JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2856-2859

Vol. 29, No. 12

0095-1137/91/122856-04$02.00/0 Copyright © 1991, American Society for Microbiology

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Antibodies to Seven Rotavirus Serotypes in Cord Sera, Maternal Sera, and Colostrum of German Woment HARALD BRUSSOW,l* JOSETTE SIDOTI,' LOTHAR LERNER,2 HASSAN RAHIM,' WIEBKE ECKSTEIN,2 HERMANN WERCHAU,3 AND CARL MIETENS2 Nestlé Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH 1000 Lausanne 26, Switzerland,' and St. Joseph Hospital2

and Institute

of Medical Microbiology and Virology,3 Ruhr-Universitat Bochum, Bochum, Federal Republic of Germany Received 15 March 1991/Accepted 4 September 1991

Forty percent of colostrum samples from German women showed neutralizing antibody titers of -50 to rotavirus (RV) serotypes 1, 3, 4, and 6. Antibody to serotypes 2, 8, and 9 was less prevalent. Titers are, however, too low to indicate an important effect of colostrum on the RV vaccine take rate. On the other hand, about 50% of the cord serum samples showed high neutralizing-antibody titers to serotypes 1, 3, and 4, which could interfere with the take rate of RV vaccines based on these serotypes in very young infants.

Rotaviruses (RV) are the single most important cause of childhood diarrhea throughout the world (13). Studies of maternally derived passive immunity to RV have measured mainly antibodies (Ab) directed at the nonneutralizing, common group antigen of the virus in colostrum, breast milk, and cord serum (9, 12, 15, 22, 24); only a few studies have reported the serotype-specific neutralizing-Ab titers to RV in human milk (1, 19). Knowledge of serotype-specific Ab titers in milk is relevant to RV vaccination strategy (13). To achieve optimal efficacy, oral RV vaccine should be administered in the neonatal period. Yet at this age breast-feeding is common and RV-specific milk Ab might interact with vaccine virus, thereby preventing vaccine take (18). In addition, the presence of maternally derived serum Ab above a given titer is known to interfere with seroconversion rates after RV vaccination (10). To assess passively acquired Ab levels to RV, we obtained maternal serum, cord serum, and colostral milk samples from 45 women at delivery and from their infants at the St. Elisabeth Hospital, Bochum, Federal Republic of Germany. Most of the colostrum samples were obtained at day 2 or 3 after delivery. The mean age of the women was 26.6 years, and their mean weight at delivery was 71 kg. Their mean weight gain during pregnancy was 11.8 kg. The mean length of gestation was 39.7 weeks, and the mean birth weight of the infants was 3,480 g. Of these women, 42% were prima para, whereas 42, 7, and 9% already had one, two, and three or more children, respectively; 44% of the women had breast-fed their previous infant. However, none of these clinical parameters could predict whether a woman's colostrum had higher or lower neutralizing-Ab titers to RV (data not shown). The total immunoglobulin G (IgG) concentration in maternal and cord serum samples and the total IgA concentration in colostrum were determined by radial diffusion (17) with commercial IgG and secretory IgA standards

(Behringwerke, Marburg, Federal Republic of Germany, and Sigma, St. Louis, Mo.). The physical integrity of the secretory IgA antibody was checked by a double-diffusion technique (17) and immunoelectrophoresis (17). We could not detect degradation of secretory IgA by the Ouchterlony test or immunoelectrophoresis (data not shown). RV-specific enzyme-linked immunosorbent assay (ELISA) and neutralizing (NT) antibody titers were determined as described previously (7). Human RV strains Wa, S-2, Hochi, 69M, and W161 representing RV serotypes 1, 2, 4, 8, and 9, respectively, were used in this study. RV serotypes 3 and 6 were, however, represented by animal RV strains SA11 and NCDV, since heterologous simian and bovine RV strains have been used in a Jennerian approach of RV vaccination (13, 14). At a 1:50 serum dilution, 70 to 96% of the maternal serum samples showed neutralizing Ab to serotypes 1, 3, 4, and 9. Only 43 and 20% of the maternal serum samples neutralized RV serotypes 2 and 8, respectively, whereas 61% neutralized heterologous bovine RV serotype 6 (Table 1). A similar pattern was observed for cord serum samples (Table 1). Diaplacentar transfer of IgG antibody thus ensured that more than 80% of the German infants tested were born with high neutralizing-Ab titers to RV serotypes 1, 3, and 4 in their serum. In fact 49, 45, and 55% of the cord serum samples showed neutralizing-Ab titers of .300 against serotypes 1, 3, and 4, respectively. These titers are known to interfere with the take rate of serotype 1 reassortant RV in 1- to 2-month-old infants (10). About 40% of the colostral milk samples showed neutralizing-Ab titers of .50 to RV serotypes 1, 3, 4, and 6 (Table 1). This prevalence was 23, 14, and 9% for Ab to serotypes 9, 2, and 8, respectively (Table 1). As in a previous report from Australia (19), milk Ab to RV serotype 1 was the most prevalent in German women; this was followed by Ab to serotypes 3, 4, and 9. Serotype 1 was also the predominant RV serotype isolated from children hospitalized with gastroenteritis in the study area during the time of colostrum sampling (3). In colostrum from German women, RV-specific antibody titers were similar to those reported for titers in colostrum of Swiss (2), British (20, 21), Norwegian (16)

