Immunoglobulins in infants of rubella-exposed mothers

1 186

December, 1969 T h e Journal o[ P E D I A T R I C S

Immunoglobulins in infants 4 rubella-exposed mothers Noninfected infants o[ women who received large doses of gamma globulin early in pregnancy for the prevention of rubella were found to have lower serum lgG levels than normal controls up until 1 year of age. This effect, however, did not create clinical problems. Infected babies, as defined by the presence of rubella antibody persisting beyond 5 months of age, had higher serum lgM and IgG concentrations than noninfected babies until age 2 years.

Carolyn C. Huntley, M.D.,* Anne D. Lyerly, B.S., Miriam V. Patterson, and John L. Sever, M.D., Ph.D. WINSTON-SALEM~

N. (3.~ AND B E T H E S D A ~

Q U E S T I O N S have been

repeatedly r a i s e d

concerning the potential dangers of isoimmunization to gamma globulin antigens. During the rubella epidemic of 1964 in North Carolina large doses of gamma globulin were given to hundreds of women during the first trimester of pregnancy in an attempt to prevent rubella following exposure. We were intrigued to find out if this pooled gamma globulin, containing most IgG antigenic determinants, would induce immunization in these mothers and suppress IgG formation in their infants. We were additionally From the Department o[ Pediatrics of the Bowman Gray School o[ Medicine, and the National Institutes of Health. Supported in part by United States Public Health Service Grants Nos. AM 11906 and FR 5404 and a grant [tom the National Foundation. *Requests for reprints: Department o] Pediatrics, Bowman Gray School ol Medicine, Winston-Salem. North Carolina 27103.

Vol. 75, No. 6, part 2, pp. 1186-1193

MD.

interested in the long-term effects of maternal and fetal rubella on the serum immunoglobulin concentrations of the child. At the beginning of the epidemic, a largescale rubella project was initiated and planned by the Department of Obstetrics and Gynecology of the Bowman Gray School of Medicine and we therefore had the opportunity to secure, for our own studies, samples of many of the serum specimens which were being collected from mothers and infants for the purpose of rubella antibody determinations. MATERIAL

AND METHODS

The mothers were all women exposed to rubella during early pregnancy. The first serum specimen was obtained on mother and baby at some time during the baby's first year of life. The second specimen was obtained on the baby between 2 and 4 years

Volume 75 Number 6. part 2

Infants of rubella-exposed mothers

1 1 87

m

A [] 1.6

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Norma[ control infants

B [ ] infants of mothers who received y globulln but did not have overt maternal rubella [ ] Infants of g?oup B omitting those with rubella antibody perslst|ng after 5 months of age

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Fig. 1. Comparison of serum IgG concentrations from normal control infants (Group A), infants of mothers who were given gamma globulin and did not develop clinically recognizable rubella (Group B), and the Group B infants after dropping out those with persistent rubella antibody (presumably infected infants). There was a slight but significant decrease in the IgG levels of noninfeeted infants of mothers who received gamma globulin compared with control infants. This effect was not noted after one year of age.

of age. T h e mothers a n d their infants were divided into 3 groups. G r o u p B c o n t a i n e d w o m e n who received g a m m a globulin after exposure but did not have clinically recognizable rubella. G r o u p C c o n t a i n e d w o m e n w h o received g a m m a globulin a n d d i d develop overt rubella. G r o u p D contained w o m e n who d i d not receive g a m m a globulin a n d d i d develop overt rubella. Babies f r o m a well baby clinic which was a t t e n d e d by one of us (C. C. H.) were used as controls. T h e c o n t r o l g r o u p of infants was designated as G r o u p A. Immunoglobulin determinations were carried o u t by a single i m m u n o d i f f u s i o n m e t h o d in gel tubes?, 2 T h e latex fixation

test 3 a n d the R i p l e y coated red blood celt agglutination test 4 were p e r f o r m e d on m a t e r n a l sera to determine the presence of a n t i - g a m m a globulin antibodies. T w o m e r e a p t o e t h a n o P a n d sucrose g r a d i e n t experiments ~ were p e r f o r m e d to determine w h e t h e r or n o t 7S ( I g G ) antibodies, c a p a b l e of crossing the placenta, were present in maternal sera. RESULTS

Effects of g a m m a globulin. W h e n serum I g G concentrations of n o r m a l control subjects were c o m p a r e d with those of babies born to m o t h e r s who h a d received g a m m a globulin b u t h a d not h a d overt rubella

1 1 88

Huntley et al.

