Respiratory Syncytial Virus: Implications for Parenteral

Transcription

1 INFECTION AND IMMUNITY, July 1982, p /82/ $02.00/0 Vol. 37, No. 1 Comparison of Enzyme-Linked Immunosorbent Assay and Neutralization Techniques for Measurement of Antibody to Respiratory Syncytial Virus: Implications for Parenteral Immunization with Live Virus Vaccine ROBERT B. BELSHE,1* LEE P. VAN VORIS,1 MAURICE A. MUFSON,' EUGENE B. BUYNAK,2 ARLENE A. McLEAN,2 AND MAURICE A. HILLEMAN2 Section ofinfectious Diseases, Department of Medicine, Marshall University School of Medicine, Huntington, West Virginia 25701,' and Division of Virus and Cell Biology Research, Merck Institute for Therapeutic Research, West Point, Pennsylvania Received 8 May 1981/Accepted 22 February 1982 The sensitivity of an enzyme-linked immunosorbent assay (ELISA) to detect low levels of antibody to respiratory syncytial (RS) virus was compared with a tube dilution neutralization test (NEUT) on sera obtained from children who received a parenteral live RS virus vaccine. Among the children who developed antibody in response to live RS virus vaccine, ELISA was as sensitive as NEUT at detecting antibody increases. Some children who did not have detectable prevaccine ELISA antibody possessed NEUT antibody; these children were generally less than 12 months pld, suggesting that they had low levels of maternal antibody. Low levels of NEUT or ELISA antibody were associated with the absence of antibody increases after injection of live RS virus vaccine. The quantity of antibody stimulated by this live RS virus vaccine was small compared with that which was stimulated by naturally acquired RS virus infection. We concluded that ELISA is a satisfactory test for determining antibody to RS virus in vaccine field trials, given the understanding that low levels of preexisting antibody are not detected in some instances. No effective vaccine has been developed to prevent infection with respiratory syncytial (RS) virus, the single most important respiratory pathogen of early life (14). RS virus vaccines previously evaluated in infants and children have included an inactivated, alum-precipitated vaccine for parenteral administration (10), a cold-adapted, live attenuated virus vaccine for intranasal administration (9), and two live attenuated temperature-sensitive mutants of RS virus for intranasal administration (8; Belshe, unpublished data). To date, none of these four vaccines has proven satisfactory for use in the immunoprophylaxis of RS virus infection. Recently, we tested a parenterally administered live RS virus vaccine (6). During the initial evaluations of this vaccine, Buynak and coworkers reported that greater than 90% of seronegative infants and children who received this live RS virus vaccine developed postvaccine serum-neutralizing antibody (4, 6). However, during the course of our evaluation of this live RS virus vaccine, we observed that one-third of seronegative infants failed to seroconvert after vaccination as measured by enzyme-linked immunosorbent assay (ELISA) (2a). ELISA, as described by Richardson et al. (15), was superior to the plaque reduction and complement fixation (CF) tests for determining antibody responses during naturally acquired RS virus infection in young infants. The tube dilution neutralization test (NEUT) used by Buynak et al. (6) to assay serum antibody levels to RS virus differed from the standard plaque reduction assay (12) generally used to measure RS virus antibody. Subsequently, Buynak et al. (4) reported that the addition of complement increased the sensitivity of their assay, analogous to the findings of Mills et al. (12), who showed that added complement increased the sensitivity of the plaque reduction assay. To determine the relative abilities of the ELISA and NEUT techniques to detect low levels of antibody to RS virus and also to detect antibody increases after parenteral vaccination with live RS virus vaccine, we tested a group of sera by both methods. We also compared the level of antibody induced by this vaccine with that induced by natural infection with RS virus. This communication summarizes the findings of our comparative studies. MATERIALS AND METHODS Vaccine. Live RS virus vaccine (lot 592; Merck & Co., Inc., Rahway, N.J.) was stored lyophilized in 160

