General discussion—Session IV

General Discussion-Session IV DR E. D. KILBOURNE. The great importance of finding virulence markers must make us quite cautious about accepting evidence that we actually have them in hand. The suggestion that inhibitor resistance may correlate with virulence for man raises the question whether, in fact, the residual (albeit lesser) sensitivity to inhibitor of the partially attenuated Hong Kong strain used in the investiga- tion reported by Dr Beare (see page 595) does not imply that this virus remains a mixture of so-called " +" and " -" particles. The elegant work of Choppin & Tamm with the early A2 strains showed quite clearly that viruses of intermediate reactivity with inhibitors are mixtures of such particles. The residual virulence of the virus may be explained in this way, if, in fact, the correlation of inhibitor sensitivity and virulence is valid. Another point relating to Dr Beare's paper con- cerns the importance of comparing the virulence of egg-passaged lines that have been subjected to selective procedures with parallel control lines at the same passage level. Even then, the numbers of viral generations may not be identical because of the use of the selection technique. DR A. S. BEARE. The residual inhibitor-sensitivity of the partially inhibitor-resistant strain of A2/Hong Kong/l/68 reflected the persistence of inhibitor- sensitive particles and the virus seed used was in fact a mixture of the 2 types of particles. The inhibitor- sensitive component could not, however, be elimin- ated, and this was why the virus could not be wholly attenuated for man by passage in the presence of horse serum. DR F. S. LIEF. In agreement with Dr Tyrrell, we, too, realized some years ago that live influenza virus vaccines may have some very definite advantages over killed virus vaccines. We were also conscious of the problems such an approach to influenza vaccina- tion might pose. As an initial point of attack on the problem we decided to seek out a viral marker which might be useful for selecting out a vaccine strain and chose sensitivity (S+) and non-sensitivity (S-) to inhibitors present in sera and nasopharyn- geal secretions as the first such markers to be studied. Accordingly, cloned S+ and S- substrains were selected out from a series of A2 viruses. Two such contrasting substrains derived from a 1957 isolate were used for preparation of candidate vaccines in leucosis-free embryonated eggs. Both substrains were at the 14th egg-passage level. The human volunteer trials were performed under conditions of isolation and tight medical surveillance in young adult female prisoner volunteers. When administered by spray into the nasopharynx at doses of 107 ElD50, neither substrain proved to be infective. However, when approximately 109 E1D50 were ad- ministered, virus could be recovered over a period of 5-6 days from all of the 12 volunteers who received the S + vaccine, but from only a portion of those given the S- strain. All the volunteers yielding virus showed marked increases in serum neutralizing antibody to the vaccine strain as well as to a series of later A2 strains including A2/Japan/ 170/62, A2/Rochdale/1/65 and A2/New Jersey/68. Disappointingly, however, on going back to these sera recently, no significant increases to the Hong Kong variant could be detected. Of interest was the finding that infection could be induced in persons with pre-existing homologous serum neutralizing antibody titres as high as I: 256. Furthermore, when volunteers sprayed with placebo were housed with the vaccinees, the S+ strain was found to be transmissible to contacts. Such transmission was not detected with the S- vaccine. These results were duplicated in 2 different trials, one carried out in the winter and the other in the summer. No influenza- like illness occurred in any of the subjects. At most, a short-lived afebrile coryza became evident in 2 S+ vaccinees and in 1 of the S- vaccinees who shed virus. Unfortunately, nasal washings for assay- ing local antibody could not be collected during these studies. Under the threat of the 1967-68 epidemic, the S+ strain was again administered under isolation to successive groups of new volunteers. About 30 per- sons were vaccinated. During this trial saliva specimens were collected for local antibedy determin- ations. However, by the time this trial was carried out, the vaccine had been stored at - 70°C for 31/2 years and had lost its infectivity. 'Ihus, no virus was recovered from any of the vaccinees. None the less, examination of their saliva specimens by haemagglutination-inhibition or hacmadsorption neutralization revealed that 60% of the vaccinees sustained increases in local antibody to the vaccine and later A2 strains. Of these subjects with local antibody rises, 30% showed concomitant rises in, serum neutralizing antibodies as well. - 611 GENERAL DISCUSSION-SESSION IV I cannot explain why our experience with the S + strain, especially with respect to pathogenicity, is at variance with the English data. Perhaps I was just fortunate in choosing the A2/2946/57, an isolate which came from a benign outbreak at Bryn Mawr College in Pennsylvania. Or perhaps it is a matter of the relatively small numbers of volunteers tested. In any event, we now have selected out similar contrasting cloned substrains from a Hong Kong virus from which vaccines are now being prepared. We hope to be able, in the very near future, to test these vaccines in further volunteer trials in order to deny or confirm the thesis that inhibitor-sensitivity may well serve as a marker for choosing an efficacious live vaccine strain. DR D. HOBSON. The methods of virus attenuation used by Dr Mills on the one hand, and by Dr Beare or Dr Maassab on the other hand, may depend on completely different fundamental processes with con- siderable practical implications in deriving efficient vaccines. Dr Mills' methods are undoubtedly producing and selecting