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BIOLOGIC PROPERTIES OF DENGUE VIRUSES FOLLOWING SERIAL PASSAGE IN PRIMARY DOG KIDNEY CELLS: STUDIES AT THE UNIVERSITY OF HAWAII

ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Serial passage at low dilution of seven different wild-type dengue (DEN) viruses into primary dog kidney (PDK) cell cultures placed selective pressure that resulted in the following changes from parental phenotype: smaller plaques in LLC-MK2 cells, absent plaque formation in green monkey kidney cells, lack of a cytopathic effect in LLC-MK2 cells, shut-off of virus replication at high temperatures (temperature sensitivity), reduced virulence for rhesus monkeys manifested by reduced or absent viremia and/or absence of a secondary-type antibody response following homotypic challenge, and progressive increase in the mean day of death following intracerebral inoculation of sucking mice. Two DEN-1 strains showed most of these changes by the 15th PDK passage. Only one of two DEN-2 strains studied was carried to the 50th PDK passage at the University of Hawaii. For the latter strain, both the temperature of viral replicative shutoff and mouse neurovirulence were reduced. Three DEN-4 strains showed similar late-passage biologic marker changes. The observations made, although not exhaustive, provide laboratory correlates for virus strains that have shown reduced virulence but retained immunogenicity in humans. Candidate human vaccines have been prepared from five of the studied strains: DEN-1 (16007) at PDK 13, DEN-2 (16681 and S-16803) at PDK 50 or above, and DEN-4 (1036 and 341750) at PDK 48 and 20, respectively.

INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In 1971, recognizing the severity of the worldwide dengue problem, the United States Armed Forces Epidemiology Board initiated a cooperative scientific effort to develop vaccines against dengue (DEN) viruses types 1–4. The Department of Tropical Medicine and Medical Microbiology at the University of Hawaii at Manoa participated in this effort receiving contracts from the U.S. Army over the period 1971–1986 to carry out various tasks assigned for the dengue vaccine development effort. The attenuation method selected by these investigators was serial tissue culture passage. To select an appropriate tissue culture system, wild type DEN-1, -2, and -4 plus prototype DEN-3 (H-87) strains were inoculated onto primary cells or cell lines that had been used to prepare vaccines licensed in the United States.¹ These included continuous WI-38 human fibroblasts, primary chick and duck embryos, kidney cells from rabbit, primary dog kidney (PDK) cells, and African green monkeys kidney (GMK) cells. DEN-1–4 viruses at a multiplicity of infection of 0.1 were inoculated on monolayers of each of these cells and incubated for one week at 32°C or 37°C. All four dengue viruses grew only in PDK and GMK cells. Low yields of DEN-1 and -2 were obtained in WI-38 cells, while none of the four DEN viruses grew in chick embryo, duck embryo, or rabbit kidney cells.¹ In 1971, when the Department assumed the responsibility to attenuate dengue 4 virus, the effort entailed serial passage in PDK and GMK cells. A series of papers report the extensive experience with a single dengue strain passaged to different levels in PDK cells and then tested for biologic markers using uncloned virus or virus cloned by a terminal dilution method.^1–5 In 1984, investigators in the Department were contracted to attenuate a second DEN-4 strain, 341750 Carib in PDK cells.⁶

Early in 1980, a collaborative dengue vaccine technology transfer project was initiated with the goal of attenuating strains of DEN-1–4 under conditions that would permit their use in humans.⁷ Support for this project was provided by the Rockefeller Foundation (RF) to the Department and by the Southeast Asia Office of the World Health Organization to Mahidol University in Bangkok, Thailand. In November 1980, a specially designed training laboratory was equipped and opened to support this effort. All virus passages in Hawaii were performed under controlled laboratory conditions.

