0031-3998/04/5606-0883
PEDIATRIC RESEARCH
Copyright © 2004 International Pediatric Research Foundation, Inc.

Vol. 56, No. 6, 2004
Printed in U.S.A.

Effect of Dietary Ribonucleotides on Infant
Immune Status. Part 1: Humoral Responses
JOSEPH P. SCHALLER, MATTHEW J. KUCHAN, DEBRA L. THOMAS,CHRISTOPHER T. CORDLE, TIMOTHY R. WINSHIP, RACHAEL H. BUCK,GERALDINE E. BAGGS, AND J. GARY WHEELER
Ross Products Division [J.P.S., M.J.K., D.L.T., C.T.C., T.R.W., R.H.B., G.E.B.], Abbott Laboratories,
Columbus, Ohio 43219, Department of Pediatrics [J.G.W.], University of Arkansas for Medical Sciences,
Little Rock, Arkansas 72204

ABSTRACTThe objective of this study was to further explore previously
identified effects of supplemental ribonucleotides on infant immune status as measured by antibody responses to routine infant
immunizations. Infants were randomized to a milk-based formulawith (FN, n ⫽ 138) or without (F, n ⫽ 147) 72 mg ribonucleotides/L. A cohort of human milk–fed (HMF, n ⫽ 192) infants
was also followed. Subjects were given Haemophilus influenzae
type b (Hib), diphtheria tetanus acellular pertussis, and oral
poliovirus vaccinations at 2, 4, and 6 mo of age, and specificantibody responses were assessed at 2, 6, 7, and 12 mo. Growthand safety data were also monitored. Using a two-group repeatedmeasures analysis (RMA), FN-fed infants had significantly
higher poliovirus type 1 neutralizing antibody (PV-VN1) responses than F-fed infants (p ⫽ 0.045). Using three-group RMA,
PV-VN1 responses in HMF infants were not different from
FN-fed infants, while HMF-fed infant PV-VN1 responses weresignificantly higher than F-fed infants at 6 (p ⫽ 0.0004) and 12
mo (p ⫽ 0.0001). FN-fed infants had responses to Hib Farr,
diphtheria, tetanus toxoid, oral poliovirus–specific IgA, and PVVN3 not significantly different from those of F and HMF infants.
Growth, gastrointestinal tolerance, and adverse events wereequivalent among the three groups. The FN-associated increase

Infants are at higher risk for morbidity and mortality resulting from infectious diseases than older children, due in part toimmaturity of the infant immune system (1,2). The mode of
feeding during infancy may influence the risk of infection asbreast-fed newborns have been reported to have a lower incidence of infections than formula-fed infants (2– 4). It has been
speculated in published reports that immune modulators inhuman milk could explain some of the protective effects ofhuman milk (3,5,6).

Received June 9, 2004;accepted June 30, 2004.
Correspondence: Joseph P. Schaller, Ph.D., Ross Products Division Abbott Laboratories, 3300 Stelzer Rd., Columbus, OH 43219; e-mail: joseph.schaller@abbott.com
Supported by grants from the Ross Products Division of Abbott Laboratories.
DOI: 10.1203/01.PDR.0000145576.42115.5C

in PV-VN1 response and nonstatistically significant trends toward increased Hib and diphtheria antibody responses wereconsistent with observations from earlier studies, indicating immune benefits of nucleotide supplementation of infant formula.
(Pediatr Res 56: 883–890, 2004)

Abbreviations
DTaP, diphtheria tetanus acellular pertussis vaccine
ExHMF, exclusive human milk-fed
F, formula without nucleotides
FN, formula with supplemented nucleotides
Hib PRP, Hib capsular polysaccharide (polyribosylribitol
phosphate)
Hib TITER, Haemophilus influenzae type b vaccine
HMF, human milk-fed
OPV, oral poliovirus vaccine
PV-VN1, poliovirus type 1 neutralizing antibody
PV-VN3, poliovirus type 3 neutralizing antibody
RCFD, reverse cumulative frequency distribution
RMA, repeated measures analysis
SWI, Similac威 with ironRibonucleotide 5'-monophosphates modulate cellular andhumoral immune function in animals as has been shown fromstudies in which nucleotides were added to a basal animal diet
low in nucleotides (7–11). Likewise, supplemental ribonucleotides increased immune responses in infants (12–16) and in
malnourished children (17). In four of these studies, antigenspecific antibody responses have been used as surrogate markers to assess the influence of ribonucleotides on immune status.
These data suggested that compositional changes to infant
formula might augment the infant’s humoral immune response.
Ribonucleotides are found in human milk (18,19) and are atmuch lower levels in cow’s milk. Cow’s milk– based infant
formulas had only trace amounts of ribonucleotides before
1996. Although most formula manufacturers now add ribonucleotides, the levels and ratios of added nucleotides vary. In

883

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SCHALLER ET AL.

