BIOLOGICAL EFFECTS OF RECOMBINANT VACCINIA VIRUS-EXPRESSED INTERLEUKIN 4 Marion
E. Andrew
and Barbara
E.H.
Coupar
Recombinant vaccinia viruses expressing murine interleukin 4 (IL-4), either alone or together with interleukin 2 (IL-2) or y interferon (y-IFN), were constructed. Unlike IL-2, IL-4 expressing viruses were not cleared from immunodeficient mice and the mice died. As they died more rapidly than immunodeficient mice inoculated with a control virus, it appeared that IL-4 contributed to their death and the IL-4 mediated toxicity was confirmed in normal immunocompetent mice. The toxicity was reversed by co-expression of either IL-2 or y-IFN, probably due to virus clearance and therefore lower levels of circulating IL-4. Vaccinia virus-expressed IL-4 did not increase antibody or natural killer cell levels and caused a slight decrease in cytotoxic T lymphocyte levels
IL-4 was first described as a B-cell growth factor,1 but it also binds to and stimulates the proliferation of T cells.zJ It is produced by the Th, subset of T cells, whereas the other T-cell stimulating factor, IL-2 is produced by the Th, subset.4 A variety of functions for IL-4 have been reported, including inhibition of the ability of IL-2 to stimulate natural killer (NK) and lymphokine activated killer cells, but not the stimulation of antigen-specific cytotoxic T lymphocytes (CTL)5$ antagonistic interaction with another product of Th, cells, y-interferon (y-IFN)7; suppression of the production of y-IFNs and increase in the production of IgE and IgGl in in-vitro assays.9 Cytokines have been expressed from recombinant vaccinia viruses and shown to have biological activity in vivo.1@12 Recombinant vaccinia viruses expressing IL-2 are cleared from immunodeficient mice (either athymic nude micelOJ1 or sublethally irradiated micel3), whereas vaccinia viruses not expressing IL-2 continue to replicate and the mice die of generalized vaccinia infection. Compared with mice inoculated with a control virus, antibody and T-cell
responses to vaccinia antigens and the co-expressed foreign antigen are lowered rather than elevated in normal or immunodeficient mice inoculated with IL2-expressing virus. Thus, co-expression of IL-2 in a recombinant vaccinia virus vaccine is likely to increase vaccine safety, but not the efficacy of the recombinant viral vaccine. As IL-4 acts on both T and B cells, it was considered possible that it could increase immune responses to foreign antigens co-expressed from a recombinant vaccinia virus. However, this paper demonstrates that IL-4 is unlikely to increase vaccine efficacy and for the first time documents that IL-4 can be lethal when administered in vivo.
RESULTS Table 1 lists the recombinant vaccinia viruses used in this study. Their construction is detailed in the Materials and methods section and in the references indicated in the table.
Effects of Vaccinia-expressedIL-4 in Immunodeficient Mice From the CSIRO Australian Animal Health Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia. Corresponding author: M.E. Andrew, CSIRO Australian Animal Health Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia Received 14 January 1992; accepted for publication 12 March 1992 0 1992 Academic Press Limited 1043-4666/921040281+06 $0.5.00/O KEY
WORDS:
CYTOKINE,
interleukin
Vol.
4, No.
Wmiceivaccinia
4 (July),
virus
1992: pp 281-286
To assess whether co-expressed IL-4 would clear vaccinia virus from immunodeficient mice, T-cell deficient nude mice and sublethally irradiated mice were inoculated with virus and observed for mortality. Groups of five outbred nude mice were inoculated intravenously with 107 pfu recombinant vaccinia virus expressing IL-4 (W-HA-IL4 and W-VP7-IL4) or with control virus, W-HA-TK. Before inoculation, 281
282 I Andrew and Coupar TABLE
1.
Construction
CYTOKINE,
of vacciuia
virus recombinants.
Single insertions TK-
Double insertion6 HSV TK’
VV-HA14
VV-HA-IL2’O W-HA-IL4 VV-HA-TK”’ VV-IL2-IL4 W-IL2-TK W-al-IL2 VV-ILCTK VV-ILC$FN vv-VPI-IL4 VV-KD-yIFN
vv-IL2” W-IL4
VV-VP7lh VV-KD”
virus suspensions were sonicated to ensure that the effective dose was not varied due to clumping of the virus particles. The mice were observed until all mice were either dead or recovered. In the control group inoculated with W-HA-TK, only one mouse survived and the mean time to death was 17.3k3.8 days (Table 2). In contrast, in the two groups of mice inoculated with IL-4-expressing virus, all mice died and the mean time to death was shorter than in the control group (P=O.O159 for both W-HA-IL4 and W-VP7-IL4 by Mann-Whitney two-sample test). When sublethally irradiated mice were inoculated intranasally with 107 pfu sonicated virus, there was 100% mortality in groups of mice inoculated with W-HA-IL4 or with W-HA-TK (Table 2). However, the mean time to death was much shorter in the group inoculated with W-HA-IL4, compared with the group inoculated with W-HA-TK (P < 0.0001 by t-test). In both types of immunodeficient mice, co-expressed IL-4 failed to mediate clearance of the vaccinia virus and appeared to contribute to the death of the mice, as they died more quickly than control mice. When virus titres were assayed in lung, ovary
Survival
Immunizing virus0
VV-HA-TK W-HA-IL4 vv-VP7-IL4
of immunodeficient Nude mice
% Survival
20 0 0
mice. Irradiated
MTIY
17.3 * 3.8 8.4 -r- 1.3 6.6 z? 0.9
4 (July
1992: 281-286)
and spleen of nude and sublethally irradiated mice at various times after inoculation with either W-HA-TK or W-HA-IL4, there was no consistent difference between the groups (Table 4 and data not shown). Thus, the more rapid death of immunodeficient mice inoculated with IL-4 expressing virus was not due to more rapid replication of the vaccinia virus.