Here we provide unpublished data from RF-supported serial passages performed in Hawaii. This includes a set of DEN-1, -2, and -4 viruses ("back-up" strains) subjected to 50 passages in PDK cells. A second set passed in Hawaii to PDK passage 15 for DEN-1, -2, and -4 and GMK passage 5 for DEN-3 were transferred to the World Health Organization-supported program in Thailand for further vaccine development. Briefly mentioned are efforts to replicate two DEN-3 strains in PDK cells. Finally, we summarize published data from our laboratory on the biologic attributes of two DEN-4 strains passaged in PDK cells: H-241, and 341750 Carib.^1,6

Although the biologic marker studies presented here are not exhaustive, their importance is enhanced because safe, relatively non-reactogenic and highly immunogenic human vaccines have been developed from several of the studied viruses. Viruses attenuated in Thailand include DEN-1 (16007) at PDK 13, DEN-2 (16681) at PDK 53, DEN-3 (16562) at GMK 33, and fetal rhesus lung (FRhL) 2 and DEN-4 (1036) at PDK 48.⁷ Partial descriptions of the biologic attributes of these viruses and results of phase I trials in human beings have been published.^8–12 Other published work includes phase I studies in human volunteers for two DEN 4 strains, the terminally-diluted H-241 and uncloned 341750 Carib.^5,13 The Walter Reed Army Institute of Research has selected PDK-passaged dengue viruses of each serotype for Phase II human trials. The results of pre-clinical and clinical studies on candidate vaccines prepared from viruses attenuated in Hawaii that include DEN 2 (S16803) at PDK 50, fetal rhesus lung (FRhL) 3 and DEN-4 (431750 Carib) at PDK passage 20, and FRhL-4 plus one each PDK-passaged DEN-1 and DEN-3 strains are reported in this supplement.

MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Virus attenuation plan. The methodologic approach adopted in the technology transfer program was as follows. Wild DEN 1–4 strains as free as possible from putative adventitious agents were selected for attenuation. A search was made for dengue-permissive tissue cultures from among those used to produce viral vaccines licensed by the U.S. Food and Drug Administration. Viruses were passaged under carefully controlled laboratory conditions and several biologic markers monitored at pre-selected passage levels.

In this approach, there was no intent to predict which, if any, biologic marker would correlate with virulence of virus in humans. As with other successful human vaccines, it was planned that passaged virus would be tested at the highest level working down. The empirical approach to evaluate reactogenicity and immunogenicity in humans at multiple passage levels is based upon the precedent of other attenuated virus vaccines. For example, rubella strains differing by only a few duck embryo cell passages varied markedly in human virulence.¹⁴

It was hoped the identification of a successful human vaccine for one DEN type might validate biologic markers of attenuation permitting early selection of other attenuated vaccine candidates.

Viruses. DEN-4, H-241. This strain was recovered from a patient with dengue fever by Hammon and others¹⁵ in Manila, the Philippines in August 1956. Viremic serum was sent to Dr. Leon Rosen in 1969 and passaged into a susceptible Macaca irus. In 1971, viremic monkey serum was serially passaged three times in adult Aedes albopictus by intrathoracic inoculation. A saline suspension of third mosquito passage was used for inoculation of PDK and GMK cells.¹

DEN-4, 341750 Carib. This strain was isolated from a dengue fever patient in Colombia in 1982.¹⁶ Viremic serum was inoculated serially two times in Toxorhynchites amboinenses and then into PDK cells.

Specially passaged Halstead prototype dengue 1–3 viruses and Indonesian dengue 4. The provenance and laboratory passage histories of these strains are shown in Table 1. DEN-1 (16007), DEN-2 (16681), and DEN-4 (1036) at PDK and GMK passage 15 were sent to Mahidol University in Bangkok, Thailand for further passage and preparation of production seeds and candidate vaccines. DEN-3 (16562) did not replicate in PDK cells and was sent to Thailand at passage GMK 5. In a parallel effort, DEN-4 (1036) was serially transferred to PDK passage 48 at the University of Hawaii.

Virus assays. Plaque assays were performed in LLC-MK2 or GMK cells by published techniques.¹ Briefly, a single agar overlay medium was used. LLC-MK2 or GMK cell monolayers were propagated in 10% calf serum and Eagle’s basal medium (BME) with Hanks’ buffered salt solution. The agar overlay consisted of a final concentration of 1% Noble agar, 10% heat-inactivated calf serum in BME with Earle’s salt solution, plus 1% neutral red. Cells were propagated in one-ounce prescription bottles. Monolayers were incubated with 0.2 mL of virus suspension for 60 minutes at 37°C. Following the addition of agar overlay medium, cells were incubated in the dark for seven days at 37°C, then for an additional 7–14 days in the dark at room temperature during which time new plaques appeared and an increase in plaque size and sharpness was noted.