earlier studies, although inherent levels in unsupplemented
formulas were not described, addition of relatively small
amounts of ribonucleotides (19.2 mg/L) were reported to beefficacious for gut health in high-risk infant populations (20),slightly higher levels (ⵑ30 mg/L) had transient effects onnatural killer cell activity (12), and no effect was observed on
antibody response or lymphocyte subsets (21). Higher levels ofribonucleotides (72 mg/L) patterned after the ratios in humanmilk have enhanced antibody response to some routine pediatric vaccines (14,15) and modified immune cell types (22) andsuggested reduced diarrheal illness in healthy Taiwanese infants (16). Thus, the benefits conferred appear to depend on
both concentration and ratio of dietary ribonucleotides, and the
population studied.
Antibody response to immunization requires synergistic activity of multiple immune components and is among the best
surrogate measures of the integrity and functional status of theimmune system. This double-blind, active treatment–
controlled, randomized trial of FN was designed to verify theeffect of supplemental ribonucleotides on vaccine responses, toexplore effects on the development of immune cells using flow
cytometric analysis, and to assess their effect on clinical morbidity. Separate reports will communicate the results of theseobjectives; the current article describes the vaccine response
data.
METHODS
Study design. This was a 12-mo, prospective, randomized,
double-blind, parallel, longitudinal study of healthy, term,
formula-fed infants carried out from fall 1996 to the spring of
1998. Infants, whose mothers had chosen to formula feed
before enrollment were enrolled before 8 d of age and randomized to the cow milk– based control formula (F) or the same
formula supplemented with ribonucleotide 5' monophosphates(72 mg/L) (FN, commercial SWI) ad libitum from enrollment
to 12 mo of age. Infants in the nonrandomized reference groupof subjects fed human milk (HMF) were enrolled concurrently
before 8 d of age, and fed human milk exclusively until at least
2 mo of age. After 2 mo, infants whose parent(s) chose to wean
were fed SWI not supplemented with ribonucleotides (SWI,
pre-1997 commercial SWI). Therefore, the HMF group consisted of a combination of infants exclusively fed human milk,
infants receiving a combination of human milk and SWI, andinfants weaned to SWI. All infants were fed their assigned
formula, or human milk (alone or with SWI), exclusively until
4 mo of age, after which other foods were permitted. The
immunization schedule was consistent with the recommendations of the American Academy of Pediatrics (23) and subsequent changes made to the schedule (24). Blood samples wereobtained at 2, 6, 7, and 12 mo of age.
Physician office visits occurred every 4 wk (⫾ 3 d) for theduration of the study. Anthropometric measurements weretaken at enrollment and at 1, 2, 4, 6, 7, and 12 mo of age as
described elsewhere (25). Tolerance responses to the formulas
were assessed using parent diaries for 3 d before the 1, 2, and4-mo visits. Serious adverse events were monitored throughout
the study.

Subjects. Eligible infants were recruited from local hospitals
and pediatric practices at 18 sites in 10 states (AR, CA, FL,
GA, IL, LA, NC, NY, OH, and PA). Subjects had a gestationalage of 37– 42 wk, weighed ⱖ2500 g at birth, and scored ⱖ7 on
a 5-min Apgar. Exclusions were made where there was evidence of significant organ system or metabolic disease andmaternal medical history known to have adverse effects on thefetus (diabetes, preeclampsia, HIV infection, or drug abuse).
Infants whose parent(s) had decided to formula feed were noteligible for the study if they had consumed colostrum or humanmilk. Infants that had been fed soy formula or milk-based
formula supplemented with ribonucleotides were not eligible
for the study. Informed consent was obtained from parent(s) or
guardian(s) of each subject before participation in the trial. The
consent process and clinical documentation for this study werereviewed and approved by the Internal Review Board at eachstudy site.
Formulas. F and FN were masked through the use of labels
differing only in a numeric code. All study formulas were in theready-to-feed form and did not require dilution. SWI was
comparable to F except for minor changes in fatty acid composition, and a different protein blend (Table 1). Formulas were
manufactured by Ross Products Division, Abbott Laboratories
and met or exceeded nutrient levels recommended by theCommittee on Nutrition of the American Academy of Pediatrics (26) and required by the Infant Formula Act of 1980 and
subsequent amendments.
Immunizations and blood collection schedule. Study sites
were instructed to administer each of the injected vaccines inseparate limbs according to manufacturer directions at 2, 4, and6 mo of age. To minimize interlot variation in the immunogenicity of the vaccines, single lots of Hib polysaccharideTable 1. Partial composition of study formulas (per liter)*Nutrient

F†,‡

FN†,‡

SWI†,§

Protein (g)Fat (g)
Linoleic acid (g)Carbohydrate (g)Iron (mg)Ribonucleotides㛳
Total (mg)
CMP (mg)
UMP (mg)
AMP (mg)
GMP (mg)

15.0 ⫾ 0.38
37.3 ⫾ 0.42
7.87 ⫾ 0.93
74.3 ⫾ 0.68
14.1 ⫾ 0.21

14.9 ⫾ 0.36
37.2 ⫾ 0.38
7.07 ⫾ 0.46
73.3 ⫾ 0.52
13.9 ⫾ 0.59

14.8
35.8
12.2
70.2
13.2

7.43 ⫾ 0.45
3.1 ⫾ 0.66
3.53 ⫾ 0.15
0.55 ⫾ 0.07
0.65 ⫾ 0.07

80.8 ⫾ 3.6
38.8 ⫾ 3.1
16.6 ⫾ 0.55
12.5 ⫾ 1.3
13.2 ⫾ .089

13.0
4.6
8.4
ND
ND

* Met or exceeded nutrient levels recommended by the Committee on
Nutrition of the American Academy of Pediatrics 26 and required by the Infant
Formula Act of 1980 and subsequent amendments. F, control; FN, ribonucleotide-supplemented; SWI, Similac with Iron; ND, not detected.
† Experimental batches were manufactured between 5/96 and 11/97. Two F
and 2 FN batches were derived from the same parent batch. Similac with Iron
was manufactured 2/97 and 3/97. Values represent mean ⫾ SD of three batches
except for AMP and GMP for F, which represents the mean ⫾ SD of two
batches.
‡ Protein, nonfat milk and whey protein; fat, high oleic safflower, soy andcoconut oils; carbohydrate, lactose.
§ SWI; protein, nonfat milk; fat, soy and coconut oils; carbohydrate, lactose.
㛳 Ribonucleotide content for the supplemented formulas is the sum ofinherent and supplemented sources. Values for Similac with Iron were notanalyzed, but are taken from analysis of representative batches.