2 VOL. 37, 1982 two-dose vials at -20 C (6). The vaccine was reconstituted with sterile water immediately before use. Each 0.5-ml dose contained o tissue culture infective doses of RS virus. Placebo vaccine consisted of tissue culture fluid and was given in 0.5-ml doses. Volunteers. At the time children visited their pediatricians for routine health maintenance, their parents were asked if they wished to enroll their child in a placebo-controlled efficacy field trial of live RS virus vaccine. Only well children who were 6 months to 3 years 11 months old were enrolled in the study. After written informed consent was obtained from the parents, either live RS virus or placebo vaccine was administered subcutaneously. The vaccines were administered according to a double-blind and randomized protocol. This investigation was approved by the Human Studies Committee of the Marshall University School of Medicine, Huntington, W. Va. Sera. Sources of sera used in this study are shown in Table 1. Serum specimens were collected from each vaccinated child before and 1 month after vaccination with either live RS virus vaccine or placebo vaccine. Sera were also collected from placebo-vaccinated children who subsequently became naturally infected with RS virus; these sera were collected before the onset of illness (as part of ongoing sero-epidemiological surveillance for naturally occurring RS virus infection) and at least 21 days after infection with RS virus. RS virus infection was confirmed in these individuals by isolation of RS virus from respiratory secretions. Serological Tests. ELISA tests were performed as previously described by Richardson and co-workers, using an RS virus-infected HEp-2 cell suspension that had been adsorbed to polyvinyl microtiter plates (Dynatech Laboratories, Inc., Alexandria, Va.) (15). After washing with phosphate-buffered saline-tween to remove unadsorbed material, test sera in dilutions of 1:100, 1:400, 1:1,600, 1:6,400, and 1:25,600 were added to duplicate wells previously adsorbed with either an RS virus-infected cell suspension (antigen) or an uninfected HEp-2 cell suspension (control). A lower dilution of serum (1:25) frequently reacted with the control cell suspension; therefore, 1:100 was the lowest dilution of serum regularly employed (15). Reac- TABLE 1. Immune stimulus Sources of sera ANTIBODY RESPONSE TO LIVE RS VIRUS VACCINE 161 No. of sera (No. of pairs) Live RS virus vaccine 112a (56) Placebo vaccine 126a (63) Live RS virus or placebo vaccine 74a ( )b Naturally acquired RS virus infectionc 40 (20) a Sera included in Fig. 1 (n = 312). b Pre- or postvaccine serum that was unpaired. The corresponding serum from the pair could not be tested by NEUT owing to insufficient quantity of available serum. c In each instance, RS virus was isolated from respiratory secretions to confirm infection with this agent. All 20 pairs were tested by ELISA, 6 pairs were tested by NEUT, and 6 pairs were tested by CF. Each pair selected for testing was from a placebo vaccine recipient undergoing infection in the first epidemic after enrolling in the vaccine study. ELISA CR.ccaO 42444' Sk25S Neutralizing Antibody Titer (Reciprocal) FIG. 1. Comparison of ELISA titers with NEUT titers among 312 sera from children given live RS virus or placebo vaccine. Sera were tested by NEUT at an initial dilution of 1:2 when sufficient volume was available; otherwise, they were tested at 1:4 or 1:8 initial dilution. tion of serum and antigen was carried out overnight at 4 C, and the unreacted serum was removed by washing. Subsequently alkaline phosphatase-conjugated goat anti-human immunoglobulin G (Miles Laboratories, Inc., Elkhart, Ind.) was added to each well, and after 2 h of incubation, substrate (p-nitrophenyl phosphate [Sigma 104]; Sigma Chemical Co., St. Louis, Mo.) was added. The test plates were incubated further for 30 mi at 37 C, and the absorbance at 400 nm was determined for each well with a Multiskan ELISA reader (Flow Laboratories, Inc., Rockville, Md.). A single reference serum was included in each group of tests as a standard. This reference serum has been used many times, both in Richardson's laboratory and in our own, for this purpose, with the antibody titer set at 1:1,600 as previously described (15). Antibody titer for each test serum was calculated as follows: a calibration line was fitted by the least-squares method to a series of log dilutions and the corresponding logit absorbances of this standard. The fitted logit absorbance (y*) of a preselected dilution (1:1,600) was derived from the slope and intercept of the calibration line. For each experimental serum, a line was fitted by the least-squares method to a series of log dilutions and the corresponding logit absorbances. The titer of the experimental serum was taken to be the antilog of the fitted log dilution on the line in question which corresponded to y*. A similar procedure which omits the use of the logit transformation of the absorbance

