Effect of Dosage on Immunogenicity of a Vi Conjugate Vaccine Injected Twice into 2- to 5-Year-Old Vietnamese Children

In a double-blind, randomized, and placebo-controlled previoustrial, the efficacy of Vi-rEPA for typhoid fever in 2- to 5-year-oldswas 89.0% for 46 months. Vi-rEPA contained 25 µg of Viand induced a greater-than-eightfold rise in immunoglobulinG (IgG) anti-Vi in all of the vaccinees tested. In this investigation,we conducted a dosage-immunogenicity study of 5, 12.5, and 25µg of Vi-rEPA in this age group. Two doses of Vi-rEPAwere injected 6 weeks apart. Blood samples were taken beforeand at 10 weeks (4 weeks after the second injection) and 1 yearlater. All postimmunization geometric mean (GM) levels werehigher than the preimmune levels (P < 0.0001). At 10 weeks,the GM IgG anti-Vi level elicited by 25 µg (102 EU/ml)was higher than those elicited by 12.5 µg (74.7 EU/ml)and 5 µg (43 EU/ml) (P < 0.004): all of the childrenhad $≥$ 3.52 EU/ml (estimated minimum protective level). One yearlater, the levels declined about sevenfold (13.3 and 11.3 versus6.43 EU/ml, P < 0.0001) but remained significantly higherthan the preimmune levels (P < 0.0001), and >96% of thechildren had a greater-than-eightfold rise. This study alsoconfirmed the safety and consistent immunogenicity of the fourlots of Vi-rEPA used in this and previous trials.

INTRODUCTION

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Introduction

The Vi capsular polysaccharide of Salmonella enterica serovarTyphi is both an essential virulence factor and a protectiveantigen (1, 7, 11). To improve its immunogenicity in children<5 years of age, a conjugate composed of the Vi capsularpolysaccharide bound to recombinant mutant Pseudomonas aeruginosaexoprotein A (Vi-rEPA) was synthesized (8, 13, 14). A doubleblind, randomized, and placebo-controlled trial with 2- to 5-year-oldVietnamese children showed Vi-rEPA to be safe and 89.0% effectivein preventing typhoid fever for 46 months (9, 10). Among 76children selected randomly from the phase 3 trial, Vi-rEPA induceda greater-than-eightfold rise in immunoglobulin G (IgG) anti-Viin all vaccinees tested. The conjugate contained $~$ 25 µgof Vi as Vi-rEPA. Because dosage-related immunogenicity hasbeen shown for other conjugates such as the polysaccharidesof Haemophilus influenzae type b and pneumococcus types, inthis study the safety and immunogenicity of various dosages(5, 12.5, and 25 µg) of Vi as Vi-rEPA were evaluated in2- to 5-year-old children (2, 3, 5, 6, 12).

MATERIALS AND METHODS

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Materials and Methods

Study design. Two hundred forty-one children, 2 to 5 years old and evenlydistributed by age and sex, were recruited from Thanh Ba District,Phu Tho Province, Vietnam. Half of the children were recruitedfrom day care centers, and the other half were from the communityof three communes. At the time of their recruitment and afterinformed consent was obtained from their parents or guardians,the children received identification numbers and were randomlyassigned to three dosage groups (25, 12.5, and 5 µg ofVi as Vi-rEPA). Two injections of the same dosage were administered6 weeks apart. Blood samples were collected before the firstinjection, 4 weeks after the second injection, and 1 year afterthe first injection.

Excluded were children with an illness requiring ongoing medicalcare, those who were immunocompromised, and those with a historyof severe vaccine-associated reactions.

Before being injected, the children were examined by a physicianand their axillary temperature was measured with a digital thermometer.Those with a temperature of $≤$ 37.5°C received an injectionin the deltoid muscle, and the vaccine code was recorded. Thechildren were observed at 30 min by the vaccination teams andat 6, 24, and 48 h by a community health worker who recordedthe axillary temperature and reactions at the injection site.

Vaccine. Vi-rEPA (lot 102970) and diluent (0.2 M NaCl, 0.01% thimerosal,and 10 mM sodium phosphate buffer at pH 7.0) were prepared bythe National Institute of Child Health and Human Development(NICHD), National Institutes of Health (NIH), and bottled bythe Pharmaceutical Development Section, Pharmacy Department,Clinical Center, NIH. Vi-rEPA, at 50 µg of Vi/ml, 2.9ml/vial, met the requirements of the U.S. Code for Federal Regulations610.

To maintain the 0.5-ml volume of conjugate for all injections,the 12.5- and 5-µg doses were prepared each day at thestudy site by dilution of the vials into color-coded vials andkept in cold boxes. Partially used vials at the end of eachday were returned to the Typhoid Vaccine Study laboratory atPhu Tho Province and returned to the NICHD for analysis.

Serum IgG anti-Vi. Blood samples (2.0 ml) were collected by venipuncture, deliveredinto 2-ml centrifuge tubes, stored in a cold box until broughtto the Typhoid Vaccine Study laboratory, and centrifuged, andthe sera were collected. The sera were stored at –70°Cand shipped in dry ice to the NICHD for blinded IgG anti-Vititration by enzyme-linked immunosorbent assay (ELISA) (8).Results are expressed as the geometric mean (GM) and 25th and75th percentiles (10). Comparisons of GMs were performed withthe unpaired or paired t test.