The Journal of Pediatrics December 1969

WI Normal control infants O Infants of mothers who received y globulin and did not have overt rubella A Group B after dropping out babies with rubella antibody persisting beyond 5 months of age

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Fig. 2. Comparison of the mean serum IgG concentrations of the same 3 groups described in Fig. 1. Note that the mean in the adjusted Group B (/x), after presumably infected babies have been omitted, was lower than that of the control group until one year of age.

(Group B), there was no significant difference between the two groups at any age (Fig. 1). However, after the babies with rubella antibody persisting beyond 5 months of age (presumably infected babies) were removed from Group B, this group then had significantly lower serum IgG concentrations than the control subjects. This difference persisted until 1 year of age, after which it was no longer present. The mean IgG concentrations for control subjects, Group B, and corrected Group B infants are charted in Fig. 2. When the individual low IgG values in the first infant samples from all patient groups were plotted (Fig. 3), it was noted that only 5 values fell below 2 standard deviations from the mean of the control group. There were no values under 250 rag. per cent and all rose to at least 600 nag. per cent by 2 years of age. There were'no life-

threatening infections in this group of babies, but a few had recurrent otitis media and other of the usual infantile diseases. There was only 1 baby (arrow) in this group with evidence of intrauterine rubella (persistence of antibody after 5 months of age). When the low IgG values in second samples (obtained from the children between 2 and 4 years of age) were plotted (Fig. 4), it was observed that many of the first specimens on these same babies had high IgG values and that there were many rubella babies (arrows) in this group. Fig. 5 is the same as Fig. 4, with the addition of low second samples for which we did not have paired first samples. There were 5 additional rubella babies in this group. Again, there were no serious or increased numbers of infections in the group of infants with low IgG concentrations in second samples.

Volume 75 Number 6, part 2

In[ants o/ rubella-exposed mothers

1 18 9

1.8 8 9

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Fig. 3. The low IgG values (more than one standard deviation below the control mean) in the first samples collected from the infants of the 3 study groups are plotted with their corresponding second or follow-up samples. The curves indicate minus 1 and 2 standard deviations from the mean of the normal control group. The circled dots indicate values more than 2 standard deviations below the control mean and their corresponding follow-up sample vMues. The arrows denote the only baby in this group with persistent rubella antibody.

T w o h u n d r e d a n d fifty-six m o t h e r s were t y p e d for the G m ( a ) factor. T h e r e were 92 G m ( a - ) mothers. A comparison was m a d e between the I g G levels of the 39 G i n ( a + ) a n d the 63 G m ( a - ) babies of G m ( a - ) m o t h ers. T h e m e a n I g G c o n c e n t r a t i o n for the G m ( a + ) babies was 793 rag. p e r cent, as opposed to 883 rag. p e r cent for the G m ( a - ) group, b u t the difference was not significant. T h e c o m p a r i s o n was r e p e a t e d a f t e r babies w i t h rubella a n t i b o d y were o m i t t e d from the group a n d the difference was even less. As a control check I g M levels were comp a r e d in t h e 2 groups of babies; there was v i r t u a l l y no difference before or after omitting babies with rubella antibody. T h e ages of the babies in the 2 groups were v e r y close.