3 162 BELSHE ET AL. has been described by Leinikki and Passila (11). The method of Gail and Green was used to determine the lower limit of the calculated ELISA titers that were considered seropositive (7). Calculated ELISA titers of <1:50 were considered seronegative, and titers of -1:50 were considered seropositive. NEUT tests were performed as previously described (4). Sera were tested by NEUT at an initial dilution of 1:2 when sufficient serum volume was available; otherwise, they were tested at 1:4 or 1:8. For this analysis, a serum was considered NEUT antibody negative when the initial dilution used for that serum was negative. Serum with added fresh guinea pig complement (10% final concentration of complement) was serially twofold diluted and mixed with an equal volume of RS virus suspension containing 10 to 30 50% tissue culture infective doses per 0.1 ml. The resulting serum-virus mixtures were incubated for 1 h at 37 C. Duplicate HEp-2 tissue cultures were inoculated with 0.2 ml of each mixture and incubated at 37 C. The antibody titer was taken as the highest initial dilution of serum that prevented viral cytopathic effects. For purposes of comparison with NEUT and ELISA titers, CF tests were performed by the standard method (13). Statistical analysis. The correlation between the log ELISA titer and the log NEUT titer was calculated with the standard formula for the classical correlation coefficient, r. NEUT antibody-negative sera (<1:2, <1:4, or <1:8) were treated as a group and assigned a value of 1 for purposes of calculating the correlation coefficient. ELISA antibody-negative sera (<1:50) were assigned a value of 25 for purposes of calculating the correlation coefficient. Two-by-two chi-square comparisons were made with Yates's correction. The t test was calculated by the standard method. INFECT. IMMUN. RESULTS Antibody titers to RS virus measured by ELISA and NEUT were compared for the 312 pre- or postvaccine sera (Fig. 1). Of the 211 ELISA antibody-negative sera (ELISA titer, <1:50), 145 were also NEUT antibody negative (<1:2, <1:4, or <1:8). Sixty-six sera contained NEUT antibody but not ELISA antibody. Of 101 ELISA antibody-positive sera (ELISA titer, -1:50), 86 contained NEUT antibody (.1:2) and 15 did not. The correlation between the log ELISA titer and the log NEUT titer was 0.71 (P < 0.01). The antibody responses of the two vaccine groups as determined by ELISA and NEUT are shown in Table 2. In the group of children who were seronegative as determined by ELISA, 31 (72%) of 43 developed ELISA antibody after live RS virus vaccine. In the group of NEUT antibody-negative children, 22 (76%) of 29 developed postvaccine NEUT antibody. The number of seronegative children who developed antibody as indicated by ELISA was not significantly different from the number of children who developed antibody as indicated by NEUT (X2 < 1). Of the recipients of live RS virus vaccine, 12 were initially seronegative by ELISA and failed to develop postvaccine ELISA antibody; 9 (75%) of these 12 vaccinated children had prevaccine NEUT antibody, and 3 (25%) were NEUT antibody negative. The three vaccinated children who were seronegative by both ELISA and NEUT were 7, 7, and 9 months of age at the TABLE 2. Serum antibody response as determined by ELISA among children receiving live RS virus or placebo vaccine compared with the serum antibody response as determined by NEUT No. of children with indicated response to vaccination with: Live RS virus Placebo ELISA NEUT NEUT NEUT ELISA antibody status antibody responsea to response' to NEUT response" to before vaccination response' vaccine vaccine response' to placebo to vaccine (sero- (seropositive placebo (sero- (seropositive negativeb) [:: 1:21b) negative ) [(1:2ob) +++ Seronegative (<1:50) - 3' Seropositive (.1:50) afourfold increase or conversion from seronegative to seropositive. b NEUT antibody status before vaccination. Sera were tested at initial dilutions of 1:2, 1:4, or 1:8, depending on the volume available for testing; thus, "seronegative" may mean <1:2, <1:4, or <1:8. c The rate of seroconversion after live RS virus vaccine among the ELISA antibody-negative children (31 of 43) was not significantly different than that among the NEUT antibody-negative children (22 of 29; x2 < 1). d The rate of seroconversion after placebo vaccine among the ELISA antibody-negative children (3 of 49) was not significantly different than that among the NEUT antibody-negative children (7 of 47; x2 = 1.1).