Cell cultures. Primary African green monkey kidney (GMK) cells. Kidneys were obtained from four young adult African green monkeys. These animals were housed individually in cages with closed sides. The animals were quarantined in separate rooms for nine months before the kidneys were removed. Precautions were taken to prevent cross-contamination from humans or animals of other species. Safety tests on animals, the preparation of cell cultures, and safety tests on them have been described.⁴

Primary dog kidney (PDK) cells. Cells were obtained from Hawaii-quarantined beagles or from beagle puppies. Cells were grown and maintained in the same media used for GMK. All PDK cells were propagated with a single lot of fetal calf serum (lot number 45533; Flow Laboratories, Rockville, MD).

Two animals were maintained from birth in a veterinary facility on the island of Lanai. Kidneys were harvested from these animals at three months of age. These dogs were in apparent good health and had no direct exposure to animals other than their sires since birth. The animals were free of canine parasites. Their blood chemistries were normal. They were clinically free of tuberculosis, infectious canine hepatitis, canine distemper, rabies, and leptospirosis. After removal of both kidneys, a veterinarian performed autopsies on each dog. By gross examination, both animals were free of detectable disease.

Serial passage of dengue 4 in PDK and GMK cells. Based upon earlier successful attempts to attenuate RNA viruses, it was decided to set the temperature for DEN-4 passage at 32°C. Preliminary studies showed that at this temperature virus yields were maximal on or about six days after inoculation of 100–10,000 plaque-forming units (PFU) of DEN-4.

Safety tests on cells and culture media. Safety tests have been described previously.^1,4 In addition, tests for the following canine microbial pathogens were performed on PDK cells by the Dow Chemical Company (Indianapolis, IN): canine distemper, infectious canine hepatitis, canine herpes, canine SV-5, and reovirus type 1.

Frozen storage of cells. Cell suspensions were prepared in Hawaii as described and frozen at -70°C in maintenance medium supplemented with 10% fetal calf serum and 10% glycerol.

Mosquito inoculation. At the National Institutes of Health Pacific Research Section in Honolulu, Hawaii, groups of five adult laboratory-reared T. amboinensis were inoculated intrathoracically with strains of dengue viruses. The inoculum was approximately 0.0003 mL. Mosquitoes were maintained on 10% sucrose solution at 28°C for 12 days. At the end of the incubation period, each group of insects was killed by freezing, and their heads were removed and triturated in 5.0 mL of phosphate-buffered saline containing 0.5% gelatin, 30% heat-inactivated calf serum, and antibiotics. After centrifugation at 5°C for 30 minutes, each supernatant fluid was inoculated into another group of five T. amboinensis. These insects were also held at 28°C for 12 days. They were killed by freezing, the whole insect was triturated, suspended in 5 mL of diluent, and used as viral seed.

Establishment standards. The preparation of primary PDK and GMK cell cultures and virus passages were done in separate isolated laboratory rooms using disposable glass or plasticware. Only a single laboratory technician had access to the vaccine development laboratory. Virus passages were done as the first procedure in the morning of a given work day by this individual. This individual worked on no other virus within a 24-hour period prior to passage of dengue virus in cell cultures.

Biologic marker studies. Plaque formation or cytopathic effect (CPE) in cell cultures. Screw-capped test tubes containing monolayers of LLC-MK2, GMK or PDK cells were inoculated with 0.1 mL of undiluted dengue virus, either parental or harvested from serial passage in PDK cells. Cells were observed daily for 7–10 days and observed for CPE. Parental or passaged viruses were also inoculated on LLC-MK2, GMK, and PDK cell monolayers and observed for plaque formation and morphology using the single agar overlay method described earlier in this report.

