271
Simultaneous Administration of Two Human-Rhesus Rotavirus Reassortant Strains of VP7 Serotype 1 and 2 Specificity to Infants and Young Children Peter F. Wright, James King,* Kazuko Araki,* Yasuo Kondo,* Juliette Thompson, Sharon J. Tollefson, Masaaki Kobayashi, and Albert Z. Kapikian
Department of Pediatrics, Vanderbilt University, Nashville, Tennessee, and Laboratory ofInfectious Diseases, National Institute ofAllergy and Infectious Diseases, National Institutes ofHealth, Bethesda, Maryland
Rotaviruses are recognized as the primary etiologic agents in infantile diarrheal illness leading to serious dehydration and death throughout the world [1]. As diarrheal illness in turn represents one of the leading causes of childhood morbidity and mortality in developing countries, rotavirus has been given the highest priority for vaccine development [2]. The initial approach taken was to develop live, attenuated, orally administered vaccines. Rotaviruses ofanimal origin, a Jennerian approach, have the advantages of host range attenuation and better tissue culture growth than the human strains [3]. For example, rhesus rotavirus strain MMU18006, isolated from a young rhesus monkey, and three additional human-rhesus rotavirus reassortants, each containing a single gene encoding VP7, a major outer capsid protein, derived from representative human rotavirus serotypes, are being evaluated as potential vaccine candidates in children. Given as single strains, the original rhesus strain, RRV, and its reassortants possess many of the attributes sought in a vaccine [4-6]. However, as four recognized human rotavirus
Received 15 November 1990; revised 5 April 1991. Presented in part: American Society of Virology, Salt Lake City, July 1990. Informed consent was obtained from parents and protocols were reviewed and approved by the Vanderbilt Committee for the Protection of Human Subjects. Financial support: contract AI-05050 (National Institutes of Health) and RR-00095 (NIH to Vanderbilt Clinical Research Center). Correspondence: Dr. Peter Wright, Department of Pediatrics. Vanderbilt University, Nashville. TN 37232. * Present addresses: Department of Pediatrics, University of Maryland. Baltimore (J.K.); Department of Pediatrics. Teikyo University, Tokyo, Japan (K.A. and Y.K.). The Journal of Infectious Diseases 1991;164:271-6 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6402-0006$01.00
group A strains are epidemiologically important, there may be need for a multivalent vaccine as well as administration of multiple doses. As a step towards the evaluation of a multivalent vaccine, studies ofthe simultaneous administration of reassortants with the VP7 of human rotavirus serotype 1 (RRVxD) or 2 (RRVxDS-l) and the remaining 10 genes from the parental rhesus strain were undertaken to assess the capability of infants and young children to allow replication and to respond immunologically to each component of the vaccine. Simultaneous oral administration of bivalent live attenuated rotavirus vaccine was evaluated with two different input ratios of the strains. The clinical, viral, and serologic approach to their evaluation did not vary significantly from previously described studies of monovalent serotype 3 (RRV) [5] and serotype 1 (RRVxD) [6] except for the necessity to identify the serotype(s) of virus recovered in the stool specimens. This was accomplished using RNA gels that differentiated the genes coding VP7 from the human strains D and DS-l.
Methods Clinical evaluation. Clinical assessment of 56 infants and young children ages 4-70 months was carried out in a day care facility in the Clinical Research Center, Vanderbilt University, between May 1987 and October 1989. There were 37 children 4-23 months of age and 19children >2 years of age in the trials. None was concurrently being breast-fed. Ten groups of3-9 children at a time were observed daily for at least 6 hours a day. For 12 consecutive days a physical examination was done by a member of the vaccine evaluation team. Efforts were made to obtain a daily stool sample. Each child returned home in the evening, where a continued record of fever, gastrointestinal symptoms, and number of stools was kept.
Downloaded from http://jid.oxfordjournals.org/ at University of Manitoba on June 20, 2015
Two rotavirus vaccine strains representing VP7 serotypes 1 and 2 derived by reassortment between a rhesus rotavirus master strain, MMU18006, and either of two human rotavirus strains were administered simultaneously to infants and young children to assess potential interactions betweenstrains. Children wereobserved in a day care setting for 10 days after vaccineadministration for clinical symptoms, evidence of vaccine transmission, and patterns of viral shedding. Serum and local antibody responses were measured. The ratio of input virus strongly influenced the amount of each strain recovered from the child. Regardless of dose of virus administered, the neutralizing antibody response to the VP7 glycoprotein, the serotype determinant, was diminished in a bivalent preparation compared with a monovalent vaccine. Additional strategies must be sought to induce immunity against the multiple serotypes of human rotavirus before broad immunity will be established.
