Reactogenicity and immunogenicity of rotavirus WC3 vaccine in 5-12-month old infants

© INSTIrUTPASTEUR/ELsEVIER Paris 1989

Res. Virol. 1989, 140, 207-217

R E A C T O G E N I C I T Y A N D I M M U N O G E N I C I T Y OF ROTAVIRUS WC3 VACCINE IN 5-12-MONTH O L D INFANTS

A. Garbag-Chenon (1), J.-L. Fontaine (1), G. Lasfargues (t), H.F. Clark (2), j. Guyot 0), G. Le Mo~ng (l), L. Hessel (3) and F. Bricout (l) (1) Hdpital Trousseau, 26 av. Dr Netter, 75531 Paris Cedex 12 (2) Wistar Institute and Children's Hospital, 34th St & Civic Center Bid, Philadelphia, PA 19104 (USA), and (3) lnstitut M6rieux, 1541 av. Marcel Mdrieux, Marcy i'Etoile (France)

SUMMARY Rotavirus is a major cause of acute gastroenteritis in infants worldwide and there is need for an effective vaccine. Rotavirus Wistar calf 3 (WC3) is a strain of bovine origin attenuated by 12 passages in cell culture. A lyophilized candidate vaccine containing 1 × 107 P F U of WC3 has been developed. An oral dose was given to 25 French infants 5-12-months old (mean age 8.6 months). No diarrhoea was observed within 2 weeks after vaccination. Unexplained vomiting was reported once and isolated fever > 37.8°C was reported 3 times during the first week. One m o n t h later, a neutralizing antibody response to serotypes tested was shown in 88 °70 of cases, with heterotypic responses to h u m a n serotype 3 (SA11 strain) in 72 070 and to type 1 (WA strain) in 48 070.The percentage of i m m u n e response was similar whether the infant had antibody prior to immunization or not, but a booster effect was observed in children who had pre-immunization rotavirus antibodies. Considering these promising results, efficacy trials are in the planning in different parts of the world. KEY-WORDS: Rotavirus, Vaccine; I m m u n e response, Infants.

INTRODUCTION Rotaviruses (RV) are recognized as the leading cause of viral gastroenteritis (GE) in infants worldwide (Flores et al., 1986). RV diarrhoea is a major con-

Submitted March 26, 1988, accepted March 17, 1989.

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tributor to infant death in developing countries (Institute of Medicine 1986). Such infections also occur within the first 3 years of life in most infants in developed countries, mainly during the winter season (Kapikian et al., 1986). RV represents at least 1/3 of the enteropathogens isolated in GE in children in France, and, between October and March, up to 80 °70 in those under 24 months of age (Drucker et al., 1981 ; Heibert and CaiUet, 1984). Rotavirus infection (RVGE) is frequently associated with fever, vomiting and dehydration (Bricout, 1978). As the only treatment consists of rehydration, the development of an effective vaccine is considered a primary goal by the WHO Diarrheal Disease Control Program (Edelman, 1987) and the US Institute of Medicine, for both developing and developed countries (Institute of Medicine, 1985 ; 1986). Human RV do not grow easily in cell culture but antigenic cross-reactivity between animal and human RV strains suggests that readily cultivated animal RV may be used as vaccines (Kapikian et al., 1980). Three candidate vaccines have been developed. One from a bovine strain, Nebraska calf diarrhoea virus (NCDV), has shown a protective effect against clinical RVGE in Finnish infants (Vesikari et al., 1984; 1985). Unfortunately, these results were not confirmed in other efficacy trials conducted in developing qountries (Hanlon et aL, 1987; Senturia et al., 1987; Vesikari et al., 1987). The rhesus rotavirus (RRV) strain MMU-18006 is highly immunogenic but causes several side effects such as fever when given at high titres (Losonsky et al., 1986; Vesikari et aL, 1986). A 68 07o protective efficacy has been reported in Venezuelan infants (Flores et al., 1987) 1 to 10 months old but has not been confirmed elsewhere (Edelman, 1987). The bovine RV strain Wistar calf 3 (WC3) was isolated in 1981 from the stools of a newborn calf and adapted to cell culture at the Wistar Institute (Clark et al., 1986). Initial studies showed that no relevant general or local side effects occurred when WC3 was injected into adults or into children aged 5 months to 6 years (Clark et al., 1986). The strain elicited an immune response to both homologous WC3 strain and human serotypes 3 and 1. The response was good to type 3 and poor to type 1. Nevertheless, a 76 °7o protective efficacy against symptomatic RV type 1 infection and a 100 070efficacy against severe infection has been obtained in a placebo-controlled study carried out in Philadelphia (Clark et al., 1987). This strain has been adapted for production at Institut M&ieux (Lyon, France) where a lyophilized batch has been prepared. We report here safety and immunogenicity data after single administration of this candidate vaccine to French infants.

