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Testosterone impairs efficacy of protective vaccination against P. chabaudi malaria
Testosterone impairs efficacy of protective vaccination against P. chabaudi malaria F. Wunderlich*, W. Maurin, W.P.M. Benten and H.-P. Schmitt-Wrede Vaccination with surface membranes isolated from P l a s m o d i u m chabaudi-infected erythrocytes can protect BIO.A mice from the lethal outcome of P. c h a b a u d i malaria. However, the efficacy depends on gender and testosterone levels. Thus, vaccination protects over 90% of female mice, but only about 55% o f male mice and only about 34% of female mice when pretreated with testosterone for 4 weeks. The suppressive testosterone effect remains imprinted in females even at 10 weeks after the testosterone treatment. These data indicate that not only genetic but also environmental factors restrict the host's immune response to a malaria vaccine. Keywords: Malaria; Plasmodium chabaudi; vaccine; gender; testosterone
First vaccination trials with subunit malaria vaccines in male human volunteers have not been successful in the recent years (for a review, see for example Ref. 1). There is increasing information available describing genetic restrictions in malaria vaccination. In particular, genes of the HLA complex have been reported to restrict the immune response to subunit vaccines against malaria 2 4. Besides genetic restrictions, however, environmental restrictions may also exist. Indeed, it is known that endogenous environmental factors such as hormones play an important role in the modulation of the immune response 5. Recently, testosterone has been found to suppress the development of protective immunity in mice against infections with the malaria parasites Plasmodium chabaudi 6 and Plasmodium berghei 7. We therefore wished to determine whether testosterone is also critical for vaccination. Vaccination experiments were conducted with the P. chabaudi model previously developed in mice of the inbred strain B 10.A a. This model uses surface membranes of P. chabaudi-infected erythrocytes as an anti-disease rather than an anti-parasite vaccine (for terminology, see Ref. 9). The surface membranes were isolated in the form of ghosts from P. chabaudi-infected erythrocytes as detailed previously 1°. Intra-erythrocytic parasites express neo-proteins in these ghosts as characterized previouslylw12. Mice were vaccinated once with 5 x 106 ghosts which were suspended in 100#1 PBS, mixed with an equal volume of Freund's complete adjuvant, and administered subcutaneously (s.c.) at the base of the tail. After 1 week, the vaccinated mice were challenged with 106 P. chabaudiinfected erythrocytes obtained from infected mice as Division of Parasitology, Institute of Zoology, Heinrich-HeineUniversity, Universit&tsstr. 1, 4000 D0sseldorf 1, Germany. *To whom correspondence should be addressed. (Received 10 October 1992; revised 4 January 1993; accepted 19 January 1993) 0264-410)(/93/11/1097-03 © 1993 Butterworth-HeinemannLtd
described previously ~. Parasitaemia was evaluated in Giemsa-stained blood smears. Testosterone (TestovironDepot-50; Schering, Berlin, Germany) suspended in sesame oil was administered s.c. in 200 #1 portions twice per week for 4 weeks, if not otherwise stated. Female B10.A mice are normally susceptible to P. chabaudi. A challenge with 106 P. chabaudi-infected erythrocytes causes a fulminant parasitaemia peaking approximately 7-9 days post challenge, after which the majority of mice succumb to infections 8. Vaccination with surface membranes isolated from P. chabaudiinfected erythrocytes did not essentially affect the course of parasitaemia, though peak parasitaemia was significantly reduced from about 50% to 38% (Table 1). However, vaccination protected mice from the lethal outcome of the infection. Thus, the survival rates increased dramatically from about l 1% to over 90% in female mice (Table 1). The same procedure of anti-disease vaccination was not as effective in male mice. Only about 55% of male mice could be converted from non-healers to self-healers, in contrast to 84% of female mice (Table 1). That testosterone is critically involved in this gender-dependence of anti-disease vaccination can be deduced from the experiments summarized in Table 2. In these experiments, B10.A females were treated with different doses of testosterone before vaccination and subsequent infection with P. chabaudi. An impairment of vaccination was already seen at the lowest testosterone dose of 30#g. Maximal impairment occurred at the highest testosterone dose of 0.9mg, which allows only about 34% of mice to be converted from non-healers to self-healers. Intriguingly, the impairing testosterone effect on vaccination persisted for a long time after terminating the testosterone treatment. For instance, when the mice were vaccinated at different times after the testosterone treatment before challenge, vaccination was never as effective as in untreated control mice (Figure 1). Even at 10 weeks after the testosterone treatment, only
Vaccine, Vol. 11, Issue 11, 1993
1097
S h o r t p a p e r . F. W u n d e r l i c h et al. Table 1 Effect of vaccination against P. chabaudi in female and male B10.A mice. Mice were vaccinated with 5 × 106 surface membrane ghosts isolated from P. chabaudi-infected erythrocytes. After 7 days, they were challenged with 106 P. chabaudi-infected erythrocytes Infection with P. chabaudi
Sex of mice
Vaccination
Number of surviving/total mice
Days until death of succumbing mice (days p.i. ±s.d.)
