immunity and antibody to influenza in mice

6 1 6 . 9 2 1 . 5 : 576 .809 . 73

IMMUNITY AND ANTIBODY TO INFLUENZA IN MICE

C. L. OAKLEY and G. HARRIET WARRACK From the Wellcome Physiological Research Laboratories, Beckenham, Kent

BURNET, Keogh and Lush, in their monograph on the immunological reactions of the filterable viruses (1937), put forward the possibility that antibody to viruses might be produced locally in the structures for which the virus has a special affinity and suggested that influenza, with its localisation, as far as was then known, in lung and turbinate, was a very suitable virus on which to work to determine this point. We therefore immunised mice in various ways in the hope of demonstrating antibody in the lungs before it appeared in the circulation.

We immunised them (1) by inoculation intranasally under anaesthetic with a dose of virus just sufficient to infect * but not to kill, (2) by vaccination with two intraperitoneal doses of living virus a t an interval of about one week and (3) by vaccination in a similar way with formolised virus. We killed them at times which we thought appropriate, bled them out from the heart, washed out the vessels with warm citrate-saline by Welcker’s method and determined the antibody in serum and organs against the homologous strain of virus, using throughout as standard the serum of a ferret convalescent from infection with this strain. We have used in all this group of experiments the W.S. strain, as it is remarkably constant in its behaviour in mice.

Antibody in ~erurn Though it is well known that antibody to influenza virus appears in the

serum after infection in the ferret (Smith, Andrewes and Laidlaw, 1933), man (Francis, 1934-35 ; Andrewes, Laidlaw and Smith, 1935 ; Francis and Magill, 1935 ; Francis, Magill, Beck and Rickard, 1937), hedgehog (Stuart- Harris, 1936), rat and guinea-pig (Stuart-Harris, 1937), Chinese mink- Musteh sibirice-(Tang, 1938), little work has been done on the time of its appearance or its persistence. Fourteen days after infection large amounts are present in the serum of ferrets (Smith and Andrewes, 1938) and traces can be detected as late as 12 months, though much of the immunity has faded (Stuart-Harris et al., 1938). Stuart-Harris et al. also found that anti- body after natural infection in man appeared in the s e m in about 8 days, reached its maximum in 9-12 days and could still be detected 52 days after infection. Rosenbusch and Shope (1939) have shown that antibody to swine influenza virus appears in pigs about 7 days after infection, reaches a maximum on the 14th-27th day and persists in some pigs though at a rather diminished titre for a t least 84 days.

* As will be seen later (p. 38) the meaning of this word is not as clear as might

JOURN. OF PATE.-VOL. L 37 c 2 appear.

38 C . L. OAKLEY AND G. H . WARRACK

A great difficulty in our work has been to determine a dilution of W.S. virus which would infect but not kill. Smith, Andrewes and Laidlaw (1935) have previously noted this difficulty. It is not unusual for a dilution of filtered virus to kill every mouse inoculated, some dying as late as 3 weeks with completely consolidated lungs, while a dilution may produce no lesions in any mice. It is only with great good luck that a dilution is hit on which will infect 75 per cent. of the mice and kill only 5-10 per cent. We have been forced to give our results for intranasal inoculation in a rather incomplete form, up to 3 months only,

FIG. 1.-Development of antibody in mice immunised in various ways to W.S. influenza, virus

I.N. = by intranasal inoculation of 10-100 M.I.D. of virus I.P.2. = with two intraperitoneal doses of 0.2 and 0.5 C.C. of living virus

I.P.1. = with one intraperitoneal dose of 0.5 C.C. of living virus ( lo7 M.I.D. per c.c.) at an interval of a week

I.P.F.2. = with two intraperitoneal doses of 0.2 and 0.5 C.C. of formolised virus

I n each group the values given are (from left to right) for serum, nose, lung, liver, ( lo7 M.I.D. per 0.0.) a t an interval of a week

spleen, kidney.

rather than use up the immense number of mice required for a complete series. Recently at Dr Andrewes's suggestion we have used the neurotropic variant of the W.S. strain with a good deal of success, but we feel that it is not quite the same as mouse-lung-

IMMUNITY TO INFLUENZA 39

adapted virus, and that comparison of mice inoculated intranasally with neurotropic influenza virus with mice vaccinated intra- peritoneally with lung virus is hardly fair. We have found in a few experiments tha6 antibody to lung virus after infection with neurotropic virus is lower than that after infection with lung virus, suggesting that differentiation is already occurring.