ELISA NCDV PAGE PBS PFU PRN

= enzyme-linked immunosorbent assay. = N e b r a s k a calf diarrhoea virus. = p olyacry lamide gel electrophoresis. = phosp hate-buffered saline. = p l a q u e - f o r m i n g unit. = plaque reduction neutralization.

RRV RV RVGE URI WC 3

= Rhesus RV. = rotavirus. = RV gastroenteritis. = upper respiratory tract infection. = Wi s t a r calf 3.

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MATERIALS AND METHODS

Subjects. Twenty-five healthy infants aged 5 to 12 months (mean age 8.6 months) were recruited after the parents and/or legal tutor had received proper information and had given written consent. To allow optimal surveillance during the study period, all the infants were housed in two nurseries in the Paris area. None of them were suffering from intestinal disease which could have interfered with the appreciation of the reactogenicity of the vaccine. Infants were not breastfed and no other vaccine was given one week before or after WC3 vaccine administration.

WC3 strain. WC3 virus was isolated in primary AGMK cells, passed 3 times in CV1, plaqued twice in MA104, and then grown 3 more times in CV1 to attain the 9th passage (Clark et al., 1986); the vaccine, lot S1764 from Merieux, underwent 6 CV1 passages, 2 in MA104, 1 in AGMK and 3 passages in tertiary monkey kidney. The vaccine was lyophilized with a mixture of lactose and saccharose. Each vial contained 107 PFU of WC3 virus.

Vaccine administration. Infants were given a bottle of formula half an hour before immunization. One dose of vaccine was diluted in 2 ml of purified water and given by dropper.

Clinical surveillance. For 7 days following vaccination, the general reaction, rectal temperatures and intestinal symptoms were recorded daily by a nurse. From day 8 to day 28, a clinical follow-up was performed and every episode of diarrhoea and/or other symptoms were recorded.

Serology. Two fingerstick blood samples were collected, one prior to vaccination and the second 4 weeks after. RV antibody titration was performed by both plaque reduction neutralization (PRN) (Offit et al., 1983) and ELISA (Yolken et al., 1977). The PRN test was set up to detect antibodies to WC3 strain, WA strain (human serotype 1) and SA11 strain (human serotype 3). Titres lower than 1/100 were considered negative. A positive antibody response was defined as a 3-fold increase in titre or, in seronegative individuals, as an increase from less than 1/50 to 1/125 or more. ELISA plates were coated with CsCl-purified WC3 antigen (0.5 ~tg of purified virus per well). Sera diluted in phosphate-buffered saline (PBS) were then added, and antibody attachment was detected by with peroxidase-labelled anti-human IgG. For each serum the test was performed in 3 wells, 2 wells coated with antigen and one not coated. The absorbance value at 492 nm was calculated as the difference between the mean absorbance value of reactive wells and the value of the control well. ELISA titres were determined by endpoint dilution as previously described (Vaur et aL, 1986). Briefly, reference sera obtained from Drs H.F. Clark and S. Plotkin were two-fold diluted from 1/400 to 1/12,800 and the absorbance value was deter-

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mined for each dilution. Ten reference sera known to be negative were included in the test and a mean curve was determined. This mean curve plus 4 standard deviations was arbitrarily defined as the reference curve of negative sera (fig. 1). Thus, the ELISA titre of a serum was taken to be the reciprocal of the endpoint dilution defined by the intersection between the curve of the tested serum and the reference curve of negative sera. The strong correlation between ELISA titres determined by endpoint dilution and the absorbance values obtained with a dilution of 1/400 allowed us to assay all the sera at this dilution. Titres lower than 1/400 were considered negative. A positive antibody response was defined as a 4-fold increase in antibody titre or, in seronegative subjects, as an increase from less than 1/400 to 1/800 or more.