point mutants. However, selection of attenuants by time-consum- ing serial passage may depend on mutants arising during the course of the serial procedures, or on selecting pre-existing mutants by a process of selective enrichment from a mixed initial virus population (as Dr Kilbourne has just mentioned in the case of Dr Choppins' R-15 experiments). As Dr Ikic has said (see page 608), speed is of the essence in deriving safe attenuated vaccines from newly emerging antigenic strains. A possible rapid method of screening for suitable variants is to adapt the well-known bacteriophage technique of replica plating under a variety of selective conditions, start- ing from the original strain treated with mutagenic agents. However, one wonders what the views of vaccine control authorities would be on a living vaccine virus which has been exposed to a chemical mutagen. DR H. A. BLOUGH. Has Dr Mills examined his ts mutants of influenza to see if they have reverted to the wild type, especially following animal passage? DR J. MILLS. Reversion frequencies of these mutants have not yet been studied in detail, but the procedure used in isolating them selected against unstable mutants. Tests to determine reversion frequencies are in progress. DR D. A. J. TYRRELL. Mackenzie has observed that all temperature-sensitive strains became attenu- ated for mice. Viruses which have a temperature- sensitive step in the haemagglutinin might not be useful as donors of attenuation if we wished to obtain an attenuated strain by recombining a new virus with a known attenuated strain. DR J. MILLS. The two mutants we have tested do not produce haemagglutinin at the restrictive temperature of 39°C, yet prior infection with either mutant offers protection against subsequent chal- lenge. This suggests that neutralizing antibody to the haemagglutinin is produced. It may be, however, that a mutant which was able to produce haemag- glutinin protein would offer even better protection against subsequent challenge. DR J. C. MCDONALD. In earlier trials of live influenza vaccine considerable trouble was taken to use volunteers without neutralizing antibody to the vaccine strain. Without this, it was extremely difficult to interpret the clinical and serological find- ings. I am concerned that, in the absence of this precaution, work in volunteers may not be closely relevant to the results to be expected in susceptible persons exposed to natural infection. Would Dr Tyrrell or Dr Beare comment on this problem? DR D. A. J. TYRRELL. The first volunteers were given Hong Kong virus by Dr Beare at a time when the virus was not epidemic in Britain and most people had no detectable antibody. DR F. M. DAVENPORT. When selecting mutants for candidate vaccine strains, attention should also be paid to speed in adaptation to growth in eggs, since eggs would be the ultimate production source. Dr Kilbourne's recombination efforts to produce strains for killed vaccines might usefully be tried with live mutants. SIR CHRISTOPHER ANDREWES. In 1948 and 1949 it became possible to see that prevention of polio- myelitis by immunization was not just a vague dream; with the publication of Dr Francis' report less than a decade later, we knew that it could be achieved. Similar goals are in sight for measles and rubella. We have now, however, been striving to immunize against influenza for 35 years, and while we can protect particular groups with moderate success, we cannot halt nation-wide epidemics. 612 GENERAL DISCUSSION-SESSION IV As we all know, the antigenic instability of influ- enza A virus is a major obstacle. Perhaps an even greater one is the unwillingness of people to have injections at intervals, and not once or twice for all, against a disease which does not frighten them (they forget 1918-19). If we could but find the ideal attenuated live influenza vaccine, it would solve many problems. Dr Tyrrell pointed out that it could be produced more quickly in adequate amounts; it would ensure that effective antibody was right there in the nose, where it was wanted; it could be administered rapidly, without injection, and would thus be more acceptable. One would have, however, to be certain that the attenuation was sufficient, even for children; otherwise we should not have sufficiently wide acceptance. I believe that one important clue to the epi- demiology of influenza or other respiratory infections lies in the existence of relatively few persons who are lavish excretors and spreaders of virus. It is likely that many children are such people and that effective immunization of children might be a most rewarding undertaking, as Dr Davenport and Dr Monto have urged. Even if our vaccines did not wholly protect, they might eliminate these dangerous spreaders. We could not hope, however, to persuade people to accept vaccination for purely altruistic reasons. We have heard at this session of various attempts to obtain ideal attenuated influenza viruses, by modifying their sensitivity to horse-serum inhibitors or their ability to grow at various temperatures or in other ways. We do not know which will prove best nor whether suitable markers will be found. Possibly the right sort of recombinant virus will provide the answer. If, however, we wish to arrange a marriage between a new antigenic type and an attenuated but immunizing virus, we need to make certain that the bridegroom is really steady and reliable. In other words, has our marriage bureau got available any virus which is sufficiently attenuated to be acceptable and yet antigenically potent? Or will it turn out that, to be an effective immunizing agent, a virus must retain some tendency to cause symptoms in a few particularly sensitive persons? That our search has taken us 35 years already is not wholly our fault. The influenza virus is a wily and elusive creature and probably still has a few tricks up its sleeve. Nevertheless, I think we have a right, at long last, to be optimistic. 613