In man, Francis and Magill (1937) have shown that after vaccination with living culture virus, antibody appears in the 2nd week and persists about 5 months. Stuart-Harris et al. report the development of antibody in mice after vaccination with living

FIG. 1-contiuued

virus. Our results are given in fig. 1, which shows the values for antibody in serum, nose, lung, liver, spleen and kidney of groups of 5 or more mice at various times after immunisation.

Owing to the logarithmic method of expressing the results and the great variation in the response to infection in mice, the answers obtained from pooled samples of serum tend to be weighted in favour of high values. For instance if one serum has a titre equal to standard (S) and four a titre of S/125, the result for the pooled sera will be S / 5 ; ordinarily it is not practicable either to use a large enough number of mice to avoid this difficulty, or to carry out serum titrations on individual mice.

As might be expected from the effect of a secondary stimulus on antibody titre in immunity against other antigens, the rise in

40 C. L. OAKLEY AND G. H . WARRACK

antibody is fastest in animals receiving two doses of living virus intraperitoneally, and the greater part of the rise occurs soon after the second dose. It is therefore surprising that the rise in animals receiving two doses of formolised virus is no faster than in those infected intranasally. All reach the same maximum in about 14 days,* none so far showing much advantage.

The disappearance of antibody is very different in the three groups. We have previously held the view that antibody due to intranasal inoculation is reduced to negligible proportions in about six weeks but further work has shown that this is true only of some groups of mice : at 28 days after infection two groups showed serum antibody equal to 5/25 and 515 ; in three others the titres at 42 days were 5/25, XI5 and 515. In many sera the concentration is high up to three months, beyond which time we have not been able to carry the experiment.?

By about 8 weeks antibody due to vaccination with formolised virus has largely disappeared, while that due to vaccination with living virus is still at a high level at 12 weeks-a considerable time in the life of a mouse.

As we thought that the high values for some groups at and after 6 weeks might be due to deterioration of the standard (Smith and Andrewes), we tested all the sera from the groups infected intranasally at one time against the same standard. We obtained much the same result m before.

The values given in fig. 1 for antibody after infection apply only to those animals showing lesions in the lung. Mice showing no lesions after " infection " almost invariably have no demonstrable antibody.

As a matter of interest we determined the antibody response in mice vaccinated with one dose of living virus intraperitoneally ; the antibody levels were always low, rising only on one occasion to S/5 and thereafter remaining at about 8/25,

Antibody in organs

Before examining the results some consideration of their accuracy is necessary. The method adopted was to wash out first the lungs and then the systemic circulation until the washings were colourless, grind up the organs with quartz powder and ten times their weight of broth, freeze overnight, thaw and allow the gross particles to settle, and having determined the antibody titre in the supernatant in the usual way against 100 M.I.D. of W.S. virus, multiply the answer by ten. It not infrequently happened that even undiluted organ extracts failed to protect; all that could be said of such extracts was that their titre was <S/3125 or <St15625 accord- ing to the efficiency of the standard; the corrected value for the organ

* Compare the results of Stuart-Harris et al., p. 116, for man. t Compare the irregular peaks in antibody observed by Stuart-Harris et al.

in ferrets.

IMMUNITY TO INFL WENZA 41

had to be set down as <S/300 or <S/1500, though its real titre might easily have been S/20,000. Moreover it is possible that Welcker’s method may not wash all the serum out of the vessels : if 1 per cent. is left behind and the serum value is S, the organs, even if they contained no antibody, might give a corrected titre of S/lOO.