Virus shedding. Stool samples were collected before vaccination, daily during the week following vaccination and, if diarrhoea occurred, during the 3 weeks that followed. RV was detected both by ELISA and by PAGE silver stain RNA technique (Dolan et al., 1985). ELISA plates were coated with hyperimmune goat anti-serum to bovine rotavirus. The faecal extracts were diluted 10 070 in PBS and 200-~d samples were added to each well. Rotavirus antigen was detected with a group-specific mouse monoclonal antibody and peroxidase-labelled anti-sheep IgG. For each test, purified rotavirus WC3 (undiluted, and at 10-1, 10-2 and 10-3 dilution), human rotavirus strain 403 (at 10-1, 10-2 and 10 -3 dilution) and a negative sample (faeces considered free of rotavirus by ELISA test and electron microscope examination) were used as controls. A rapid technique was used to extract RNA. Briefly, 0.2 g of faecal sample was diluted in 0.4 ml EDTA and extracted once with fluorocarbon. RNA was extracted once with phenol/chloroform/isoamyl alcohol, precipitated with ethanol and resuspended in 20 ~1 of sample buffer. Polyacrylamide gel slabs (10 °70)were prepared and run by the method described by Laemli. Within each gel, the WC3 strain RNA was used as a control. Slab gels were stained with silver nitrate.

RESULTS Reactogenicity. Diarrhoea was not observed during the 2 weeks following vaccination. Three diarrhoeal episodes o f m o d e r a t e intensity occurred during the third week, associated in 2 cases with an upper respiratory tract infection (URI). No RV was isolated f r o m the stool samples. No episode o f R V G E occurred in the other children in the nurseries not participating in the trial. Two vaccinated infants v o m i t e d once during the first week : one h a d s y m p t o m s o f U R I , the other h a d no general or local symptoms. In 4 infants, the rectal t e m p e r a t u r e exceeded 37.8°C, with a m a x i m u m o f 38.6°C in a child with U R I s y m p t o m s . Fever was isolated and did not exceed 38.2°C in the other infants.

Virus shedding. WC3 virus was isolated in the stools o f one i n f a n t , using the 2 tests, between the third and sixth day after vaccination. In another infant who presented

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vomiting and URI but not diarrhoea, a wild h u m a n RV, as indicated by electrophoretype, was found from day 3 to day 7 (and not in the pre-immunization sample).

Seroresponse.

Four weeks after immunization, the geometric mean of RV antibody titres increased significantly in both PRN and ELISA assays. P R N tests showed significant increases in geometric mean titres to the homologous vaccine strain and to the type 3 strain, but not to the type 1 strain (table I).

TABLE I. - - Geometric mean o f RV antibody titres o f 25 infants 5-12 months o f age measured before (Pre) and 1 m o n t h after (Post) 1 dose o f W C 3 candidate vaccine.

Neutralizing antibody titres WC3 Pre (95 °70 conf. limits) Post (95 07o conf. limits) Post/Pre t p

108 (66.4-175) 628 (388-1017) 5.8 5.3 < 0.001

SA 11

WA

ELISA

142 (85-237)

190(106-341)

814 (371-1789)

662 (385-1140)

385 (204-727)

6103 (2869-12982)

4.7 4.3 < 0.001

2 1.69 NS

7.5 3.81 < 0.001

NS = not significant.

Before immunization (table II), 80 °70 of the infants had RV antibodies, detected by PRN, directed equally against human serotypes 1 and 3 (56 070 each), and only 40 070 by ELISA. Four weeks after 1 dose of WC3 vaccine, a seroresponse was observed in 88 070 of infants regardless of serotype. A group of 20 infants (80 070) demonstrated a response to the vaccine strain. A heterologous response to h u m a n serotype 3 was found in 18 children (72 070), and to type 1 in 12 (48 070). By ELISA, 21 infants had an antibody titre t> 1/400 after immunization (84 %), 15 (60 070)of whom had at least a fourfold increase in their initial titre. The influence of pre-immunization antibody titres is summarized in table III. The percentage of infants developing an immune response was the same whether the subject had previous rotavirus antibodies or not, and this was observed for both homotypic and heterotypic antigens. A primary im-

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ET AL.

