Vaccinia-rotavirus VP7 recombinants protect mice against rotavirus-induced diarrhoea M a r i o n E. A n d r e w * § , D a v i d B. B o y l e * , B a r b a r a E. H . C o u p a r * , D a v i d R e d d y t , A. R i c h a r d B e l l a m y t a n d G e r a l d W . B o t h $
Recombinant vaccinia viruses expressing wild type intracellular VP7 (VP7wt) from rotavirus S A I l or VP7sc, a cell surface-anchored variant, boosted antibody titres in SAil-immune mice. Pups born to these mice were protected from diarrhoea following challenge with SA 11. In rotavirus-naive mice, two immunizations with recombinant vaccinia virus expressing VP7sc stimulated protective immunity that could be transferred to pups, whereas viruses expressing VP7wt did not stimulate protective immunity. Recombinant vaccinia viruses expressing intracellular or cell surface-anchored VP6, the rotavirus group-reactive antigen from the inner capsid, did not stimulate protective immunity. These experiments demonstrate that a live viral vector expressing cell surface anchored VP7 may represent a strategy for the development of safe, effective vaccines against rotavirus-induced diarrhoea. Keywords: Recombinant;vaccinia; rotavirus; rotavirus-induceddiarrhoea; mice
INTRODUCTION Diarrhoeal disease resulting from rotavirus infection is a major problem worldwide, particularly in young children. In developing countries, malnutrition may be exacerbated and high levels of infant mortality occur ~'2. In developed countries, dehydration resulting from rotavirus infection is a common reason for hospitalization of children with diarrhoea. The problem is of sufficient magnitude that the development of a rotavirus vaccine has been identified as a priority area by the World Health Organization. Rotaviruses are also widespread in the animal kingdom and epidemics are responsible for production losses in some livestock species, particularly where they are farmed intensively. Young animals are most at risk in the first few days or weeks of life, depending on the species. This poses a problem for vaccination as active immunity cannot be acquired in time to prevent infection. Thus, the best strategy to provide protection may be to boost immunity in the mother so that protection is passively transferred to the suckling offspring. Similarly, passive transfer of immunity may provide protection in humans before weaning, although a narrow window for vaccination may exist in humans, as children become most susceptible to infection from about 6 months of age and remain susceptible until about 5 years of age. *CSIRO Australian Animal Health Laboratory, PO Bag 24, Geelong, Vic 3220, Australia. tDepartment of Cellular and Molecular Biology, University of Auckland, Auckland, New Zealand. :~CSIRO Division of Biomolecular Engineering, PO Box 184, North Ryde, NSW 2113, Australia. §To whom correspondence should be addressed. (Received 27 May 1991 ; revised 1 October 1991 ; accepted 3 October 1991 ) 0264-410X/92/030185-07 © 1992 Butterworth-HeinemannLtd
Vaccine trials with live, attenuated rotaviruses have not been totally successful 3'4. Candidate vaccine strains for human trials have included animal viruses and non-pathogenic strains isolated from asymptomatic neonates. However, they have proven to be either too virulent, producing unacceptable side-effects, or too attenuated, not providing protective immunity. Empty viral capsids 5 also have been shown experimentally to provide protective immunity, but they are likely to be costly to produce. There are many rotavirus serotypes, but some are restricted to particular host species. Serotype specificity is conferred mainly by the outer capsid protein VP7, but the minor capsid protein VP4 also contributes. Monoclonal antibodies to both these antigens neutralize the virus and can confer passive protection against challenge 