This investigation was approved by the Institutional ReviewBoard of the NICHD (OH98-CH-N002), NIH; the Center for BiologicsEvaluation and Research, FDA (BB IND 6990); and the NationalInstitutes of Hygiene and Epidemiology (NIHE) of the Ministryof Health, Vietnam.

RESULTS

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Results

Adverse reactions. There were no serious adverse reactions. Table 1 reports thetemperatures of the vaccinees after the two injections. Elevatedtemperatures were infrequent, mild, and resolved within 24 h.After the first injection, a recipient of the 12.5-µgdose had a temperature of 39.0°C at 24 h. After the secondinjection, a recipient of the 5-µg dose had a temperatureof 39.0°C.

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TABLE 1. Axillary temperatures after injection of 2- to 5-year-old Vietnamese children injected with 5, 12.5, or 25 µg of Vi as Vi-rEPA twice 6 weeks apart

None of the vaccinees had swelling or erythema at the injectionsite after the first injection. After the second injection,one recipient of the 25-µg dose had a 6-cm swelling for24 h, 15 had erythema <1 cm in diameter at 6, 12, or 24 h.No local reactions were detected at 48 h.

After the second injection, three vaccinees complained of mildheadache and one complained of nausea at 6 h: both of thesesymptoms subsided within 24 h.

Serum IgG anti-Vi. Table 2 lists the GM and 25th to 75th percentiles prior to thefirst injection, 4 weeks after the second injection, and 1 yearafter the first injection. One participant in each dosage groupwas lost to follow-up at 1 year. The antibody data are clear:there was a dose-related response, with the highest IgG anti-Vilevels elicited by the 25-µg dosage and the lowest elicitedby the 5-µg dosage, although this difference was not statisticallysignificant for the 25-µg dosage versus the 12.5-µgdosage at 1 year.

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TABLE 2. Serum IgG Vi antibodies elicited in 2- to 5-year-old Vietnamese children injected with Vi-rEPA^a

Four weeks after the second injection, all of the vaccineeshad a $≥$ 13-fold rise in IgG anti-Vi (P < 0.0001). The 25-µgdosage of Vi-rEPA elicited the highest level (102 EU/ml), the12.5-µg dose elicited a level of 74.7 EU/ml, and the 5-µgdosage elicited a level of 43.0 EU/ml. The differences betweenall of these values are significant (P < 0.004). All recipientshad $≥$ 3.52 EU of IgG anti-Vi/ml, the estimated minimal protectivelevel based on the efficacy trial (9).

The GM IgG anti-Vi levels declined at similar rates in all threegroups during the first year: 6.7-fold in the 5-µg doserecipients (43.0 to 6.43 EU/ml), 6.6-fold in the 12.5-µgdose recipients (74.7 to 11.3 EU/ml), and 7.7-fold in the 25-µgdose recipients (102 to 13.3 EU/ml). At 1 year, 17 (23%) ofthe 75 5-µg dose recipients, 4 (5%) of the 79 12.5-µgdose recipients, and 4 (5%) of the 77 25-µg dose recipientshad <3.52 EU of IgG anti-Vi/ml, the estimated minimal protectivelevel (9).

DISCUSSION

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Discussion

As observed in three earlier trials with three separate lots,Vi-rEPA was safe and highly immunogenic (8-10, 14). Only 13(2.7%) of 479 injections elicited a mild fever, which lastedno more than 24 h. Local reactions were similarly infrequentand mild.

It is our limited experience that larger polysaccharide dosesof conjugates elicit antibodies of longer duration (4). We recommendthe 25-µg dose of Vi as Vi-rEPA for 2- to 5-year-old childrenbecause this is the dose that gives the strongest antibody responseand conferred protection for at least 4 years (9). Larger doses(>25 µg), although potentially more immunogenic, werenot evaluated here.

In children <2 years of age, conjugates of H. influenzaetype b and pneumococcus types had optimal immunogenicity ata dose of $~$ 5 µg of polysaccharide (2, 4, 6). Because at1 year in both the 12.5- and 25-µg dosage groups the GMIgG anti-Vi levels were not significantly different and 95%of the vaccinees had IgG anti-Vi levels considered to be protective,we plan to evaluate both doses of Vi-rEPA injected concurrentlywith diphtheria-pertussis-tetanus vaccine in infants for optimalimmunogenicity, as well as the duration of IgG anti-Vi.

ACKNOWLEDGMENTS

We are grateful to Jeanne Kaufmann and Loc Trinh, who contributedto the preparation of Vi-rEPA and to translation of the clinicalprotocol, and to Tran Thi Kim Chi, Aventis Pasteur, Vietnam,who prepared dilutions of Vi-rEPA for the second injection.Except for Marie-Claude Bonnet and Dominique Schulz, we do nothave commercial or other associations that might pose a conflictof interest.

FOOTNOTES

* Corresponding author. Mailing address: National Institutes of Health, Building 31, Room 2A29, Bethesda, MD 20892-2324. Phone: (301) 496-4524. Fax: (301) 402-9108. E-mail: szus{at}mail.nih.gov.

This article is dedicated with affection and admiration to thelate Dang Duc Trach, Chairman of the Vietnam General Associationof Medicine and Pharmacy and Director of the Extended Programon Immunization, Vietnam.

Editor: D. L. Burns

REFERENCES

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