Dengue-2 Vaccine: Virological, Immunological, and Clinical Responses of Six Yellow Fever-Immune Recipients

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1 INFECTION AND IMMUNITy, Feb. 1981, p Vol. 31, No /81/ $02.00/0 Dengue-2 Vaccine: Virological, Immunological, and Clinical Responses of Six Yellow Fever-Immune Recipients WILLIAM H. BANCROFT,`* FRANKLIN H. TOP, JR.,' KENNETH H. ECKELS,' JAMES H. ANDERSON, JR.,2 JACK M. McCOWN,' AND PHILIP K. RUSSELL' Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Washington, DC 20012,' and Division ofmedicine, United States Army Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland Six male volunteers, previously immunized with yellow fever vaccine, were inoculated subcutaneously with a live, attenuated dengue-2 virus (PR-159/S-1) candidate vaccine. Five recipients developed viremia 8 or 9 days after vaccination, which lasted 1 to 10 days. The onset of viremia was followed by fever in three people, transient leukopenia in four, and an erythematous rash in one. One volunteer developed an oral temperature of 38.8 C with headache, myalgia, fatigue, and photophobia suggestive of mild dengue fever. All five viremic volunteers developed fourfold or greater rises in serum neutralizing antibody. The sixth volunteer, who had a low titer of preexisting dengue-2 neutralizing antibody, had no viremia, no symptoms, and a modest rise in hemagglutination inhibiting antibody. Virus isolates obtained from plasma retained the small-plaque and temperature-sensitive growth characteristics of the vaccine virus in vitro. In this study, the vaccine virus was genetically stable and immunogenic and seemed sufficiently attenuated for additional testing in humans. Dengue-2 (DEN-2) (PR-159/S-1) is a candidate live virus vaccine which is temperature sensitive, produces uniform small plaques in cell culture, and has reduced neurovirulence for mice and subhuman primates (4, 5, 8). Undiluted vaccine virus stimulated neutralizing antibody in only 10 of 15 rhesus monkeys and produced viremia in only one animal (16). The evidence suggests that this DEN-2 vaccine is highly attenuated for animals and raises a question about its potential infectivity and immunogenicity in humans. In naturally acquired dengue infections, ultimate antibody titers to infecting virus are higher in persons previously infected with a flavivirus (14). Schlesinger and associates (13) reported higher neutralization indices in volunteers immunized with yellow fever (YF) vaccine than in nonimmune volunteers. There is also evidence that disease caused by dengue is more severe after secondary rather than primary dengue infections (11). We deemed it important to test the DEN-2 vaccine strain both in persons with previous flavivirus infections (YF immunition being the most common in a military population) and in persons not previously infected with flaviviruses. Because of the possibility of severe disease caused by the dengue vaccine candidate strain, we elected to seek volunteers among investigators and technicians knowledgeable in infectious diseases, dengue in particular. All volunteers were military and had previously received YF immunizations. This report describes the first trial of the safety and immunogenicity of the DEN-2 (PR-159/S-1) vaccine in six volunteers previously immunized with YF vaccine. MATERIALS AND METHODS Candidate vaccine. DEN-2 virus, PR-159 strain, was isolated from the serum of a patient with dengue fever in Puerto Rico on 19 August Sequential passage in primary green monkey kidney cells permitted the selection of a stable virus clone (S-1) which produced only small plaques in continuous monkey kidney cells (LLC-MK2) and replicated only at temperatures below 39 C (4). For production of a vaccine, the S-1 clone was passaged an additional four times in fetal rhesus lung cells (5). The final virus preparation, designated DEN-2 (PR-159/S-1) lot no. 1, was lyophilized in 3.0-ml portions in the Department of Biologics Research, Walter Reed Army Institute of Research, Washington, D.C. For this study, each vial of lyophilized vaccine was reconstituted with sterile water for injection, USP, immediately before use. Each volunteer received 0.5 ml of vaccine subcutaneously in the upper arm on study day 0. The virus in 0.2 ml of the unused portion of vaccine was titrated within 3 h of rehydration by plaque assay in LLC-MK2 cells at 35 C (4). The first two volunteers (group A) received 4.5 x 105 plaqueforming units (PFU); the remaining four volunteers (group B) received 2.5 x 105 PFU. Human volunteers. Six adult males, knowledgea- 698