We feel that in organs only titres greater than SjlOO should be allowed any weight; and almost the only organs with this or a higher titre are the lungs, with occasionally the nose or spleen. The spleen is notoriously difficult to wash out, so that, though the result is interesting, we do not lay much stress on it. The only other organs with a significant if small amount of antibody are the lungs and nose, the ordinary sites of election of the virus. It will be observed, however, that when the lungs are rich in antibody so is the serum ; we have thus no evidence that antibody is produced in the lung. If it is, it must be very rapidly removed from the lungs to the circulation.

We see no way of getting over the difficulty that, as the animal is washed out, antibody normally more or less fixed in the tissues may be removed: our methods of estimating antibody are far too inaccurate to permit of any determination of this point.

Antibody and the persistence of virus in tissues In animals infected intranasally, W.S. virus persists in

diminishing concentration in lung and turbinate for 7-14 days. At this time antibody reaches its highest concentration in the serum; later no virus can be demonstrated and no further rise in antibody occurs.

After intraperitoneal vaccination with sufficiently large amounts of living virus, virus can be demonstrated in the lungs ; typical influenza1 lesions are sometimes produced and occasionally death (Rickard and Francis, 1938). Virus persists for 7-11 days after the first inoculation and for a shorter time after the second. No increase in antibody occurs after the disappearance of virus.

After intraperitoneal inoculation with formolised virus no living virus can be detected, but antibody reaches its maximum at about the same time as in infection or vaccination with living virus. The apparent connection between time of maximum antibody level and time of disappearance of virus may therefore be coincidence.

Antibody and immunity Infection or vaccination of the susceptible animal is followed

by the development of antibody and immunity, but this immunity varies much in quality as well as in degree. Ferrets convalescent from intranasal infection with a given strain are apparently immune for a considerable time to all other strains, though they may have

42 0. L. OAKLEY AND G. H . WARRACK

:&:, withstood

significant amounts of antibody only for the homologous strain. Mice vaccinated with living or formolised virus show greatest immunity to the homologous and closely related strains and very little to serologically unrelated strains (Smith and Andrewes). The picture is much complicated by questions of the titre of virus used for vaccination and test inoculation (Francis, 1939).

We have therefore investigated the relationship between immunity and antibody by immunising groups of mice with W.S. virus either by infecting them intranasally with small doses of living virus or by vaccinating them intraperitoneally with two doses of formolised virus, to avoid the possible effect of the localisation of living virus in the lung. At appropriate times thereafter some of them were bled for antibody, the blood from those showing lesions in the lungs being kept separate from that of those showing none. Suitable numbers of the rest were inoculated intranasally with falling tenfold dilutions of the four main strains, W.S., Talmey, Gatenby and Christie, similar procedures being carried out on each occasion with equal numbers of controls of the same age. The results are expressed as the number of minimal infecting doses of virus (M.I.D.) withstood by the immunised groups. For instance, if the highest dilution of virus producing lesions in the controls was and lesions were produced in the immunised animals at but not at the group was said to resist lo4 M.I.D. The method is rough and ready, but fairly satisfactory in practice. We are not satisfied with the use of death and survival as indicators, as much depends on the time the experiment is allowed to continue, the number of mice used and the virulence of the strain.

At the same time the serum from the batch was titrated for antibody against the same four strains, using specific serum from convalescent ferrets as standards. It should be noted that the sera used as standards cannot readily be compared with one another ; our Christie standard, for instance, is certainly very poor against Christie virus as compared with W.S. serum against W.S. virus, and the determinations of Christie antibody are corre- spondingly too high.

The results for intraperitoneal vaccination with formolised virus are given in table I.

