Anti-IFN-γ-treated mice — a model for testing safety of live Salmonella vaccines

Anti-IFN-y-treated m i c e - a model for testing safety of live Salmonella vaccines Anna Muotiala Salmonella enterica serovars Typhimurium and Enteritidis are facultative intracellular pathogens which cause in mice a disease similar to human typhoid fever caused by serovar Typhi. An essential phase in the infection process is bacterial replication inside cells of the liver and spleen; the rate of replication is restricted by interferon-y (IFN-~) produced early in the infection ~. In the present study the effect of IFN-7 was neutralized in vivo with monoclonal antibody and the fate of bacteria in the liver and spleen in these mice compared with that in control mice after intravenous challenge. The depletion of IFN-7 remarkably sensitized the mice to Typhimurium infection. Five different Typhimurium and Enteritidis candidate vaccine strains were tested. Only one of them, the aroA mutant SL3261, was avirulent also in anti-IFN-7 treated mice. This finding may have important implications for the safety of live attenuated Salmonella vaccines since immunosuppression is likely to cause a state of reduced production of IFN-~. Keywords:Salmonellaenterica;livevaccine;safety;attenuation;interferon-7;immunosuppression; mice

INTRODUCTION An increasing interest in live viral and bacterial vaccines can be seen in research for the development of new vaccines 2. Their main advantage is their ability to induce cell-mediated immunity, which is believed to be needed for protection from intraceUular pathogens. The possibility of administering live vaccines by the oral route is an additional advantage 3. However, a key issue before accepting a live vaccine for human use is the possible risk of disease when inadvertently immunizing immunocompromised individuals necessarily present in any large population. Because of the general advantages of live vaccines, recombinant live vaccines as carriers of new antigens are of special interest4'5. The two most studied vectors for such recombinant vaccines are vaccinia virus and Salmonella enterica serovar Typhi, both of which have a large capacity to incorporate foreign genetic materiaP '6. They both have the advantage over other possible vectors that they have already been extensively used as live human vaccines 2. However, both are secreted by vaccinees, with the risk of further spread to unimmunized persons. The large scale use of vaccinia virus in the smallpox control programmes proved very effective, but was associated with more severe local and general reactions than any vaccine now in use 2. Because of this there is a strong reluctance to reintroduce vaccinia for human use. On the other hand, the present possibilities of attenuation Molecular Biology Unit, National Public Health Institute, Mannerheimintie 166, SF-00300 Helsinki, Finland. (Received 18 June 1991 ; revised 30 August 1991 ; accepted 14 October 1991) 0264-410X/92/040243-04 © 1992 Butterworth-HeinemannLtd

of the virus by genetic manipulation may result in new vaccinia-based live vaccines that could be much safer than the traditional smallpox vaccine 7-x2. It will, however, be difficult to prove their safety. There have even been alarming reports of fatal vaccinia infection in patients with acquired immunodeficiency syndrome (AIDS) after inoculation with a new recombinant vaccinia-based vaccine preparation 13-15. Oral Salmonella vaccines are of particular interest because they can stimulate a mucosal antibody response 16. The Typhi strain Ty21 a has been used in extensive clinical trials without adverse reactions attributable to the vaccine 17'~s. A problem with this strain is that the mechanism and genetic basis of its attenuation are uncertain 19-2~. Therefore there is much interest in developing new genetically well defined attenuated strains 4,22. Salmonella serovars Typhimurium and Enteritidis are pathogens which cause a disease in mice (mouse typhoid) that closely resembles human typhoid fever. The basic development of Salmonella vaccines has been carried out in mice, using mouse-pathogenic serovars since Typhi infects only humans. An essential phase in the bacteraemic infection process in both human and mouse typhoid is intracellular bacterial replication, primarily in the liver and spleen. The rate of bacterial replication in these organs is a crucial determinant of the outcome of the infection. We have recently shown that interferon-gamma (IFN-~) produced during Salmonella infection influences this rate of intracellular replication, and that depletion of IFN-7 in the mice greatly enhances the virulence of Typhimurium ~. Comparable depletion of IFN-~ may occur in humans in some types of immune deficiency states 23, and might lead in these individuals to an increased virulence of attenuated Salmonella vaccines.