6. Laboratory experiments and vaccine trials have demonstrated that some heterotypic immunity can be induced 1. VP4 and VP7 carry cross-reactive epitopes 7 ; however, the immunogenicity of these epitopes appears to be restricted and may depend on prior exposure to rotavirus s. The group reactive inner capsid antigen, VP6, also shows extensive cross-reactivity amongst rotaviruses, but antibody specific for VP6 does not neutralize the virus and no role for VP6 in protection has been shown. We have investigated the use of recombinant vaccinia viruses expressing rotavirus antigens to stimulate immune responses in rabbits and mice. Vaccinia virus-expressed VP7 induced low levels of neutralizing antibodies in rabbits 9, and in mice antibody levels were at, or below, the level of detection, even after secondary immunization 1o. Subsequently, a novel, cell surface-expressed form of VP7
Vaccine, Vol. 10, Issue 3, 1992 185
Recombinant vaccine against rotavirus-induced diarrhoea: M.E. Andrew et al. Table 1
Recombinant vaccinia viruses
Virus
Foreign gene
Cellular location of antigen
VV-VP7wt VV-VP7wt-TK VV-VP7d47 VV-VP7sc
Rotavirus SA11 VP7 Rotavirus Rotavirus
SA11 VP7 + HSV TK SA11 VP7 SA11 VP7
Endoplasmic reticulum Endoplasmic reticulum Secreted Cell surface anchored
VV-VP7sc-TK VV-VP6wt
SA11 VP7 + HSV TK Rotavirus SA11 VP6
Cell surface anchored Cytoplasmic
VV-VP6sc
Rotavirus SA11 VP6
Cell surface anchored
VV-HA6 VV-HA-TK
Influenza A/PR/8/34 HA HA + HSV TK
Cell surface anchored Cell surface anchored
PROTECTION PROTOCOL Primary immunization of female mice
Secondary immunization
Immunized females mated 21 days or later Pups born 7 days
Pups challenged orally with S A l l . In litters of more than five pups, one pup killed for collection of milk plug
Modification
Reference
None TK + phenotype Deletion of sequence for aa 47-61 Replacement of signal sequence with one from influenza HA + addition of influenza HA anchor sequence TK ÷ phenotype None
9 16 9 11
Addition of influenza HA signal sequence and IgG 1 anchor sequence None TK + phenotype
16 Reddy et al. (unpublished results) Reddy et al. (unpublished results) 17 16
by assessing cytopathic effects in an end-point dilution assay on MA104 cell monolayers and was expressed in IU m1-1 (Ref. 14). The ratio between IU and plaque forming units is 1:1 (unpublished observation). Recombinant vaccinia viruses were constructed by insertion of foreign genes into the thymidine kinase ( T K ) gene of vaccinia virus, using established techniques 15. The foreign genes were expressed under the control of the vaccinia virus 7.5 K promoter and the recombinant viruses had a T K - phenotype. T K ÷ viruses were constructed by inserting the herpes simplex virus (HSV) T K gene into the HindlII F region 16. A summary of recombinant vaccinia viruses used is shown in Table 1. V V - T K - is a T K - mutant of WR strain vaccinia virus.
4 days Pups monitored daily for signs of diarrhoea Dams bled on day 4
Figure 1
Protocol for protection experiments
was engineered and this had greatly improved immunogenicity ~1. We also have produced vaccinia virus recombinants carrying genes for intracellular wild-type and cell surface-expressed VP6 (Reddy and co-workers, unpublished results). In the present study, the abilities of these vaccines to provide protective immunity have been compared using a murine disease model that assesses protection from diarrhoea induced by oral challenge with the simian rotavirus, SAIl 12. MATERIALS AND METHODS
Mice C B A / H mice were obtained from the specific pathogenfree colony at Animal Resources Centre, Perth, W.A., Australia. Mice were screened by ELISA for antibodies to rotavirus before use and found to be rotavirus seronegative. Female mice were ~ 5 weeks of age at primary immunization and male mice were at least 8 weeks old when used for mating.