TABLE I Immunity and antibody in mice immunised with fomzoliaed W.S. virus

Antibody in Serum

Time since immunisation

(days)

_ _ _ ~

0 10

100 100

10 0

7 14 21 28 42 56

Sl625 S/125 S/625 Sj625 S/125 51625

- Strains of influenza virus used in tests

~ _ _

0 10 10

W.S.

Sjl25 S/25 S/6

100 103-104

103-104 103-104

I- 51125

sp S/5

S/5

0 10 10 10

100 0

Talmey

s/3125 S/625 S/625 S/625 S/625 S/125

Antibody withstood in Serum

Gatenby

$2:. Ant,ibody withstood ln serum

Mice immunised with 2 doses of formolised W.S. virus (lo7 M.1 interval of 1 week

- Christie

7;:. Antibod withstood in serun

IMMUNITY TO INFLUENZA 43

It is clear that there is a very rough correlation between antibody and immunity and that both are better developed to the homologous than to the heterologous strain. As antibody fades so does immunity. In no case can the degree of immunity be regarded as very high, especially as compared with that following intranasal inoculation.

Burnet (1938) has recently reported that vaccination as well as infection with W.S. virus gives rise to a much more generalised immunity than is produced by the use of most other strains ; he used living virus and the possibility of localisation of virus in the lung cannot be ruled out.

Immunity after intranasal inoculation is much more complicated. A sample experiment given in fig. 2 makes it clear that there is a

10' /o-' /a 10 -4 /O -5-

-~ ~

FIQ. 2.-Lungs of mice inoculated intranasally with W.S. virus and tested for immunity with Christie virus intranasally 21 days later. Black = recent lesions (Christie) ; stippled = old lesions (W.S.). The Christie virus used produced lesions in two out of three control mice at 10-4 ; no lesions at

sharp difference in immunity between animals showing old lesions due to the immunising intranasal inoculum and those showing none. In this experiment a batch of mice was immunised by intranasal inoculation under anasthetic with 0.05 C.C. of lo-' W.X. virus. Three weeks later a sample of Christie virus was titrated in these immunised animals and in a batch of controls of the same age. When the immunised animals were killed on the sixth day after inoculation their lungs, if they showed anything at all, showed either old grey lesions or recent plum-coloured ones, never both.

44 C. L. OAKLEY AND B. H. WARRACK

.---_______

0 S/625 0 513125 >lo5 S/125 >lo6 51625 >lo4 s p 5 >lo4 s/m >lo8 <s'ji25 >lo4 c. Sji25 ... > 104

In other words, mice surviving intranasal inoculation with W.S. virus with the production of lesions are immune to Christie virus of as high a titre as is available ; mice similarly treated without the development of lesions have no advantage over the controls. That this dependence of immunity on the production of lesions is not Iimited to one strain is shown by the fact that mice inoculated with W.S., with its neurotropic variant (neuroflu) obtained through the kindness of Drs Andrewes and Stuart-Harris, with Talmey or with Gatenby, if they develop lesions, all show complete immunity for at least 2 months to all four strains tested, and usually to a degree far higher than is attained after intraperitoneal inoculation with formolised virus (tables I1 and 111). Mice showing no lesions after intranasal inoculation have no immunity whatever.

withstood

TABLE I1 Immunity and antibody in mice showing old lesions after intranmal

infection with W.S. virus

Antibody in serum

Time since previous infection

(days)

>lo5

>lo4 >lo5

>lo4

> 102

>lo2

I 14 21 28 56

5/26 5/25

siifL5

s / i 2 5 5/25

Strain used in

prelimipary infection

Tdmey

Neuroflu

,,

,, ,,

14

14

28 56

28 56

Reaction to different strains of influenza virus I

>lo7

>lo7

> lo6 >lo6 >lo6 > lo6

1 W.S.

S/625 51125

s/5:S/25 515 i 515

I Talmey 1 Gatenby I

'lo5 :I5 >lo6 >lo3 >lo5 s i i i 5

>lo3 5/25 >lo6 5/25

-I I I

I withstood serum withst,ood in withstood in sen'm 1 Antibody 1 %k? 1 Antibody 1 1 AnMbody 1