RIBONUCLEOTIDES AND INFANT IMMUNE STATUS

recombinant diphtheria toxin (CRM) vaccine (HibTITER, Lederle-Praxis, Wayne, NJ) and diptheria-tetanus-acellular
pertussis (DTaP) vaccine (Tripedia, Connaught, Swiftwater,PA) were supplied to all sites. Two lots of oral trivalent polio
vaccine (OPV; Orimune, Lederle, Pearl River, NY) were used.
The first lot was used for the 2- and 4-mo immunizations, andthe second for the 6-mo immunization. Use of study vaccine
lots was verified using vaccination records. Administration ofother immunizations was at physician discretion and was not a
protocol deviation.
Blood samples were obtained at the 2, 6, 7 and 12 mo study
office visits to 1) establish baseline antibody levels before thefirst immunization, 2) capture peak antigen-specific antibody
response by measuring antibody titers before and 28 d after thethird immunization, and 3) provide an assessment of long-term
antibody responses by measuring antigen-specific titers at 12
mo of age.
Antibody and immunoglobulin determinations. Analysis ofsera for anti-Hib PRP antibody was done using a modified Farr
assay as described elsewhere (27). Analyses of sera for antidiphtheria toxoid IgG, anti-tetanus toxoid IgG, and poliovirusspecific IgA to monovalent polio vaccine MP12–97, type 1(Statens Seruminstitut, Copenhagen, Denmark) were done using enzyme-immune assay methods previously described (15).
PV-VN1 and PV-VN3 were measured as reported elsewhere(28) and are presented as the reciprocal of the highest serum
dilution that inhibited visible poliovirus-induced cytopathology. Total IgE was performed using DPC Immunlite Chemiluminescent Technology (Diamond Diagnostics, Holliston,MA) and cow milk–specific IgE were assessed using Immuno
CAP system fluorescence enzyme immunoassay (Kabi Pharmacia Diagnostics, Piscataway, NJ). Total serum IgG and IgA
were measured by radial immunodiffusion kits (The Binding
Site, San Diego, CA). Total IgG and IgA were then used tonormalize antigen-specific IgG and IgA antibody titers expressing these as specific activities.
Growth and safety. The number of visits to the physician
was analyzed as an exploratory outcome. Medical personnel
blinded to study products, used medical records to identify
newly diagnosed conditions. Routine well visits and follow-up
visits for previously diagnosed conditions were identified andexcluded from the analysis. Diagnoses were categorized as
otitis media, respiratory infection other than otitis media, noninfectious, and total. Visit diagnosis hierarchy was as follows:
any mention of otitis media (except chronic otitis media,
resolved otitis media, follow-up or recheck otitis media, etc.)
resulted in the visit being categorized under otitis media. Visitsin which both otitis media and another illness were diagnosed
were categorized as otitis media. Mention of upper or lower
respiratory tract infections, respiratory syncytial virus, cough,or congestion without otitis media resulted in classification as
respiratory tract infection. Physician visits were verified bymonitoring the review of each subject’s medical records during
routine monitoring at frequent and predetermined intervals.
Statistics. Subjects were randomized into formula groups tomaintain balance among groups within each site, using aschedule derived from a standardized SAS program (SASInstitute, Cary, NC), with permuted blocks of size 2. The

885

randomization was stratified by gender. Sample size was basedon the number of subjects required to detect differences in the
Hib-Farr response reported by Pickering et al. (14). A sample
size of 77 evaluable subjects per group in a two-group parallel
design with one-sided alternatives was expected to give an
80% power to detect at least a 0.4 SD difference between groupmeans for log-to-base10 transformed Hib-Farr values.
All outcomes were reported as intent-to-treat analyses except for antibody responses to OPV. PV-VN1, PV-VN3, andpoliovirus-specific IgA responses were reported on an evaluable subset excluding subjects that received injected inactivated poliovirus vaccine instead of the per protocol live oral
poliovirus immunization. Criteria for evaluability targeted responses to mucosal immunization (OPV) and were established
before unblinding data.
Hib-Farr responses were evaluated using one-tailed tests
comparing the FN group to the F group. All other comparisonswere two-tailed. Comparisons of the two formula groups were
specified as primary analyses in the protocol. Two-group statistical tests were considered significant at the 0.05 level.
Although it is not a truly randomized comparison group, theHMF group was compared with the formula-fed groups using
post hoc three-group comparisons. Group comparisons of the
Hib-Farr, anti-diphtheria IgG, anti-tetanus IgG, PV-VN1, PVVN3, and poliovirus-specific IgA were performed by fitting
RMA models using SAS PROC MIXED. Site was used as a
blocking factor and sites with low enrollment were combinedin these assessments. Vaccine response data were transformed
using base-10 logarithms before analyses. Undetectable levels
were replaced by half the assay threshold value. Adjustments
for multiple comparisons were made using Holm’s step-down
procedure (29). The smallest p value was tested at 0.05/3 ⫽0.0167, the intermediate p value at 0.05/2 ⫽ 0.025, and thelargest p value at 0.05.
Because of the large proportion of individuals who failed togenerate cow milk-protein-specific IgE (60 – 81%), one categorical variable (response/no response, ⬍0.10 KU/L) was created and Cochran-Mantel-Haenszel test was performed on thederived variables by using SAS PROC FREQ to compare thefeeding effect.
Growth and safety data, and the number of physician visitswith each illness category while on study feeding, were compared for the three groups using Poisson regression.
RESULTS
Subjects. A total of 477 infants (147 F, 138 FN, and 192
HMF) were enrolled, and 381 (115 F, 113 FN, and 153 HMF)
completed the 12-mo study. The most common reasons for
dropping from the study included intolerance (n ⫽ 23), missing
visits (n ⫽ 19), and removal by the investigator (n ⫽ 9).
Tolerance problems reported by parents included fussing, gas,diarrhea, and spit-up. Demographics did not significantly differ
between the formula-fed groups (Table 2), but in the HMF
group, maternal education level was higher and smoking lesscommon. There were no differences in maternal parity among
the three feeding groups. The proportion of HMF subjects
whose milk source was exclusively HM or HM in combination