Viruses SA11 rotavirus was grown in MA104 cells and purified on caesium chloride gradients ~3. Virus titre was measured
186
Vaccine, Vol. 10, Issue 3, 1992
Immunization and challenge protocols A plan of the immunization and challenge schedule is shown in Figure 1 and details of dose and route of inoculation are provided for each experiment in the results section. For intravenous inoculations, 0.2 ml virus was injected into the tail vein. Mice inoculated intranasally were lightly anaesthetized with ether to suppress their gag reflex and 50/d virus was delivered to the nostrils with an Eppendorf pipette. For intraperitoneal inoculation, the dose of virus was suspended in 0.2 ml phosphate-buffered saline. When mice were obviously pregnant, they were caged separately and assigned a code number by a technician who was not involved in the challenge of pups and assessment of diarrhoea. Mice were observed daily for delivery of litters and between days 6 and 8 after birth, the pups were challenged orally with SA 11. Immediately before challenge, a small quantity of purified SA11 (5-10 #1) was dialysed against phosphatebuffered saline containing 0.1% calcium chloride and 0.1% magnesium chloride (PBS/CaMg). Dialysis was performed for 30 min by placing the virus on a Millipore filter (Cat. no. VSWP 02500) which had been floated on P B S / C a M g in a petri dish. After dialysis, the virus was diluted in P B S / C a M g + 1:80 green food colouring (tartrazine, blue, ethryosine (2.3% total dye stuff)+ potassium sorbate preservative). Each pup was inoculated with 25/A SA11, which was delivered into the stomach with a 26G needle from which the bevel had been removed. The food colouring permitted visual confirmation that the virus had reached the stomach, as it could
Recombinant vaccine against rotavirus-induced diarrhoea: M.E. Andrew et al. 1.0 0.9 7
0.8
7
0.7 "o E
7 0.6 0.5
tIL £3
0.4 0.3 0.2 6 0.1 0 SAIl
VP6wt
VP6sc
VP7wt
VP7sc
TK--
Figure 2 Diarrhoea in litters from SAil-immune mice reimmunized with vaccinia-rotavirus recombinant viruses. SAil-immune mice were reimmunized intravenously with SA11 or with recombinant vaccinia virus expressing the indicated antigen. Each bar represents the mean diarrhoeal index for that group of litters following challenge with 4 x 105 IU SA11, with the error bar indicating 1 standard error. The number of litters in each group is indicated at the top of each bar
be seen through the abdominal and stomach walls. Pups were observed once daily over the following 4 days for signs of diarrhoea following gentle palpation of their abdomen. Diarrhoeal index (DI) for each litter was calculated as :
were remated and the second litter was challenged with SA11. The time between secondary immunization of the dams and challenge of litters ranged between 53 and 107 days, due to variation in the time taken to conceive and to produce a live litter. Pups from dams that were reimmunized with SAIl were protected from diarrhoea (p < 0.001). The mean DI was 0.03 and only one of the nine litters tested developed diarrhoea (DI = 0.20) (Figure 2). If the mice were reimmunized with V V - T K - instead of SA11, there was no protective immunity transferred to the pups and the mean DI of the group of seven litters was 0.77 (Figure 2), a value similar to that obtained from pups born to naive mice (data not shown) or to mice immunized with only V V - T K - (Figure 3) (p =0.7505, t test). Reimmunization with VV-VP6wt or VV-VP6sc failed to stimulate protective immunity. However, significant protection from diarrhoea was observed in litters born to dams reimmunized with VV-VP7wt (p < 0.001) or VV-VP7sc (p < 0.001) (Figure 2). For VV-VP7wt, only one of three litters had diarrhoea (DI = 0.10) and for VV-VP7sc, only one of six litters had diarrhoea (DI = 0.63). Transfer of protection to neonates from dams immunized with vaceinia-rotavirus recombiants Groups of five (VV-VP6wt and VV-VP6sc) or ten (remaining groups) female mice were immunized
Sum of number of pups with diarrhoea on each of days 1-4
1.0
Sum of total number of pups on each of days 1-4
0.9 2
2 0.8
Antibody assays of milk and serum When there were more than five pups per litter, one pup was killed on the day of challenge and the milk plug removed from its stomach. The milk plug was weighed and saline (10/d mg -x milk plug) added. The milk was resuspended by sonication and assayed for rotavirus-specific antibody by ELISA, using whole SA11 virus as antigen 11. At the end of the 4-day observation period, dams were bled and the sera assayed for antibody by ELISA 11. All ELISA assays were performed in the presence of calcium and magnesium to preserve the VP7-containing outer coat on the virus.