272
Wright et al.
following cell lines: primary human embryonic kidney (HEK), HEp-2 (a continuous epithelial cell line), primary rhesus monkey kidney, and WI-38 (a human fibroblast line). Enteroviruses were typed in the Tennessee State Virology Laboratory. Stools were examined from each child for bacterial pathogens-Salmonella, Shigella, Campylobaeter, and Yersinia species-by conventional methods before and 4 and 7 days after vaccine administration. Stools were examined for ova and parasites at the beginning of each trial and midway through its course. Serologic determinations. Serum neutralizing antibodies to rotavirus were measured by plaque reduction assay as previously described [5]. Titers are expressed at a 50%inhibition of plaque formation. To define the breadth ofthe response, antibody titers were determined to prototype human strains (Wa, VP7 serotype 1; DS-l, VP7 serotype 2; P, VP7 serotype 3) and to the rhesus strain RR V (VP7 serotype 3). Antibodies were also measured using an ELISA assay with cesium chloride gradient-purified RRV as antigen to look at serum IgG and IgA antibodies and IgA antibodies in stools [5]. A hemagglutination inhibition (HAl) assay was used that measures antibody to RR V VP4, a surface glycoprotein with hemagglutinating properties [6]. Purified RRV was used as antigen and adjusted to 4 hemagglutination units/0.025 ml in PBS. Serum was treated with receptor-destroying enzyme (WHO Collaborating Center for Influenza, Centers for Disease Control, Atlanta) overnight at 37°C and incubated at 56°C for 30 min to remove complement. A 0.5% suspension of fresh adult human erythrocytes from a single type a-negative donor was used throughout. Serial twofold dilutions of serum, 25 ,uleach, from a starting dilution of 1:8 were made in plates and 0.025 ml of antigen was added for 1 h at 37°C. Next, 50 ,ul of erythrocytes was added and incubated at room temperature for I h. HAl titers were determined by dilution to an end point with complete inhibition of hemagglutination.
Results Vaccine infectivity. Of42 vaccinees, 32 shed rotavirus. In six cases in three separate trials, wild-type rotavirus was identified that was intercurrently circulating among trial participants. Wild-type virus was differentiated from vaccine virus by its ready detection using antigen capture methods, poor tissue culture growth, inability to form plaques, and RNA gel pattern distinct from that of either vaccine strains (figure 1). Rotavirus with growth characteristics ofvaccine was recovered from 25 of 36 vaccinees (the 6 vaccinees infected with wild-type rotavirus were excluded from this analysis). There was a tendency for recovery of virus to be age-dependent, with 4 of 10 > 2 years and 21 of26 < 2 years shedding virus (P = .08, Fisher's exact test). Viral recovery was decreased by preexisting serum antibodies, as measured by plaque neutralization, ELISA for IgA or IgG, and HAl, and by IgA cOProantibody. Five of the children with preexisting HAl and RRV antibody were children who had previously received RRV or RRVxD vaccine; only one of these children was
Downloaded from http://jid.oxfordjournals.org/ at University of Manitoba on June 20, 2015
Illness was defined as a fever of ~38.3°C, diarrhea (three or more stools per day or any stools characterized as watery or unformed), vomiting (more than once a day), or respiratory infection as defined by respiratory signs or symptoms leading to a clinical diagnosis of upper respiratory illness or acute otitis media. Vaccine administration. Vaccine was initially administered to older children, with progression in 10 separate trials to children < 1 year of age. Children were randomly assigned to receive vaccine or placebo, and the code remained blinded to the investigators and participants until the end of the study. Six of the older children had previously received monovalent RR V (VP7 serotype 3) or RR VxD (VP7 serotype 1) vaccine. After 2 days of preliminary studies and clinical observation, vaccine was administered orally in a volume of 1 ml in a water suspension. Virus was given after 300 mg of sodium bicarbonate to neutralize gastric acidity. Feedings were withheld for 30 min before and 30 min after vaccine administration. In the initial six trials, children received a 1:100 dilution of the stock suspension of RR VxD (mean titer administered per child, 1.1 X 105 pfu) and undiluted stock suspension ofRRVxDS-l (mean titer, 1.8 X 106 pfu). Twenty-four children received this bivalent vaccine with the infectivity ratio ofRRVxD (VP7 serotype 1) to RRVxDS-l (VP7 serotype 2) of I: 16. Twelve children served as placebo controls to assess intercurrent illness and possible transmissibility of vaccine virus to individuals in close daily contact with vaccine recipients. In four subsequent trials RRVxD was diluted 1:10 (mean titer administered per child, 1.1 X 106 pfu) as was RR VxDS-l (mean titer, 8.3 X 104 pfu). Eighteen children received this bivalent vaccine with an infectivity ratio of RR VxD to RR VxDS-l of 13:1. There were two controls. The total