Christie

::$. Antibody withstood in

0 S/625 >lo4 1 615 >lo4 s p Yi05 I 0:s.

TABLE I11 Immunity and antibody in mice ahowing old lesions following pyevious

infection with in$uenza virus

Reaction t o different strains of infiuenza virus

Days ' I W.S. 1 Talineg sinceArst I , I infection

MLD' virus Antibody :::' Antibody withstood in Serum withstood in SeNm

I- 1-1-1-

Gatenby Christie

Andrewes and Smith (1937) record that most of their mice possessing immunity t o W.S. after intranasal inoculation showed old lesions in the

IMMUNITY TO INFLUENZA 46

lungs ; Burnet (1937) states that virus passaged for many generations through eggs, though it produces in mice only minute patches of consolidation in the lungs or no lesions at all, still immunises perfectly and gives rise to high antibody titres. More recently (1938) he has shown that M.E. virus gives much greater immunity against W.S. if old lesions are produced than if they are not. Some strains, however, when inoculated intranasally without producing lesions, yet immunised against the homologous strain. Shope (1935) has also found that a recently isolated strain which produced extensive lesions in mice only after repeated passage would, in its early passages when no evident lesions were produced, immunise mice completely against mouse- lung-adapted virus. This variation between strains needs much further investigation.

The old lung lesions were in some cases exceedingly small, and might have eluded casual observation. It may therefore be suggested that the extensive recent pneumonia in tested animals without obvious old lesions may really conceal minute or even microscopic old pneumonic patches. We have searched carefully for these and think it very improbable that they were present, but this point can be proved only by serial section of the lungs. Notwithstanding Burnet’s (1938) statement to the contrary, it is sometimes exceedingly difficult to distinguish old lesions from recent, especially with ‘‘ weak ” viruses (e.g. Gatenby). In general, old lesions are grey in colour and deeply sunken beneath the surface ; if the whole lobe is involved it is much reduced in size and somewhat granular on the surface. Recent lesions are a deep plum colour ; they are sharply separated from the unaffected lung, and if the whole lobe is involved it is usually though not always much enlarged. After infection with “ weak ” viruses, however, grey “ recent ’’ lesions may be produced, and very careful consideration of such matters as collapse and the continuity of general appearance and colour of the lesions may be necessary to determine the point. We have, however, never had a sufficient number of difficult cases to invalidate the general argument; by about 2 months after infection the difficulty hardly ever arises.

Although animals possessing no immunity have no antibody, there appears to be no correlation between immunity and antibody in animals showing old lesions. Indeed the antibody response to intranasal inoculation with development of lesions appears much the same as that to vaccination with formolised virus, though the immunity produced is much more striking and entirely different in its lack of strain specificity.

The interference phenomenon As a result of extensive experiments Rickard and Francis feel that

antibody is insufficient to account for the immunity developed after intra- peritoneal inoculation with living virus. They argue as follows. Mice inoculated intraperitoneally with large doses of living virus show after about 24-48 hours quantities of virus in the lungs sufficient to kill large

46 C. L. OAKLEY AND G. H . WARRACK

numbers of mice if inoculated intranasally under anesthetic. Notwithstand- ing this, such mice do not die, though small lesions may be produced, but become in time solidly immune. Indeed they show considerable immunity to intranasal inoculation with homologous virus as early as two days after intraperitoneal vaccination, although there is no demonstrable circulating antibody. They suggest that virus accumulated in the lung from the intraperitoneal inoculation interferes in some way with the entrance of virus inoculated intranasally, and thereby reduces the degree of infection.

With their view that virus can be recovered in large quantity from the lungs of mice inoculated intraperitoneally with sufficiently large amounts of living virus we are in entire agreement ; but though we have repeated their experiments on the immunity developed thereafter on several occasions on a large scale with W.S. and P.R.8 strains, we have been entirely unable to confirm their finding that immunity develops before antibody is demonstrable in the circulation.

