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1. (WO1980002504) VACCIN DE BACTERIES GENETIQUEMENT ATTENUEES AVEC MUTATIONS MULTIPLES DU MEME PHENOTYPE
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AMENDED CLAIMS
(received by the International Bureau on 24 November 1980 (24.11.80))

19. The strain of any of Claims 17 or 18, wherein said bacterium is H. influenzae.

20. The strain of Claim 17, wherein said bacterium is H. influenzae A214-A/3.

21. The strain of Claim 17, wherein said bacterium is H. influenzae A214-A/3-C2/13.

22. A method of preparing a bacterial vaccine which comprises altering a virulent bacterial strain by introducing therein two or more mutations which convey the same phenotype, thereby genetically attenuating said strain by rendering said strain avirulent while allowing said strain to retain immunogenicit .

23. The method of Claim 22, wherein said phenotype is a temperature-sensitive phenotype.

24. A method of protecting an animal against
infectious diseases which comprises inoculating said animal with a vaccine composition according to any of Claims 1, 2, 5, 8 or 9.

25. A method of protecting a human against infection by H. influenzae which comprises inoculating said human with a vaccine composition according to any of Claims 10-12.

11. The vaccine composition of Claim 10, wherein said strain of H. influenzae contains at least two different mutations of the same temperature-sensitive phenotype.

12. The vaccine composition of Claim 11, wherein said ts phenotype suppresses the growth of said H.
influenzae stra'in at normal human body temperature.

13. The vaccine composition of Claim 10, wherein said bacterium is H. influenzae A214-A/3.

14. The vaccine composition of Claim 10, wherein said bacterium is H. influenzae A214-A/3-C2/13.

15. The vaccine composition of Claim 2, wherein said phenotype is such that replication of said bacterium will continue for at most seven divisions after
transfer to the non-permissive temperature.

16. The composition of Claim 15, wherein the phenotype of said bacterium is such that replication is allowed for three divisions after transfer to the non-permissive temperature.

17. An avirulent bacterial strain derived from a virulent bacterial strain by containing at least two different mutations of the same phenotype, which render said strain avirulent while permitting said strain to retain immunogenicity.

18. The strain of Claim 17, wherein said phenotype is a temperature-sensitive phenotype which suppresses growth at normal human body temperature. Claims

1. A vaccine composition comprising a genetically-attenuated bacterial strain which contains at least two mutations of the same phenotype which strain is rendered avirulent while retaining immunogenicity, together with an inert, imrnunologically-acceptable carrier.

2. The vaccine composition of Claim 1, wherein said phenotype is a temperature-sensitive phenotype.

3. The vaccine composition of Claim 2, wherein said strain contains three mutations of the same temperature-sensitive phenotype.

4. The vaccine composition of Claim 2, wherein said temperature-sensitive phenotype suppresses the replication of said strain at normal human body temperature.

5. The vaccine composition of Claim 1, wherein said phenotype suppresses the replication of said strain in the absence of a growth factor.

6. The vaccine composition of Claim 5, wherein said growth factor is selected from the group consisting of antibiotics, and non-host metabolites.

7. The vaccine composition of Claim 6, wherein said growth factor is an antibiotic.

8. The vaccine composition of Claim 1, wherein said phenotype suppresses the formation of a toxic toxin from said strain.

9. The vaccine composition of Claim 1, wherein said phenotype suppresses the invasiveness of said strain.

10. The vaccine composition of Claim 1, wherein said bacterium is Haemophilus influenzae.

11. The vaccine composition of Claim 10, wherein said strain of H. influenzae contains at least two different mutations of the same temperature-sensitive phenotype.

12. The vaccine composition of Claim 11, wherein said ts phenotype suppresses the growth of said H. influenzae strain at normal human body temperature.

13. The vaccine composition of Claim 10, wherein said bacterium is ik influenzae A214-A/3.

14. The vaccine composition of Claim 10, wherein said bacterium is H. influenzae A214-A/3-C2/13.

15. The vaccine composition of Claim 2, wherein said phenotype is such that replication of said bacterium will continue for at most seven divisions after transfer to the non-permissive temperature.

16. The composition of Claim 15, wherein the phentoype of said bacterium is such that replication is allowed for three divisions after transfer to the non-permissive temperature.

17. A bacterial strain which contains at least two different mutations of the same phenotype, which strain is rendered avirulent while retaining immunogenicity.

18. The strain of Claim 17, wherein said phenotype is a temperature-sensitive phenotype which suppresses growth at normal human body temperature.

19. The strain of any of Claims 17 or 18, wherein said bacterium is \_ _ influenzae.

20. The strain of Claim 17, wherein said bacterium is H. influenzae A214-A/3.

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21. The strain of Claim 17, wherein said bacterium is H. influenzae A214-A/3-C2/13.

22. A method of preparing a bacterial vaccine which comprises altering a virulent bacterial strain by introducing therein two or more mutations which convey the same phenotype, thereby genetically
attenuating said strain by rendering said strain avirulent while allowing said strain to retain immunogenicity.

23. The method of Claim 22, wherein said phenotype is a temperature-sensitive phenotype.

24. A method of protecting an animal against infectious diseases which comprises inoculating said animal with a vaccine composition according to any of Claims 1, 2, 5, 8 or 9.

25. A method of protecting a human against infection by H. influen-zae which comprises inoculating said human with a vaccine composition according to any of Claims 10-12.

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