2 VOL. 31, 1981 DENGUE-2 VACCINE IN HUMANS 699 ble of microbiological research by training and experience, provided informed consent for this study. All volunteers had received YF vaccinations in the past (Table 1). Four men had previously resided for 3 or more years in countries with epidemic dengue, and volunteer 3 (vol 3), in retrospect, had a history of DEN-4 infection established by virus isolation 11 years earlier. None of the volunteers had detectable neutralizing antibody to a prototype strain of DEN-2 at a serum dilution of 1:10. Before being accepted for the study, each volunteer underwent a complete physical examination and had normal values for a complete blood count, coagulation time, prothrombin time, activated partial thromboplastin time, fibrin split products, serum complement factor 3, blood urea nitrogen, creatinine, serum glutamic oxalacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), lactic dehydrogenase, bilirubin, and urinalysis. Study design. The volunteers were divided into two study groups, A and B. Group A consisted of two people who were vaccinated on 20 February 78. Group B included four people who were vaccinated on 3 April 78 in the absence of any untoward responses by the first two recipients. The date of vaccination was designated study day 0. To avoid exposure to any extrinsic infections, volunteers were placed in continuous protective isolation on a closed research ward in the U. S. Army Research Institute of Infectious Disease, Fort Detrick, Md., for 14 days, starting 3 days before immunization. Each volunteer was examined daily by a physician for 10 days after immunization and then twice a week until day 21. Oral temperatures were recorded three times a day and blood pressure was checked daily during the period of confinement. Daily blood samples were collected from days 0 to 21 for studies of viremia, antibody responses, complete blood counts, platelet counts, serum biochemistries (SGOT, SGPT, lactic dehydrogenase, blood urea nitrogen, creatinine), serum complement factor 3 determinations, and the presence of fibrin split products. Routine urinalysis was performed every 2 days until day 20. Detection of viremia. Heparinized plasma samples were assayed for virus by inoculation of LLC-MK2 cell monolayers in 25-cm2 plastic flasks. Each plasma sample was tested by plaque assay (4) and also by passage of the plasma in LLC-MK2 cells for amplification of low levels of virus (1). Three cell culture flasks were inoculated with 0.2 ml of plasma each and incubated at 350C for 1 to 2 h to permit virus adsorption. After washing, two flasks received agar overlay medium for plaque assay; one flask was incubated at a permissive temperature (35 C) for the vaccine virus, and the other was incubated at a nonpermissive temperature (39.3 C). After 6 days, a second overlay containing neutral red stain was added and the plaques were counted. In the third flask, the inoculum was diluted in 5.0 ml of liquid medium to minimi the effect of plasma inhibitors. After incubation at 350C for 7 days, the culture medium was replaced. Seven days later, the supernatant was tested for virus by plaque assay as described above. Serology. Antibody responses to immunization were measured by hemaggutination inhibition (HI) and complement fixation (CF), using microtiter modifications of standard procedures (3, 10). The prototype strains of flaviviruses used for these tests included DEN-1, Hawaii; DEN-2, New Guinea-C; DEN-3, H- 87; DEN-4, H-241; Japanese encephalitis (JE), Ml/ 