In fact up to the 4th day after intraperitoneal inoculation the lesions produced by intranasal infection in vaccinated animals at all dilutions were more extensive and deaths were more frequent than in the controls (fig. 3). No doubt the lesions at the very high dilutions were due to the vaccinating dose of virus. By the fifth day traces of antibody could be demonstrated in the circulation and slight differences in favour of the vaccinated animals could be made out.

We communicated our findings to Dr Francis who very kindly sent us the following suggestions.

(1) Our immunising dose was too small. As Rickard and Francis use the M.L.D. as their unit and we prefer the M.I.D. as less affected by the time of duration of the experiment and the virulence of the virus, i t is d;fficult to compare values. Unfortunately following our usual practice we killed all survivors on the sixth day after inoculation, while Rickard and Francis allowed their experiments to continue for ten days. In a recent experiment with W.S. virus we have determined the effect on the estimation of the number of M.L.D. per C.C. of a sample produced by varying the duration of the experiment from six to nine days. On the sixth day the titre was lo4 M.L.D., on the 7th day 1@, on the eight and ninth 106 ; the M.I.D. per C.C. remained the same at los. I n other words by waiting a few more days it is possible to increase the estimate of the number of M.L.D. of a virus sample a hundredfold, while the M.I.D. remains unchanged. We think this explains the difference between us and Rickard and Francis, whose M.L.D. titre (up to the tenth day) was lo6 while ours at the sixth day is only 1O8-1O4.

In any event our immunising dose produces lesions in the lungs of about half the mice injected and if the views of Rickard and Francis on the amount of virus which it is necessary to inject intraperitoneally to produce these lesions are correct, the titre of our virus cannot be much lower than theirs.

( 2 ) Our results, especially the somewhat irregular distribution of lesions in the controls, might be due to secondary bacterial infection. To avoid this we have throughout used virus filtered through gradocol membranes capable of retaining bacteria (0.6-0.8 p). On the one occasion when unfiltered P.R.8 virus was used for vaccination to duplicate the experiments of Rickard and Francis more accurately, large numbers of mice died in 24 hours with

FIG

. 3.-I

mm

unity

ag

ains

t in

tran

asal

inf

ectio

n w

ith

vary

ing

dilu

tion

s of

W

.S.

viru

s a

t va

riou

s ti

mes

aft

er i

ntra

peri

tone

al i

nocu

latio

n w

ith

The

res

ults

fo

r th

e fi

fth

day,

whi

ch

one

dose

of

0.5

C.C

. of

liv

ing

W.S

. vi

rus

(lo7

M.I.

D.

per

c.c.

). ar

e m

ore

favo

urab

le t

o th

e va

ccin

ated

ani

mal

s, h

ave

been

om

itte

d, t

o s

ave

over

crow

ding

. E

ach

squa

re r

epre

sent

s a

mou

se.

I3 =

eat

en b

y ot

her

mic

e

48 C. L. OAKLEY AND G. H . WARRACK

lesions in no way resembling influenza and the survivors showed exactly the same distribution of immunity as mice inoculated with filtered virus,

(3) It is still necessary to explain why mice inoculated intraperitoneally with large amounts of living virus do not die although they show two days thereafter an amount of virus in the Iung which would kill many thousands of mice if inoculated intranasally.

It appeared to us possible that the development of lesions in the lung might not be an essential sequel to the multiplication of influenza virus in them. We therefore inoculated batches of mice with falling tenfold dilutions of filtered influenza virus intranasally under anaesthetic. Half of these mice were killed at 2 days ; the rest were allowed to survive for ten days t o determine the highest dilution of virus producing lesions in the lungs. The lungs of those killed at 2 days were ground up with quartz and broth-saline and centrifuged. Falling tenfold dilutions of the supernatant fluid were inoculated intranasally under anaesthetic into small mice.