Temperature sensitivity studies. Temperature of replication shut-off was measured by two methods as described: the growth curve and efficiency of plating (EOP) methods.¹ To measure a growth curve, virus was added to LLC-MK2 monolayers in screw-capped test tubes that were incubated in a circulation-type water bath accurate to +0.1°C. Growth of virus at various temperatures was compared with that at 37°C. Absence of growth on five consecutive days of incubation was taken as temperature sensitivity. The EOP was measured by immersing watertight boxes in water baths at various temperatures. Boxes held one-ounce prescription bottles containing a LLC-MK2 monolayer, virus dilutions and an agar overlay. Plaque counts at various temperatures were compared with those at 37°C. Temperature sensitivity was scored when plaque counts were equal to or greater than 90% lower than controls.

Monkey viremia and antibody responses. Male or female Macaca mulatta, generally weighing 2–3 kg and free of DEN-1–4 neutralizing antibodies were inoculated subcutaneously with virus on the volar surface of the right mid-forearm. Animals were bled daily for 12 days following inoculation. Plasmas for virus isolation attempt were stored at -70°C. Later, blood samples for antibody studies were obtained as described in this report. Research on monkeys was carried out in compliance with the Animal Welfare Act.

Antibody tests. Antibody tests were performed on lightly heparinized plasma (20 IU/ml final concentration). The micro-hemagglutination inhibition (HI) test was performed using standard methods.¹⁹ Plasmas were tested at an initial dilution of 1:20 versus 4–8 units of hemagglutinin. Plaque-reduction neutralizing antibodies were quantitated in one-ounce prescription bottles at the 50% plaque reduction end point using the single agar LLC-MK2 assay.¹ Serial four-fold dilutions were tested from an initial plasma dilution of 1:10 versus 30–50 PFU of DEN virus.

Suckling mouse neurovirulence. One hundred PFU of virus were inoculated intracerebrally into 24 hour-old BALB/c mice. Mice were observed daily for deaths to 21 days and average survival time was calculated. For the purposes of this calculation, mice surviving 21 days or longer were scored as dying on day 21.

RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Passage of dengue 1–4 viruses. LLC-MK2 plaque morphology of PDK-passaged viruses. Data in Table 2 illustrates the emergence of plaque size changes when DEN-1 (16007) was passaged successively in PDK cells. Table 3 displays LLC-MK2 plaque size changes during PDK cell passage for all studied viruses.

Biologic marker studies. Tables 4–6 summarize biologic marker studies on all dengue strains studied. Limited observations were made up to PDK passage 15 for Halstead prototype strains, but across a wider range of PDK passage levels for DEN-4 H-241 and 341750 Carib viruses and Halstead back-up strains. When 4.6 x 10⁴ PFU of 341750 Carib PDK 15 virus was inoculated into two susceptible rhesus monkeys, no viremia was detected, although both animals developed HI and DEN-4 neutralizing antibodies. At passage PDK 30, no viremia or neutralizing antibody response was observed in two monkeys. When challenged with parental virus, these monkeys developed a secondary HI and neutralizing antibody responses, but no viremia. Limited biologic marker studies were done at various PDK passages and on the candidate vaccine PDK 20, FRhL 4. Vaccine virus was less infectious for susceptible rhesus monkeys than parental virus. It was determined that 40 PFU of parental virus contained one monkey 50% infectious dose (ID₅₀) while for vaccine virus 1.8 x 10⁵ PFU was required.

DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

By passage PDK 15, both strains of DEN-1 showed significant plaque size changes, temperature sensitivity, absence of LLC-MK2 CPE, an immune response in monkeys that did not protect against secondary neutralizing antibody response to homologous challenge, and reduced suckling mouse neurovirulence. It is significant that candidate DEN-1 (16007) is administered to humans at passage 13.⁷

The two DEN-2 viruses behaved similarly over 15 passages in PDK cells, but differed from DEN-1 viruses. By PDK 15, plaque size differences appeared, but LLC-MK2 CPE was retained through seven passages, there was no change in shutoff temperature, only a modest change in suckling mouse neurovirulence and solid protection was induced in monkeys to homologous challenge. For the S16803 strain, shut-off temperature changes and reduced suckling mouse neurovirulence were observed at passage 50. However, when monkeys were inoculated with passage-40 virus, solid immunity to homologous challenge did not occur. Both DEN-2 (16681) and (S16803) are successfully attenuated for humans at approximately PDK 50.⁸

In this series of experiments, a DEN-3 strain was passaged 29 times in GMK cells and was inoculated in PDK cells at many levels. All attempts to grow GMK-passaged virus in PDK cells failed. The inability to obtain consistent replication of DEN 3 in PDK cells suggests this virus may belong to a different biotype than DEN-1, -2, and -4 viruses.

