vaccine was developed in cell culture that appears to have overcome some of ... dioimmunoassay (RIA) exhibits increased sensitivity as well as the capability to ...
JOURNAL
OF
Vol. 19, No. 5
CLINICAL MICROBIOLOGY, May 1984, p. 651-659
0095-1137/84/050651-09$02.00/0
Antibody Response to Dengue-2 Vaccine Measured by Two Different Radioimmunoassay Methods PETER L.
SUMMERS,'* KENNETH H. ECKELS,' JOEL M. DALRYMPLE,2 ROBERT M. SCOTT,3 AND V.
ANN
BOYD4
Department of Biologics Research' and Department of Virus Diseases,3 Walter Reed Army Institute of Research, Washington, D.C. 20307, and Department of Viral Biology, U.S. Army Research Institute of Infectious Diseases, Fort Detrick,2 and Department of Biomedical Sciences, Hood College,4 Frederick, Maryland 21701 Received 23 September 1983/Accepted 30 January 1984
Two different radioimmunoassays were used to detect virus-specific antibodies in sera from human volunteers inoculated with an attenuated dengue type 2 (DEN-2) vaccine (PR-159/S-1). An indirect radioimmunoassay required purified DEN-2 virions for optimal reactivity but was 10 to 500 times more sensitive than neutralization or hemagglutination inhibition tests. An antibody capture radioimmunoassay was able to utilize crude antigens from either DEN-infected mouse brains or Aedes albopictus cell culture supernatants. When the two radioimmunoassay techniques were compared, the indirect method appeared to be the best assay for immunoglobulin G (IgG), whereas the antibody capture method was more sensitive for IgM detection. Selected human sera were examined for IgG, IgM, and IgA responses by using both techniques at various intervals after immunization. Although there were differences in magnitude, yellow fever immune as well as flavivirus nonimmune volunteers responded to DEN-2 vaccination by demonstrating IgG, IgM, and IgA antibody responses. In the nonimmune group, the most prevalent immunoglobulin detected was IgM, whereas in the yellow fever immune group, the predominant post-DEN-2 vaccine immunoglobulin was IgG. The preponderance of DEN-2-specific neutralizing antibodies were associated with either IgM or IgG according to the immune status of the volunteer. All classes of immunoglobulins attained maximum levels between 21 and 60 days postvaccination. In the majority of volunteers, IgM responses were relatively transient and could not be detected 6 months after immunization, whereas IgG and IgA antibodies were still detectable after this period.
Dengue (DEN) is a mosquito-transmitted viral disease afflicting mankind in the tropical and subtropical areas of the world. The association of DEN infection with hemorrhagic and shock syndromes have caused DEN to become a serious and sometimes fatal disease (11). Several live attenuated DEN vaccines developed after 1945 were effective in protecting individuals against experimental challenge (13, 19, 24). However, these vaccines were undesirable for human use because they were prepared from infected mouse brain material. Recently, a live attenuated DEN type-2 (DEN-2) vaccine was developed in cell culture that appears to have overcome some of these difficulties (6). To determine the efficacy of the vaccine, several studies have been conducted to evaluate vaccinee antibody response to immunization (1, 21). To date, 145 volunteers have been vaccinated with the DEN-2 (PR-159/S-1) vaccine, and serological responses have been measured by hemagglutination inhibition (HI) and plaque reduction neutralization (PRN) tests. Conventional serological tests such as HI and PRN do not distinguish the different immunoglobulin classes without additional serum fractionation procedures. The radioimmunoassay (RIA) exhibits increased sensitivity as well as the capability to detect and measure individual immunoglobulin subclasses. This report describes two different RIA methods used to quantitate the immune response of human DEN-2 vaccinees.