886

SCHALLER ET AL.

Table 2. Demographic variables in the F, FN, and HMF groups*
F
Subjects (n) (%)
Male (n) (%)
Female (n) (%)
Ethnicity (n) (%)
Caucasian (%)
African-American (%)
Hispanic (%)
Asian (%)
Other (%)
Gestational age (wk)†
Weight (g) at 10 d of age (g)‡MaleFemaleMaternal education, n (%)
Some high school
High schoolSome college
College or more

FN

HMF

147
62
85

138
61
77

192
104
88

126
14
1
0
6
39

119
14
1
1
3
39

170
12
4
0
6
39

3528 ⫾ 66
3535 ⫾ 59

3561 ⫾ 67
3397 ⫾ 48

3625 ⫾ 51
3509 ⫾ 49

11 (8)
38 (27)49 (34)45 (31)9 (7)35 (26)53 (39)39 (28)5 (3)30 (16)64 (33)93 (48)* F, control; FN, ribonucleotide-supplemented; HMF, fed exclusively human milk for at least 2 mo followed by human milk or commercial Similac
with Iron at weaning.
† Values are mean.
‡ Values are mean ⫾ SEM.

with formula, respectively, was 54% and 73% at 3 mo, 35%
and 60% at 5 mo, 22% and 34% at 8 mo, and 15% and 30% at
12 mo.
Compliance. Compliance with the feeding regimen wasassessed using parent diaries of formula intake completed for3 d before specified visits, monthly interviews with the parents,
review of medical records, and formula disposition records.
Only four formula-fed subjects consumed a nonstudy formulafor more than a total of 7 d over the first 4 mo of exclusive
formula or HM feeding. Thirty-seven formula-fed infants were
given nonstudy formulas before 4 mo of age; volumes were
small in each case and only 4 infants were given formulas
potentially containing ribonucleotides that were consumed for
longer than 7 d. Fifty-two infants had nonformula foods (15had juice, 44 had cereal, 1 had meat, 7 had vegetables, 8 had
fruit) before the wk 16 visit, and no feeding group-related
statistical significance was associated with these feedings.
Mean ages at the blood collection time points were not different between groups and very few subjects were outside specified visit windows. Compliance with the vaccination protocol
was verified using subject vaccination records and was found
to exceed 93% of active subjects at all time points for HibTITER and Tripedia. Despite the use of inactivated poliovirus
vaccine in some subjects, compliance to the Orimune lots
provided exceeded 90% at all time points.
Serologic and immunoglobulin responses. From 2 to 6 moof age, mean serum PV-VN1, PV-VN3, and poliovirus-specificIgA responses increased by 12-, 43-, and 22-fold, respectively,
for all three feeding groups combined (Table 3). Poliovirusspecific antibody responses continued to increase at 7 mofollowed by a decline in PV-VN1 and PV-VN3 at 12 mo.
Poliovirus-specific IgA responses followed a similar pattern
but were little changed from 7 to12 mo of age.
Comparing differences between groups the PV-VN1 response of the FN group was significantly higher than that of theF group when analyzed by the per-protocol two-group RMA(FN ⬎ F, p ⫽ 0.045) (Table 3). When compared using a post
hoc three-group RMA, and after adjustment for multiple comparisons, a statistically significant difference was observed
between HMF and F at 6 and 12 mo (HMF ⬎ F, p ⱕ 0.0004),
whereas no statistically significant difference was seen between
HMF and FN. Because of a possible effect of weaning toformula without nucleotides, a post hoc repeated measures
analysis was also performed on 6-, 7-, and 12-mo data, comparing vaccine responses in exclusively HMF subjects (ExHMF) with F and FN. Results showed that ExHMF infants had
significantly higher PV-VN1 titers than F (p ⫽ 0.0105), FN
marginally higher than F (p ⫽ 0.0438), and FN not statisticallydifferent from ExHMF (p ⫽ 0.332). Reverse cumulative distribution curves (RCFD, data not shown) plotting the distribution of infants achieving each neutralizing antibody titer, supported these group differences. Comparison of the RCFD
curves revealed statistically significant distribution differences
only at 12-mo (HMF different from F, p ⫽ 0.023) using theKolmogorov-Smirnov test (30). Consistent with this observation, an additional post hoc analysis of PV-VN1 geometricmean titers by time point confirmed that differences detected
between FN and F using RMA analyses were driven primarily
by the difference at 12 mo (FN ⬎ F, p ⫽ 0.0086) (Fig. 1).
Formula had no consistent or significant effect on PV-VN3
responses and frequency distributions indicated no clear differences among feeding groups. Two-month RCFD curves
revealed that 50% of infants had 4-fold higher maternalderived neutralizing antibody levels against PV1 than against
PV3.
Poliovirus-specific IgA titers (Table 3) in FN-fed infants
were lower than those in the HMF group at 6 mo of age (p ⫽0.0001) but reached similar levels at 7 and 12 mo. Further,
PV-IgA levels in HMF infants were higher than F-fed infants
at both 6 (p ⫽ 0.0066) and 12 (0.0003) mo. Although no othersignificant differences were detected among feeding groups atany other time points, anti-poliovirus IgA titers for the FN
group were 24% and 32% higher than F levels at 7 and 12 mo.
Similar nonstatistically significant trends were also observed ina post hoc analysis of 12 mo data with ExHMF ⬎ F (p ⫽0.0330), and FN ⬎ F (p ⫽ 0.0552).
All three groups had strong (ⵑ10-fold) antibody responses
to Hib at 7 mo following the 6-mo immunizations. At 12 mo,
Hib antibody titers had dropped by approximately 4-fold tolevels still considered protective. Although the anti-Hib antibody titers of the F and FN groups were not significantly
different, the mean anti-Hib antibody titer for the FN group was
33% and 17% higher than the F group at 6 and 7 mo,respectively, and was similar to the HMF group (Table 3).
There were no clinically relevant differences detected in Hib
protection rate indicators (31) (anti-Hib antibody ⬎1.0 ␮g/mL
at 7 mo, F, 61%, FN, 63%, HMF, 61%; anti-Hib antibody⬎0.15 ␮g/mL at 12 mo, F, 78%, FN, 83%, HMF, 82%),
although a transient nucleotide-associated increase in Hib protection rate was noted at 6 mo.
Serum anti-diphtheria IgG titers followed a normal patternof response in each of the three feeding groups (Table 3).
Following the second booster immunization at 6 mo, serum