x "o
0.7
._c
0.6
¢-
0.4
r~
0.3
._~
0.2 0.1 'P6wt
RESULTS Transfer of protection to neonates from rotavirus-immune dams reimmunized with vaccinia rotavirus recombinants Groups of ten female C B A / H mice were immunized intraperitoneally with 4 × 107 p.f,u. SAIl rotavirus and 75 days later reimmunized intravenously with the same dose of SAIl, with 107 p.f.u, recombinant vaccinia virus expressing rotavirus antigen, or with 107 p.f.u, non-recombinant vaccinia virus ( V V - T K - ) . The immunized mice were mated 32 days later. The offspring were challenged orally with 4 x 105 IU SAIl when 7 days old and diarrhoea recorded over the next 4 days. When litters did not survive until day 7, the female mice
VP7wt
VP6sc
d47
VP7sc
TK--
1.0
Statistical analysis Data were analysed using Instat software from Graphpad (San Diego, CA, USA). One-way analysis of variance was used to test for differences between groups in each experiment. Bonferroni p values are given.
0.5
L
0.gb
T
0.8 × 0) -o
0.7
~
0.5
..c L L ro
0.4
.E
, 2
5
3
0.6
~5 0.3 0.2 0.1 0
VP6wt Figure 3
Diarrhoea
VP6sc
VP7wt
d47
VP7sc
TK--
in litters f r o m rotavirus-naive mice i m m u n i z e d
twice with vaccinia rotavirus recombinant viruses. Each bar represents the mean diarrhoeal index for litters from a group of mice that received two doses of the indicated virus. (a) Litters challenged with 4 x 105 IU SA11 between days 54 and 102 after secondary immunization and (b) only those litters challenged
Recombinant vaccinia viruses expressing wild type intracellular VP7 (VP7wt) from rotavirus S A I l or VP7sc, a cell surface-anchored variant, boosted antibody titres in SAil-immune mice. Pups born to these mice were protected from diarrhoea following challenge with SA 11. In rotavirus-naive mice, two immunizations with recombinant vaccinia virus expressing VP7sc stimulated protective immunity that could be transferred to pups, whereas viruses expressing VP7wt did not stimulate protective immunity. Recombinant vaccinia viruses expressing intracellular or cell surface-anchored VP6, the rotavirus group-reactive antigen from the inner capsid, did not stimulate protective immunity. These experiments demonstrate that a live viral vector expressing cell surface anchored VP7 may represent a strategy for the development of safe, effective vaccines against rotavirus-induced diarrhoea. Keywords: Recombinant;vaccinia; rotavirus; rotavirus-induceddiarrhoea; mice
INTRODUCTION Diarrhoeal disease resulting from rotavirus infection is a major problem worldwide, particularly in young children. In developing countries, malnutrition may be exacerbated and high levels of infant mortality occur ~'2. In developed countries, dehydration resulting from rotavirus infection is a common reason for hospitalization of children with diarrhoea. The problem is of sufficient magnitude that the development of a rotavirus vaccine has been identified as a priority area by the World Health Organization. Rotaviruses are also widespread in the animal kingdom and epidemics are responsible for production losses in some livestock species, particularly where they are farmed intensively. Young animals are most at risk in the first few days or weeks of life, depending on the species. This poses a problem for vaccination as active immunity cannot be acquired in time to prevent infection. Thus, the best strategy to provide protection may be to boost immunity in the mother so that protection is passively transferred to the suckling offspring. Similarly, passive transfer of immunity may provide protection in humans before weaning, although a narrow window for vaccination may exist in humans, as children become most susceptible to infection from about 6 months of age and remain susceptible until about 5 years of age. *CSIRO Australian Animal Health Laboratory, PO Bag 24, Geelong, Vic 3220, Australia. tDepartment of Cellular and Molecular Biology, University of Auckland, Auckland, New Zealand. :~CSIRO Division of Biomolecular Engineering, PO Box 184, North Ryde, NSW 2113, Australia. §To whom correspondence should be addressed. (Received 27 May 1991 ; revised 1 October 1991 ; accepted 3 October 1991 ) 0264-410X/92/030185-07 © 1992 Butterworth-HeinemannLtd