amount of virus given was similar to that in the initial trials, but the ratio ofRRVxD to RRVxDS-l was changed from 1:16 to 13: 1. Rotavirus isolation. Each stool was tested for rotavirus shedding by an antigen detection ELISA system (ROTAZYME II; Abbott Laboratories, North Chicago). For viral isolation a 10% suspension of stool was made in Hanks' balanced salt solution and clarified by centrifugation. The suspension was then incubated with 20 ,ug/ml trypsin for 30 min and inoculated onto MA-I04 cells using methods previously described for rotavirus isolation and identification [5]. Stool suspensions that were positive on primary culture for rotavirus were thawed, serially diluted, and inoculated onto MA-I04 cells to determine the titer of virus shed, which was expressed as plaque-forming units (pfu) per milliliter of 10% stool suspension. At the terminal dilution, plaques were picked and regrown in MA-I 04 cell culture for determination by RNA gels of the virus present. RNA was extracted from the tissue culture harvest using phenol and chloroform as previously described [7]. Electrophoresis of segmented double-stranded RNA was done on 10% polyacrylamide slab gels with a 3.6%stacking gel in a TRIS-glycine-SDS buffer system using 20-25 rnA for 5-6 h. RNA bands were stained with silver (Bio-Rad Laboratories, Richmond, CA). Identification of other enteric pathogens. Other enteric and respiratory viruses were sought in nasal and stool specimens every third day during the trial with virus isolation using the
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Simultaneous Administration of Two Human-Rhesus Rotavirus Reassortant Strains of VP7 Serotype 1 and 2 Specificity to Infants and Young Children Peter F. Wright, James King,* Kazuko Araki,* Yasuo Kondo,* Juliette Thompson, Sharon J. Tollefson, Masaaki Kobayashi, and Albert Z. Kapikian
Department of Pediatrics, Vanderbilt University, Nashville, Tennessee, and Laboratory ofInfectious Diseases, National Institute ofAllergy and Infectious Diseases, National Institutes ofHealth, Bethesda, Maryland
Rotaviruses are recognized as the primary etiologic agents in infantile diarrheal illness leading to serious dehydration and death throughout the world [1]. As diarrheal illness in turn represents one of the leading causes of childhood morbidity and mortality in developing countries, rotavirus has been given the highest priority for vaccine development [2]. The initial approach taken was to develop live, attenuated, orally administered vaccines. Rotaviruses ofanimal origin, a Jennerian approach, have the advantages of host range attenuation and better tissue culture growth than the human strains [3]. For example, rhesus rotavirus strain MMU18006, isolated from a young rhesus monkey, and three additional human-rhesus rotavirus reassortants, each containing a single gene encoding VP7, a major outer capsid protein, derived from representative human rotavirus serotypes, are being evaluated as potential vaccine candidates in children. Given as single strains, the original rhesus strain, RRV, and its reassortants possess many of the attributes sought in a vaccine [4-6]. However, as four recognized human rotavirus
Received 15 November 1990; revised 5 April 1991. Presented in part: American Society of Virology, Salt Lake City, July 1990. Informed consent was obtained from parents and protocols were reviewed and approved by the Vanderbilt Committee for the Protection of Human Subjects. Financial support: contract AI-05050 (National Institutes of Health) and RR-00095 (NIH to Vanderbilt Clinical Research Center). Correspondence: Dr. Peter Wright, Department of Pediatrics. Vanderbilt University, Nashville. TN 37232. * Present addresses: Department of Pediatrics, University of Maryland. Baltimore (J.K.); Department of Pediatrics. Teikyo University, Tokyo, Japan (K.A. and Y.K.). The Journal of Infectious Diseases 1991;164:271-6 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6402-0006$01.00
group A strains are epidemiologically important, there may be need for a multivalent vaccine as well as administration of multiple doses. As a step towards the evaluation of a multivalent vaccine, studies ofthe simultaneous administration of reassortants with the VP7 of human rotavirus serotype 1 (RRVxD) or 2 (RRVxDS-l) and the remaining 10 genes from the parental rhesus strain were undertaken to assess the capability of infants and young children to allow replication and to respond immunologically to each component of the vaccine. Simultaneous oral administration of bivalent live attenuated rotavirus vaccine was evaluated with two different input ratios of the strains. The clinical, viral, and serologic approach to their evaluation did not vary significantly from previously described studies of monovalent serotype 3 (RRV) [5] and serotype 1 (RRVxD) [6] except for the necessity to identify the serotype(s) of virus recovered in the stool specimens. This was accomplished using RNA gels that differentiated the genes coding VP7 from the human strains D and DS-l.