887

RIBONUCLEOTIDES AND INFANT IMMUNE STATUS
Table 3. Serum antibody responses to oral poliovirus, Hib, diphtheria, and tetanus vaccinations and cow milk protein*
Geometric Mean (95% CI) No. of Subjects
Immune Indicator
Anti-poliovirus VN1(neutralizing antibody titer)
F†
FN†
HMF
Anti-poliovirus VN3(neutralizing antibody titer)F
FN
HMF
Anti-poliovirus IgA(⫻ 106 PAEU)
F
FN
HMF
Anti-Hib Ig (␮g g/mL)
F
FN
HMF
Anti-diphtheria IgG (U/mL)
F
FN
HMF
Anti-Tetanus IgG (U/mL)
F
FN
HMF
Anti-cow milk IgE (kU/L)**
F
FN
HMF

2 Mo
(at 1st dose)

6 Mo(2 mo after 2nd dose)

7 Mo(1 mo after 3rd dose)

12 Mo(6 mo after 3rd dose)

28 (22–35) 97
31 (23– 41) 98
46 (37–58) 139

265 (179 –392) 102‡358 (248 –518) 92
646 (484 – 862) 136§

643 (463– 892) 99
834 (590 –1177) 88
886 (670 –1171) 133

367 (283– 476) 96‡
616 (449 – 845) 92
817 (629 –1061) 125§

11 (9 –14) 97
12 (10 –15) 98
17 (14 –21) 138

606 (444 – 827) 102
463 (315– 681) 92
623 (492–789) 136

985 (758 –1280) 99
858 (626 –1175) 88
744 (611–907) 133

501 (408 – 614) 96
550 (437– 694) 92
467 (377–578) 125

0.00 (0.00 – 0.00) 102
0.00 (0.00 – 0.00) 98
0.00 (0.00 – 0.00) 144

1.92 (1.59 –2.31) 102㥋
1.55 (1.28 –1.87) 95㥋
2.73 (2.35–3.17) 139¶

2.39 (2.03–2.83) 100
2.97 (2.43–3.62) 92
2.77 (2.36 –3.26) 136

2.02 (1.64 –2.49) 98㥋
2.66 (2.16 –3.27) 93
3.26 (2.73–3.89) 129¶0.15 (0.13– 0.19) 113
0.15 (0.12– 0.18) 113
0.14 (0.12– 0.17) 160