Positive tests for a n t i - g a m m a globulin factors in m a t e r n a l s e r a are c h a r t e d in Fig. 6. T h e r e were a p p r o x i m a t e l y equal percentages of positive tests (14 to 16 p e r cent) in the 3 study groups of mothers (B, C, a n d D ) . I n G r o u p B there a p p e a r e d to be an association between a n t i - g a m m a globulin factors in the m o t h e r a n d low g a m m a globulin values in the infant, b u t numbers were too small in each age g r o u p for statistical analysis. I t is not p r a c t i c a l to do inhibition experiments w i t h the latex test, but we were able to inhibit the a n t i - g a m m a globulin factors in 2 of the Ripley-positive sera. T h e a n t i b o d y of the m o t h e r in G r o u p C with the R i p l e y titer of 1:32 was i n h i b i t e d by her own b a b y ' s serum as well as by pooled g a m m a globulin.

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Fig. 4. The low ( < 8 0 0 mg. per cent) IgG values in second samples collected from the infants of all 3 study groups are plotted with their corresponding first sample values. T h e arrows denote babies with rubella antibody in the first sample. There were 10 in this group.

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Volume 75

In[ants o[ rubella-exposed mothers

Number 6, part 2

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Mother did not receive ~, globulin - had overt rubella 16% of 48 patient sera positive Open symbol indicates positive Ripley and closed symbol positive latex titer

Fig. 6. Positive latex a n d R i p l e y a n t l - g a m m a globulin titers in m a t e r n a l sera f r o m the 3 s t u d y groups. T h e p e r c e n t a g e of positives was 14 to 16 per c e n t in e a c h group. All antibodies were I g M .

The anti-Ripley factor in the Group D mother was inhibited by pooled g a m m a globulin only. All a n t l - g a m m a globulin factors in maternal sera disappeared after incubation of the sera with 2-mercaptoethanol, indicating that they were all I g M antibodies. Seven latexpositive sera with high titers were separated on a 10 to 40 per cent sucrose gradient. In

I 19 1

each case the antibody was found only in the first or high molecular weight peak. Only one of the Ripley-positive sera had a high enough titer to be detected in a sucrose gradient experiment. This factor was also found in the first or high molecular weight peak. A latex factor was uncovered in the heavy peak after separation of this serum (from a woman in Group D) which could not be detected in whole serum. This was presumably due to binding of the a n t i - g a m m a globulin factor with the patient's own 7S g a m m a globulin in whole serum. Since we had no normal 5 to 12 month postpartum sera to compare with the maternal sera in the 3 study groups, we tested 49 sera from women 3 days to 6 weeks post partum using the latex test and found only 1 positive reaction. This resulted in an incidence of 2 per cent positives among the control subjects, as compared with 14 to 16 per cent positives in the study mothers. Effects of rubella. We were also interested in the effect of the disease on the immunoglobulin levels of these infants and so compared I g G and I g M concentrations in babies with and without rubella antibody persisting beyond 5 months of age. The antibody-positive group had significantly higher values for both IgG and I g M until 2 years of age, after which time the effect disappeared (Figs. 7 and 8). Quantitative IgA concentrations were not measured in the babies; however, 243 infants in the study were screened for IgA deficiency and all sera tested contained IgA. This was done because we had previously found the complete absence of IgA in a patient with dysgammaglobulinemia and congenital rubella.6, 7 Since the description of our original patient, a similar patient has been encountered. DISCUSSION

We believe that our data indicate that the large doses of g a m m a globulin given to mothers during the first trimester m a y have depressed the infant's own g a m m a globulin levels up to 1 year of age. This was not marked and did not appear to result in life-threaten-

I 19 2

Huntley et al.