4 VOL. 37, 1982 time of vaccination. Among the nine vaccinated children who were seronegative by ELISA but seropositive by NEUT, six were less than 8 months old at the time of vaccination. We compared the ELISA antibody response to naturally acquired RS virus infection with the ELISA antibody response of children who received live RS virus vaccine (Table 3). The group of children with natural RS virus infection consisted of 20 children who had received placebo vaccine and became infected in the first RS virus epidemic subsequent to enrollment into the study. The ages of the children who were naturally infected were not significantly different from those of the live RS virus vaccinees. Natural infection resulted in high titers of ELISA antibody. The level of ELISA antibody stimulated by natural infection was 10 times higher than that stimulated by live RS virus vaccine. Additionally, we compared the level of NEUT and CF antibody after live RS virus vaccine to that occurring after natural infection. Low levels of NEUT and CF antibody developed in some vaccinated children in response to vaccine (Table 3), but of the naturally infected children, all those tested developed high levels of NEUT or CF antibody. DISCUSSION In this report, we describe the serum antibody responses to a parenteral live RS virus vaccine as determined by the ELISA and NEUT procedures. There was good correlation between the two assays for quantitating the amount of anti- TABLE 3. ANTIBODY RESPONSE TO LIVE RS VIRUS VACCINE 163 body to RS virus. ELISA was as efficient as NEUT in detecting antibody increases after live RS virus vaccine and required a small amount of serum (0.01 ml per test). In contrast, the NEUT test required a relatively large volume of serum (0.3 ml per test). Considering the technical difficulty of venipuncture in infants and young children, ELISA was preferable because only small amounts of serum were needed. Furthermore, ELISA was suitable to run en masse and did not require living tissue culture cells. The NEUT test was laborious and required large numbers of tissue cultures to perform. We found ELISA suitable for use in a field situation, with the reservation that seronegative children (ELISA titer, <1:50) who fail to seroconvert after live RS virus vaccine may possess low levels of NEUT antibody. Of the 12 children who were ELISA antibody negative and did not develop postvaccine antibody, 9 possessed preexisting NEUT antibody. These data suggested that most ELISA antibody-negative children who failed to respond to live RS virus vaccine possessed low levels of circulating antibody which inhibited vaccine virus replication (and therefore prevented antibody stimulation). That this preexisting antibody was of maternal origin in most instances was suggested by the young ages of these vaccinated children and the observation that RS virus had not circulated in the community within the lifetime of five of the nine children. Two of the children were sufficiently old to suggest that they were probably infected with RS virus previ- Comparison of the antibody response of children after vaccination with live RS virus vaccine or natural RS virus infection Immune stimulus Mean age Geometric mean (reciprocal) serum and method of No. of serum (mo) + SE antibody titer to RS virus determining antibody pairs tested at time of immune stimulus' Prevaccination Postvaccination or acute or convalescent RS virus vaccine ELISA 21b 17.7 ± 1.8 < NEUT 21b 17.7 ± 1.8 <2 48 CF 20c 21.0 ± 2.3 <2 4.8 Natural infection ELISA 20d 22.1 ± ,000 NEUT 6d 26.0 ± CF 6d <2 18 a There was no significant difference in age between the groups tested by the same method of antibody determination (t test). b Same serum pairs. In each instance, the prevaccine serum was antibody free by ELISA and NEUT, and antibody developed after vaccine. c Different serum pairs than those described in footnote b. Each serum pair selected for testing by CF had ELISA antibody increase. d Only placebo vaccine recipients who were infected with RS virus in the first winter after enrollment into the vaccine study were included in this comparison. The serum pairs tested by NEUT and CF were among those serum pairs tested by ELISA.