* Corresponding author. t Dedicated to the memory of C. Mietens and L. Lerner, whose help was essential in providing clinical samples in this and previous

studies. C. Mietens died in May 1990 and L. Lerner died in June 1991. 2856

VOL. 29, 1991

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and Ethiopian (16) women. Substantially higher titers have, however, been reported for Australian women (19). All maternal and cord serum samples showed RV-specific ELISA IgG Ab titers of 50 or greater. Also, 100 and 69% of the maternal serum samples, respectively, showed RVspecific IgM and IgA Ab, which were not detected in cord serum samples (Table 2). All colostral milk samples showed RV-specific IgA antibody at a 1:10 dilution. With an ot-chainspecific and a secretary component-specific conjugate, 64 and 70%, respectively, of the colostral milk samples were still positive at a 1:50 dilution (Table 2). Secretory Ab were revealed by a mouse monoclonal Ab to human secretory component (GA-1; Sigma) followed by incubation with phosphatase conjugated to goat antibody directed against mouse IgG (Sigma). Serum and colostrum ELISA Ab titers to RV were higher than Ab titers to the lipopolysaccharides of the most common O serogroups (026, 055, 0111, 0127, and 0128) identified in enteropathogenic Escherichia coli diarrhea (Table 2). Lipopolysaccharide-specific ELISA was performed as described previously (5). In this study, we also found a pronounced difference in immunogenicity between a dietary protein, P-lactoglobulin, commonly found in cow's milk products, and bacterial and viral antigens (Table 2). P-Lactoglobulin-specific ELISA was performed as described previously (6). This observation concurs with that for an animal model, in which rats produced high serum and moderate milk Ab titers to antigens of E. coli, but only very low serum and milk Ab titers to P-lactoglobulin after oral exposure to both antigens (23). On the basis of our results, rhesus RV serotype 3 and reassortant RV serotypes 1 and 4, which are current RV vaccine candidates (13), are expected to experience a partial neutralization by colostrum of German women. Titers are, however, too low in the majority of the colostrum samples to indicate an important effect on the vaccine take rate. Reassortant RV serotype 2 vaccine will experience only a negligible neutralization by colostrum of German women. Serotype 2-neutralizing Ab were also the least prevalent in colostrum and milk of Australian women (19). Notably, the heterologous bovine RV NCDV, which is serologically similar to another RV vaccine candidate (8), was neutralized by more than one-third of German colostrum samples. This has also been reported for milk of women in the United States (1). It is not surprising to find Ab to heterologous bovine RV serotype 6 in adults. It has been shown that adults infected with a human RV develop Ab responses to NCDV as well as other heterotypic RV antigens (11). This was also observed after repetitive natural RV infections in children (4). Our data support the conclusions of a recent meta-analysis of rotavirus vaccine trials. A significant, although not very strong, adverse effect of breast-feeding was observed with respect to rotavirus vaccine seroconversion in North America (18). It is important to measure Ab titers to the different rotavirus serotypes in milk samples from mothers in developing countries. This will help to determine the vaccine serotype and the age of the children that will lead to optimal take rates of RV vaccine in the areas where it is most needed. We thank W. Strunz and the midwives of the St. Elisabeth Hospital, Bochum, Federal Republic of Germany, for careful collection of the samples and Q. Genoud and F. Theulaz for typing the manuscript.

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Prospects for development of a rotavirus vaccine against rotavirus diarrhea in infants and young children. Rev. Infect. Dis. 11:S539-S546. 15. McLean, B., and I. H. Holmes. 1980. Transfer of antirotaviral antibodies from mothers to their infants. J. Clin. Microbiol.

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