Observations were made on two DEN-4 strains passaged to approximately PDK-50 and a third strain, 341750 Carib, to PDK passage 30. Small plaques appeared at earlier passage levels with DEN-4 1036 than H-241. CPE in LLC-MK2 cells was observed through passage 30 for 1036. However, both viruses demonstrated a change in shut-off temperature, a marked decrease in suckling mouse neurovirulence at passage 50, and failure to provide solid protection against homologous challenge at approximately passage 30. DEN-4 (1036), passaged from PDK 15 in Thailand, is used as candidate vaccine at PDK 48.⁷ DEN-4(341750 Carib) PDK-20 FRhL 4 was inoculated in 12 human volunteers, seven of whom developed viremia, HI, and DEN-4 neutralizing antibodies, but with minimal signs and symptoms of dengue fever.¹³

In this series biologic marker data are absent for DEN-2 above PDK passage 15 and DEN-3 at various GMK cell passages. This limits our ability to identify biologic marker correlates of successful attenuation for humans for each dengue virus type.

Earlier in the 20th century, when many live-attenuated viruses were successfully obtained using the empirical process of serial passage in cells from vertebrates other than human beings, it was obvious that a very sophisticated process was at work. Even a single passage of virus in certain vertebrate cells has a powerful selective effect on biologic properties.²⁰ The underlying selection process is incompletely understood. It has been conjectured that serially passaged live-attenuated vaccines may emerge because such passage creates a heterogenous viral population that interacts in vivo to immunize without producing disease symptoms.⁵ Such a virus population might contain a mixture of wild-type and defective interfering viruses, for example. It is clear that one or more amino acid changes differentiate parental virus and serially passaged attenuated strains.^21,22 Tests in humans of live-attenuated viruses prepared from cDNA constructs could provide a method to evaluate these hypotheses. However, the current experience with genetically defined live-attenuated vaccines in humans is too limited to permit conclusions to be drawn.

For separately passaged DEN-1, -2, and -4 viruses, a surprising consistency was observed in the PDK cell level at which changes in biologic properties were observed. PDK passage provides an excellent model for those who wish to study the empirical process of cell-passage attenuation. However, just as PDK cell serial passage exerts a cumulative selection process, further passage in another cell substrate may generate other genetic and phenotypic changes. These are insufficient data on human responses to DEN viruses passaged in PDK only or in PDK plus FRhL to rule out the possibility that FRhL cell passage selects for phenotypic attributes different than those previously selected by PDK cell passage.

Efforts are under way to certify Vero cells as a vaccine substrate. Vero cells have been used for preparation of seed dengue virus pools in laboratories for many years without placing obvious selective pressure on wild dengue viruses (Gubler DJ, unpublished data). It is possible that use of Vero cells for production of live-attenuated viruses may preserve desireable biologic properties of viruses derived during serial passage in PDK cells.

Acknowledgments: Thanks are due to Laddawan Srisukonth, Ravithat Putvatana, Suwanna Vithanomsat, C. N. Venkateshan, and Kay Larsen for technical support, to Dr. Leon Rosen for providing the original viremic sera for DEN-2 (S16803) and DEN-3 (40957), to Dr. Duane J. Gubler for providing infected Aedes aegypti suspensions containing DEN-1 (1009) and DEN-4 (1036), and to Drs. Leon Rosen and Robert Tesh for amplifying viruses by mosquito inoculations.

Financial support: This work was supported by grants HS-8011, 8140 and 8304 from the Rockefeller Foundation and Contract DADA 17-69-C9146 from the U.S. Army Medical Research and Development Command.

^* Deceased.

Author’s address: Scott B. Halstead, Department of Preventive Medicine and Biometrics, Uniformed Services University of Health Sciences, 5824 Edson Lane, North Bethesda, MD 20852, Fax: 301-984-8042, E-mail: halsteads{at}erols.com.

REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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