Average survival rate (%)
+
3/28 37/39
11.3_+ 1.8 11.0
0/37 21/38
10.2_+ 1.8 12.0_+2.2
Female
Male
+
Peak parasitaemia % ±s.d.
days p.i. _+s.d.
11 95
50.4_+9.4 38.1 _+_5.4 (p%0.01)
6.8_+0.5 7.5 _+ 1.2
0 55
65.2_+ 15.2 47.4_+ 14.5 (p ~<0.005)
7.3_+ 1.3 7.8_+ 1.0
p.i., postinfection
Table 2 Effect of testosterone on efficacy of vaccination against P. chabaudi malaria. Female B10.A mice were treated with testosterone twice a week and challenged with 106 P. chabaudi-infected erythrocytes at 2 days after the last testosterone application. Mice were vaccinated with 5 x 106 surface membrane ghosts isolated from P. chabaudi-infected erythrocytes at 7 days before infection. Values for non-vaccinated control mice are given in parentheses
100
Dose (mg)
Period (weeks)
Number of total mice
Average survival rate (%)
0.0 0.03 0.3 0.6 0.9 0.9 0.9
0 4 4 4 4 3 1
19 (18) 10 (7) 16 (8) 17 (6) 38 (7) 20 (6) 20 (7)
95 (11 ) 70 (0) 50 (0) 41 (0) 34 (0) 55 (0) 90 (0)
A
1098
V a c c i n e , Vol. 11, I s s u e 11, 1993
-T, +vacc.
i
> •~
60
(8)
+T, +vacc.
(10)
+T,-vacc.
(10)
(m)
/
40
20
(19) (10)
(10)
Q
about 60% of the testosterone-pretreated mice could be converted to self-healers, in contrast to 100% of the control mice. The mechanisms by which vaccination with surface membranes of P. chabaudi-infected erythrocytes brings about protection in mice from the lethal consequences of P. chabaudi infections are still totally unknown. At least these data provide the first (though only phenomenological) evidence that the environmental factor testosterone can impair the efficacy of protective vaccination against P. chabaudi. Indeed, the higher the testosterone doses applied, the less effective is the vaccination-induced conversion from non-healers to self-healers. It can be deduced from our findings that testosterone does not deteriorate the protective capacity of the membranes used as the anti-disease vaccine. Obviously, testosterone impedes the vaccine from activating those immune mechanisms in mice which mediate self-healing of P. chabaudi infections. Intriguingly, this impairing effect of testosterone remains imprinted for a long time. At present, the suppressive effect of testosterone on vaccination is not yet understood and its possible relevance for the human situation is not clear. In this context, however, it is noteworthy that immunity against malaria is under similar hormonal control in mice and humans, which becomes evident, for example, in a loss of acquired immunity against malaria during pregnancy (for a review, see for example Ref. 13). Whatever the mechanisms of the testosterone-induced impairment in the efficacy of anti-disease vaccination
A
(8)
80
Infection with P. chabaudi Testosterone pretreatment
A
(10)
0
•
2
•
4
6
8
10
Weeks after T-treatment Figure 1 Vaccination of female B10.A mice after different weeks of testosterone treatment. Mice aged 10-12 weeks were treated with 0.9 mg testosterone applied twice a week for 4 weeks. At different weeks after this testosterone treatment, mice were vaccinated with 5 x 106 surfacemembrane ghosts isolated from P. chabaudi-infected erythrocytes at 7 days before challenge with 106 P. chabaudi-infected erythrocytes (©). In addition, mice treated with testosterone were not vaccinated before challenge (O), and mice not treated with testosterone were vaccinated before infection (A). Parentheses indicate total number of infected mice
prove to be, these data indicate that not only genetic but also environmental restrictions have to be overcome by an efficacious 'vaccine' against malaria. REFERENCES 1 2
3 4
5
Siddiqui, W.A. Where are we in the quest for vaccines for malaria? Drugs 1991, 41, 1-10 Patarroyo, M.E., Vinasco, J., Amador, R., Espejo, F., Silva, Y., Moreno, A. et al. Genetic control of the immune response to a synthetic vaccine against Plasmodium falciparum. Parasite Immunol. 1991, 13, 509-516 Pink, J.R.L. and Sinigaglia, F. Characterizing T-cell epitopes in vaccine candidates. Immunol. Today 1989, 10, 408-409. Quakyi, I.A., Otoo, L.N., Pmbo, D., Sugars, L.Y., Menon, A., de Groot, A.S. et al. Differential non-responsiveness in humans of candidate Plasmodium falciparum vaccine antigens. Am. J. Trop. Med. Hyg. 1989, 4, 125-134 Schuurs, A.H.W.M. and Verheul, H.A.M. Effect of gender and sex steroids on the immune response. J. Steroid Biochem. 1990, 35, 157-172
Short paper: F. Wunderlich et al.