O.D 1.8-

1.7 16 1.5 1.4

1.2 11

1.C

.7. .6. ,5. .4.

.3 .2 .1 I

400

800

I

1600

I

I

3200

6400

I

12800

I

25600

Reciprocal of serum dllutlon

FIG. 1. - - Reference curve for determination of ELISA R V antibody titres. Endpoint dilution ELISA for RV antibodies. Results of 4 positive sera are shown. The lower boundary of the hatched area represents the mean curve for negative sera, whereas the upper boundary is the. reference curve for negative sera (mean curve plus 4 SD). ELISA titres (arrows) are the reciprocal of endpoint dilution defined by the intersection between the curve of the tested serum and the reference curve of negative sera: • = 1/25,600; ~ = 1/12,800; • = 1/6,400; A = 1/3,200.

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TABLE II. - - RV antibody status before (Pre) and after (Post) 1 dose of WC3 vaccine of 25 infants 5 to 12 months of age.

Nb (07o) with neutralizing antibody titres

Pre (*) Post (*) Seroresponse (**)

WC3

SA11

WA

Any

ELISA

9 (36) 23 (92) 20 (80)

14 (56) 22 (88) 18 (72)

14 (56) 17 (68) 12 (48)

20 (80) 25 (100) 22 (88)

10 (40) 21 (84) 15 (60)

(*) Neutralizing antibody I> 1/100; ELISA antibody titres /> 1/400. (**) t> 3-fold increase in P R N ; /> 4-fold increase in ELISA.

m u n e response was clearly d e m o n s t r a t e d as the five triple negative children all developed antibodies. In t w o children, antibodies were only directed to W C 3 ; in 2 others, to b o t h W C 3 and SA11, and the last one r e s p o n d e d to all 3 strains. These children were also negative b y E L I S A b e f o r e vaccination and an i m m u n e response was f o u n d in 4 o f them afterwards. A b o o s t e r effect was also observed, indicated b y the rise o f geometric m e a n titres in previously seropositive infants (table III).

TABLE III. - - Antibody response to WC3 vaccine by pre-vaccine antibody status in 25 children 5-12 months old.

Antibody status WC3

Pre + + +

9 16 14 11 14

-

11

+ + -

20 5 10 15

-

SAll WA Any SN antibody ELISA

Nb (070) with antibody response to : the same any serotype serotype 7 13 10 8 8

G M T = geometric mean titre. SN = seroneutralizing.

(78) (81) (71) (73) (57)

4 (36)

8 14 12 10 11

(89) (88) (86) (91) (79)

11 (100)

17 5 5 10

(85) (100) (50) (67)