0.15 (0.13– 0.19) 111
0.20 (0.17– 0.25) 108
0.18 (0.16 – 0.22) 151

1.62 (1.19 –2.21) 107
1.89 (1.40 –2.55) 106
1.87 (1.43–2.44) 247

0.40 (0.31– 0.51) 106
0.41 (0.33– 0.52) 104
0.44 (0.36 – 0.53) 140

0.05 (0.04 – 0.07) 109
0.06 (0.05– 0.07) 110
0.07 (0.06 – 0.09) 156

0.12 (0.10 – 0.14) 107
0.15 (0.13– 0.18) 104
0.15 (0.12– 0.17) 149

0.68 (0.58 – 0.79) 106
0.77 0.65– 0.91) 101
0.76 (0.65– 0.88) 138

0.18 (0.15– 0.21) 103
0.19 (0.16 – 0.23) 103
0.19 (0.16 – 0.22) 138

0.29 (0.23– 0.35) 108
0.29 (0.23– 0.35) 109
0.35 (0.30 – 0.41) 157

0.27 (0.23– 0.31) 107
0.25 (0.21– 0.30) 104
0.28 (0.25– 0.31) 149

1.30 (1.13–1.48) 105
1.35 (1.15–1.57) 102
1.30 (1.13–1.50) 142

0.41 (0.35– 0.48) 102
0.38 (0.32– 0.45) 102
0.33 (0.25– 0.42) 138

0.13 (0.05– 4.03) 100
0.10 (0.05– 0.71) 103
0.06 (0.05– 0.39) 146

0.08 (0.05– 0.79) 103
0.08 (0.05– 0.49) 98
0.22 (0.05–17.30) 143

0.10 (0.05–2.73) 106
0.09 (0.05– 0.52) 94
0.23 (0.05–15.50) 132

0.14 (0.05–2.56) 99
0.11 (0.05– 0.83) 99
0.15 (0.05–5.64) 128* Significant differences among feeding groups are indicated by different symbols and bold numbers F, control; FN, ribonucleotide-supplemented; HMF, fed
exclusively human milk for at least 2 mo followed by human milk or commercial Similac with Iron at weaning. Repeated measures analyses were performedby two-group (†) or three-group (‡,§,㛳,¶) methods. Poliovirus data is from a prospectively identified evaluable subset that excluded subjects who received IPV
instead of the per protocol OPV (5 in F, 6 in FN, and 11 in HMF). OPV, oral poliovirus vaccine; Hib, Haemophilus influenzae type b polysaccharide; CI,
confidence interval; VN, virus neutralization; PAEU, anti-poliovirus specific immunoglobulin A enzyme immunoassay.
† Different by a per protocol two-group RMA on the evaluable subset (p ⫽ 0.045).
‡,§ Different by a post hoc three-group RMA on the evaluable data subset for 6, 7, and 12 mo group ⫻ time interaction p ⬍ 0.0285.
After adjustment for multiple comparisons, HMF ⬎ F at 6 mo (p ⫽ 0.0004) and 12 mo (p ⫽ 0.0001).
㛳¶ Different by a post hoc three-group RMA on the evaluable data subset for 6, 7, and 12 mo groups by time interaction (p ⫽ 0.0001). After adjustment for
multiple comparisons, HMF ⬎ FN at 6 mo (p ⫽ 0.0001), HMF ⬎ F at 6 mo (p ⫽ 0.0066), and HMF ⬎ F at 12 mo (p ⫽ 0.0003).
** Values are arithmetic mean (minimum, maximum) number of subjects.

anti-diphtheria responses increased approximately 5- to 6-fold
1 mo later. There were no significant differences detected
between formula-fed groups, and results were similar to thoseof the breast-fed group. However, as seen in the Hib-Farr
results, the anti-diphtheria antibody titer group means for theFN group trended nonsignificantly higher than the F group at 6
and 7 mo (Table 3, Fig. 1).
Tetanus toxoid–specific IgG responses were similar in all
feeding groups with a peak response at 7 mo followed by areturn to near-baseline levels by 12 mo of age (Table 3). Unlike
antibody responses to diphtheria toxoid, anti-tetanus responses
were slightly lower at 6 mo compared with baseline values at
2 mo. The three feeding groups had similar serum IgA and IgGlevels, except at the 2-mo time point when the HMF group had
higher IgG levels (data not shown). Formula and HMF groups
had similar plasma cow milk–specific IgE (Table 3) and total
IgE concentrations (data not shown). A small number of HMF

infants weaned from human milk appeared to have high outlier
values that drove trends for cow milk–specific IgE to be higher
in the HMF group.
Growth and safety. Formula-fed infants did not differ in any
growth measure from entry to 4 mo of age or at 12 mo of age.
Growth was comparable to that of the HMF group. Average
daily formula and energy intakes were similar for F and FN.
Formula tolerance including stool consistency and average
daily percent of feedings with spit up, were also similar for theformula groups. As expected, the HMF group experienced
more frequent yellow stools and less odoriferous stools (32).
The mean number of physician visits due to infectious or
noninfectious episodes (3.8 ⫾ 0.2 and 2.0 ⫾ 0.2, respectively)
was not different between HMF and formula groups. This was
also the case for the number of visits with a diagnosis of otitis
media (HMF, 1.7 ⫾ 0.2; FN, 1.6 ⫾ 0.2; F, 1.8 ⫾ 0.2),
respiratory tract infections independent of otitis media (HMF,888

SCHALLER ET AL.

Figure 1. Antigen-specific antibody responses at 6, 7, and 12 mo fromprevious study (14) (A) and current study (B) expressed as ratios (nucleotide
supplemented/nonsupplemented formulas). Statistically significant and marginal differences presenting the effect of nucleotide supplementation are shownfor Hib total antibody (Farr), Hib IgG (ELISA), diphtheria, and poliovirus
antibody responses. Statistically significant differences (*p ⬍ 0.05, two-groupcomparisons; **p ⫽ 0.045, two-group RMA). Reverse cumulative distributions were different at 12 mo (¶FN ⬎ FP ⫽ 0.023). Three-group per time point
analysis comparing F, FN, and HMF (†FN ⬎ F, nonstatistically significant
multiplicity adjusted trend p ⫽ 0.0086 at 12 mo). Three-group RMA comparing F, FN, and ExHMF (§, FN ⬎ F, nonstatistically significant trend p ⫽0.0438), (‡FN⬎F, nonstatistically significant trend p ⫽ 0.0552). In the study
by Pickering et al. (14), infants received two OPV immunizations (2 and 4 mo)
whereas infants in current study received three OPV immunizations (2, 4, and6 mo).