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Safety of live S a l m o n e l l a vaccines: A. Muotiala Table 1 Description of the Salmonella enterica serovars Typhimurium and Enteritidis strains used Strain

Relevant properties

Reference

SL2965 SL2982 SL2996 SL1292

Carries virulence plasmid Virulence plasmid-cured Enteritidis plasmidless Q1 prototroph his-rfb deletion aroA mutant

33 33 34 28 29 31

his-515

SL3261

MATERIALS AND METHODS Bacteria

S. enterica serovars Typhimurium and Enteritidis strains used are shown in Table 1. All strains were passaged by intravenous (i.v.) injection of ~105 bacteria/mouse and reisolated from the liver 3-4 days later. The mouse-passaged strains were stored in 1% skim milk 25 at -70°C until used. The bacteria were grown at 37°C on L-agar plates or in L-broth 26 under aeration into late exponential phase. Mice

7

6

Balb/c/Han/HY female mice bred at the Laboratory Animal Unit of the University of Helsinki, Helsinki, were used at the age of 8-10 weeks.

5

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Anti-IFN- 7 treatment

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0

jl~

I 2

I 3

I 4

I 5

I 6

I 7

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Days postchallenge

Figure I Increased virulence of Typhimurium in anti-IFN-7-treated Balblc mice. Mice were injected I h before challenge i.p. with 25/~g of anti-IFN-7 in a volume of 200/~I ( ) or with the same amount of diluent only (. . . . ). Arrow shows the challenge dose of SL2965 used i.v. Mice were killed I, 2, 4 and 7 days after challenge and the numbers of bacteria in the liver determined by culture. Each point represents the geometric mean of three mice, bars indicate the s.e. By day 7 all the treated mice had died (deaths occurred on days 5, 6 and 7)

Hamster anti-mouse IFN- 7 monoclonal antibody (25/~g) (Gentzyme Corporation, Boston, MA, USA) diluted in 200/~1 0.1% bovine serum albumin in phosphate-buffered saline was injected intraperitoneally (i.p.) 1 h before challenge. Control mice received the diluent only. Infection The i.v. challenge was prepared and given in a volume of 200 #1 as described previously27. The outcome of the infection was evaluated by counting the bacteria in the liver and spleen 1, 2, 4 and 7 days after challenge. Each point shown in Figures I and 2 represents the geometric mean of three mice with the standard error of the mean (s.e.) indicated. The statistical significance of differences was calculated using Student's t test on log-transformed data.

7

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SL1292

his-515

SL3261

SL2982

RESULTS Increased virulence of Typhimurium in anti-lFN-7-treated Baib/c mice

SL2996

Fifum 2 The effect of anti-IFN-7 treatment on bacterial numbers in mice challenged with attenuated strains. Balb/c mice were injected 1 h before challenge i.p. with 25 #g of anti-IFN- 7 in a volume of 200/~1 ( [ ] ) or with the diluent only (i-]). Arrows show the challenge dose given i.v. for each strain. The strains were the naturally avirulent SL1292, rough his-515, aroA SL3261 and plasmidless Typhimurium SL2982 and plasmidless Enteritidis SL2996. Each column represents the geometric mean of the numbers of viable bacteria in the liver of three mice on day 4 after challenge, bars indicate the s.e.

Whether such restoration of virulence can indeed take place, and if so, whether some ways of attenuation would be safer than others remains to be answered. The author has now tested this with several strains attenuated by different mechanisms. Balb/c mice, which are highly susceptible to Typhimurium infection24, were used for these experiments instead of the previously used relatively resistant mice1; it is shown here that IFN-7 has a major impact on the outcome of the disease also in the susceptible strain.

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The mice were challenged with the virulent Typhimurium strain SL2965 at a dose of 102.8 (50% lethal dose is < 10). One group was injected 1 h before challenge with anti-IFN-),, the control group with the diluent only. The outcome of the infection was followed by enumerating viable bacteria from liver and spleen on days 1, 2, 4 and 7 after challenge. At each time point, the numbers of bacteria in the liver (Figure 1) and spleen (not shown) of the mice treated with anti-IFN- 7 were higher than those of untreated, mice. The difference was statistically significant (p < 0.001 ) by day 4 when the anti-IFN-?treated mice had 500-fold (2.7 logs) more bacteria in the liver than the controls. By day 7 after challenge all the anti-IFN-?-treated mice had died, whereas all the untreated mice were alive. Effect of anti-IFN- 7 treatment on the multiplication of attenuated Salmonella strains in the mice On the basis of the kinetic study described above we chose day 4 after challenge as the time point for assessing the virulence of the attenuated strains. Four different types of attenuated strains were used. SL1292 was the type strain of line Q1, an avirulent natural isolate2S ; the

