigen by a direct fluorescent antibody technique (FA)..Monkeys immunized witheithe-r 'the: can.didate vaccine or"the parent deng\:le 2 strains were challenged with the.candidate vaccine strain or with low passage. Southeast Asian wild type strains of dengue 2 (BM-5-7 LLCMk2-2) or dengue 3 (C LLCMk2-1). Dengue 2 (BM-5-76, LLC-Mk -2) was isolated fm an Aedes aegypti mosquito collected in the home of a DnF patient; denge 3 (C , :LLC-Mk2-1) was isolated fm a DF patient. The monkeys. were handled and samples were collected for isolation and selogy in a manner similar to that used in the initial infections. RESULTS: F.ifteen monkeys were inoculated with.5 1 of dengue 2 virus prepa-rations. Ten of these received the candidate vaccine strain (PR-159 (81) PGMK- 19, FRL-4, Lot 111) and 5 received the parent strain (PR-159, PGMK-6) (Table 1). At.the time of inocu1atio these virusgreparations were titered at 37:.C and found to contain 6.7 x 1 and 1.3 x 1 PFU/m respectively. Examination of the 15 monkeys thughout the course of the experiment showed no significant physical or hematological changes. Using direct and de1aye.d plaque assay at 37 C and 35 C.no virus was recovered fm the plasma of any of the ten:monkeys who received the candidate vaccine virus. Antibodies aga1nst dengue 2 virus had developed by 3 days following inoculation in six of these monkeys and antibody was"-found in three. 77

Viremia was documented in all five of the monkeys inoculated with the parent strain.

most monkeys thugh day eight but had cleared by day nine. Dengue 2 virus was isolated fm leukocytes taken fm monkeys on days eight and nine, late in the course of infection.

Both and antibody to dengue 2 developed by the 15th day after immunization in all of the monkeys inoculated with the parent strain.

3 Viremia was documented in all five of the monkeys inoculated with the parent strain. Virus was isolated fm the plasma of one monkey by the second day following inoculation and was found in all monkeys on days six and seven. The viremia persisted in.most monkeys thugh day eight but had cleared by day nine. Dengue 2 virus was isolated fm leukocytes taken fm monkeys on days eight and nine, late in the course of infection. On two occasions virus was isolated f'om leukocytes but could not be detected in plasma. Both and antibody to dengue 2 developed by the 15th day after immunization in all of the monkeys inoculated with the parent strain. The antibody titers at the time of measurement 'did not appear to be related to the time or duration of viremia. Dengue virus was not detected by either isolation or FA in mosquitoes that fed on monkey inoculated with the candidate vaccine strain. nfection was detected by direct and delayed plaque isolation and by FA only in mosquitoes fed on monkeys inoculated with the parent virus. Virus was intermittently isolated fm mosquitoes that fed on these monkeys on day three thugh six following inoculations. solations could be obtained fm mosquitoes at times when no virus could be detected in the plasma, and virus was usually isolated fm mosquitoes before or early in the stage of detectable viremia. Challenge experiments were carried out 126 days following initial inoculation (Table 2). The monkeys which had received the candidate vaccine strain were divided into gups based upon the antibody titer to dengue 2 antigen at 3 days. Three pairs were established each contained one monkey with an, antibody titer 21:2 and one with a titer of > 1:2. These pairs were inoculated with the candidate vaccine strain, and low passage Southeast Asian wild type strains of dengue 2 (BM-SO-76, LLC-Mk2-2) and dengue 3 (C , LLC-Mk2-1) respectively. Of the remaining four monkeys who did not developed antibody following inoculation with the candidate vaccine strain, one was inoculated with the candidate vaccine strain, two were challenged with the wild type dengue 2 strain and one received the wild type dengue 3 virus. Plasma collected fm all monkeys just prior to challenge were retested for and antibodies. Of the monkeys which had received the candidate vaccine strain, only one monkey had and/or antibody at this time, suggesting that the antibody detected at 3 days might have been of the gm class. All the monkeys which had received the parent strain retained both and antibodies. The titers of nocu1um at the time of injection, determined by p1que assay at 35 C were 5 x 1 PFU/m1 for the candidate vaccine strin, 1.1 x 1 PFU/m1 for the wild type dengue 2 strain (BM-5-76) and 4.6 x 1 PFU/m1 for the wild type dengue 3 strain (C, ). n the monkeys reimmunized with the candidate vaccine strain, there was no detectable viremia; none the less all of these monkeys developed appreciable titers of dengue 2 and antibody by day 141, 15 days after reimmunization. These antibodies persisted at essentially the same titer for at least one month after immunization. Of the four vaccinated monkeys challenged with the dengue 2 strain (BM-5-76), three developed viremia and leukocyte infections on the fourth thugh the sixth day after challenge. The fourth which developed no demonstrable infection was the one monkey which had appreciable amounts of dengue 2 and antibody 78

4 1. Eckels, just prior to challenge. Challenge of two monkeys that received the parent strain of dengue 2 virus (PR-l59, PGMK-6), with wild type dengue 2 (BM-5-76) resulted in titers rises but no demonstrable viremia. antibody was not tested at a level nece.ssary to detect a titer rise, it was already> 1:64 at the time of challenge. - Four monkeys were cija11enged with the dengue 3 wild type virus (C ) Two of these, having previously received candidate dengue 2 vaccine vrus, developed dengue 3 viremia and css reactive and antibodies. The other two were initially inoculated with the dengue 2 parent strain (PR 159, PGMK-6). Of these, only one developed a viremia. This occurred at appximately the same time as the viremia occurred in the previous immunization with the dengue 2 parent strain. Both of these monkeys developed css reactive and anti-bodies. This work is now ompleted and will prepared for publication. REFERECE K.., Brandt, W.E., arrison, V.R. McCown, J.M., and Russell, P..K.,1976. solation of TemperatuTe-Sensitive Dengue-2 Virus under Conditions Suitable for Vaccine Development. nf. & mm., :

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Dengue-2 Vaccine: Viremia and Immune Responses in Rhesus Monkeys

INFECTION AND IMMUNITY, Jan. 1980, p. 181-186 0019-9567/80/01-0181/06$02.00/0 Vol. 27, No. 1 Dengue-2 Vaccine: Viremia and Immune Responses in Rhesus Monkeys ROBERT McN. SCOTT,'t* ANANDA NISALAK,' KENNETH