Male and female CBA/H mice were inoculated either intraperitoneally or intravenousIy with 107 pfu recombinant vaccinia virus expressing IL-4 or with control virus and assessed for mortality (Table 3). In the first experiment, female mice, but not male mice, inoculated with W-HA-IL4 died. In contrast, female mice inoculated with W-HA-TK all survived (Table 3) and, in fact, showed no signs of morbidity (data not shown). Mice that were inoculated with W-HA-IL4 intraperitoneally died slightly more quickly than mice inoculated intravenously (P = 0.0556 by Mann-Whitney two-sample test). To confirm that vaccinia-expressed IL-4 was lethal in immunocompetent female mice, a second experiment was performed, using two IL-4-expressing viruses. Again, female mice inoculated with either W-HA-IL4 or W-VP7-IL4 all died, whereas female mice inoculated with W-HA-TK survived (Table 3, experiment 2). As vaccinia virus replicates to high titre in ovaries,18 the IL-4 mediated death of female, but not male mice may have been due to the highest amount of IL-4 produced in female mice. To test this, male mice were inoculated with a higher dose (108 pfu) of either W-HA-IL4 or W-HA-TK. At that dose the male mice inoculated with W-HA-IL4 died, whereas there were no deaths in the W-HA-TK inoculated mice (Table 3, experiment 3).
TABLE
3.
Survival
Immunizing Virus0
2.
4, No.
Effects of Vaccinia-ExpressedIL-4 in Normal Mice
u Foreign gene inserted into the vaccinia virus TK gene to create a TKnegative recombinant virus expressing a single foreign gene. h Foreign gene plus HSV TK gene inserted into the Hind111 F region of the TK- single insertion recombinant to create an HSV TK’ recombinant virus expressing two foreign antigen genes plus HSV TK.
TABLE
Vol.
% Survival
0 0 ND
E. Andrew
and Barbara
E.H.
Coupar
Recombinant vaccinia viruses expressing murine interleukin 4 (IL-4), either alone or together with interleukin 2 (IL-2) or y interferon (y-IFN), were constructed. Unlike IL-2, IL-4 expressing viruses were not cleared from immunodeficient mice and the mice died. As they died more rapidly than immunodeficient mice inoculated with a control virus, it appeared that IL-4 contributed to their death and the IL-4 mediated toxicity was confirmed in normal immunocompetent mice. The toxicity was reversed by co-expression of either IL-2 or y-IFN, probably due to virus clearance and therefore lower levels of circulating IL-4. Vaccinia virus-expressed IL-4 did not increase antibody or natural killer cell levels and caused a slight decrease in cytotoxic T lymphocyte levels
IL-4 was first described as a B-cell growth factor,1 but it also binds to and stimulates the proliferation of T cells.zJ It is produced by the Th, subset of T cells, whereas the other T-cell stimulating factor, IL-2 is produced by the Th, subset.4 A variety of functions for IL-4 have been reported, including inhibition of the ability of IL-2 to stimulate natural killer (NK) and lymphokine activated killer cells, but not the stimulation of antigen-specific cytotoxic T lymphocytes (CTL)5$ antagonistic interaction with another product of Th, cells, y-interferon (y-IFN)7; suppression of the production of y-IFNs and increase in the production of IgE and IgGl in in-vitro assays.9 Cytokines have been expressed from recombinant vaccinia viruses and shown to have biological activity in vivo.1@12 Recombinant vaccinia viruses expressing IL-2 are cleared from immunodeficient mice (either athymic nude micelOJ1 or sublethally irradiated micel3), whereas vaccinia viruses not expressing IL-2 continue to replicate and the mice die of generalized vaccinia infection. Compared with mice inoculated with a control virus, antibody and T-cell
responses to vaccinia antigens and the co-expressed foreign antigen are lowered rather than elevated in normal or immunodeficient mice inoculated with IL2-expressing virus. Thus, co-expression of IL-2 in a recombinant vaccinia virus vaccine is likely to increase vaccine safety, but not the efficacy of the recombinant viral vaccine. As IL-4 acts on both T and B cells, it was considered possible that it could increase immune responses to foreign antigens co-expressed from a recombinant vaccinia virus. However, this paper demonstrates that IL-4 is unlikely to increase vaccine efficacy and for the first time documents that IL-4 can be lethal when administered in vivo.