Vaccine trials with live, attenuated rotaviruses have not been totally successful 3'4. Candidate vaccine strains for human trials have included animal viruses and non-pathogenic strains isolated from asymptomatic neonates. However, they have proven to be either too virulent, producing unacceptable side-effects, or too attenuated, not providing protective immunity. Empty viral capsids 5 also have been shown experimentally to provide protective immunity, but they are likely to be costly to produce. There are many rotavirus serotypes, but some are restricted to particular host species. Serotype specificity is conferred mainly by the outer capsid protein VP7, but the minor capsid protein VP4 also contributes. Monoclonal antibodies to both these antigens neutralize the virus and can confer passive protection against challenge 6. Laboratory experiments and vaccine trials have demonstrated that some heterotypic immunity can be induced 1. VP4 and VP7 carry cross-reactive epitopes 7 ; however, the immunogenicity of these epitopes appears to be restricted and may depend on prior exposure to rotavirus s. The group reactive inner capsid antigen, VP6, also shows extensive cross-reactivity amongst rotaviruses, but antibody specific for VP6 does not neutralize the virus and no role for VP6 in protection has been shown. We have investigated the use of recombinant vaccinia viruses expressing rotavirus antigens to stimulate immune responses in rabbits and mice. Vaccinia virus-expressed VP7 induced low levels of neutralizing antibodies in rabbits 9, and in mice antibody levels were at, or below, the level of detection, even after secondary immunization 1o. Subsequently, a novel, cell surface-expressed form of VP7
Vaccine, Vol. 10, Issue 3, 1992 185
Recombinant vaccine against rotavirus-induced diarrhoea: M.E. Andrew et al. Table 1
Recombinant vaccinia viruses
Virus
Foreign gene
Cellular location of antigen
VV-VP7wt VV-VP7wt-TK VV-VP7d47 VV-VP7sc
Rotavirus SA11 VP7 Rotavirus Rotavirus
SA11 VP7 + HSV TK SA11 VP7 SA11 VP7
Endoplasmic reticulum Endoplasmic reticulum Secreted Cell surface anchored
VV-VP7sc-TK VV-VP6wt
SA11 VP7 + HSV TK Rotavirus SA11 VP6
Cell surface anchored Cytoplasmic
VV-VP6sc
Rotavirus SA11 VP6
Cell surface anchored
VV-HA6 VV-HA-TK
Influenza A/PR/8/34 HA HA + HSV TK
Cell surface anchored Cell surface anchored
PROTECTION PROTOCOL Primary immunization of female mice
Secondary immunization
Immunized females mated 21 days or later Pups born 7 days
Pups challenged orally with S A l l . In litters of more than five pups, one pup killed for collection of milk plug
Modification
Reference
None TK + phenotype Deletion of sequence for aa 47-61 Replacement of signal sequence with one from influenza HA + addition of influenza HA anchor sequence TK ÷ phenotype None
9 16 9 11
Addition of influenza HA signal sequence and IgG 1 anchor sequence None TK + phenotype
16 Reddy et al. (unpublished results) Reddy et al. (unpublished results) 17 16
by assessing cytopathic effects in an end-point dilution assay on MA104 cell monolayers and was expressed in IU m1-1 (Ref. 14). The ratio between IU and plaque forming units is 1:1 (unpublished observation). Recombinant vaccinia viruses were constructed by insertion of foreign genes into the thymidine kinase ( T K ) gene of vaccinia virus, using established techniques 15. The foreign genes were expressed under the control of the vaccinia virus 7.5 K promoter and the recombinant viruses had a T K - phenotype. T K ÷ viruses were constructed by inserting the herpes simplex virus (HSV) T K gene into the HindlII F region 16. A summary of recombinant vaccinia viruses used is shown in Table 1. V V - T K - is a T K - mutant of WR strain vaccinia virus.