1.4 ⫾ 0.1; FN, 1.5 ⫾ 0.2; F, 1.4 ⫾ 0.1) and other infections.
No serious adverse events attributable to feeding groups wereobserved.
DISCUSSION
This study was designed to increase understanding of supplemental ribonucleotide effects on the immune status of infants. The working hypothesis was that ribonucleotide supplementation supports development of the infant immune system.
This hypothesis was based on infant feeding studies showing
ribonucleotide-associated increases in antibody responses(14,15) and observations from animal studies (7–11).
Based on established differences in immune development
between infants fed human milk and those fed formula (2), we
proposed that monitoring antibody responses to pediatric vaccines might reveal differences in selected immune parameters
in infants fed formulas differing in ribonucleotide concentra-tion. Antibody responses to vaccination are used to measureintegrated immune responses (33) and, in this study, were used
to reflect immune status during early development. Antibody
responses are end products of complex processes and interruption of any step can reduce the magnitude of response. Attribution of differences in specific antibody response to dietaryribonucleotides requires that the response not be due to differences in vaccine potency or the immunologic ability to respondto vaccine challenge. For these reasons single lots of vaccine
were selected and antibody responses were measured in infantsof similar ages.
Pickering et al. (14) showed that ribonucleotide fortification(72 mg/L) of cow milk– based infant formula elevated peak
antibody responses to Hib PRP and diphtheria toxoid, but not
to tetanus toxoid or oral poliovirus vaccine. In the current
study, primary analyses revealed trends for ribonucleotideassociated elevations of antibody to Hib PRP and diphtheria
toxoid that failed to reach statistical significance. Primary
analyses also revealed statistically significant ribonucleotideassociated increases in PV-VN1 response. Differences between
ribonucleotide effects on PV-VN1 and PV-VN3 responses may
reflect lower baseline PV-VN3 titers (maternal antibody) and amore rapid response after immunization with OPV. Changes tostandard vaccination practice dictated that two aspects of the
Pickering et al. (14) study could not be duplicated. Whereas theoriginal study used whole cell Bordetella pertussis vaccine(DTwP) and OPV immunization at 2 and 4 mo of age, thecurrent study used an acellular pertussis vaccine (DTaP) and an
additional OPV immunization at 6 mo of age. These changes in
immunization regimen may have contributed to vaccine response differences in results between Pickering et al. (14) andthe current study.
Use of acellular pertussis vaccines is associated with reduced, but protective, antibody responses to diphtheria toxoid,
tetanus toxoid, and the PRP antigen in HibTITER (34,35) whencompared with whole-cell pertussis vaccines. Acellular Tripedia vaccine contains less diphtheria toxoid, two isolated pertussis components in place of killed whole cell pertussis, andless alum adjuvant than the DTwP version of Tripedia used by
Pickering et al. (14). The antibody response to PRP is driven
by the immunogenicity of the carrier (CRM197, recombinantdiphtheria toxoid) (36). Less diphtheria toxoid may have led tolower Hib carrier immunogenicity resulting in lower Hib PRP
antibody responses. In addition, a general lowering of Hib
antibody titers after vaccination during the last decade has beenreported (37). Thus, a plausible explanation for the discordance
between the current Hib PRP and diphtheria toxoid antibody
responses and those previously reported is that the current
responses were limited by the intensity of the vaccine-specificresponse itself rather than the availability of ribonucleotides.
Antibody responses to Hib PRP, diphtheria toxoid, andtetanus toxoid were substantially reduced in the current study
compared with those reported earlier using similar study designs. Pickering et al. (14) and Ostrom et al. (15) reported
mean ribonucleotide supplemented peak anti-Hib PRP antibody responses of 7.24 ␮g/mL and 6.99 ␮g/mL, respectively,
compared with 1.89 ␮g/mL in the current study. Similar lower
titers were also observed in diphtheria and tetanus toxoid

RIBONUCLEOTIDES AND INFANT IMMUNE STATUS

antibody responses. Demographic descriptors and parental
smoking rates were similar in the two studies and there wassignificant geographic overlap in the participating sites. Unfortunately, Pickering et al. (14) did not determine maternal
education as a proxy for socioeconomic status. Populationdifferences are unlikely to have accounted for the statistical
discrepancies in the antibody observations made in this study
and Pickering et al. (14). Considering all available information,
it is likely that the known lower potency of DTaP comparedwith DTwP used by Pickering et al. (14) contributed to thedifferences observed in antibody response with supplemental
ribonucleotides. Despite these differences, the data are directionally consistent for Hib-Farr responses in both studies.
The current effect of ribonucleotides on PV-VN1 response isconsistent with, but not identical to, previous reports (14,15).
The primary two-group RMA evaluating change over time
revealed a ribonucleotide-mediated elevation in PV-VN1 response, and a post hoc three-group RMA revealed that the
HMF group had a significantly higher response than the F, but
not the FN group, suggesting that ribonucleotides modified the
PV-VN1 response to levels more like those of the HMF group.
Similarly, Ostrom et al. (15) reported that 12-mo PV-VN1
responses in infants fed ribonucleotide-supplemented soy formula were intermediate between those of HMF and control
groups. Perhaps because of the inherently high polymericribonucleotide levels in soy formulas, no significant differenceswere observed in antigen-specific vaccine responses between
study formulas. Consistent with the current data, Pickering etal. (14) observed higher 6-mo PV-VN1 titers in the HMF
group compared with both F and FN, and the same trend at 7
and 12 mo. The Pickering study infants received only 2- and4-mo immunizations with OPV, and is likely responsible forthe inability to detect a statistically significant difference between F and HMF at 7 and 12 mo. Here, RCFD curves and post
hoc per time point comparisons indicated that differences
between formula groups were driven primarily by higher
12-mo PV-VN1 titers in the FN group compared with the Fgroup. Nonsignificant trends at 6 and 7 mo, however, appeared
to contribute to the group differences as determined by RMA.
Trends showing a nucleotide associated effect on polio IgAtiters (polio type 1 ELISA) were also observed, the highest
ratio occurring at 12 mo, consistent with that of the PV-VN1
response. It is interesting that although ribonucleotides were
fed for the duration of the study, the most significant change invaccine response occurred at 12 mo. Current data suggest thatthe decline in PV-VN1 titers between 7 and 12 mo was slower
in FN and HMF groups than that of the F group.
Pickering et al. (14) and the present study have each identified statistically significant nucleotide-mediated improvement
in immune response to some vaccine antigens. Results from thetwo cow milk– based studies are summarized in Figure 1 asratios of antibody responses (FN/F) to the various vaccine
antigens used. Antibody response data from these studies show
statistically significant nucleotide associated differences for
Hib (Hib-Farr and Hib IgG ELISA) and diphtheria in study 1(14), poliovirus VN1 responses in study 2, and other trends inboth studies supportive of effects of nucleotides on immune
responses. Although the studies have not indicated statistically889

