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RISES IN TITERS OF ANTIBODY TO HUMAN CORONA VIRUSES OC43 AND 229E IN SEATTLE FAMILIES DURING 1975–1979

Abstract Sequential serum specimens were obtained every four months during 1975–1979 from 44 children and adults of 10 Seattle families. The 419 specimens were tested for antibody to human coronaviruses OC43 and 229E by enzyme-linked immunosorbent assay (ELISA). Antibody titers were found to increase with age, and titers as well as frequency of rises were greater for OC43 than for 229E virus in all age groups. Significant antibody rises were most frequent in specimens bracketing the winter interval, but some also occurred in the spring-summer and summer-fall intervals. Concurrent significant antibody rises to OC43 virus in different members of the same family were observed in 15 instances, to 229E virus in seven instances, and to OC43 virus in some members and 229E virus in others in eight instances. Significant antibody rises to OC43 or 229E virus indicating reinfections were frequently observed throughout the three-year period but were always separated by at least two four-month intervals. Concurrent significant antibody rises to both 229E and OC43 viruses were seen only in three persons. Finally, the frequency of significant antibody rises in children, about one per person-year, was almost three times higher than in adults.

results to results obtained with virus neutralization, complement fixation, and hemagglutination inhibition. In this report we describe the application of ELISA in seroepidemiologic studies of coronaviruses in 10 Seattle families under intensive continuing surveillance for influenzavirus infections during 1975-1979 (22).

Preparation of viral antigens
The source of human coronavirus strains 229E and OC43 and their propagation have been described previously (18). Briefly, plaque purified virus strains were grown in human rhabdomyosarcoma cell cultures, purified by rate zonal and isopycnic centrifugation, and concentrated to 4 mg/ml of protein (19,20). Whole virus antigens were prepared from purified virions by dissociation of concentrated virus samples in 1 per cent sodium dodecyl sulfate at 4 C for 10 minutes and subsequent dilution in coating buffer (0.1 M carbonate-bicarbonate, pH 9.6) immediately before coating of ELISA plates (15).

Enzyme-linked immunosorbent assay
ELISA was carried out in Immulon M-129 microtiter plates with flat-bottomed wells, as previously described (15). All reagents were dispensed in aliquots of 110 fA per well. Plates were coated with antigens (0.8 ng of protein per well) diluted in carbonate-bicarbonate coating buffer (21) and serial 0.5 logi 0 serum dilutions were prepared directly in the antigen-coated plates in phosphate-buffered saline containing 0.2 per cent Tween 20 and 100 Mg/ml of gelatin. Affinity purified heavy chain-specific goat anti-human immunoglobulin G alkalinephosphatase conjugate (Sigma Chemical Co., St. Louis, MO) was added at a dilution of 1:1,000. Para-nitrophenylphosphate (1 mg/ml) in 10 per cent diethanolamine buffer was used as substrate solution (21) and optical densities were determined with a "Titertek Multiskan" filter photometer (Flow Laboratories, McLean, VA) at 405 nm. Human sera devoid of antibody to human coronaviruses 229E and OC43 as determined by virus neutralization and radioimmune precipitation (15) were used as appropriate negative serum controls. Assays were carried out in duplicate and all specimens obtained from the same family were tested in the same run to avoid differences attributable to run-to-run variation.
Serum antibody titers were expressed as the reciprocal of the highest serum dilution which yielded optical densities of 0.35. These cutoff points were safely in the linear portion of the antibody titration curves of all sera tested and were significantly increased (at least two standard deviations) over nonspecific reactions (15). Antibody rises were expressed as ELISA ratios, which were defined as the ratios of maximum to minimum optical densities between sequential serum specimens at the same dilution. ELISA ratios of 1.8 or greater (i.e., a 1.8-fold increase in optical density) were accepted as indicating significant rises. These ratios were found to correlate with fourfold or greater increases in virus neutralizing and complement-fixing antibody titers in paired sera from 200 unrelated patients with respiratory disease (unpublished data).

Study population
Of some 200 Seattle families entering into continuing observation for influenzavirus infections during 1975-1979, 10 were selected because each had at least one preschool child at entry into the study in fall 1975. The study population and methods of observation have been described in detail elsewhere (22). Nose-throat specimens were collected for culture biweekly and additional specimens were obtained when illness was observed. Blood specimens were collected three times yearly: late fall (October/November), spring (March/April), and late summer (July/August). A total of 419 blood specimens were examined. Of the 44 persons studied, nine were in the 0-4year age group at entry, 11 in the 5-9-year group, four in the 10-14-year group, and 20 were adults.

Age-specific antibody titers to coronauiruses
OC43 and 229E Only two children, both less than two years old, lacked antibody to 229E virus and one also lacked antibody to OC43 virus. All other persons had antibodies to both viruses. Titers ranged from 100 (reciprocal of endpoint serum dilution) in the very young to 60,000 in persons with a recent antibody rise. The 419 sera tested were distributed according to the age of the donor when blood samples were collected and the geometric mean titers for each age group were calculated. As seen in table 1, the geometric mean titers increased directly with age. Also, in each age group, the geometric mean titer for OC43 virus exceeded that for 229E virus, the relative difference decreasing with age from 2.8-fold (0-4 years) to 1.2-fold (adults).