4 days Pups monitored daily for signs of diarrhoea Dams bled on day 4
Figure 1
Protocol for protection experiments
was engineered and this had greatly improved immunogenicity ~1. We also have produced vaccinia virus recombinants carrying genes for intracellular wild-type and cell surface-expressed VP6 (Reddy and co-workers, unpublished results). In the present study, the abilities of these vaccines to provide protective immunity have been compared using a murine disease model that assesses protection from diarrhoea induced by oral challenge with the simian rotavirus, SAIl 12. MATERIALS AND METHODS
Mice C B A / H mice were obtained from the specific pathogenfree colony at Animal Resources Centre, Perth, W.A., Australia. Mice were screened by ELISA for antibodies to rotavirus before use and found to be rotavirus seronegative. Female mice were ~ 5 weeks of age at primary immunization and male mice were at least 8 weeks old when used for mating.
Viruses SA11 rotavirus was grown in MA104 cells and purified on caesium chloride gradients ~3. Virus titre was measured
186
Vaccine, Vol. 10, Issue 3, 1992
Immunization and challenge protocols A plan of the immunization and challenge schedule is shown in Figure 1 and details of dose and route of inoculation are provided for each experiment in the results section. For intravenous inoculations, 0.2 ml virus was injected into the tail vein. Mice inoculated intranasally were lightly anaesthetized with ether to suppress their gag reflex and 50/d virus was delivered to the nostrils with an Eppendorf pipette. For intraperitoneal inoculation, the dose of virus was suspended in 0.2 ml phosphate-buffered saline. When mice were obviously pregnant, they were caged separately and assigned a code number by a technician who was not involved in the challenge of pups and assessment of diarrhoea. Mice were observed daily for delivery of litters and between days 6 and 8 after birth, the pups were challenged orally with SA 11. Immediately before challenge, a small quantity of purified SA11 (5-10 #1) was dialysed against phosphatebuffered saline containing 0.1% calcium chloride and 0.1% magnesium chloride (PBS/CaMg). Dialysis was performed for 30 min by placing the virus on a Millipore filter (Cat. no. VSWP 02500) which had been floated on P B S / C a M g in a petri dish. After dialysis, the virus was diluted in P B S / C a M g + 1:80 green food colouring (tartrazine, blue, ethryosine (2.3% total dye stuff)+ potassium sorbate preservative). Each pup was inoculated with 25/A SA11, which was delivered into the stomach with a 26G needle from which the bevel had been removed. The food colouring permitted visual confirmation that the virus had reached the stomach, as it could
Recombinant vaccine against rotavirus-induced diarrhoea: M.E. Andrew et al. 1.0 0.9 7
0.8
7
0.7 "o E
7 0.6 0.5
tIL £3
0.4 0.3 0.2 6 0.1 0 SAIl
VP6wt
VP6sc
VP7wt
VP7sc
TK--
Figure 2 Diarrhoea in litters from SAil-immune mice reimmunized with vaccinia-rotavirus recombinant viruses. SAil-immune mice were reimmunized intravenously with SA11 or with recombinant vaccinia virus expressing the indicated antigen. Each bar represents the mean diarrhoeal index for that group of litters following challenge with 4 x 105 IU SA11, with the error bar indicating 1 standard error. The number of litters in each group is indicated at the top of each bar
be seen through the abdominal and stomach walls. Pups were observed once daily over the following 4 days for signs of diarrhoea following gentle palpation of their abdomen. Diarrhoeal index (DI) for each litter was calculated as :