Safety of live Salmonella vaccines: A. Muotiala

basis of its attenuation is not known. The strain his-515 is a Typhimurium mutant, which is rough, of chemotype Ra, because of a deletion in its rfb gene cluster 29. This strain has been shown to survive but not replicate in the liver and spleen of mice and to induce protective immunity3°. SL3261 is an aroA Typhimurium mutant, which is unable to replicate in mouse tissues because of its inability to synthesize several nutritionally important metabolites of the aromatic aminoacids pathway, including dihydroxybenzoate and enterochelin31'32. It has been widely used as a carrier of recombinant antigens for immunization. The fourth type of attenuated strains were derivatives of Typhimurium (SL2982, Ref. 33) and Enteritidis (SL2996, Ref. 34) cured of their virulence plasmid. The ability of these and several other serovars of Salmonella to grow in the liver and spleen of mice has been shown to be dependent on the presence of a large virulence plasmid 34'a5, and plasmid-cured strains have been suggested as vaccine candidates 36. We challenged groups of anti-IFN-7-treated and control Balb/c mice with ~ 103 bacteria of each of the attenuated strains. The numbers of bacteria recovered in the livers of the control mice 4 days after challenge were at most !.4 logs above the challenge dose indicating little or no replication, consistent with the previously demonstrated lack of virulence of these strains (Figure 2). Anti-IFN-7 pretreatment of the mice increased significantly (p < 0.01 ) the virulence of SL1292, his-515 and both plasmidless strains. Bacterial numbers in the livers of the anti-IFN-7-treated mice on day 4 were 16-(SL1292) to 1600-fold (SL2982) higher than in the control mice. By contrast, the aroA strain SL3261 showed no increase in virulence, as indicated before in studies , using Salmonella-resistant mice ~.

DISCUSSION Treatment of mice with anti-IFN- 7 was found to increase the virulence of several attenuated Salmonella strains. IFN-~, is an important component of the host defence system for restricting the intracellular replication of Salmonella in the liver and spleen 1'37'38. The effect of IFN-~ has been assumed to be bacteriostatic, because anti-IFN-7 treatment did not sensitize mice to infection by a non-replicating nutritional mutant strain of Salmonella 1. The strains that in the present study showed increased virulence in the anti-IFN-7-treated mice included a naturally avirulent strain, a rough strain and strains cured of their virulence plasmid. The functional basis of the attenuation of these strains is not known ; a cluster of four genes on the virulence plasmid has been identified as required for virulence39-42 but their gene products are not yet known. It seems, however, clear that none of these attenuated strains has a nutritional defect as the basis of its attenuation. Anti-IFN-7 did not increase the virulence of all attenuated strains tested: the aroA mutant SL3261 still remained avirulent. The nutritional defect of the aroA mutant makes the strain unable to replicate in mammalian tissues where suitable intermediates or end products of the aromatic pathway are not available 32. The avirulence of SL3261 in the anti-IFN-7-treated mice is consistent w i t h o u r .previous findings in Salmonella-resistant (CBA x C57B1/6) Fl-hybrid mice i. Mice with -other types of immunodeficieneies - caused by irradiation or

the inherited functional B-lymphocyte defect in xid mice have likewise been shown to be resistant to this attenuated strain 43. The rough Ra-strain was able to replicate in the anti-IFN-7-treated mice. The Ty21a vaccine strain was originally thought to be attenuated because of its rough character based on the 9alE mutation. However, it has later been shown that the strain also contains several other mutations all of which have not been defined~9. Furthermore, a new galE mutant of Typhi has been found virulent 2~. Another lipopolysaccharide mutant, a semirough double mutant rfc pmi has recently been suggested as a vaccine candidate44. According to the data presented here one would need to be very cautious with this type of attenuated strain. The mode of attenuation indeed seems to be decisive for the behaviour of putative Salmonella vaccine strains, and the present results show that such strains should be tested for attenuation in immunodeficient animals. The anti-IFN-~-treated mice are one possible model for testing the safety of live Salmonella vaccines. Several inherited and acquired defects cause IFN-~ deficiency in humans 23. T cells of newborns secrete considerably less IFN- 7 upon mitogen stimulation than do T cells from adults. Patients with rheumatoid arthritis, systemic lupus erythematosus, psoriasis, multiple sclerosis, viral diseases such as AIDS and viral hepatitis show deficient production of IFN-7. AIDS patients have been described to have an increased incidence rate of bacteraemic salmonellosis, notably with opportunistic Salmonella serovars that cause only local enteric infections in healthy persons 45. The data in this paper suggest a likely mechanism for this: lack of IFN- 7 production in the patients would increase the virulence of otherwise non-virulent Salmonellae. In summary, the results presented call for caution in accepting attenuated Salmonella strains for use as vaccines against Typhi infection or as carriers of other antigens. Attenuation should be carefully selected as caused by factors, e.g. nutritional requirements, that are unlikely to be affected by immunosuppression. -

ACKNOWLEDGEMENT The author is grateful to Professor P. Helena M~ikel~i for critical reading of the manuscript. REFERENCES 1 2 3 4

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