Laboratory characteristics of attenuated influenza viruses

Bull. Org. mond. Sante 1969, 41, 595-598 Bull. Wld Hlth Org. Laboratory Characteristics of Attenuated Influenza Viruses A. S. BEARE 1 The selection of attenuated mutants of a virus related to A2/Tokyo/3/67 and of the subtype A2/Hong Kong/1/68 is described. By passing the former in the presence of heated horse serum it was possible to obtain a strain wholly resistant to thermostable horse-serum inhibitor. A2/Hong Kong/1/68 was, however, rendered only partially resistant by this technique. In subsequent volunteer trials it was clear that inhibitor-resistance was a marker of attenuation. Residual inhibitor-sensitivity was associated with some remaining patho- genicity. Inhibitor-resistant viruses were infective and antigenic. Inhibitor-sensitive viruses were also attenuated by repeatedpassage at 250C. When this was attempted with the partially inhibitor-resistant form ofA2/Hong Kong/1/68, the virus became over-attenuated. In the laboratory, inhibitor-resistant viruses were shown to elute more rapidly from red blood cells than their inhibitor-sensitive parents and to grow better at 40°C. There are as yet no wholly satisfactory methods of attenuating influenza viruses for use as living vaccines. Because of their reputed genetic lability (Burnet & Bull, 1943) there have been frequent attempts at attenuation by serial passage in embryon- ated eggs. In our hands, however, this method was relatively ineffective (Beare, Bynoe & Tyrrell, 1968). Subsequently, greater success was achieved by selecting inhibitor-resistant strains of influenza A viruses and by passing virulent viruses repeatedly at low temperatures (Beare & Bynoe, 1969). By these means it was possible to produce viruses which caused only minor clinical reactions but which remained infective and potentially protective. Since then, some of the attenuated viruses have been studied further both in the laboratory and in volun- teers, and, in this paper, I shall present additional data which are relevant to the problems of live vaccine preparation. MATERIALS AND METHODS Viruses and methods of attenuation Two viruses were used; one of them, known as A2/England/501/68, was antigenically related to A2/Tokyo/3/67, and the other was the antigenic subtype A2/Hong Kong/1/68. Their origins and 1 Common Cold Research Unit, Salisbury, Wilts., England. modes of attenuation have been described previously (Beare & Bynoe, 1969). The latter consisted, briefly, in the selection of inhibitor-resistant strains of both viruses by serial egg-passage in the presence of gradually increasing amounts of inactivated horse serum, and by the repeated passage of inhibitor- sensitive A2/Hong Kong/1/68 at 25°C after a preliminary process of adaptation to growth at low temperatures. A strain of A2/England/501/68, totally resistant to thermostable horse-serum inhibitor, was obtained with relative ease. However, the Hong Kong virus could be rendered only partially resistant in spite of prolonged efforts. All attenuated viruses were repropagated at limit dilution in broth saline at 33°C before being given to volunteers. Viruses grown at 25°C usually produced no detectable haemag- glutinin, but this reappeared in normal amounts after the 33°C limit-dilution passage. Trials at the Common Cold Research Unit When 2 or more viruses were given to volunteers in the same trials, the volunteer groups were of comparable susceptibility to infection. The effects of the virus inoculations were assessed by the incidence and severity of the clinical reactions, virus excretion and rises of haemagglutination-inhibiting (HI) anti- body 2-3 weeks later, according to methods previ- ously described (Tyrrell, 1963; Beare, Bynoe & Tyrrell, 1968). 2409 595- A. S. BEARE TABLE 1 COMPARISON OF THE EFFECTS PRODUCED IN VOLUNTEERS 'BY INHIBITOR-SENSITIVE AND INHIBITOR-RESISTANT A2 VIRUSES a Viruses b A2/England/501 /68 (inhibitor-sensitive) A2/England/501/68(inhibitor-resistant) A2/Hong-Kong/1 /68(inhibitor-sensitive) A2/Hong Kong/1/68(after 3 passages with (horse serum) A2/Hong Kong/1/68 (after 11 passages with horse serum) Virus dose Virus(EID54) excretion 105.5 105.5 105.0 105-5 105.5 7/8 6/9 3/5 c 10/15 9/16 Hi Severe or moderate antibody rises clinical symptoms 6/8 5/8 3/5 c 13/15 12/16 6/8 0/9 3/5 c 5/15 1/16 a EID5o = 50 % egg-infecting doses. Numerators are numbers of responses, denominators numbers of volunteers providing specimens (Tables 1-4). b Complete passage histories of viruses are given by Beare & Bynoe (1969). c 2 people had initial antibody and were not infected. Laboratory studies of viruses The following characters of original viruses and of their attenuated mutants were examined, (1) neura- minidase activity, (2) thermostability of haemag- glutinin and neuraminidase, (3) resistance to inhibi- tors present in heated horse serum, (4) rate of elution from human red blood cells and (5) replication at 40°C and 25°C. The methods used were those of Simpson & Hirst (1961), Laver & Kilbourne (1966) and Willers & Beare (1969). RESULTS Volunteers were inoculated with inhibitor-sensitive :and inhibitor-resistant viruses, and the effects are shown in Table 1. Clearly the inhibitor-resistant strain of A2/England/501/68 was less pathogenic than its parent, although it remained infective and stimulated antibody formation. The 2 strains of A2/Hong Kong/l/68, which had been passed with horse serum, were also attenuated but incompletely so; and the strain which had had only 3 