Methods Clinical evaluation. Clinical assessment of 56 infants and young children ages 4-70 months was carried out in a day care facility in the Clinical Research Center, Vanderbilt University, between May 1987 and October 1989. There were 37 children 4-23 months of age and 19children >2 years of age in the trials. None was concurrently being breast-fed. Ten groups of3-9 children at a time were observed daily for at least 6 hours a day. For 12 consecutive days a physical examination was done by a member of the vaccine evaluation team. Efforts were made to obtain a daily stool sample. Each child returned home in the evening, where a continued record of fever, gastrointestinal symptoms, and number of stools was kept.
Downloaded from http://jid.oxfordjournals.org/ at University of Manitoba on June 20, 2015
Two rotavirus vaccine strains representing VP7 serotypes 1 and 2 derived by reassortment between a rhesus rotavirus master strain, MMU18006, and either of two human rotavirus strains were administered simultaneously to infants and young children to assess potential interactions betweenstrains. Children wereobserved in a day care setting for 10 days after vaccineadministration for clinical symptoms, evidence of vaccine transmission, and patterns of viral shedding. Serum and local antibody responses were measured. The ratio of input virus strongly influenced the amount of each strain recovered from the child. Regardless of dose of virus administered, the neutralizing antibody response to the VP7 glycoprotein, the serotype determinant, was diminished in a bivalent preparation compared with a monovalent vaccine. Additional strategies must be sought to induce immunity against the multiple serotypes of human rotavirus before broad immunity will be established.
272
Wright et al.
following cell lines: primary human embryonic kidney (HEK), HEp-2 (a continuous epithelial cell line), primary rhesus monkey kidney, and WI-38 (a human fibroblast line). Enteroviruses were typed in the Tennessee State Virology Laboratory. Stools were examined from each child for bacterial pathogens-Salmonella, Shigella, Campylobaeter, and Yersinia species-by conventional methods before and 4 and 7 days after vaccine administration. Stools were examined for ova and parasites at the beginning of each trial and midway through its course. Serologic determinations. Serum neutralizing antibodies to rotavirus were measured by plaque reduction assay as previously described [5]. Titers are expressed at a 50%inhibition of plaque formation. To define the breadth ofthe response, antibody titers were determined to prototype human strains (Wa, VP7 serotype 1; DS-l, VP7 serotype 2; P, VP7 serotype 3) and to the rhesus strain RR V (VP7 serotype 3). Antibodies were also measured using an ELISA assay with cesium chloride gradient-purified RRV as antigen to look at serum IgG and IgA antibodies and IgA antibodies in stools [5]. A hemagglutination inhibition (HAl) assay was used that measures antibody to RR V VP4, a surface glycoprotein with hemagglutinating properties [6]. Purified RRV was used as antigen and adjusted to 4 hemagglutination units/0.025 ml in PBS. Serum was treated with receptor-destroying enzyme (WHO Collaborating Center for Influenza, Centers for Disease Control, Atlanta) overnight at 37°C and incubated at 56°C for 30 min to remove complement. A 0.5% suspension of fresh adult human erythrocytes from a single type a-negative donor was used throughout. Serial twofold dilutions of serum, 25 ,uleach, from a starting dilution of 1:8 were made in plates and 0.025 ml of antigen was added for 1 h at 37°C. Next, 50 ,ul of erythrocytes was added and incubated at room temperature for I h. HAl titers were determined by dilution to an end point with complete inhibition of hemagglutination.