6
Wunderlich, F., Mossmann, H., Helwig, M. and Schillinger, G. Resistance to Plasmodium chabaudi in B10 mice: influence of the H-2 complex and testosterone. Infect. Immun. 1988, 56, 2400-2406 7 Kamis, A.B. and Ibrahim, I.B. Effect of testosterone on blood leucocytes in P/asmodium berghei-infected mice. Parasito/. Res. 1989, 75, 611-613 8 Wunderlich, F., Brenner, H.-H. and Helwig, M. Plasmodium chabaudi malaria: protective immunization with surface membranes of infected erythrocytes. Infect. Immun. 1988, 56, 3326--3328 9 Playfair, J.H.L., Taverne, J., Bate, C.A.W. and de Souza, J.B. The malaria vaccine: anti-parasite or anti-disease? Immunol. Today 1990, 11, 25-27 10 Wunderlich, F., Helwig, M., Schillinger, G., Vial, H., Philippot, J. and
Spoth, V. Isolation and characterisation of parasites and host cell ghosts from erythrocytes infected with Plasmodium chabaudi. Mol. Biochem. Parasitol. 1987, 23, 103-115 11 Wunderlich, F., Helwig, M., Schillinger, G. and Speth, V. Cryptic disposition of antigenic parasite proteins in plasma membranes of erythrocytas infected with Plasmodium chabaudi. Mol. Biochem. Parasitol. 1988, 30, 55-66 12 Wunderlich, F., Helwig, M, Schillinger, G., Speth, V. and Wiser, F. Expression of the parasite protein Pc90 in plasma membranes of erythrocytes infected with P/asmodium chabaudi. Eur. J. Cel/+ Bio/. 1988, 47, 157-164 13 Alexander, J. and Stimson, W.H. Sex hormones and the course of parasitic infection. Parasitol. Today 1988, 4, 189-193
Vaccine, Vol. 11, Issue 11, 1993
1099
First vaccination trials with subunit malaria vaccines in male human volunteers have not been successful in the recent years (for a review, see for example Ref. 1). There is increasing information available describing genetic restrictions in malaria vaccination. In particular, genes of the HLA complex have been reported to restrict the immune response to subunit vaccines against malaria 2 4. Besides genetic restrictions, however, environmental restrictions may also exist. Indeed, it is known that endogenous environmental factors such as hormones play an important role in the modulation of the immune response 5. Recently, testosterone has been found to suppress the development of protective immunity in mice against infections with the malaria parasites Plasmodium chabaudi 6 and Plasmodium berghei 7. We therefore wished to determine whether testosterone is also critical for vaccination. Vaccination experiments were conducted with the P. chabaudi model previously developed in mice of the inbred strain B 10.A a. This model uses surface membranes of P. chabaudi-infected erythrocytes as an anti-disease rather than an anti-parasite vaccine (for terminology, see Ref. 9). The surface membranes were isolated in the form of ghosts from P. chabaudi-infected erythrocytes as detailed previously 1°. Intra-erythrocytic parasites express neo-proteins in these ghosts as characterized previouslylw12. Mice were vaccinated once with 5 x 106 ghosts which were suspended in 100#1 PBS, mixed with an equal volume of Freund's complete adjuvant, and administered subcutaneously (s.c.) at the base of the tail. After 1 week, the vaccinated mice were challenged with 106 P. chabaudiinfected erythrocytes obtained from infected mice as Division of Parasitology, Institute of Zoology, Heinrich-HeineUniversity, Universit&tsstr. 1, 4000 D0sseldorf 1, Germany. *To whom correspondence should be addressed. (Received 10 October 1992; revised 4 January 1993; accepted 19 January 1993) 0264-410)(/93/11/1097-03 © 1993 Butterworth-HeinemannLtd