elevated antibody response(s) to the same vaccines, consistent
trends have been observed in cases when statistical significancewas not detected. When examined collectively, these study
results are consistent with increased immune responses in
infants fed ribonucleotide-supplemented formula (Fig. 1).
Further, no differences in the number of physician visits for
infection or other reasons were observed among the threegroups studied, suggesting equivalent safety outcomes. With
respect to otitis media, although there appear to be more
published studies reporting a protective role for breast-feeding(2), approximately one-third of such studies reported no such
effect (38). It is possible that study population or study design
differences might explain interstudy outcome differences. Differences between currently used infant formulas and those usedin past studies might also be partially responsible. Data presented here were collected only for the first year of life and it
is possible that observed nucleotide associated benefits (developmental pattern of cell mediated as well as modestly improved responses to some vaccines, closer to that of breast-fed
infants) could be apparent during the second year and thereafter. Finally, similar to results reported earlier (14), no differences in cow’s milk-specific or total IgE levels were observed
between breast-fed and formula-fed infants.
We have shown that infants fed ribonucleotide-supplemented
formula have improved humoral immune responses, and shifts in
T-cell populations indicative of improved immune cell maturation(22). Although the impact of ribonucleotides on infant health is
not firmly established at present, it is quite possible that these
improvements in immune function could influence morbidity
outcomes in infants to help close the morbidity gap identified
between breast- and formula-fed infants (2). No gap was identified
in the infant population studied in the current report. Had thepresent study been performed in a different infant population, it ispossible that nucleotide-supplemented formula might provide aclinical profile closer to that of the breast-fed infant relative tounsupplemented formula. In this regard, it is most interesting thatsignificant differences in key indicators of immune function (poliovirus VN1 responses and subjects of T and NK cells) wereidentified not only between formula groups but also more often
between breast-fed infants and those fed formula withoutribonucleotides.
The mechanism by which ribonucleotides enhance immune
status is unknown. The finding of enhanced T-cell immune
maturation reported in infants fed FN (22) complements vaccine antigen response data and may provide insight into a
possible functional role in immune function as evidenced by
the increase of some antibody responses. Ribonucleotides appear to play an important role in intestinal physiology. Dietaryribonucleotides are rapidly catabolized (39) in the intestinal
epithelium (40,41), are efficiently absorbed into enterocytes as
nucleosides and free bases (42), and were also shown to havediscernible effects on intestinal vascular blood flow (43). Immune response to OPV primarily linked with efficient functioning of gut-associated immune cell populations may havebeen influenced by the presence of dietary ribonucleotides.
This observation may explain the effect of FN on the PV-VN1
response, and, in this regard, modification of peripheral immune responses has also been reported (11,14). As others have

890

SCHALLER ET AL.

postulated, gut-associated immune cell function appears to bemodified by dietary ribonucleotides, leading to changes in
gut-associated and peripheral immune responses (44).
CONCLUSIONS
These and previous data indicate that supplemental ribonucleotides, at levels (72 mg/L) and ratios similar to those in
human milk, enhance immune status in formula-fed infants
when assessed by antigen-specific antibody response to immunization. This effect occurs without modification of the safety
profile associated with control formula. When considered together with data reported elsewhere (11,22) evidence indicatesthat supplemental ribonucleotides cause shifts in the development of T-cell populations, improves infant humoral immune
status, and may help provide some of the immunologic benefits
associated with breast-feeding.
Acknowledgments. The authors thank the following people
for technical support: Marc Masor, Ph.D., Ross Products Division, Abbott Laboratories; Howard Faden, M.D., Division of
Infectious Diseases, Children’s Hospital, Buffalo, NY; andDaniel M. Granoff, M.D., Children’s Hospital/Oakland Research Institute, Oakland, CA. We thank Pam Newton, R.N.,
Tim Murphy, MS, John Lasekan, Ph.D., Peter Hu, Ph.D., Amy
Bates, Joe Laco, Pete Yakimovich, and Melissa Nameth, all of
Ross Products Division, Abbott Laboratories, for technicalassistance. We also thank the following participant physicians:
Wilson Andrews, Mark Blatter, Iqbal Choudhry, David Ferrera, William Forti, Martin Greenberg, Terry Jefferson, Susan
Kerns, Jacob Lohr, Edgardo Malacaman, Michael Pichichero,
Bruce Pistorius, Robert Reis, Allan Robinson, Laurie Schnell,and Michael Smith.
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