It is clear from table IV that mixed lungs from mice after intranasal infection with a dose of W.S. virus which produces minimal lesions or none at all may contain a t two days quantities of virus capable of producing extensive lesions or even of killing large numbers of mice. We have also frequently observed that mice inoculated with lungs from mice killed two days after infection die sooner than those of the infected batch allowed to survive.

TABLE IV Virus in lungs (mixed) 2 days after intranasal inoculation of W.S. virus

D = died before 10th day ; typical influenzel lesions L = killed on 10th day ; 0 = ,, ,, ,, ,, ; no lesions

, I

Twelve mice were inooulated intranasally with W.S. virus. At two days 6 were killed and their lungs collected. The rest died in about 8 days from influenza. Suspensions of lungs of those killed on the second day were inoculated intranasally under anaesthetic into mice ; most of these died on the 3rd or 4th day, i.e. 2-3 days before the death of these allowed to survive in the original group. Smorodintseff and Ostrovskaya (1937) have obtained results much more striking than ours ; lungs of mice inoculated with virus diluted to IO-l2, though themselves showing no lesions, would after repeated passage produce typical influenza in mice. We think it likely that the difference is due to the high degree of mouse adaptation of our viruses, for Stuart-Harris et al. have shown that recently isolated strains can be propagated for several generations in mice before producing lesions. Burnet’s results (see p. 45) support this view.

I M M U N I T Y T O INFLUENZA 49

Virustitre; M.L.D. (10 days)

( 10 days) Virus titre; M.I.D.

1

We have also determined the titre of influenza virus in individual lungs of mice two days after intranasal infection. The results (table V) show that the lungs can be divided sharply into two

TABLE V Tdre of inJuenza virus in individual lungs 2 days after

intramsal infection with lo-' W.S. virus

o 1 0 5 1 108 o o o o o o 105 j 10' 0 0 0 0 0 0 ~ 1 0 . 1 0

I

/Mouselungno. . 1 1 ! 2 ~ 3 I 4 I 5 1 6 ~ I 1 8 I 9 1 1 0 1 I---- ____-____- i

Of 28 mice of same group allowed to survive 10 days, 13 developed lung lesions.

groups, those showing large quantities of virus and those showing none. Since the lungs of those mice killed at ten days can similarly be divided into two groups, those showing lesions and those showing none, we think it likely that those showing large quantities of virus at 2 days would have developed lesions, while those with no virus would not have done so if allowed to survive for 10 days.

In order to prove that the lesions and deaths were due to influenza virus, a matter of considerable importance where unfiltered virus is used, influenza1 antibody was mixed with some of the dilutions of two-day lungs and inoculated intranasally into mice under anaxthetic. None died and very few developed lesions, though at the corresponding dilutions of virus alone almost all the mice had died. There seems no doubt that in the main the deaths and lesions were due to influenza virus, and we are left with the paradox that two days after intranasal inoculation of mice with high dilutions of influenza virus the lungs may contain virus sufficient to kill thousands of mice, and yet at ten days they show only minimal lesions.

It is we think probable that when only a few virus particles are inoculated, each can proliferate locally to about 1 O6 particles before the mass of virus becomes greater in volume than the cell in which it is parasitic. Consequently, though large quantities of virus may be present, they may be strictly localised to a few cells in the lung, and only negligible lesions be produced. Certainly in this case the interference phenomenon cannot be invoked.

If this is so, antibody will explain the immunity developed after intraperitoneal inoculation with living virus without any further mechanisms. The remarkable spread of immunity to heterologous strains after intranasal infection is as yet unexplained ; virus-neutralising antibody is certainly insufficient and we have as yet no information on complement-fixing antibody in mice.