were remated and the second litter was challenged with SA11. The time between secondary immunization of the dams and challenge of litters ranged between 53 and 107 days, due to variation in the time taken to conceive and to produce a live litter. Pups from dams that were reimmunized with SAIl were protected from diarrhoea (p < 0.001). The mean DI was 0.03 and only one of the nine litters tested developed diarrhoea (DI = 0.20) (Figure 2). If the mice were reimmunized with V V - T K - instead of SA11, there was no protective immunity transferred to the pups and the mean DI of the group of seven litters was 0.77 (Figure 2), a value similar to that obtained from pups born to naive mice (data not shown) or to mice immunized with only V V - T K - (Figure 3) (p =0.7505, t test). Reimmunization with VV-VP6wt or VV-VP6sc failed to stimulate protective immunity. However, significant protection from diarrhoea was observed in litters born to dams reimmunized with VV-VP7wt (p < 0.001) or VV-VP7sc (p < 0.001) (Figure 2). For VV-VP7wt, only one of three litters had diarrhoea (DI = 0.10) and for VV-VP7sc, only one of six litters had diarrhoea (DI = 0.63). Transfer of protection to neonates from dams immunized with vaceinia-rotavirus recombiants Groups of five (VV-VP6wt and VV-VP6sc) or ten (remaining groups) female mice were immunized
Sum of number of pups with diarrhoea on each of days 1-4
1.0
Sum of total number of pups on each of days 1-4
0.9 2
2 0.8
Antibody assays of milk and serum When there were more than five pups per litter, one pup was killed on the day of challenge and the milk plug removed from its stomach. The milk plug was weighed and saline (10/d mg -x milk plug) added. The milk was resuspended by sonication and assayed for rotavirus-specific antibody by ELISA, using whole SA11 virus as antigen 11. At the end of the 4-day observation period, dams were bled and the sera assayed for antibody by ELISA 11. All ELISA assays were performed in the presence of calcium and magnesium to preserve the VP7-containing outer coat on the virus.
x "o
0.7
._c
0.6
¢-
0.4
r~
0.3
._~
0.2 0.1 'P6wt
RESULTS Transfer of protection to neonates from rotavirus-immune dams reimmunized with vaccinia rotavirus recombinants Groups of ten female C B A / H mice were immunized intraperitoneally with 4 × 107 p.f,u. SAIl rotavirus and 75 days later reimmunized intravenously with the same dose of SAIl, with 107 p.f.u, recombinant vaccinia virus expressing rotavirus antigen, or with 107 p.f.u, non-recombinant vaccinia virus ( V V - T K - ) . The immunized mice were mated 32 days later. The offspring were challenged orally with 4 x 105 IU SAIl when 7 days old and diarrhoea recorded over the next 4 days. When litters did not survive until day 7, the female mice
VP7wt
VP6sc
d47
VP7sc
TK--
1.0
Statistical analysis Data were analysed using Instat software from Graphpad (San Diego, CA, USA). One-way analysis of variance was used to test for differences between groups in each experiment. Bonferroni p values are given.
0.5
L
0.gb
T
0.8 × 0) -o
0.7
~
0.5
..c L L ro
0.4
.E
, 2
5
3
0.6
~5 0.3 0.2 0.1 0
VP6wt Figure 3
Diarrhoea
VP6sc
VP7wt
d47
VP7sc
TK--
in litters f r o m rotavirus-naive mice i m m u n i z e d
twice with vaccinia rotavirus recombinant viruses. Each bar represents the mean diarrhoeal index for litters from a group of mice that received two doses of the indicated virus. (a) Litters challenged with 4 x 105 IU SA11 between days 54 and 102 after secondary immunization and (b) only those litters challenged