Results Vaccine infectivity. Of42 vaccinees, 32 shed rotavirus. In six cases in three separate trials, wild-type rotavirus was identified that was intercurrently circulating among trial participants. Wild-type virus was differentiated from vaccine virus by its ready detection using antigen capture methods, poor tissue culture growth, inability to form plaques, and RNA gel pattern distinct from that of either vaccine strains (figure 1). Rotavirus with growth characteristics ofvaccine was recovered from 25 of 36 vaccinees (the 6 vaccinees infected with wild-type rotavirus were excluded from this analysis). There was a tendency for recovery of virus to be age-dependent, with 4 of 10 > 2 years and 21 of26 < 2 years shedding virus (P = .08, Fisher's exact test). Viral recovery was decreased by preexisting serum antibodies, as measured by plaque neutralization, ELISA for IgA or IgG, and HAl, and by IgA cOProantibody. Five of the children with preexisting HAl and RRV antibody were children who had previously received RRV or RRVxD vaccine; only one of these children was
Downloaded from http://jid.oxfordjournals.org/ at University of Manitoba on June 20, 2015
Illness was defined as a fever of ~38.3°C, diarrhea (three or more stools per day or any stools characterized as watery or unformed), vomiting (more than once a day), or respiratory infection as defined by respiratory signs or symptoms leading to a clinical diagnosis of upper respiratory illness or acute otitis media. Vaccine administration. Vaccine was initially administered to older children, with progression in 10 separate trials to children < 1 year of age. Children were randomly assigned to receive vaccine or placebo, and the code remained blinded to the investigators and participants until the end of the study. Six of the older children had previously received monovalent RR V (VP7 serotype 3) or RR VxD (VP7 serotype 1) vaccine. After 2 days of preliminary studies and clinical observation, vaccine was administered orally in a volume of 1 ml in a water suspension. Virus was given after 300 mg of sodium bicarbonate to neutralize gastric acidity. Feedings were withheld for 30 min before and 30 min after vaccine administration. In the initial six trials, children received a 1:100 dilution of the stock suspension of RR VxD (mean titer administered per child, 1.1 X 105 pfu) and undiluted stock suspension ofRRVxDS-l (mean titer, 1.8 X 106 pfu). Twenty-four children received this bivalent vaccine with the infectivity ratio ofRRVxD (VP7 serotype 1) to RRVxDS-l (VP7 serotype 2) of I: 16. Twelve children served as placebo controls to assess intercurrent illness and possible transmissibility of vaccine virus to individuals in close daily contact with vaccine recipients. In four subsequent trials RRVxD was diluted 1:10 (mean titer administered per child, 1.1 X 106 pfu) as was RR VxDS-l (mean titer, 8.3 X 104 pfu). Eighteen children received this bivalent vaccine with an infectivity ratio of RR VxD to RR VxDS-l of 13:1. There were two controls. The total amount of virus given was similar to that in the initial trials, but the ratio ofRRVxD to RRVxDS-l was changed from 1:16 to 13: 1. Rotavirus isolation. Each stool was tested for rotavirus shedding by an antigen detection ELISA system (ROTAZYME II; Abbott Laboratories, North Chicago). For viral isolation a 10% suspension of stool was made in Hanks' balanced salt solution and clarified by centrifugation. The suspension was then incubated with 20 ,ug/ml trypsin for 30 min and inoculated onto MA-I04 cells using methods previously described for rotavirus isolation and identification [5]. Stool suspensions that were positive on primary culture for rotavirus were thawed, serially diluted, and inoculated onto MA-I04 cells to determine the titer of virus shed, which was expressed as plaque-forming units (pfu) per milliliter of 10% stool suspension. At the terminal dilution, plaques were picked and regrown in MA-I 04 cell culture for determination by RNA gels of the virus present. RNA was extracted from the tissue culture harvest using phenol and chloroform as previously described [7]. Electrophoresis of segmented double-stranded RNA was done on 10% polyacrylamide slab gels with a 3.6%stacking gel in a TRIS-glycine-SDS buffer system using 20-25 rnA for 5-6 h. RNA bands were stained with silver (Bio-Rad Laboratories, Richmond, CA). Identification of other enteric pathogens. Other enteric and respiratory viruses were sought in nasal and stool specimens every third day during the trial with virus isolation using the
JID 1991; 164 (August)
1ID 1991 :164 (August)
Bivalent Rhesus Rotavirus Vaccine z
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