JOURN. OF PATE.-VOL. L D

50 C . L. OAKLEY AND G. H . WARRACK

Lastly, in the light of our experiments and those of Rickard and Francis on the development of lesions in the lung after intra- peritoneal inoculation with living virus in sufficient amount, it is interesting to speculate on the strain immunity developed by such animals. Will it be the same whether lesions are developed or not 1 Or, if lesions are developed, will the immunity spread to heterologous strains ! Possibly the somewhat divergent results obtained after immunisation with living virus may find their solution here.

Summary

1. After intranasal inoculation with living virus or intraperitoneal vaccination with two doses of living or formolised virus, mice develop both antibody and immunity to influenza of varying quality and degree.

2. The rise in antibody is fastest in the animals given two doses of living virus intraperitoneally ; all three groups reach the same maximum two weeks after the first inoculation. In some mice infected intranasally antibody has disappeared by six weeks ; in others it may remain more than 3 months. Antibody persists about 8 weeks in animals vaccinated with two doses of formolised virus, 12 weeks in those vaccinated with living virus. Mice vaccinated with one dose of living virus intraperitoneally show very poor antibody levels.

3. In mice vaccinated intraperitoneally with formolised virus antibody titre and immunity to the main strains run parallel. Animals infected intranasally, though their antibody shows no striking difference from those vaccinated with formolised virus, are immune to all four strains.

4. After intranasal inoculation with W.S., its neurotropic variant, Talmey and Gatenby, the production of lesions is essential for the development of antibody and immunity.

5. Circulating antibody can account for all the characters of the immunity to the homologous strain possessed by animals vaccinated intraperitoneally with living virus.

REFERENCES

ANDREWES, C. H., LAIDLAW, P. P., AND SMITH, W.

W. ANDREWES, c. H., AND SMITH,

BURNET, F. M. . . . . .

BURNET, F. M., KEOGH, E. V.,

FRANCIS, T., Jr. . . . .

. . . . . I,

AND LUSH, D.

1935.

1937. Ibid., xviii, 43.

Brit. J. Exp. Path., xvi, 566.

1937. Ibid., xviii, 37. 1938. Ibid., xix, 388. 1937. Austr. J . Exp. Biol. and Med. Sci.,

1934-35. Proc. SOC. Exp. Biol. and Med., xv, 227.

xxxii, 1172.

I M M U N I T Y TO I N F L U E N Z A 51

FRANCIS, T., J r . . . . . FRANCIS, T., Jr., AND MAGILL,

T. P. 1 , I ,

FRANCIS, T., Jr., MAGILL. T. P., BECK, M. D., AND RICKARD, E. R.

RICKARD, E. R., AND FRANCIS, T., Jr.

ROSENBUSCH, C. T., AND SHOPE, R. E.

SHOPE, R. E. . . . . . SMITH, W., AND ANDREWES,

SMITH, W., ANDREWES, C. H., C. H.

AND LAIDLAW, P. P. ,, 1 ,

SMORODINTSEFF, A. A., AND

STUART-HARRIS, C. H. . . .

STUART - HARRIS, C. H., ANDREWES, C. H., SMITH, W., CHALMERS, D. K. M., COWEN, E. G. H., AND HUGHES, D. L.

TANG, F. F. . . . . . .

OSTROVSKAYA, S. M.

,, . . .

1939. 1935. Ibid., lxii, 505.

1937. Ibid., lxv, 251. 1937.

J . Exp. Med., Ixix, 283.

J . Amer. Med. Assoc., cix, 566.

1938.

1939. Ibid., lxix, 499.

1935. Ibid., lxii, 561. 1938.

1933. Lancet, ii, 66.

1935. 1937. This Journal, xliv, 559.

1936. Brit. J . Exp. Path., xvii, 324. 1937. Ibid., xviii, 485. 1938. Medical Research Council, Spec.

J . Exp. Med., Ixvii, 953.

Brit. J . Exp. Path., xix, 293.

Brit. J . Exp. Path., xvi, 291.

Rep. Ser., no. 228. London.

1938. Brit. J . Exp. Path., xix, 179.