5 164 BELSHE ET AL. INFECT. IMMUN. TABLE 4. Antibody response to four live-virus vaccines relative to the antibody response after naturally acquired infection with homologous virus Ratio of antibody titer Live virus vaccine (strain) Type of antibody after vaccine to antibody assay titer after natural Reference infection Live RS virus (Merck lot 592) ELISA 0.10 This study Measles (Moraten) Hemagglutination 0.49 Buynak et al. (5) inhibition Mumps (Jeryl Lynn) Neutralization 0.13 Weibel et al. (16) Rubella (RA27/3) Hemagglutination 0.60 Black et al. (3) inhibition ously, and two children may or may not have been previously infected. To ensure that vaccinated children no longer possess maternal antibody, this live RS virus vaccine would have to be administered after children reach their first birthday, a situation analogous to that observed with measles virus vaccine (1). Since the majority of severe RS virus infections occur during the first 12 months of life, our findings pose a dilemma for use of a parenterally administered live virus vaccine (2, 14). The vaccine must protect against an infection which occurs at a time when maternal antibody may inhibit the replication of vaccine virus. The geometric mean ELISA titer after natural RS virus infection was large (1:2,000); this is close to the figure (1:2,600) previously reported (data adapted from Richardson et al. [15]). The magnitude of the antibody response was limited after administration of live RS virus vaccine. In contrast to natural infection, only very low titers of ELISA, NEUT, or CF antibody were stimulated by this vaccine. The ratio of geometric mean antibody titer after vaccination to geometric mean antibody titer after natural infection was small (0.10). Although the levels of serum antibody after other parenteral live virus vaccines may be less than that stimulated by natural infection, the relative antibody level induced by live RS virus vaccine was less than that induced by measles or rubella vaccines as compared with the respective natural infections (Table 4). Of the licensed parenterally administered live viral vaccines, mumps vaccine stimulates the least amount of antibody relative to natural infection, yet this vaccine has been quite successful at preventing illness from naturally acquired mumps. The determination of efficacy of the low levels of antibody stimulated by this RS virus vaccine will require clinical evaluation. ACKNOWLEDGMENT We thank David W. Alling of the National Institute of Allergy and Infectious Diseases, Bethesda, Md., for his assistance in adapting his method of generating endpoint titers for use in this study and Beverly Pofahl of Marshall University for technical assistance. This research was supported in part by a grant from the Merck Research Foundation, Inc. LITERATURE CITED 1. Albrecht, P., F. A. Ennis, E. J. Saltzman, and S. Krugman Persistence of maternal antibody in infants beyond 12 months: mechanism of measles vaccine failure. J. Pediatr. 91: Belshe, R. B., L. P. Van Voris, M. A. Mufson, and L. Hyler Epidemiology of severe respiratory syncytial virus infections in Huntington, West Virginia. W. Va. Med. J. 77: a.Belshe, R. B., L. P. Van Voris, and M. A. Mufson Parenteral administration of live respiratory syncytial virus vaccine: results of a field trial. J. Infect. Dis. 145: Black, F. L., S. H. Lomm, J. E. Enmons, and F. P. Pinheiro Durability of antibody titers induced by RA 27/3 rubella virus vaccine. J. Infect. Dis. 137: Buynak, E. B., R. E. Weibel, A. J. Carlson, A. A. McLean, and M. R. Hilleman Further investigations of live respiratory syncytial virus vaccine administered parenterally. Proc. Soc. Exp. Biol. Med. 160: Buynak, E. B., R. E. 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6 VOL. 37, 1982 ANTIBODY RESPONSE TO LIVE RS VIRUS VACCINE Mufson, M. A., and R. B. Belshe Respiratory syncytial virus and the parainfluenza viruses, p In N. R. Rose and H. Freidman (ed.), Manual of clinical immunology, 2nd ed. American Society for Microbiology, Washington, D.C. 14. Parrott, R. H., H. W. Kim, J. 0. Arrobio, D. S. Hodes, B. R. Murphy, C. D. Brandt, E. Camargo, and R. M. Chanock Epidemiology of respiratory syncytial virus infection in Washington, D.C. II. Infection and disease with respect to age, immunologic status, race and sex. Am. J. Epidemiol. 98: Richardson, L. S., R. H. Yolken, R. B. Belshe, E. Camargo, H. W. Kim, and R. M. Chanock Enzymelinked immunosorbent assay for measurement of serological response to respiratory syncytial virus infection. Infect. Immun. 20: Weibel, R. E., E. B. Buynak, A. A. McLean, and M. Hilleman Long-term follow-up for immunity after monovalent or combined live measles, mumps, and rubella virus vaccines. Pediatrics 56: