DR. UNGAR AND OTHERS: REFERENCES—continued

Gepts, W. (1965) Diabetes, 14, 619.Gottlieb, C., Lau, K.-S., Wasserman, L. R., Herbert, V. (1965) Blood,

25, 875.Harris, H. (1950) Ann. Eugen. 15, 95.Irvine, W. J. (1965) New Engl. J. Med. 273, 432.

— (1966) Clin. exp. Immun. 1, 99.— Davies, S. H. (1963) Lancet, ii, 838.— Teitelbaum, S., Delamore, I. W., Wynn Williams, A. (1965) Ann,

N.Y. Acad. Sci. 124, 657.Landing, B. H., Pettit, M. D., Wiens, R. L., Knowles, H., Guest, G. M.

(1963) J. clin. Endocr. Metab. 23, 119.Lau, K.-S., Gottlieb, C., Wasserman, L. R., Herbert, V. (1965) Blood,

26, 202.Lawrence, C., Klipstein, F. A. (1967) Ann. intern. Med. 66, 25.LeCompte, P. M., Steinke, J., Soeldner, J. S., Renold, A. E. (1966) Diabetes,

15, 586.Mancini, A. M., Costanzi, G., Zampa, G. A. (1964) Lancet, i, 726.Martin, F. I. R., Stocks, A. E. (1967) Australas. Ann. Med. 16, 289.Moore J. M., Neilson, J. McE. (1963) Lancet, ii, 645.Raven, J. L., Barkhan, P. (1966) ibid. ii, 1078.Schilling, R. F. (1953) J. Lab. clin. Med. 42, 860.Simpson, N. E. (1964) Diabetes, 13, 462.te Velde, K., Abels, J., Anders, G. J. P. A., Arends, A., Hoedemaeker, Ph.J.,

Nieweg, H. O. (1964) J. Lab. clin. Med. 64, 177.Ungar, B. (1967) Aust. J. exp. Biol. med. Sci. 45, 317.

— (1968) Australas. Ann. Med. 17, 107.— Stocks, A. E., Martin, F. I. R., Whittingham, S., Mackay, I. R.

(1967) Lancet, ii, 77.— Whittingham, S., Francis, C. M. (1967) Australas. Ann. Med. 16, 226.

Wangel, A. G., Callender, S. T., Spray, G. H., Wright, R. (1968) Br. J.Hœmat. 14, 161, 183.

Witts, L. J. (1963) Israel med. J. 22, 294.Wood, I. J., Doig, R. K., Motteram, R., Hughes, A. (1949) Lancet, i, 18.

418

A COMPARISON OF LIVE AND KILLED

INFLUENZA-VIRUS VACCINES

Report to the Medical Research Council’s Committeeon Influenza and other Respiratory Virus Vaccines

A. S. BEAREM.B. N.U.I., M.C.Path., D.T.M. & H.

OF THE COMMON COLD RESEARCH UNIT, HARVARD HOSPITAL,SALISBURY, WILTSHIRE

D. HOBSONM.D. Sheff., M.C.Path.

SENIOR LECTURER IN BACTERIOLOGY, UNIVERSITY OF LIVERPOOL

SYLVIA E. REEDM.B. Cantab.

D. A. J. TYRRELLM.D. Sheff., F.R.C.P., M.C.Path.

OF THE COMMON COLD RESEARCH UNIT, HARVARD HOSPITAL,SALISBURY, WILTSHIRE

Summary 98 volunteers were vaccinated with for-malin-inactivated influenza-B virus intra-

muscularly or with living influenza-B virus intranasally.They, and a group of control volunteers, were sub-sequently challenged with live virus administered intra-nasally. 71% of those given killed vaccine developed anantibody response but 15% were infected on challenge.51% of those who had received live virus had an antibodyresponse and 2% were infected on challenge. Vaccinationalso reduced the frequency of clinical reactions. The livingvaccine used in this trial produced too many reactions tobe used on a large scale.

Introduction

FOR many years, killed-influenza-virus vaccines adminis-tered parenterally have been used in Europe and theU.S.A. while living intranasal vaccines have been pre-ferred in the U.S.S.R. Both are believed to protect, butno direct comparison between the two types has yet beenpublished. Our own experience confirmed that live vaccineprotects against a challenge with the same virus strainalthough titres of circulating antibody are not raised to thesame extent as when intramuscular killed vaccines are

used (Beare et al. 1968). We have compared two vaccinesprepared from the same virus strain, one a living vaccineand the other a killed vaccine, and describe here theclinical reactions and serum-antibody titres which theyinduced and their ability to protect against a challenge ;with live virus.

Volunteers, Material, and Methods148 employees of the Esso Oil Refinery, Fawley, Hampshire,

volunteered to take part in the trial. 134 were male and 14 werefemale. They were unselected and their ages ranged fromseventeen to sixty-four.The volunteers were allocated at random to one of four

different groups according to the types of vaccine they were toreceive: (A) 49 to be inoculated with living influenza-B vaccineintranasally, (B) 49 to receive formalin-inactivated vaccine

intramuscularly prepared from the same virus strain as thatused for vaccine A; (C), a control group of 25, to receive killedvaccine intranasally of the same strain as that of vaccine A; and(D), a second control group of 25, to be given a formalin-killedvaccine intramuscularly made from a heterologous virus withno antigenic relationship to that of vaccine A.

Blood-samples were taken and vaccines given on the first dayof the trial. Blood-sampling was repeated four weeks later andall volunteers were challenged with intranasal spray of livingvirus of the type used in vaccines A, B, and C. Three daysafter the challenge, throat swabs were taken for virus isolation.A final blood specimen was collected about three weeks afterthe challenge.

Vaccines

An influenza virus B/England/13/65 (Beare et al. 1968) wasused in the preparation of vaccines A, B, and C. The livingvaccine A was made from a 20th egg passage and was adminis-tered intranasally in 0-5 ml. amounts with a coarse hand spray.Each dose contained 105 egg-infecting doses (E.I.D.5,,) of virus.The formalin-killed vaccine B was prepared from the samevirus strain by Evans Medical Ltd. and was given intra-

muscularly in 1-0 ml. doses. Vaccine C was prepared by heatingan ampoule of B/England/13/65 at 56°C for forty-five minutesand diluting it to the same extent as the virus of vaccine A. Theheterologous vaccine D was prepared by Glaxo Ltd. from thevirus strain AJEngland/1/61 and was given intramuscularly in1-0 ml. amounts.The challenge virus consisted of a sixth egg passage of

B/England/13/65 given in the same dosage and by the sametechnique as vaccine A.

Isolation of Virus after ChallengeMaterial from throat swabs was inoculated into three tubes

of primary cultures of monkey kidney which were tested forhasmadsorption after four days’ incubation in a rolling-drumat 33°C.

Clinical Observations

A simple medical-history card was given to each volunteerat the times of vaccination and challenge. Replies were

requested to questions on specific symptoms during the follow-ing eight days and a brief general account of reactions was alsoasked for. Reactions were subsequently graded nil, mild,moderate, or severe (Tyrrell 1963).

SerologyHEemagglutination-inhibiting (H.I.) titres to the virus

B/England/13/65 were measured simultaneously in the threesera. The technique was that of the World Health Organisation(1953) modified by the use of 1% human group-0 erythrocytesinstead of 0-5% fowl erythrocytes.

Results

Serological responses to B/England/13/65 after vaccina-tion are shown in table i and the figure. The mean andthe range of antibody titres at the beginning of the trial

419

TABLE I-SEROLOGICAL RESPONSES TO VACCINATION

were similar in the two main volunteer groups. After the

vaccinations, most volunteers in groups A and B produceda response, but rises were more frequent in those who hadhad formalin-inactivated vaccine than in those who hadhad live vaccine, and final mean titres were also higher.There were 5 antibody rises in the control group C, whichhad been given dilute heat-killed influenza B virus intra-nasally, and this may be significant.

H.I. titres, before vaccination and before and after challenge, ingroups given live vaccine or killed vaccine.Horizontal axes show titres of antibody; vertical axes show numbers

of volunteers.

The frequency of clinical reactions after vaccination isshown in table 11. Sore arms were experienced only bypeople vaccinated intramuscularly and the incidence ofthis symptom was satisfactorily low. Respiratory symp-toms of varying grades, some with low fever and malaise,

were most frequent in the group receiving live vaccine inwhich the component virus had been only partiallyattenuated. The respiratory symptoms complained of bythose vaccinated parenterally or given dilute killed virusintranasally are presumed to have been caused by inter-current infections. This suggests that of the 37 volunteersshown in table 11 as having respiratory symptoms after livevaccine in only 14 were these due to the virus given.There were, however, 17 antibody rises in 22 volunteerswho had reported moderate or severe reactions after live

TABLE II-CLINICAL REACTIONS AFTER VACCINATION

Numbers who provided details of their clinical reactions.Figures in italic type show expected incidence of reactions, calculated from

the frequency of respiratory and general reactions in the other three groups.

vaccine, but only 6 in 27 volunteers whose reactions hadbeen graded nil or mild. There was therefore a correlationbetween numbers of reported reactions and antibody risesas has been shown before in a larger study with the samevirus (Beare et al. 1968).

In the second stage of the trial, the volunteers werechallenged with live virus and their resistance was

evaluated by three criteria-virus isolation, antibodyresponse, and respiratory and general symptoms (seefigure and table ill). A few volunteers dropped out forreasons unconnected with the trial or because they hadexperienced unpleasant reactions after vaccination. Only1 volunteer vaccinated with live virus was infected by thechallenge (as evidenced by a rise in antibody titre), butthe two control groups showed the expected frequency ofinfection, in spite of the limited antibody response pro-duced by group C after vaccination. Those who had beengiven formalin-killed B/England/13/65 were less effectivelyprotected than those given live vaccine and virus wasisolated from 3 of them. Since all the volunteers werechallenged, there remained no group to provide a

" base-line " incidence of reactions, but it seems clear that therewere few reactions in the group given live vaccine. Poolingthe figures for clinical symptoms in groups C and Denabled us to derive an expected incidence, however. On

TABLE III-RESPONSES OF VACCINATED VOLUNTEERS TO INTRANASAL CHALLENGE WITH LIVE B/ENGLAND/13/65

Numerators are numbers of responses, denominators are numbers providing data.

420

this basis, the incidence of clinical symptoms was halvedin the 39 volunteers who were given formalin-killed virus(expected 26, observed 13) and reduced by 82% (28expected, 5 observed) in the group given live vaccine.Infections diagnosed by laboratory tests were reduced by62% and 94%, respectively.

Discussion

An important object of the trial was to find a procedurewhich would compare the protective power of vaccines ofradically different types such as those administered

parenterally and intranasally. We believe that a deliberatechallenge with live virus is a satisfactory means of achiev-ing this, although this virus challenge is admittedly some-what different qualitatively and quantitatively from thatencountered under natural conditions. The practical con-venience of being able to arrange one’s own experimental" epidemic " on a chosen day is, however, considerable,and the degree of protection induced by the killed

parenteral vaccine accorded well with that often observedin carefully performed epidemiological trials with thiskind of vaccine. The volunteers themselves were mostcooperative, but the frequency of moderate or severereactions after the live vaccine was probably too high to beacceptable except in the face of a bad epidemic. This is initself of interest, since the same vaccine given underdifferent conditions at the Common Cold Research Unit

produced fewer ill-effects.We conclude that the live vaccine gave better protection

than the killed vaccine and that this was almost certainlybecause it induced higher titres of local antibody (Mannet al. 1968). However, its reaction-rate was too high. Weare now trying to induce protection without reactions byintroducing killed virus into the upper respiratory tract asdescribed by Waldman et al. (1968). The antibodyresponses in 5 volunteers given dilute killed virus intra-nasally in our own trial may have been due to a. slightantigenic stimulus by the inoculum, and this possibility isstrengthened by the fact that the volunteers of this groupproduced fewer antibody rises after the challenge thanthose who had been vaccinated with the inactivated

heterologous virus A2/England/1/61.Attention is also being given to the attenuation of living

viruses. The observation in this trial that a similarincidence of reactions occurred after 6th and 20th eggpasses of B/England/13/65 lends support to other workwhich showed that the virus remained incompletelyattenuated after 30 egg passes at 33°C (Beare, Bynoe, andTyrrell 1968).We thank the volunteers at the Esso Refinery, Fawley, tjants, for

their wholehearted cooperation. The trial was much helped byDr. A. Ward Gardner and his staff. We also thank Dr. D. C. Breezeof Evans Medical Ltd. and Dr. A. J. Beale of Glaxo Ltd. for thepreparation of two of the vaccines and of the challenge virus, Dr.Beale for supplies of leucossis-free eggs, and Miss P. D. Ball andMrs. Maria Gregory for technical assistance.