RESULTS Table 1 lists the recombinant vaccinia viruses used in this study. Their construction is detailed in the Materials and methods section and in the references indicated in the table.
Effects of Vaccinia-expressedIL-4 in Immunodeficient Mice From the CSIRO Australian Animal Health Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia. Corresponding author: M.E. Andrew, CSIRO Australian Animal Health Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia Received 14 January 1992; accepted for publication 12 March 1992 0 1992 Academic Press Limited 1043-4666/921040281+06 $0.5.00/O KEY
WORDS:
CYTOKINE,
interleukin
Vol.
4, No.
Wmiceivaccinia
4 (July),
virus
1992: pp 281-286
To assess whether co-expressed IL-4 would clear vaccinia virus from immunodeficient mice, T-cell deficient nude mice and sublethally irradiated mice were inoculated with virus and observed for mortality. Groups of five outbred nude mice were inoculated intravenously with 107 pfu recombinant vaccinia virus expressing IL-4 (W-HA-IL4 and W-VP7-IL4) or with control virus, W-HA-TK. Before inoculation, 281
282 I Andrew and Coupar TABLE
1.
Construction
CYTOKINE,
of vacciuia
virus recombinants.
Single insertions TK-
Double insertion6 HSV TK’
VV-HA14
VV-HA-IL2’O W-HA-IL4 VV-HA-TK”’ VV-IL2-IL4 W-IL2-TK W-al-IL2 VV-ILCTK VV-ILC$FN vv-VPI-IL4 VV-KD-yIFN
vv-IL2” W-IL4
VV-VP7lh VV-KD”
virus suspensions were sonicated to ensure that the effective dose was not varied due to clumping of the virus particles. The mice were observed until all mice were either dead or recovered. In the control group inoculated with W-HA-TK, only one mouse survived and the mean time to death was 17.3k3.8 days (Table 2). In contrast, in the two groups of mice inoculated with IL-4-expressing virus, all mice died and the mean time to death was shorter than in the control group (P=O.O159 for both W-HA-IL4 and W-VP7-IL4 by Mann-Whitney two-sample test). When sublethally irradiated mice were inoculated intranasally with 107 pfu sonicated virus, there was 100% mortality in groups of mice inoculated with W-HA-IL4 or with W-HA-TK (Table 2). However, the mean time to death was much shorter in the group inoculated with W-HA-IL4, compared with the group inoculated with W-HA-TK (P < 0.0001 by t-test). In both types of immunodeficient mice, co-expressed IL-4 failed to mediate clearance of the vaccinia virus and appeared to contribute to the death of the mice, as they died more quickly than control mice. When virus titres were assayed in lung, ovary
Survival
Immunizing virus0
VV-HA-TK W-HA-IL4 vv-VP7-IL4
of immunodeficient Nude mice
% Survival
20 0 0
mice. Irradiated
MTIY
17.3 * 3.8 8.4 -r- 1.3 6.6 z? 0.9
4 (July
1992: 281-286)
and spleen of nude and sublethally irradiated mice at various times after inoculation with either W-HA-TK or W-HA-IL4, there was no consistent difference between the groups (Table 4 and data not shown). Thus, the more rapid death of immunodeficient mice inoculated with IL-4 expressing virus was not due to more rapid replication of the vaccinia virus.
Male and female CBA/H mice were inoculated either intraperitoneally or intravenousIy with 107 pfu recombinant vaccinia virus expressing IL-4 or with control virus and assessed for mortality (Table 3). In the first experiment, female mice, but not male mice, inoculated with W-HA-IL4 died. In contrast, female mice inoculated with W-HA-TK all survived (Table 3) and, in fact, showed no signs of morbidity (data not shown). Mice that were inoculated with W-HA-IL4 intraperitoneally died slightly more quickly than mice inoculated intravenously (P = 0.0556 by Mann-Whitney two-sample test). To confirm that vaccinia-expressed IL-4 was lethal in immunocompetent female mice, a second experiment was performed, using two IL-4-expressing viruses. Again, female mice inoculated with either W-HA-IL4 or W-VP7-IL4 all died, whereas female mice inoculated with W-HA-TK survived (Table 3, experiment 2). As vaccinia virus replicates to high titre in ovaries,18 the IL-4 mediated death of female, but not male mice may have been due to the highest amount of IL-4 produced in female mice. To test this, male mice were inoculated with a higher dose (108 pfu) of either W-HA-IL4 or W-HA-TK. At that dose the male mice inoculated with W-HA-IL4 died, whereas there were no deaths in the W-HA-TK inoculated mice (Table 3, experiment 3).
TABLE
3.
Survival
Immunizing Virus0
2.
4, No.
Effects of Vaccinia-ExpressedIL-4 in Normal Mice
u Foreign gene inserted into the vaccinia virus TK gene to create a TKnegative recombinant virus expressing a single foreign gene. h Foreign gene plus HSV TK gene inserted into the Hind111 F region of the TK- single insertion recombinant to create an HSV TK’ recombinant virus expressing two foreign antigen genes plus HSV TK.
TABLE
Vol.
% Survival
0 0 ND