GMT Pre

GMT Post

381 53 311 52 523 52 --9152 209

1347 409 1170 320 854 140 --11942 3901

GMT Post/Pre 3.5 7.7 3.8 6.2 1.6 2.7

1.3 18.7

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DISCUSSION

These data confirm (Clark et al., 1986) that 107 PFU of WC3 candidate vaccine is safe and does not cause diarrhoea; other symptoms were mild and generally associated with concomitant URI. Shedding of the virus was found only in one child. In Clark's study, though none of 22 infants shed virus at a concentration detectable by PAGE, the WC3 strain was isolated by plaque assay 7 times (32 %). Although more frequently found with RRV candidate vaccine, the virus titres were usually low. In the trials recently reported by Wright et al. (1987), the vaccine strain was recovered from stools of 7 1 % of vaccinees by tissue culture, but no specimens were positive with the " R o t a z y m e " test. However, it has not been demonstrated that virus shedding is indicative of the efficacy of RV vaccine. WC3 appears to be highly immunogenic. Beyond the 80 % rate of homologous seroresponse, the importance of cross-reactivity must be underlined. As serotypes 3 and 1 represent the main causes of RVGE, the ability of WC3 to produce such a heterotypic response i~ very promising (Edelman, 1987). These results are more favourable than those reported in Philadelphia (Clark et al., 1986) but one must consider the higher percentage of RV-positive individuals before vaccination, perhaps related to the conditions of life in the nursery (Garbag-Chenon et al., 1983). Nevertheless, analysis according to the pre-immunization antibody titres (table III) revealed no differences in immune responses between seronegatives and seropositives with respect to either homologous or heterologous strains. However, the overall booster effect of the WC3 strain is of interest. As young infants seem to elicit a weaker immune response (Clark et al., 1986), it is clear that multi-dose administration has to be tested (Edelman, 1987). A placebo-controlled study of WC3 candidate vaccine given in a 3-dose schedule to infants 2 to 6 months of age is in progress. Lastly, the ELISA antibody test is less sensitive than the P R N assays but the correlation seems good and all children who responded by ELISA were also found to respond by P R N assay. Even though these results are encouraging, many problems remain before a vaccine using WC3 strain as the definitive RV vaccine is implemented. Is serum immune response indicative of RVGE protection ? This has been shown in experimental studies (Offit and Clark, 1985) and recently in humans. The study reported by Chiba et al. (1986), suggested that natural protection against RVGE was related to homotypic circulating antibody titres. However, local immunity or cell-mediated immune responses are probably involved in protection against RVGE but are not easy to assess (Edelman, 1987). Other points need to be clarified, such as the role of naturally attenuated human " n u r s e r y " strains (Flores et al., 1986) involved in asymptomatic infections of newborn (Perez-Schael et aL, 1984), the influence of maternal pro-

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tection (Berger et al., 1984) or breast-feeding and possible interference between the vaccine and other enteric viruses (Clark et al., 1986) or oral polio vaccine ( V o d o p i j a et al., 1986). Thus, assessing the protective effect o f W C 3 strain against s y m p t o m a t i c R V infections under different conditions is p r o b a b l y the best practical w a y to obtain some answers. Several field trials are being u n d e r t a k e n in different epidemiologic contexts b o t h in developing and industrialized countries.

RI~SUMI~ TOLI~RANCEET IMMUNOGI~NICITI~DU CANDIDATVACCINROTAVIRUS WC3 VIVANTATTI~NUIP.CHEZ DES ENFANTS,~GI~SDE 5 A 12 MOIS

WC3 est un rotavirus bovin att6nu6 par 12 passages sur diff6rents milieux cellulaires. Une forme lyophilis6e titrant 10 7 PFU/dose a 6t6 mise au point et administr6e par voie orale en 1 dose chez 25 enfants de 5 h 12 mois (age moyen: 8,6 mois). Aucune r6action diarrh6ique n'a 6t6 observ6e pendant les 2 semaines suivant la prise vaccinale. Un vomissement inexpliqu6 et 3 616vations isol6es de la temp6rature rectale comprise entre 37,8°C et 38,2°C ont 6t6 not6es pendant la premi6re semaine. Un mois apr~s l'administration, 88 °70 des enfants ont d6velopp6 une r6ponse immunitaire s&ologique, homologue pour la souche vaccinale dans 80 07o des cas, crois6e avec la souche SA11 (s6rotype 3) dans 72 07odes cas et avec la souche WA (s6rotype 1) dans 48 07o des cas. Le taux de r6ponses immunitaires 6tait identique, que l'enfant soit ou non positif ant&ieurement. Devant ces r6sultats encourageants, plusieurs essais de protection contre les infections symptomatiques ~ rotavirus sont envisag6s. MOTS-CLI~S; Rotavirus, Vaccin WC3; R6ponse immunitaire, Nourrissons.

ACKNOWLEDGEMENTS

The authors would like to thank Drs S.A. Plotkin and M. Cadoz for review of the text; F. Boutitie for technical and statistical assistance; and M.-A. Guinet for assistance in preparation of the manuscript. REFERENCES BERGER, R., HADZISELIMOVIC,F., JUST, M. et al. (1984), Influence of breast milk on

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