Medical Research Institute of Infectious Diseases, Ft. Detrick, Frederick, Md.) were inoculated with DEN-2 strain PR-159 (PGMK-6, FRhL-3) at a multiplicity of infection of 0.1. The A. albopictus cell line was maintained in Dulbecco modified Eagle medium containing 10% fetal bovine serum and supplemented with 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 U of penicillin, and 100 pLg of streptomycin per ml. The cells were incubated at 28°C. Infected cell cultures were harvested at 2- to 5-day intervals beginning on day 5 postinoculation. Virus harvest titers averaged 107 PFU/ml. Antigen purification. Supernatant fluids harvested from infected C6/36 cell cultures were clarified by centrifugation at 600 x g for 20 min. DEN antigens were precipitated from the supernatant fluids by polyethylene glycol 6,000 (Fisher Scientific, Pittsburgh, Pa.). This procedure consisted of adding 2.2 g of NaCl and 7.0 g of polyethylene glycol 6,000 per 100 ml of clarified virus followed by mixing on a magnetic stirrer for 4 h at 4°C. After centrifugation at 15,000 x g for 50 min, the antigen pellet was resuspended to 1/100 of the original volume in 0.01 M Tris-hydrochloride buffer (pH 7.8) containing 0.15 M NaCl and 0.005 M EDTA. Four milliliters of the virus concentrate was layered on 8 ml of a 30 to 50% potassium tartrate-glycerol gradient and centrifuged in a Beckman SW41 rotor for 16 h at 286,000 x g. After centrifugation, 24 0.5-ml fractions were collected from each gradient and screened for hemagglutination and RIA activity. The fractions that gave the highest hemagglutination or RIA activity or both were pooled and frozen at -70°C. The optimal concentration of antigen for both RIA techniques was determined by titration against DEN-2 mouse hyperimmune ascitic fluid (2) or from volunteer DEN-2 postvaccination serum or both.
MATERIALS AND METHODS Propagation of virus. Thirty-two-ounce prescription bottles containing confluent monolayers of Aedes albopictus (C6/36 clone) obtained from Pat Repik (United States Army *
Corresponding author. 651
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Test sera. A group of human volunteers was screened for the presence of HI antibodies to Japanese encephalitis, yellow fever, DEN 1 through 4, and neutralizing antibodies to YF and DEN-2 viruses. Only volunteers with no detectable DEN-2-specific antibodies were selected for vaccination with the live attenuated DEN-2 S-1 vaccine. The study group consisted of volunteers with previous yellow fever immunization (YFI) and volunteers with no prior history of YF immunization (NI). Sera were collected before and at various intervals after vaccination and were stored frozen at -20°C until serological studies could be performed. HI and PRN tests. Antibody responses to immunization were measured by HI and PRN tests. Sera for the HI test were acetone extracted, absorbed with goose erythrocytes, and tested by the methods of Clarke and Casals (5). PRN tests were performed by the techniques of Russell et al. (18) in LLC-MK2 cell cultures by using the DEN-2 vaccine parent virus strain PR-159. The neutralizing antibody titer was expressed as the serum dilution that resulted in a 50% reduction in the number of plaques compared to a negative control serum. IRIA procedure. The indirect RIA procedure (IRIA) was developed by Zollinger et al. (25) and modified by our laboratory for a DEN-2 RIA. A- schematic of the IRIA procedure is shown in Fig. 1. A drop (25 ,ul) of antigen (40 ,ug) diluted in Dulbecco phosphate-buffered saline (PBS; pH 7.4) was added to the wells of a flexible polyvinyl chloride microtiter plate (Dynatech Laboratories, Alexandria, Va.) and air dried overnight at 35°C. The microtiter wells were washed four times with 100 RI of "filler" (Dulbecco PBS containing 20% calf serum, 0.2% sodium azide, and 0.01% phenol red). After washing, the wells were again filled with 100 pl of filler and incubated for 1 h at 350C. The filler was aspirated and the wells were refilled with 25 ,lI of the test serum dilution and incubated overnight at room temperature. The next day, the wash procedure was repeated, and 25 RI of 125I-radiolabeled protein A or 125I-labeled anti-globulin
4
with a specific activity of 300 to 1,200 cpm/ng was added and incubated at room temperature overnight. The immunoglobulin and protein A preparations (kindly provided by Wendell D. Zollinger, Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C.) were labeled by the chloramine T method (10). After washing, the plates were air dried and the individual wells were cut from the plate and counted for 1-min intervals in a Nuclear Chicago model 1185 gamma counter. DEN-2 IRIA endpoint titers were calculated by a method similar to that described by Rosenthal et al. (17). The IRIA titer was the reciprocal of the highest serum dilution resulting in a binding ratio .1.5 times the control (day 0) serum. IRIA results were also presented as positive/negative (P/N) values. P/N values were expressed as the mean counts of a test serum divided by the mean counts of the appropriate dilution of control (preimmunization) serum. Because of prozone effects in low serum dilutions (