Requests for reprints should be addressed to A. S. B.

REFERENCES

Beare, A. S., Bynoe, M. L., Tyrrell, D. A. J. (1968) Unpublished.— Hobson, D., Howells, C. H. L., Pereira, M. S., Pollock, T.

M.. Tyler, L. E., Tyrrell, D. A. J. (1968) J. Hyg., Camb. (in thepress).

Mann, J. J., Waldman, R. H., Togo, Y., Heiner, G. G., Dawkins, A. T.,Kasel, J. A. (1968) J. Immun. 100, 726.

Tyrrell, D. A. J. (1963) Am. Rev. resp. Dis. 88, 128.Waldman, R. H., Mann, J. J., Small, P. A., Jr. (1968) New Engl. J. Med.

(in the press).World Health Organisation (1953) Tech. Rep. Ser. no. 64.

MAGNESIUM/CALCIUM RATIO IN URINE OFPATIENTS WITH RENAL STONES

D. G. OREOPOULOSM.D. Athens

MEDICAL REGISTRAR

M. A. O. SOYANNWOM.B. Lond., M.R.C.P.I.

RESEARCH FELLOW

M. G. MCGEOWNM.D., Ph.D. Belf., M.R.C.P.E.CONSULTANT NEPHROLOGIST

From the Department of Nephrology, Belfast City Hospital andthe Department of Medicine, Queen’s University of Belfast

Summary The urinary magnesium/calcium ratio wasfound to be significantly lower in patients

with renal stones than in healthy controls. There areindications that a decrease in this ratio is an importantfactor in the process of stone formation and it would

appear that an attempt to increase it might be worth while,at least in those patients with recurrent renal stones inwhom this ratio is low.

Introduction

WE have found that patients from whom a parathyroidadenoma had recently been removed had a striking increasein urinary magnesium/calcium (Mg/Ca) ratio, despite apostoperative decrease in magnesium excretion (Oreo-poulos, Soyannwo, and McGeown 1968). Since thesewere patients with renal stones we have investigatedfurther the significance of this ratio in patients with renalcalculi without obvious cause.

Patients and Methods

The urinary Mg/Ca ratio was studied in six patients withhyperparathyroidism before operation and 1 week and 3-6months postoperatively. In five other patients, 2 units of

parathyroid hormone (P.T.H.) per kg. body-weight were givenintravenously and the Mg/Ca ratio was estimated on hourlyspecimens for 5 hours after the injection. The Mg/Ca ratio wasalso measured in fifty-four patients with renal stones confirmedradiologically at the time of the observation and in twentyhealthy volunteers with no history of renal stones (controls).The collection of the urine for the estimation of the ratio

was done as follows: after an overnight fast from 6 P.M. theprevious day, the patient emptied his bladder at 6 A.M. (thisspecimen being discarded), fasting was continued until 9 A.M.and the Mg/Ca ratio was estimated on the total urine passedat that time. Using this technique it can be assumed that theinfluence of recently ingested calcium and magnesium on theurinary excretion is minimal (Heaton and Hodgkinson 1963).If this assumption is correct this ratio should be constant underthe same conditions. We therefore estimated this ratio in ten

patients with renal stones on three different days.Calcium was estimated with the permanganate-titration

method (Clark and Collip 1925), and magnesium was measuredon an atomic absorptiometer (Evans Electroselenium Ltd.).

Results

The table shows the Mg/Ca ratio in the 3-hour urine often patients with renal stones, under the " basal "

conditions, as described above, on 3 different days. Therewas no significant difference between them.The Mg/Ca ratio in fifty-four stone formers and in

twenty controls is shown in fig. 1. While the mean innormals was 89-3%, for the stone formers it was 448;0,a highly significant difference (P<0-001). It is worthwhile to note that, expressed as Mg/Ca ratio, the data ofMukai and Howard (1963) on the magnesium andcalcium concentration of the " evil " urines whichcalcified the cartilage of the rachitic rat, and " good "