described previously ~. Parasitaemia was evaluated in Giemsa-stained blood smears. Testosterone (TestovironDepot-50; Schering, Berlin, Germany) suspended in sesame oil was administered s.c. in 200 #1 portions twice per week for 4 weeks, if not otherwise stated. Female B10.A mice are normally susceptible to P. chabaudi. A challenge with 106 P. chabaudi-infected erythrocytes causes a fulminant parasitaemia peaking approximately 7-9 days post challenge, after which the majority of mice succumb to infections 8. Vaccination with surface membranes isolated from P. chabaudiinfected erythrocytes did not essentially affect the course of parasitaemia, though peak parasitaemia was significantly reduced from about 50% to 38% (Table 1). However, vaccination protected mice from the lethal outcome of the infection. Thus, the survival rates increased dramatically from about l 1% to over 90% in female mice (Table 1). The same procedure of anti-disease vaccination was not as effective in male mice. Only about 55% of male mice could be converted from non-healers to self-healers, in contrast to 84% of female mice (Table 1). That testosterone is critically involved in this gender-dependence of anti-disease vaccination can be deduced from the experiments summarized in Table 2. In these experiments, B10.A females were treated with different doses of testosterone before vaccination and subsequent infection with P. chabaudi. An impairment of vaccination was already seen at the lowest testosterone dose of 30#g. Maximal impairment occurred at the highest testosterone dose of 0.9mg, which allows only about 34% of mice to be converted from non-healers to self-healers. Intriguingly, the impairing testosterone effect on vaccination persisted for a long time after terminating the testosterone treatment. For instance, when the mice were vaccinated at different times after the testosterone treatment before challenge, vaccination was never as effective as in untreated control mice (Figure 1). Even at 10 weeks after the testosterone treatment, only
Vaccine, Vol. 11, Issue 11, 1993
1097
S h o r t p a p e r . F. W u n d e r l i c h et al. Table 1 Effect of vaccination against P. chabaudi in female and male B10.A mice. Mice were vaccinated with 5 × 106 surface membrane ghosts isolated from P. chabaudi-infected erythrocytes. After 7 days, they were challenged with 106 P. chabaudi-infected erythrocytes Infection with P. chabaudi
Sex of mice
Vaccination
Number of surviving/total mice
Days until death of succumbing mice (days p.i. ±s.d.)
Average survival rate (%)
+
3/28 37/39
11.3_+ 1.8 11.0
0/37 21/38
10.2_+ 1.8 12.0_+2.2
Female
Male
+
Peak parasitaemia % ±s.d.
days p.i. _+s.d.
11 95
50.4_+9.4 38.1 _+_5.4 (p%0.01)
6.8_+0.5 7.5 _+ 1.2
0 55
65.2_+ 15.2 47.4_+ 14.5 (p ~<0.005)
7.3_+ 1.3 7.8_+ 1.0
p.i., postinfection
Table 2 Effect of testosterone on efficacy of vaccination against P. chabaudi malaria. Female B10.A mice were treated with testosterone twice a week and challenged with 106 P. chabaudi-infected erythrocytes at 2 days after the last testosterone application. Mice were vaccinated with 5 x 106 surface membrane ghosts isolated from P. chabaudi-infected erythrocytes at 7 days before infection. Values for non-vaccinated control mice are given in parentheses
100
Dose (mg)
Period (weeks)
Number of total mice
Average survival rate (%)
0.0 0.03 0.3 0.6 0.9 0.9 0.9
0 4 4 4 4 3 1
19 (18) 10 (7) 16 (8) 17 (6) 38 (7) 20 (6) 20 (7)
95 (11 ) 70 (0) 50 (0) 41 (0) 34 (0) 55 (0) 90 (0)
A
1098
V a c c i n e , Vol. 11, I s s u e 11, 1993
-T, +vacc.
i
> •~
60
(8)
+T, +vacc.
(10)
+T,-vacc.