The Journal o[ Pediatrics December 1969

SERUM IgG iN INFANTS WITH [ ] 2,0

AND WITHOUT [ ]

[ ] Pts. wlth rubella antibody persisting after 2 years of age (last column) p= ~ . 005

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Fig. 7. Serum IgG concentrations in infants with and without rubella antibody compared to control infants. Those with antibody have significantly higher values until age 2 years. The p values on the chart indicate differences between infants with and without antibody. The serum IgG concentrations were also significantly higher in babies with antibody than in control babies until age 2 years.

ing or increased n u m b e r s of infections. T h e e x p l a n a t i o n for this effect was not obvious since we found no 7S a n t i - g a m m a globulin factors in a n y of the m a t e r n a l sera tested. Specimens were obtained, however, m o n t h s following delivery. I n our study, the G m (a+) baby of the G m ( a - ) m o t h e r was not found to be at a greater disadvantage t h a n the G m ( a - ) baby, even w h e n the m o t h e r was given pooled g a m m a globulin early in pregnancy. I n t r a u t e r i n e rubella, as defined by the persistence of rubella antibody in the i n f a n t after 5 months of age, resulted in elevated serum I g G a n d I g M concentrations to 2 years of age in the group as a whole, although individual levels were low.

We are grateful to Doctors Eugene B. Linton, Frank R. Lock, and Robert S. Altman and to Mrs. Ruth Jarrett for generously sharing with us the serum specimens from their rubella study. REFERENCES 1. Ituntley, C. C.: Simple gel diffusion micromethod for gamma-globulin determination, Pediatrics 31: 123, 1963. 2. Huntley, C. C., and Lyerly, A.: Immune globulin determinations in allergic children, Am. J. Dis. Child. 106: 545, 1963. 3. Watson, R. G.: A more specific method for detecting and quantitating rheumatoid factors using a simple modification of the RA-test, Am. J. Clin. Path. 43: 152, 1965. 4. Huntley, C. C., Costas, M. C., Williams, R. C., Lyerly, A. D., and Watson, R. G.: Anti-yglobulin factors in visceral larva migrans, J. A. M. A. 197: 552, 1966. 5. Huntley, C. C., Lyerly, A. D., and Patterson,

Volume 75

In[ants o] rubella-exposed mothers

Number 6, part 2

SERUMIgMIN INFANTS WITH [ ]

AND WITHOUT [ ]

119 3

RUBELLA ANTIBODY PERSISTING AFTER5 MONTHS OF AGE

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[]Pts.

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Fig. 8. Serum IgM concentrations in infants with and without rubella antibody. Those with antibody have significantly higher values until age 2 years. In the follow-up samples (2 to 4 year age group) there was no significant difference between those whose first samples were negat{ve and those whose first samples were positive. However, there was a difference between the follow-up samples of the entire group of infants whose first samples were positive and the infants whose first and second samples were positive (last 2 bars).

IV[. V.: Isohemagglutinins in parasitic infections, J. A. M. A. In press. 6. Huntley, C. C., Hancock, M. P., and Sever, J. L.: Immunoglobulin disorder associated with congenital rubella, Program and Abstracts Society Pediat. Res., April 29-30, 1966, p. 142. 7. Hancock, M. P., Huntley, C. C., and Sever, J. L.: Congenital rubella syndrome with immunoglobulin disorder, J. PEDIAT. 72: 636, 1968. DISCUSSION DR. FRANK. How much gamma globulin did the mothers receive? DR. HUNTLEY. This was variable, usually 20 to 30 ml. DR. SEVER. Yes. DR. FRANK. Could you correlate the amount of g a m m a globulin with antibody response?

DR. HUNTLEY. I haven't tried to do that. DR. ALFORD. Did you screen for antibody at 5 months or did you do end-point titrations at the 5 month level? DR. SEVER. They were titered. DR. BUCKLEY. I would like to ask if Dr. tIuntley could correlate the presence of a positive rheumatoid factor in the mother with a low level of immunoglobulin in her infant? DR. ttUNTLEV. We didn't have enough values in any one age group to do a statistical analysis, but in the Group B mothers there appeared to be some correlation between rheumatoid factors in the mother and low gamma globulin levels in the baby. This was in the group of mothers who did not have overt rubella but did receive gamma globulin.