passes with horse serum was appreciably more pathogenic than that which had had 11 such passes. When the inhibitor-sensitive form of the Hong Kong virus had been passed 6 times at 25 C, its pathogenicity was again much modified although it still produced a few undesirable reactions (Table 2). Subsequently these passages were continued, and this apparently improved the adaptation since haemag- glutinin appeared at the 12th pass. The later passes have not yet, however, been given to volunteers, nor have their basic and antigenic characters been defined. Efforts were made to enhance the attenuation of the inhibitor-resistant form of A2/Hong Kong/l /68 TABLE 2 EFFECTS PRODUCED IN VOLUNTEERS BY INHIBITOR-SENSITIVE STRAINS OF A2/Hong Kong/1/68 ADAPTED TO GROWTH AT 250C Virus dose Virus excretion Hi antibody Severe or moderate(EIDso) I rises clinical symptoms 596 Number of passes at 25"C LABORATORY CHARACTERISTICS OF ATTENUATED INFLUENZA VIRUSES TABLE 3 THE EFFECT OF LOW-TEMPERATURE PASSAGE ON THE VIRULENCE OF AN INHIBITOR- RESISTANT STRAIN OF A2/Hong Kong/1/68 a Number N1me iu ds iu H nioy Severe orof passes with Number Virus dose Virus HI antibody moderate clinical horse serum ofpasses (EID50) excretion rises symptoms at 251C 11 2 106.0 10/19 8/20 1/20 0 6 1Os.5 10/18 14/20 6/20 a The trials with the 2 viruses were performed simultaneously and the volunteer groups were initially of similar susceptibility to infection. by adapting it to growth at 25°C. This proved difficult, and the virus was lost several times in the course of these experiments. Eventually, a virus which had had 11 passes with horse serum and 2 at 25°C was given to volunteers in 2 separate trials which are shown together in Table 3. Although it produced 1 unpleasant reaction, it may nevertheless have been over-attenuated since it produced no serological change in 12 people, 6 of whom had been without initial circulating antibody. An inhibitor- sensitive low-temperature mutant, which was used simultaneously for comparison, was antigenically more effective. When original and attenuated viruses were exam- ined in the laboratory, most of the properties studied were found to be similar (Willers & Beare, 1969). No differences were seen in the thermostability of haemagglutinins and neuraminidases; the main positive findings were more rapid elution from red blood cells by inhibitor-resistant viruses than by their inhibitor-sensitive counterparts and greater yields of the former at 40°C (Table 4). There were, however, no evident dissimilarities in neuraminidase activities when assayed with fetuin substrate. DISCUSSION The experiments described provide considerable evidence that resistance to thermostable horse-serum inhibitor is a marker of attenuation in influenza A viruses. Choppin & Tamm (1960) considered most seeds of these viruses to be mixtures of inhibitor- sensitive and inhibitor-resistant particles, and the success achieved in the attenuation of A2/England/ TABLE 4 LABORATORY CHARACTERISTICS OF NON-ATTENUATED AND ATTENUATED INFLUENZA A VIRUSES a Reciprocal HI titre Haemagglutinin Virus yield Virus of inactivated horse erythfrocytes per 0.1 ml at 400Cserum ~~in 2 hours(EDo A2/England/501/68(inhibitor-sensitive) > 100 000 18 4.7 A2/England/501/68(inhibitor-resistant) <1 50 7.0 A2/Hong Kong/1/68(inhibitor-sensitive) >1 000 18 3.6 A2/Hong Kong/1/68(3 passes with horse serum) 120 25 5.5 A2/Hong Kong/1/68(11 passes with horse serum) 50 37 4.4 a Much of this work was performed by Dr Hildegard Willers, a visiting WHO Research Fellow at the Common Cold Research Unit. 597 598 A. S. BEARE 501/68 would seem to have been due to the virtually complete neutralization of the former. If this is so, then the residual pathogenicity of A2/Hong Kong/l/ 68 after 11 passes with horse serum can be attributed to the survival of inhibitor-sensitive particles which, for some reason, cannot always be eliminated by this technique. So far we have not attempted to measure the epidemiological effectiveness of inhibitor-resistant viruses against naturally infecting strains. This has, however, been done on a large scale in the USSR and, assuming that the strains are antigenically suitable, there seems to be every reason to expect that they should give substantial protection. Never- theless, alternative methods of producing vaccine viruses are clearly essential since the selection of inhibitor-resistant viruses cannot always be accom- plished (Choppin & Tamm, 1960; Lief, 1964). The somewhat empirical method of attenuating viruses by adapting them to growth at reduced temperatures which I have described could certainly be greatly improved by the use ofplaquing techniques (Maassab, 1967, 1969). Much, however, remains to be done in influenza virus genetics to ensure that laboratory mutants would be simultaneously infective, attenu- ated and antigenically appropriate. Attempts to adapt an inhibitor-resistant strain of A2/Hong Kong/1/68 to growth at 25°C encountered difficulties. Presumably this was related to the different growth properties of inhibitor-sensitive and inhibitor-resistant viruses (Table 4) and it may perhaps be possible to complete the attenuation of the virus at a somewhat higher temperature. In the immediate future, fundamental. work on the pro- perties of inhibitor-resistant viruses and on factors which determine influenza virus pathogenicity would probably contribute most to the further development of live influenza vaccines. 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