(10)
(m)
/
40
20
(19) (10)
(10)
Q
about 60% of the testosterone-pretreated mice could be converted to self-healers, in contrast to 100% of the control mice. The mechanisms by which vaccination with surface membranes of P. chabaudi-infected erythrocytes brings about protection in mice from the lethal consequences of P. chabaudi infections are still totally unknown. At least these data provide the first (though only phenomenological) evidence that the environmental factor testosterone can impair the efficacy of protective vaccination against P. chabaudi. Indeed, the higher the testosterone doses applied, the less effective is the vaccination-induced conversion from non-healers to self-healers. It can be deduced from our findings that testosterone does not deteriorate the protective capacity of the membranes used as the anti-disease vaccine. Obviously, testosterone impedes the vaccine from activating those immune mechanisms in mice which mediate self-healing of P. chabaudi infections. Intriguingly, this impairing effect of testosterone remains imprinted for a long time. At present, the suppressive effect of testosterone on vaccination is not yet understood and its possible relevance for the human situation is not clear. In this context, however, it is noteworthy that immunity against malaria is under similar hormonal control in mice and humans, which becomes evident, for example, in a loss of acquired immunity against malaria during pregnancy (for a review, see for example Ref. 13). Whatever the mechanisms of the testosterone-induced impairment in the efficacy of anti-disease vaccination
A
(8)
80
Infection with P. chabaudi Testosterone pretreatment
A
(10)
0
•
2
•
4
6
8
10
Weeks after T-treatment Figure 1 Vaccination of female B10.A mice after different weeks of testosterone treatment. Mice aged 10-12 weeks were treated with 0.9 mg testosterone applied twice a week for 4 weeks. At different weeks after this testosterone treatment, mice were vaccinated with 5 x 106 surfacemembrane ghosts isolated from P. chabaudi-infected erythrocytes at 7 days before challenge with 106 P. chabaudi-infected erythrocytes (©). In addition, mice treated with testosterone were not vaccinated before challenge (O), and mice not treated with testosterone were vaccinated before infection (A). Parentheses indicate total number of infected mice
prove to be, these data indicate that not only genetic but also environmental restrictions have to be overcome by an efficacious 'vaccine' against malaria. REFERENCES 1 2
3 4
5
Siddiqui, W.A. Where are we in the quest for vaccines for malaria? Drugs 1991, 41, 1-10 Patarroyo, M.E., Vinasco, J., Amador, R., Espejo, F., Silva, Y., Moreno, A. et al. Genetic control of the immune response to a synthetic vaccine against Plasmodium falciparum. Parasite Immunol. 1991, 13, 509-516 Pink, J.R.L. and Sinigaglia, F. Characterizing T-cell epitopes in vaccine candidates. Immunol. Today 1989, 10, 408-409. Quakyi, I.A., Otoo, L.N., Pmbo, D., Sugars, L.Y., Menon, A., de Groot, A.S. et al. Differential non-responsiveness in humans of candidate Plasmodium falciparum vaccine antigens. Am. J. Trop. Med. Hyg. 1989, 4, 125-134 Schuurs, A.H.W.M. and Verheul, H.A.M. Effect of gender and sex steroids on the immune response. J. Steroid Biochem. 1990, 35, 157-172
Short paper: F. Wunderlich et al.
6
Wunderlich, F., Mossmann, H., Helwig, M. and Schillinger, G. Resistance to Plasmodium chabaudi in B10 mice: influence of the H-2 complex and testosterone. Infect. Immun. 1988, 56, 2400-2406 7 Kamis, A.B. and Ibrahim, I.B. Effect of testosterone on blood leucocytes in P/asmodium berghei-infected mice. Parasito/. Res. 1989, 75, 611-613 8 Wunderlich, F., Brenner, H.-H. and Helwig, M. Plasmodium chabaudi malaria: protective immunization with surface membranes of infected erythrocytes. Infect. Immun. 1988, 56, 3326--3328 9 Playfair, J.H.L., Taverne, J., Bate, C.A.W. and de Souza, J.B. The malaria vaccine: anti-parasite or anti-disease? Immunol. Today 1990, 11, 25-27 10 Wunderlich, F., Helwig, M., Schillinger, G., Vial, H., Philippot, J. and
Spoth, V. Isolation and characterisation of parasites and host cell ghosts from erythrocytes infected with Plasmodium chabaudi. Mol. Biochem. Parasitol. 1987, 23, 103-115 11 Wunderlich, F., Helwig, M., Schillinger, G. and Speth, V. Cryptic disposition of antigenic parasite proteins in plasma membranes of erythrocytas infected with Plasmodium chabaudi. Mol. Biochem. Parasitol. 1988, 30, 55-66 12 Wunderlich, F., Helwig, M, Schillinger, G., Speth, V. and Wiser, F. Expression of the parasite protein Pc90 in plasma membranes of erythrocytes infected with P/asmodium chabaudi. Eur. J. Cel/+ Bio/. 1988, 47, 157-164 13 Alexander, J. and Stimson, W.H. Sex hormones and the course of parasitic infection. Parasitol. Today 1988, 4, 189-193
Vaccine, Vol. 11, Issue 11, 1993
1099