Antibody rises in relation to age
Examination of the 419 sera available revealed 92 significant antibody rises, 56 to OC43 virus and 36 to 229E virus. Overall, each study member experienced 0.7 significant antibody rises (to either OC43 or 229E virus) per year. However, as seen in table 2, the annual frequencies of rises to each  virus tended to decline with age. In all but the 10-14-year age group, antibody rises to OC43 virus were more frequent than were rises to 229E virus.

Temporal patterns of antibody rises
Antibody rises to at least one virus (usually both) were recognized in all 10 sampling intervals (figure 1). With OC43 virus, the peak rises in each year occurred in the period between the October/November and March/April samplings and are reflected in the sera collected each spring. No similar pattern for 229E virus was observed.

Concurrent antibody rises
As seen in figure 2, concurrent significant antibody rises to both viruses were observed in three children (one each in families E, I, and J). This number does not differ significantly from the number (five) expected on the basis of the observed frequencies of rises to each antigen. Concurrent significant antibody rises to OC43 virus in at least two members of the same family were observed in 15 instances. With 229E virus there were seven such instances. Significant antibody rises to OC43 virus in some family members and to 229E virus in others were observed in eight instances.

Reinfection with human coronaviruses
Primary infection, defined as seroconversion to either virus, was seen in only two of   the three children less than two years of age. In one child (family C), seroconversion to OC43 virus was observed between August and November 1976, followed by seroconversion to 229E virus between November 1976 and March 1977. The other child (family D) had antibody to OC43 virus initially but expressed seroconversion to 229E virus between November 1976 and March 1977. Apparent reinfections with coronaviruses as indicated by repeated significant antibody rises to the same virus were seen in 21 instances with OC43 virus and in eight with 229E virus. In each instance, these were separated by at least eight months. Two significant antibody rises in succession were observed in 14 persons, in each instance to different viruses.

Antibody rises in relation to illness
Because of the four-month intervals between collection of blood specimens, it was not possible to relate specific illnesses to observed rises in antibody titers. However, it was noted that 36 per cent of significant antibody rises in adults and 40 per cent in children had occurred during intervals in which no respiratory or gastrointestinal illness was reported. The two children with primary coronavirus infections had experienced respiratory illnesses unrelated to isolation of or detectable rises in antibody to other viruses. Further, OC43 viral antigens were detected in nose-throat specimens from one of the two children by ELISA (unpublished data).

DISCUSSION
The observed high prevalence of antibodies in Seattle families (prevalences from 30 to 100 per cent have been reported for other populations) probably reflects both the increased sensitivity of the assay and the age of the study population since, in other studies, children less than two years of age, also often seronegative, were relatively more numerous. We also found antibody levels to be directly related to age as has been reported by others (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36), presumably reflecting the cumulated effects of repeated infections. Antibody levels, after initial increases with age, remained generally stable within individuals except when presumed reinfection caused a sharp but temporary increase in titers with usual return to baseline levels within four to eight months.
The intervals between bleedings did not coincide with the conventionally defined four seasons since the original intent was to bracket influenza seasons. However, marked temporal differences in the frequency of significant antibody rises to OC43 virus were seen with the highest frequencies being observed in the predominantly winter interval between the October/November and March/April samplings. No such pattern was evident for 229E virus.
The combined infection rate, approaching one per person-year, was similar to that reported for an adult population in London, England (32). However, it is probable that the rates reported in this study underestimate the true frequency of infections. Our definition of a significant antibody rise, ELISA ratios of 1.8 or greater, was rather conservative. This ratio was accepted based on its correlation with fourfold or greater rises in virus neutralizing and complementfixing antibody titers in paired sera from 200 unrelated persons. ELISA ratios of between 1.5 and 1.8 also probably represented increases in specific antibody levels since many of these correlated with fourfold rises in virus neutralizing antibody titers and, in this study, 18 of 32 occurred in family clusters of two or more or coincidentally with 1.8 ratios in other family members. Our findings are in agreement with the present knowledge of the epidemiology of human coronaviruses but they also suggest that coronavirus infections in families are more frequent than has been thought.
Based on their independent frequencies, concurrent antibody rises in the same person to both 229E and OC43 viruses should occur only rarely. Both our observation of three such instances as well as findings reported by others (32,33) conform with this expectation. Given the length of the periods between bleedings (four months), the concurrent rises could reflect either consecutive or concurrent infections. On the other hand, concurrent antibody rises to each virus in different family members were seen frequently and in all seasons. Obviously, the two viruses circulated in the population simultaneously as well as independently, as has been reported by others (2,23,26,27,32,34,(36)(37)(38)(39)(40)(41).
Antibody rise to OC43 virus in one season was followed frequently by a rise to 229E virus or vice versa in the same person in the next season, but recurrent rises to the same virus were always separated by at least eight months. These results suggest that infection with one virus may convey short-term immunity to homologous reinfection.
Primary infections were observed in only two children. Both children had reported illness episodes. On the other hand, reinfections as indicated by significant antibody rises to either virus were common but their association with specific illnesses was not possible. Indeed, about 40 per cent of all significant antibody rises occurred without reported temporally related illness. We have presumed that these rises reflected reinfections with viruses related to one or the other prototype but it remains possible that at least some reflect chronic infections accompanied by antibody rises when host defenses weakened. Prolonged coronavirus antigen detection in throat and nose specimens from persons in London, England, over periods of up to two months has been reported (42). Preliminary studies of antigen detection in nasalpharyngeal specimens from persons in the Seattle study have yielded similar results (unpublished data). If these findings can be confirmed, our present understanding of the epidemiology of human coronaviruses would be considerably expanded.