311; and YF, 17-D. HI tests were done by using 4 to 8 U of suckling mouse brain antigens; CF tests used 4 U of sucrose-acetone-extracted suckling mouse brain antigens. Plaque reduction neutralization tests were performed in LLC-MK2 cells (12) with the same strains of dengue virus mentioned above, the vaccine parent DEN-2 virus, PR-159, and a French neurotropic strain of YF virus. Equal volumes of serum and virus were mixed to give a final concentration of 30 to 100 PFU/ 0.1 ml. Virus-serum mixtures were incubated at 350C for 30 min (vaccine parent virus, PR-159) and 60 mi (prototype dengue strains). Cells were inoculated and overlaid with agar after 90 min. Plaques were counted after 7 days. Neutralizing antibody (N) titers were determined by calculating the serum dilution that gave a 50% reduction in plaques. RESULTS Preliminary immune status of volunteers. Each volunteer was initially negative for N antibody to DEN-2 (NG-C) virus. Subsequent tests of the prevaccination sera revealed that vol 2 had HI antibody titers of 1:10 to JE and 1:40 to YF. Vol 5 had a CF antibody titer of 1:8 to DEN-2 (NG-C). These antibody reactions could be attributed to previous YF immunizations. The broadest antibody reactivity was found in vol 3, who had N antibody titers of 1:33 to DEN- TABLE 1. Volunteer recipients of DEN-2 vaccine Flavivirus infections Study Volunteer Sex Age YFvaccine Historyofdengue DEN-2vaccine group no. YF vaccine History of dengue DEN-2 vaccin. (latest) A 1 M 41 May 1976 No Feb M 43 Oct 1961 No Feb 1978 B 3 M 50 July 1969 DEN-4, 1967 Apr M 39 Oct 1968 No Apr M 32 Apr 1974 No Apr M 28 Jan 1978 No Apr 1978

3 700 BANCROF'T ET AL. 1, 1:20 to another strain of DEN-2 (PR-159), and 1:10 to DEN-4 and broadly reactive HI antibody to DEN-3, DEN-4, JE, and YF. With a previous history of a natural dengue infection obtained retrospectively, and broadly reactive dengue N antibody, vol 3 must be considered a heterologous dengue immune. Since his response to the DEN-2 vaccine may not be comparable to that of the five dengue nonimmune volunteers, vol 3 will be described separately. Virus isolation studies in dengue nonimmune volunteers. Viremia was detected in all five dengue nonimnmune volunteers. Twentyfour virus isolates were recovered from plasma. Only one virus isolate was obtained by direct plaque assay of plasma; the rest were detected in cell culture supemnatants, after virus amplification under liquid medium culture. All virus isolates formned uniform small plaques of 2-mm diameter or less. No plaques were produced by 16 viruses tested at 39.30C. The virus isolates retained the growth characteristics of the vaccine virus (Table 2). Viremia began 8 days (one volunteer) and 9 days (four volunteers) after immnization and lasted from 1 to 10 days (Fig. 1). The latest virus isolate was recovered on day 18 from vol 2. C woc < 4000 VIREMIA WIC 375. ~ VOL VACCINE '0 ii IS I i3 i4 i5 i6 i? I VOL 3 VACCINE I w365,aa1 360 wsc < 4000 VIREMIA TABLE awec 0 ' 4000 VIREMIA INFECT. IMMUN. Characteristics of representative DEN-2 virus isolatesa Volunteer no. Day of virus Virus titration8b at isolation 350C x X x X x 10' X x x10' x 10O X X104 lla 1.3 X 1O" llb 1.5 x x 10" x x104 aall virus isolates were obtained from first passage LLC-MK2 cell culture except for vol 5, day lla, which was obtained by direct plaque assay of plasma. Virus was identified as DEN-2 by plaque reduction neutralization test, using antibody to DEN-2 (NG-C). All isolates were small plaque, and no plaques formed at 39.31C, indicating viruses were temperature sensitive. b PFU/0.2 ml of inoculum from first passaged virus. a: VOL 4 VACCINE VOL VOL w2 3,1... r~ v'' SPEfV I I <~~~~~~~~~~~~~ lO-I I213.14I5I1617I 8I VRAI -107-:1- I I- DAY I14I3I FIG. 1. Relationship of viremia to fever, leukopenia, and rash in recipients of a DEN-2 vaccine. Shaded portions of horizontal bars represent days when WBC was less that 4,000/mm3 (leukopenia) and virus was isolated from plasma (viremia). A RAA-iJV\I\ ~ w8c 4000~ VIlE MIA DAY RASH -3-2I io i ' I\~ IIA. IkCCINE I m r...

4 VOL. 31, 1981 Antibody response to vaccination. All five viremic volunteers developed fourfold or greater CF, HI, and N antibody titers to DEN-2 (Table 3). The earliest antibody response was seen on day 10 (one volunteer) and day 17 (four volunteers). Peak titers were achieved by day 30 for each person except vol 6, whose peak CF and N antibody responses were delayed until 6 months. Vol 3, who had previous dengue antibody, showed the least antibody response to vaccination (Table 4). In contrast to the other five people, he showed no increase in N antibody and only small rises in HI and CF antibodies. All six volunteers developed broadly reactive HI and N antibody responses (Table 5). Clinical response to vaccination. Only one volunteer reported discomfort at the injection site; vol 2 complained of mild localized pruritus with focal erythema from days 1 to 3. None of the vaccinees developed axillary lymph node enlargement. Four volunteers developed symptoms during the period of viremia which may be attributed to dengue virus infection. The complaints included headache (three), oral temperature of >37.80C (1000F) (two), myalgia (two), fatigue (two), photophobia (two), eye pain (one), and rash (one). Vol 6 developed the highest temperature at 38.8 C. Each of the five viremic volunteers had a fall in leukocyte (WBC) count after the onset of viremia; four individuals had WBC count of less that 4,000/mm3, which was the criterion for leukopenia. The temporal relationships between viremia, fever, and leukopenia are illustrated in Fig. 1. The clinical response of each volunteer is described below. Vol 1 was viremic from days 8 to 14 but remained afebrile and completely asymptomatic throughout the study. Leukopenia was present from days 13 to 16. Vol 2 experienced mild pruritus and slight redness at the site of vaccine inoculation from DENGUE-2 VACCINE IN HUMANS 701 TABLE 3. DEN-2 serum antibody titers of five YFimmune volunteers with no initial dengue antibody Antibody titera at given time after Antibody test Volun- vaccination (antigen) teer no. Day m 2 mo 6 m m 2mo6MO M CF 1 < <8 <8 (NG-C) 2 < <8 <8 4 < <8 <8 < GMT? < <8 HI 1 < (NG-C) 2 <10 2, <10 2, < < GMT < N 1 < (PR-159) 2 < < < < GMT < Reciprocal serum dilution. b GMT, Geometric mean titer. TABLE 4. DEN-2 serum antibody titers of a YFimmune vaccine recipient (vol 3) with a previous dengue infection Antibody titer at given time after Antibody test vaccination (antigen) 12 DayO 1 mo 2 mo 6 mo 12 CF <8 8 < (NG-C) HI < (NG-C) N (PR-159) TABLE 5. Heterologous flavivirus antibody responses after DEN-2 vaccination Volunteer HI antibody N antibody n. Study day no. DEN-1 DEN-3 DEN-4 JE YF DEN-1 DEN-3 DEN-4 YF 1 0 <10 <10 <10 <10 <10 <10 <10 < ,560 1,280 5, <10 <10 < <10 <10 < ,280 2,560 10,240 20,480 20, < < < <10 <10 <10 <10 <10 <10 <10 < ,560 5,120 5,120 2, <10 <10 <10 <10 <10 <10 <10 < ,560 1, <10 <10 <10 <10 <10 <10 <10 <

5 702 BANCROFT ET AL. days 1 to 3. Throughout the first 10 days of study he experienced wide daily changes in temperature, with peaks between 37.2 and 37.80C. After the onset of viremia on day 9, his daily temperatures fluctuated at a slightly higher level and reached a maximum of 38 C on day 15. On day 12 he complained of mild fatigue and photophobia. On days 13 and 14 he was leukopenic. Vol 3 had no fever, viremia, or leukopenia. His only complaint was mild fatigue on day 15. Vol 4 remained afebrile throughout the study. Viremia was detected from days 9 to 14. He complained of a bitemporal headache on days 9 and 10. His total WBC count tended to be lower than the others throughout the study. On Day 7, the WBC count was 3,600/mm3; this was the only low WBC count that preceded the onset of viremia. Leukopenia returned from days 15 to 21. Vol 5 was viremic from days 9 to 13. On day 12 his temperature rose to 37.7 C, and he complained of mild chills, headache, and pain in the shoulders, knees, and eyes. A fine erythematous macular rash of the trunk and abdomen which extended into the axillae was observed from days 12 to 17. He was not too ill to miss work despite these findings. Vol 6 had detectable viremia on day 9 only. On the evening of day 10 his temperature was 37.8 C and reached 38.80C on day 11. He reported fatigue on day 11 followed by headache, muscle pain, and photophobia and was too ill to work. After the onset of fever and viremia, his WBC count dropped to a minimum of 4,000/ mm3 on day 11. None of the volunteers developed a bleeding tendency, petechiae, regional or generalized lymph node enlargement, hepatomegaly, splenomegaly, or conjunctival suffusion. There was a tendency for a slight reduction in systolic and diastolic blood pressures during periods of confinement. Pulse rates rose during periods of fever. There were no changes or abnormal values in platelet counts, hemoglobin, hematocrit, serum biochemistries, or serum complement values which could be attributed to the vaccine. Daily determinations of fibrin split products were uniformly negative. Urinalyses were normal throughout the study for all volunteers. DISCUSSION This study evaluated the immunogenicity, reactogenicity, and genetic stability of the DEN- 2 vaccine in heterologous flavivirus-immune people. Five volunteers, who had no preexisting dengue antibodies, seroconverted after vaccination and developed a peak geometric mean titer of DEN-2 neutralizing antibody of 1:180 at 30 INFECT. IMMUN. days which declined to 1:104 by 12 months. N antibody titers of this magnitude in monkeys were associated with protection from wild DEN- 2 virus challenge (16). Vol 3 had a low titer of DEN-2 N antibody at the beginning of the study which probably effectively inhibited infection with the vaccine virus. Nevertheless, the rise in CF and HI antibody in vol 3 was probably a response to limited virus replication. In this study, the DEN-2 vaccine showed satisfactory immunogenicity for people with preexisting heterologous flavivirus antibody. There were no local reactions after vaccination, but some recipients developed systemic symptoms which may have been caused by the vaccine virus. Vol 6 developed symptoms of fever, myalgia, headache, and fatigue which were compatible with mild dengue fever. Virus was isolated from his blood for only 1 day immediately before his maximum oral temperature of 38.8 C. It is possible his viremia was abbreviated by the febrile response, since replication of the S-1 clone of vaccine virus is restricted at 38.50C (5). Fever, leukopenia, and the single erythematous rash were all temporally associated with viremia and may be attributed to infection with the vaccine virus. With the exception of vol 6, the observed symptoms were mild and, in each volunteer, of short duration (3 days or less). Schlesinger and associates (13) previously administered an attenuated DEN-2 virus obtained from mouse brain to 17 people including 4 volunteers who had been immunized for YF 7 years earlier. All four YF-immune recipients developed high neutralization indices for DEN-2 virus, and one of them complained of headache, chilliness, backache, and eye pain between 8 and 11 days after immunization. His temperature rose to over 101 F (38.30C), and he developed a generalized maculopapular rash and leukopenia. The four volunteers did not have detectable viremia by intracerebral inoculation of mice. The clinical responses reported by Schlesinger and associates (13) were very similar to those observed in this study. In this study, viremia was detected in five volunteers. Direct plaque assay detected virus in only one instance, a result similar to that reported in earlier studies (13, 16). The addition of a virus amplification step before plaque assay yielded many more virus isolates for the evaluation of virus growth characteristics. All viruses isolated from our volunteers retained the in vitro markers of dengue attenuation, i.e., small plaque size and temperature sensitivity, giving evidence that the vaccine virus is genetically stable in vivo. In an earlier report, Brandt and associates (2)

6 VOL. 31, 1981 attempted to isolate dengue virus from daily fresh monocyte preparations prepared from each volunteer for 21 days postinoculation. No virus was isolated by plaque assay and infectious center assay of supernatant fluid from cultures of mononuclear cells. In contrast, wild strains of dengue virus have been isolated from similkr mononuclear cell cultures of persons with dengue fever (15). The inability to infect circulating monocytes in vivo may be another indicator of attenuation of the vaccine virus (2). As was shown by Schlesinger and associates (13), preliminary YF vaccination did not inhibit infection with the DEN-2 vaccine. Dilute flavivirus antibodies have been shown to enhaeice Murray Valley encephalitis virus infection in cell culture (9) and unattenuated dengue virus infections in human WBC cultures (2, 7) and rhesus monkeys (6). Whether the presence of YF antibody contributed to the responses to the DEN- 2 immunization can be evaluated only after the vaccine has been administered to volunteers who have not received YF immunization. The DEN- 2 (PR 159/S-1) vaccine is sufficiently safe, infectious, and immunogenic to merit additional testing in humans to determine the optimal dosage and the response of people with no preexisting flavivirus immunity. ACKNOWLEDGMENTS We thank many people at the U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Md., including Nathan Woodruff for directing the clinical laboratory procedures, Deanna McGookin for supervising nurse care, and Karen A. Bostian for the serum complement factor 3 determinations. We thank Walter E. Brandt and Robert McNair Scott for reviewing this manuscript. LITERATURE CITED 1. Bancroft, W. H., J. M. McCown, P. ML Lago, W. E. Brandt, and P. K. Russell Identification of dengue viruses from the Caribbean by plaque-reduction neutralization test, p In Dengue in the Caribbean, Scientific publication no Pan American Health Organization, Washington, D.C. 2. Brandt, W. E., J. M. McCown, F. H. Top, Jr., W. H. Bancroft, and P. K. Russell Effect of passage DENGUE-2 VACCINE IN HUMANS 703 history on dengue-2 virus replication in subpopulations of human leukocytes. Infect. Immun. 26: Clarke, D. H., and J. Casals Techniques for hemagglutination and hemagglutination-inhibition with arthropod-bome viruses. Am. J. Trop. Med. Hyg. 7: Eckels, K. H., W. E. Brandt, V. R. Harrison, J. M. McCown, and P. K. Russell Isolation of a temperature-sensitive dengue-2 virus under conditions suitable for vaccine development. Infect. Immun. 14: Eckels, K. H., V. R. Harison, P. L Summers, and P. K. Russell Dengue-2 vaccine: preparation from a small plaque virus clone. Infect. Immun. 27: Halstead, S. B In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody. J. Infect. Dis. 140: Halstead, S. B., N. J. Marchette, J. S. S. Chow, and S. Lolekha Dengue virus replication enhancement in peripheral blood leukocytes from immune human beings. Proc. Soc. Ezp. Biol. Med. 151: Harrison, V. R., K. H. Eckels, J. W. Sagartz, and P. K. Russell Virulence and immunogenicity of a temperature-sensitive dengue-2 virus in lower primates. Infect. Immun. 18: Hawkes, R. A Enhancement of the infectivity of arboviruses by specific antisera produced in domestic fowls. Aust. J. Exp. Biol. Med. Sci. 42: Kent, J. F., and E. H. Fife, Jr Precise standardization of reagents for complement fization. Am. J. Trop. Med. Hyg. 12: Russell, P. K., and W. E. Brandt Immunopathologic processes and viral antigens associated with sequential dengue virus infection. Perspect. Virol. 8: Russell, P. K., A. Nisalak, P. Sukhavachana, and S. Vivona A plaque reduction test for dengue virus neutralizing antibodies. J. Immunol. 99: Schlesinger, R. W., I. Gordon, J. W. Frankel, J. W. Winter, P. R. Patterson, and W. R. Dorrance Clinical and serologic response of man to immunization with attenuated dengue and yellow fever viruses. J. Immunol. 77: Scott, R. McN., J. ML McCown, and P. K. Russell Human immunoglobulin specificity after group B arbovirus infections. Infect. Immun. 6: Scott, R. McN., A. Nisalak, U. Cheamudon, S. Seridhoranakul, and S. Nimmannitya Isolation of dengue viruses from peripheral blood leukocytes of patients with hemorrhagic fever. J. Infect. Dis. 141: Scott, R. McN., A. Nisalak, K. H. Eckels, M. Tingpalapong, V. R. Harrison, D. J. Gould, F. E. Chapple, and P. K. Russell Dengue-2 vaccine: viremia and immune responses in rhesus monkeys. Infect. Immun. 27: