i*USELLA C0NF23EMCE SIYSIOK 0? iJiOLOGiCS STAADAA&S ■;a'v;oi;al institutes of health STENOGRAPHIC TRANSCRIPT DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE PUBLIC HEALTH SERVICE NATIONAL INSTITUTES OF HEALTH DIVISION OF BIOLOGICS STANDARDS CONFERENCE ON RUBELLA Bethesda, Maryland 30 April 1964 DIVISION OF BIOLOGICS STANDARDS CONFERENCE ON RUBELLA APRIL 30, 1964 1 DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE PUBLIC HEALTH SERVICE NATIONAL INSTITUTES OF HEALTH DIVISION OF BIOLOGICS STANDARDS CONFERENCE ON RUBELLA Assembly Room Building 29 National Institutes of Health Bethesda, Maryland Thursday, April 30, 1964 The meeting was convened at 9:10 o'clock a.m., Dr. Meyer presiding. 2 INDEX REMARKS OF: PAGE Fetal Infection and the Rubella Syndrome( Dr. Alford 4 Dr. Heggie 18 Dr. Balsamo 21 Dr. Schiff 23 Dr. Parkman 27 Rubella Infection of Experimental Animals: Dr. Schiff 30 Dr. Cabasso 40 Dr, Phillips 45 Experimental Infection of Man with Rubella Virus. Dr. Green 50 Dr. Balsamo 60 Formalin Inactivation of Rubella Virus Dr. Sever 81 Dr. Hok 81 Discussion 107 PROCEEDINGS 3 DR, MEYER: If you will please come to order, we wil! try to get underway. We have two or three announcements to makti before v/e start the scientific part of the program. (Announcements.) DR. MEYER: I think Dr. Murray may have announcements. DR. MURRAY: I just want to rectify an omission in my remarks yesterday. I assumed perhaps that you all appreciated this fact but a meeting of this kind, although it is by invita- tion, is nevertheless an open meeting inasmuch as there are a diversity of interests represent ed here and there is no privileged character to the information that is passed around. Anything you say is your own responsibility. Now, I am sure that there are things that are known that are not being said. This is the privilege of the persons involved. Our main interest here is to get as much of the general feeling of the information so that we can approach this problem of eventually working on standards and standard methods so that should any immunizing agency develop there will be no holdup in the general availability of these at a later date merely because of failure to start working on standards and methods at an early enough date. Let me emphasize again that this is in fact in the technical sense an open meeting. Dr. Meyer? HT/c 3a Rubella neutralizing antibody studies Age distribution 1 Patients 5 rno- 5 yr 1 i ? ! b - 12 yr \ 13+ i yr Congen. rub. 9 ? V Neut. AB + 11 t f 5 1 f 3 Neut. AB - • 1 2 t 9 1 ? ! ? 0 Controls % ! • 1 ! V Neut.AB + 1 I ? f 2 1 ! i 0 Neut. AB - | L i ' 1 18 ! t 3 1 1 0 1 1 • 0 t • Oj Determination of Rubella Neutralising Antibody in simultaneous maternal, fetal, cord serums and amniotic fluid from patients without clinical rubella » Antibody Titer Gestational Age Maternal Fetal Amniotic Fluid 10 wks - 30 U wks >256 180 108 11 wks 775 55 <1 12 wks - 20 12 wks 512 <4 <4 12 wks - <4 12 wks - 20 ■ 14 wks - 30 3-4 moos - 60 Birth (Cord) 156 >256 78 1000 78 500 75% 325 54 150 51 100 33 78 <4 <4 1 Reciprocal of 50% N«v£rallata« mA p1it HT/c 3b Determination of Rubella Neutralizing Antibody in simultaneous maternal, fetal, cord serums and amniotic fluid from patients with clinical rubella 1 Antibody Titer Gestational /tge Maternal Fetal Amniotic Fluid 11 wks 214 20 1 12 wks 640 16 < 4 12 wks 640 < 4 10 12 wks 10, 240 640 160 13 wks 4, 096 21 2 16 wks > 4, 096 - 11 Birth (Cord) Neutralizing Index > 1. 5 >1. 5 * > 2 > 2 * > 1. 5 > 1. 5 % ♦ > 2. 5 > 2. 5 * Reciprocal of 50% Neutralizing end point * Rubella Syndrome present at birth 1963 Epidemic Rubella Isolates (P roducts of Conception) Total Number of Specimens 20 Positive 6 Equivocal 1 Negative 13 % Positive = 30% HT/c 3c 1964 Epidemic Interfering Agents (Products of Conception) Total Number 12 Positive 7 Negative 5 % Positive = 58% Total Rubella Isolates 1961, 1963, 1964 (Products of Conception) Total Number Specimens 36 Positive 13 Equivocal 1 Negative 22 % Positive = 36% HT/c 3d Rubella Isolates from Product8 of Conception Neg. Pos. 1964 Rubella Epidemic Wc. Pos. Temporal Relationship Virus Isolates from Products of Conception to Termination of Rash Birth Intfaute rine 4 DR. MEYER: We start off this morning with a considera- tion of fetal infection and the rubella syndrome. There are a number of individuals who have indicated that they have informa- tion that comes under this general classification. We have no1 allotted time because I know at least one or two of you have nothing more than briefcase reports type of material. On the other hand, some of the others have more extensive material. I trust you can time yourself as appropriate. I think our first speaker is Dr. Alford. REMARKS OF DR. ALFORD DR. ALFORD: The material to be presented today represents a composite work that has been presented over the past two years in collaboration and under the very capable guidance of Dr. Thomas Weller and Dr. Franklin Neva, Some of this work, such as the neutralizing antibody studies in sera in patients with the rubella syndrome have been completed and is now in publication. Other parts are in various stages of completion. Because of the amount of material, a detailed description of the material and methods would be impossible, so these will be given in a very general manner. Suffice to say that the techniques employed have been presented repeatedly in this meeting. The teratogenic potential of Rubella Virus has been recognized since the initial surveys of Gregg. However, the mechanism whereby Rubella Virus exerts its deleterious effect bl 1 5 on the fetus have remained undefined because of the elusive nature of the virus. Since the advent of tissue cultures techniques for the isolation and maintenance of rubella virus this agent has been demonstrated in both placental and fetal tissues from an aborted human product of conception. The studies to be presented today were undertaken during recent rubella epidemics in the spring of 1963 and 1964 in the Boston area, primarily to elucidate certain of the virologic and serologic mechanisms that occur in the human fetus following maternal clinical rubella. In 1962, prior to the rubella epidemics, sera were collected from neutralizing antibody content, patients with thk stigmata of the rubella syndrome. These sera were obtained from patients under study in the cardiac, hearing and neurological clinics at Children’s Hospital in Boston. Patients from the same clinic, in the same age categories, and with anomalies not considered to be due to rubella constituted the control material. The neutralization procedure was performed on primary human amnion cell cultures employing suppression of Rubella CPE as the endpoint. Could I have th€* first slide, please? (Slide.) In the age group from 5 months to 5 years, 11 congenital rubella sera were positive and two were negative, bl 2 6 whereas 1 control serum was positive and 18 negative for rubella neutralizing antibody. In the total group, 19 con- genital rubella sera were positive and 3 negative, whereas 3 control sera were positive and 21 negative and 1 equivocal for rubella neutralizing antibody. These figures agree well with those reported by Plotkin and Dudgeon from their congenital rubella series and rule out classical immunologic tolerance in most of these patients. Further, these findings indicate that intrauterine infection with rubella virus usually induces a significant antibody response that persists for years, and that the retrospective serologic diagnosis of the rubella syndrome in the pre-school child is feasible, at least in inter- epidemic periods. May I have the lights, please? Further, the presence of this antibody implies the persistence of antigen until the period of immunologic compe- tency, or that the first trimester human fetus, itself, is immunologically competent. In order to investigate this latter problem, simultaneous maternal serum, fetal serum, and amniotic fluid were obtained from patients with recent clinicajl rubella who had their pregnancies voluntarily interruped by hysterotomy. Usually the intact product of conception was delivered by this means. Amniotic fluid was first aspirated, bl 3 7 then fetal blood was obtained via the umbilical cord and dil- uted with ventricular fluid from the same fetus, This latter material will be referred to in this discussion as fetal serum, but it is to be emphasized that this material was initially more dilute than the simultaneous maternal serum. Comparable specimens from hysterotomies obtained for reasons other than rubella constituted the control material. Utilizing an indirect neutralization test, employing the ab- sence of Sindbis virus interference in rubella infected primary human amnion as the endpoint, the neutralizing anti- body content of these materials was determined. The next slide, please. (Slide.) It might be a bit difficult to see. The fetal sera are down to this point. Most of these represent first trimester. This is the maternal column, fetal column, and amniotic fluid. These are cord sera from this point, sim- ultaneous maternal and fetal. In the control serum neutralizing antibody was dem- onstrated in low titer in most of the fetal sera and in one amniotic fluid specimen obtained during the first trimester. The titer of this substance is very low, relative to that of the maternal serum. It seems unlikely that the initial dilution factor could account for the difference in the titer between the maternal serum and the fetal serum, bl 4 By the time of birth, however, as previously reported by Plotkin and Dudgeon, the titer of the antibody is substantial in the cord serum and, in our series, the titer in cord serum is higher per unit volume than that found in the simultaneous maternal serum. The next slide, please. (Slide.) These are the titers obtained following recent maternal clinical rubella, the same quantitative distribution of neutralizing antibody was found in maternal serum,fetal ser| um, and amnlotic fluid during the first trimester, as noted in the control group. Again the titer in the fetal serum is less than the maternal serum but greater than that of amniotic fluid. Rubella virus has been recovered from some of the fetal specimens from which these sera were derived and not from others, yet there is no quantitative difference in the distribution of the titers of the neutralizing antibody in these cases. This antibody may be found in fetal serum as early as one week following clinical rubella in the mother. There is substantial neutralizing antibody in cord sera obtained from infants who were delivered of mothers with clinical rubella during the first trimester. In three of these infants, stigmata of the rubella syndrome were present at or shortly after birth. Interfering agents have been recovered from these infants. To date, no quantitative difference in bl 5 9 antibody content between the maternal serum and cord serum has been demonstrated in these cases, although the neutraliz- ation endpoints have not yet been reached. Only one of these infants appears perfectly normal and has remained so for the past five months. The level of neutralizing antibody in the cord serum of this infant is as high as in those sera from infants with rubella stigmata. These finds indicate that active transplacental transfer of rubella neutralizing antibody does occur during pregnancy, but during the first trimester. This placental mechanism does not maintain levels of rubella neutralizing antibody in fetal serum comparable to that in the maternal serum. Further, following infection of the mother with rubella virus and subsequent infection of the fetus, that the level of this antibody remains very low. These data do not rule out the possibility of fetal antibody production at some time during pregnancy but indicate that the physio- chemical nature of the neutralizing substance will have to be determined to resolve this question. In order to investigate the persistence of antigen during the prenatal period — the other possibility for the neutralizing antibody in congenital rubella sera — 95 specimens of various products of conception were obtained from 36 women whose pregnancies were voluntarily interrupted bl 6 10 because of recent clinical rubella. The figure is now up to 45, but incomplete. The interruptions were done by Dilatation and Curretage, Abdominal hystratomy, amenacintisis, and assisted delivery. Those specimens obtained during the 1963 rubella epidemics in the Boston area were cultured in Primary Human Amnion and African green monkey kidney. To be accepted as a definite rubella isolate, each had to produce typical CPE and Sindbis virus interference which could be maintained on serial passage in Primary Human Amnion, typical cytopathology in stained sheets of Primary Human Amnion, interference to 1,000 of virus on African green monkey kidney, which could be maintained on serial passage and neutralization by specific rubella rabbit antisera in African Green Monkey Kidney. Isolates obtained during the 1964 epidemic are not yet completely identified as rubella virus, according to criteria given above, and the data to be presented represents recovery of interfering agents in African Green Monkey Kidney, though most are producing typclal CPE in primary human amnion. The next slide. (Slide.) This is a summary of the isolation from the 1963 epidemic. And, as you can see, rubella virus was isolated from 30 percent of the products of conception in this par~ ticular epidemic. This material, however, represented primarily bl 7 11 the "D” and "C" material and we cannot say for sure that this necessarily resides in either the fetus or the placenta. It could have been in the maternal tissue. This is some pathological evidence from these tissues that there was involvement in each case, in fact in a wider percentage than we get virus isolation. The next slide, please. (Slide.) The 1964 epidemic, which is still underway and still under study, the percent positive isolation is 58 per- cent at the present time. The next slide. (Slide.) This is a composite for both epidemics. Some of these agents have not yet been identified as rubella. So far, rubella has been recovered from all the interfering material in the past and we suspect will be in these cases. There is 36 percent positive rubella isolation, we will say, in this group. The next slide, please. (Slide.) This slide shows the temporal relationships of the rubella isolates from the 1963 epidemic to the termination of the rash in the mother. This line represents the termination of the rash, the number of specimens. Positive specimens are bl 8 12 the black and the white are negative specimens, according to our techniques that are employed. In this group, virus could be isolated from the products of conception up to 35 days following the termination of the rash in the mother. The next slide, please, (Slide.) During this year’s epidemic, interfering agents have been isolated from the products of conception as late as 42 days following termination of the rash in the mother. This is the last specimen up here. Further, interfering agents with characteristics of rubella virus have been recovered from the throat and urine from three infants with stigmata of the rubella syn- drome at 2 days, 10 days and 2 and a half months of age. Lights, please. At the present time we cannot rule out the possi- bility of the acquisition of rubella virus post-natally in thijs latter group of infants. However, one of these from whom an interfering agent has been repeatedly cultured from the throatj and urine was placed in the premature nursery under strict isolation technique immediately following birth. These findings plus the presence of neutralizing antibody in patients with the rubella syndrome lend credence to the persistence of rubella virus in the fetus throughout pregnancy and after birth for an undetermined period of time. In this regard, bl 9 13 rubella infection of the human fetus could resemble fetal infection with cytomegalo virus. To date, virus has been isolated from 8 placental, six fetal, and five placental-fetal admixture specimens. It has been recovered from fetal brain, heart, and skin muscle specimens. In every case when virus was isolated from fetal tissues, it has been isolated from the simultaneous placetal tissue; however, in two instances it has been iso- lated from placental tissue when it could not be recovered from multiple fetal specimens from the same case, which were treated in an identical manner. I think that is enough for the present time. (Applause.) rel DR* MEYER: I am sure you will agree with me this is an extremely interesting report. I am equally sure there will be many questions* DR* PLOTKIN: The child from whom you repeatedly recovered agents, I take it he had neutralizing antibody as well? \ DR* ALFORD: Yes, they all had neutralizing anti- body* VOICE; Would you repeat that, Dr* Alford* Was that throat and urine? DR* ALFORD: Throats and urine* We have also cultured placenta from two of those cases* The placenta were negative by the time of birth, in spite of the fact that we could recover virus from the infant itself, DR* KRUGMAN: The blood was negative? DR. ALFORD: The placenta* Perhaps — we had recovered placenta isolates, for instance, in the interuterine group in every case* But in the new borns it wasn't in their placenta even though it was in the infant at that time. DR* FELDMAN: Was that fetal antibody level constant or decreasing? DR* ALFORD: Well, we don't know yet* These are very recent acquisitions* We intend to follow it* For the first three months it seems to be about the same. DR* FELDMAN: Well, if you follow the fetal or r«2 infant antibody types, after congenital infection with toxo- plasma, it is about 120 days that you begin to get a very marked difference in maternal and infant antibody* But I think it would be quite crucial in these fluid antibody condi- tions, perhaps around four months in order to separate passive transfer antibody — DR* ALFORD: Yes, actually we are planning to identify the physiochemical nature of whatever material is produced in this neutralization, if possible* DR. SEVER: Have you been able to titer the virus in various organs? DR. ALFORD: We haven’t tried yet. One problem is that we were not attempting a quantitative study originally* We were just attempting viral isolates, so I am not certain that the weight to volume basis of material — for instance when we ground material by hand that isolate virus will allow this* It is quite obvious that certain of these specimens have considerably more virus than others* For instance, the fetal brain in one case is in excess of that of the placenta, which is in excess of that of the skin muscle and so forth* I don’t know what meaning it has at the moment. DR. GREEN: Was the diagnosis of rubella established by laboratory means in the mothers from whose fetus rubella was not isolated? i DR. ALFORD: No, and this is one reason we have not I re3 16 tried to make anything from the percentages in these cases. The mothers in this series were all seen by physicians. The diagnosis was made, their histories were reviewed and often- times seen by three OB men at Boston Hospital before these are undertaken. They all have neutralizing antibody. That’s about all that we can say. Some of them show rises in antibody. We isolated interfering agents from the throats of a few but we were unable to recover any agent from the blood of these mothers. DR. MEYER: That touches on a question that 1 had. You did not get viremia from any of the mothers at the time of the D and C? DR. ALFORD: No. DR. MEYER: Did you try for viremia? DR. ALFORD: We tried, not in every case, but we tried in a number of cases. We could never get any virus out of urine or blood so we abandoned it after we had done so many. DR. MEYER: So insofar as you are showing that the virus at that time is not circulating actually in the fetal — DR. ALFORD: That’s right. We consider it might be in the uterine tissue and are attempting to study menses from normal females. DR. MEYER: One other question that I think is re4 17 interesting that you just touched on, you mentioned that there were pathological findings, 1 assume microscopic pathological findings in many of these fetuses* DR* ALFORD: Primarily the findings are in the placenta and this is someone else's work so I feel I shouldn't go into it into detail but there have been some pathological findings in every case from which we recover and the percent- age if you take the morphological figure would be higher than those that I have presented for virus recovery* DR* KRUGMAN: May I ask one more question* Did you have an opportunity to test blood from the new born infant? DR. ALFORD: Yes, but one of our problems, Dr. Krugmjm, was that ventricular dilution factor. We have also isolated virus from brain. We did isolate it from fetal red blood cells on one occasion, but again it had been diluted with ventricular fluid. DR* robbins: Why did you dilute the blood with ventricular fluid? DR. ALFORD: Well primarily, at the outset of this we were going to study the coagulate, the nature of the globulin in some of these sera, to determine exactly what its molecular weight was and so forth and we assumed that there is a possibility of having some globulin in ventricular fluid. Certainly there would not have been in Hank's and we had to re4 MATERNAL RUBELLA WITH FETAL INFECTION DAY 0: Onset of LMP DAY 13: Conception DAY 25: Lymphadenopathy DAY 2%; Onset of Rash Day 30: Rubella Virus from TW Neut ABY 1 iter (1:2 Gamma Globulin given DAY 31 32: Rash faded DVY 45: Neut ABY Titer 1:8 * DAY 86: Uterine curettage Rubella Virus from Fetus re5 18 have some dilution in order to carry out the procedures. There is an insufficient quantity of serum that would be obtained to perform these tests. DR. MEYER: Thank you Dr. Alford. I think next we have Dr. Heggie with more of the same. REMARKS OF DR. HEGGIE DR. HEGGIE: I would like to report a case from which we were able to isolate rubella virus from the throat early in pregnancy and from the fetal tissue after about three months gestation when the pregnancy was interrupted. I think I can do it all from this one slide. (Slide) This lady had determined her basal body temperature during her previous menstral cycle, having intended to become pregnant at this time, and so we were able to plot from this chart she had maintained, the probable date of conception. If we call today zero the onset of the last menstral period from this chart she had kept she had conceived approx- imately on date 13. On day 25 she was noted by her physician to have lymphadenopathy of the posterior occipital type and she had the onset of the rubella rash on the 29 day after the onset of her last menstral period. She appeared in the emergency room of our hospital on day 30 having been sent in by this physician to receive re6 19 gamma globulin, it being one of the responsibilities of this hospital to give gamma if the physician sends the patient in requesting it* She received at that time 20 milliliters of ordinary commercial gamma globulin and throat washing and acute serum were obtained from her* From the throat washing we were able to isolate rubella virus and her neutralizing antibody titer, this all being done in the African green monkey system. Her neutralizing antibody titer was less than one to two. Day 31-32 her rash faded* After her rash faded we were able to obtain a convalescent serum and her titer had gpne up from one to eight* She was lost to follow up on* We did not hear from her again until she consulted an obstetrician for pregnancy care and he after consulting with her decided to terminate the pregancy by and C". And from these fetal tissues which we received, we made an extract and we made some explant cultures. The tissue is not specifically identifiable except for a piece of a lower extremity, why I presume that these cultures we were able to grow were essentially skin muscle preparations* And from both the extracts made from this fetal tissue and from the supernatant fluids overlayed the explant cultures we were able to isolate rubella virus. Hie virus from the fetus was neutralized by the re7 maternal convalescent serum and not by her acute serum* And the virus from the mother compared to the virus from the fetalj were both neutralized to the same degree by a convalescent serum from a third laboratory proven case of rubella* We feel this is interesting because the lapse of time between the onset of the maternal difficulties and the isolation of rubelli virus from the fetus was 57 days* (Applause) DR. MEYER: Thank you, Doctor. Are there any questions? DR. KEMPE; Can you give us the dosage of gamma globulin? DR. HEGGIE: It was 20 ML. That was given after the onset of rash. DR. MEYER: Any other questions? (No response) DR. FELDMAN: I think it would be terribly important if somebody, perhaps some of you do have already cases where the rash or the clinical difficulties occurred just prior to j conception. Again, if I go back to the old model we worked with, the toxoplasma, when this happens there is no infection of the fetus as one might expect. But it would be nice to | be able to corroborate it with another. I - You are almost at that point and it is a little difficult to understand actually when you think in terms re7 20a ISOLATION OF RUBELLA VIRUS FROM THE FETUS FOLLOWING MATERNAL RUBELLA SUBJECT WEEKS PREGNANT AT TIME OF RASH 1 WEEKS BETWEEN 1 RASH AND ABORTION RUBELLA VIRUS ISOLATED r GAMMA GLOBULIN M.V. 7 1 YES YES J.W. 8 1 YES NO M.P. 9 1 YES NO C. N. 6 3 NO *; NO L.B. 2 4 YES • NO 0>S. 6 4 YES NO B.Z. 2 6 % YES NO C.V. 3 6 \ YES NO * 20CC GAMMA GLOBULIN ON DAY OF EXPOSURE + CLINICAL DIAGNOSIS OF RUBELLA CONFIRMED BY ISOLATION OF VIRUS FROM PHARYNX r©8 21 of the fetus or the embryo just how the virus gets to the embryo itself* If somebody can find one or you can get one where the infection occurs just prior to a case like this, where records exist for it, it would be very helpful. One would expect the embryo would be made infection free. It would be nice to corroborate it. DR. HEGGIE: I have no such case. DR. FELDMAN: I think it would be one to keep an eye open for. VOICE: There is one in progress right now. Dr. Balsamo is studying one. DR. MEYER: Dr. Balsamo. REMARKS OF DR* BALSAMO DR* BALSAMO: For the past year we have had the opportunity to study 12 specimens obtained by therapeutic abortion for first trimester rubella* All specimens were obtained by dilation and curettage* Thus far we have corapletejl viruses isolation studies in eight of these specimens. All virus isolation studies were carried out by the Interference technique utilizing the green monkey kidney* The results of virus isolation studies are summarized on the following slide* (Slide) As you can see the weeks pregnant at the time of the rash varied from 2 to 9. The weeks between the development of re9 22 rash and therapeutic abortion varied from 1 to 6* We were able to isolate the rubella virus by the interference technique in 7 out of the 8 patients* In patient CN, from whom we were not able to isolate the virus, she was seen at the time of rubella rash* She had the characteristic rash and lymphadeno-l pathy seen in rubella and as the rash faded she developed what is described as rubella arthritis* This lasted for a period of 4 or 5 days and then cleared spontaneously* So it is of interest that this patient in whom this diagnosis of rubella was definitely established we could not isolate virus from the time of conception* I should mention also that most of the specimens obtained by us were a small fragment of tissue and we could not distinguish in most cases from fetal and placental tissue* The third patient on the list, MP, it was possible to clearly differentiate between placental and fetal tissue* Then the virus is isolated from the fetus and the placenta* The next and last point of interest on this slide is that only one of the 8 patients received gamma globulin* This patient was of particular Interest because she had a very definitive date of exposure when her sister returned from college with a rash* She saw her obstetrician on that day and received 20 ce's of gamma globulin* Twenty days later she developed tyical rubella rash and she had a thera- peutic abortion performed one week later* re 10 23 It was possible as you see on the chart to Isolate an interfering agent from the product of conception in this patient* I should mention that the first two patients on the chart had their rubella and the therapeutic abortion performed in the spring of 1963* The remaining six contracted their difficulties dur- ing the present epidemic* Thank you* (Applause) DR* MEYER: There seem to be several questions* DR* NOVAK (Syracuse): I wonder if you have been able to determine the level of antibody gamma globulin in that first patient? DR# BALSAMO: We have not# This was done in the hospital outside of New York City proper and it was gamma globulin supplied by the Department of Health. We have other j charts# I have data on the Department of Health gamma globulih which I will present later this morning. DR. MEYER: Are there any other questions? (No response) Dr. Schiff? REMARKS OF DR. SCHIFF DR. SCHIFF: Could I have the slide, please? (Slide) These are some results from human volunteer studies 23a RECOVERY OF RUBELLA VIRUS FROM BLOOD Days after Inoculation V 0 L u N T E E R rell 24 which wc have done over a year ago, I show this to characterize the period of viremia in the patient* The patients have received an internasal inoculation of tissue culture passage virus* This is 100TCID 50, what we consider a high dose as compared to nature* | Vt / The rash occurred in these patients on the 12 date but we were able to recover virus from the serums as early as the 6 day* At the time of the rash, or when the rash had r *r "■ ‘ ; . f gone, the viremia had disappeared* At the same time anybody at low levels was detected, the antibody rose significantly over the next three weeks* Slide off. For this reason we were very interested to learn of the studies of Dr. Selzer where she had recovered virus from the fetus at a time relatively long after it seems to dis- appear from the maternal blood* Soon after we were able to obtain the fetus — that was last January — the history of this fetus is as follows: from a patient, a 21 year old white woman, who had her LMP on ' the 30 of November, was exposed on the 5 of January, received gamma globulin six days later and developed rubella on the 18 of January or 13 days after exposure, seven days after receiv- ing gamma globulin* Yet she received 12 cc's of gamma globuli i. Two and a half weeks after the development of the clinical difficulties she underwent a D and C* We were able to be rel2 25 present at the D and Cf were presented with the scrapings* From this we were able to identify only a very small piece of fetal tissue, of fetal heart* This was washed carefully in Hank's,balanced salt, washed five times, frozen in Hank's with gelatine at the site of the procedure. Later it was made into a ten percent suspension, diluted in half log dilution and inoculated in the monkey kidney tissue* Through the interference technique we were able to recover ten fco the two logs of virus from this particular specimen. At the same time a piece of placenta or decidua, or unidentifiable description «— which we have received that cannot be identified as being fetal tissue — was also inoculated and we recovered one and a half logs of virus* Thii is all the first, from the raw specimen* A sera collected at that time of the D and C had an antibody of 1:16. A throat swab at the time of the D and C was tested for the virus and also the sera was tested for the virus. Unfortunately we had no pre-bleed on this particular patient. We were quite impressed from the results of this particular adventure on the great chance of mixing maternal fetal tissue. So we then have placed most emphasis on getting the larger in toto fetuses. In recent weeks with the fine cooperaj. tion of the 16 hospitals and universities which are members of the perinatal research project and under the interested re 13 obstetricians we have been able to collect 14 additional fetuses* Many of these, or the majority of these have been done by vaginal and cervical packing, hypertonic salt instillation of the uterus and "C" section. There are several which have also been done by md and C". In most of these cases one of the personnel from our laboratory, one of the four divisions of our laboratory, were present at the procedure, were able to handle the tissues in a manner which would be most optimistic to recover virus. At the time of the procedure attempts have been made to collect maternal cord blood, blood from the fetal heart, araniotib fluid, placenta* In most patients in the perinatal study, a pre-blood has also been collected* The results from these fetuses are not finished* Several are on test but none have been com- pletely worked up* I can only report one additional findingy something which I think would add to the information by Dr. Alford* This was a woman who had rubella in the second week of pregnancy and then seven days later underwent a C section on which materials were collected* At the time of procedure the fetus was delivered in toto* The fetal membranes were intact* And so different materials were separated quite rel4 carefully. From the amniotic fluid of this particular fetus we were able to recover a virus. None of the other studies have been completed. (Applause) DR. MEYER: Thank you. Are there any questions on Dr. Schiff's presenta- tion? DR. PARKMAN: In that amniotic fluid were you able to titer how much virus was present in that? DR. SCHIFF: This is being done. We have many analyses of this and we got only recovery first on this and we are now back on it. DR. MEYER: That brings us down to Dr. Parkman. REMARKS OF DR. PARKMAN DR. PARKMAN: I would just like to mention briefly our experience with two human embryos in therapeutic abortion. The first of these abortions was performed four and a half weeks after the onset of rubella during the first months of pregnancy* We confirmed the diagnosis of the rubella in the mother by serologic studies. We attempted to isolate virus from ten percent suspension from fragments of placenta, decidua and fetal tissue. We were able to Identify the fetal fragments as coming from a fetus of about 30 to 40 days by the facial characteristics of the fetus. We did not recover any viruses rei4 27a RUBELLA IN PREGNANCY Pt» M. K - - - Onsetof-rash: 1/12/64 • -acute -serum- (1/14) -.• -conv* serum 1:8 (2/8) - Iherepeutic-abortion on 3/9/64 Rubella virus recovered from fatal fragments. FETAL RUBELLA INFECTION Tissue Virus Titer 10% susp. l°glo/gm of tissue Face & eye 2.8 Mandible- 2.5 Ribs 2.5 Fo6t-& hand 2.1 Intestine 3.5 Liver 1.5 Placenta 0.6 r*15 from this first attempt in the greater monkey kidney or LLCMK 4 ■ cultures* May I have the first slide? (Slide) The second patient had the onset of rash in January of this year during the first month of her pregnancy. Sera drawn on the 2nd and 24th days of disease shows a signi- ficant antibody increase. On the 57th day after infection, or at about two and a half to three months of pregnancy, a thera- peutic abortion was performed. May I have the next slide? (Slide) This abortion was performed by a dilation and curettage. ah specimens ware collected in one container. Therefore all specimen were floating in the same fluid. We were able to divide the fetal specimens into individual specimens. Each specimen was washed several times salt with cold balanced/solution and made up as a ten percent suspension in balanced salt containing one percent bovine plasma albumin. Each of these specimens was then inoculated into primary African greenraonkey kidney cultures. Virus was recovered from each of the fetal and placental specimens. No virus was recovered from the fluids which were collected at the outset of the procedure. This was mixed maternal blood re!6 29 and amniotic fluid* As you can see, the titers of virus per gram of feta| tissue ranged from 1*5 for delivered specimens to 3#5 for the small fragments of intestines and attached mesentery. The virus titer from the placental specimens collected in the same container were significantly lower than those of the fetus itself* And as I have said the initial fluid mis negative for virus isolation. These by the evidence may suggest that the uniformity of virus titers is not simply the result of contamination of one specimen by another. If we can assume that this is true it appears that the infection of fetal tissues appears to be generalized and does not appear to be confined to those involved in the manifest congenital anomalies^. (Applause) DR* MEYER: Thank you Dr. Parkraan. Are there any questions of Dr. Parkman? DR. SEVER: Dr. Parkman, on the placenta, that has a lower titer than any fetal tissue? DR. PARKMAN: Yes. DR. SEVER:: Then the mixture of embryonic fluid in maternal blood — DR. PARKMAN: That did not yield virus. DR* SEVER: I wondered if this might possibly be a function of the presence of high neutralizing antibody titer rel6 29a Table 1. Experimental Rubella Infection in Ferrets 0.5 ml 50 RV strain, Passage 7-Intranasal Day Animal No. Virus Recovery Anterior Turbinate Boney Turbinate Lung Serum 3 1 0 0 0 0 2 0 0 0 0 6 3 x 0 0 + (P) 0 4 0 0 + (P) 0 9 5 0 0 0 0 6 + (0.5) + (0) + (P) 0 12 7 0 0 0 0 8 + (1.5) + (P) 0 0 14 9 + (P) 0 0 0 10 + (P) 0 + (P) 0 Totals 4/10 2/10 4/10 0/10 Figure 1; Rubella Infection in Ferrets + = A.T. * = B.T. o = Lung rel6 29b Study 3; Experimental Rubella Infection - Weanling Ferrets Sacrificed 12th Day No. Inoculum Amount Inoc. Route Virus Recovery 1 6A 1 ml 100 TCID50 IM 0 2 n ii II 4- Turb.,Spleen 3 m ii SC + Turb.,Spleen 4 ii 0.3 ml 100 TCID50 IN + Turb.,Lung,Sp1een 5 1! II II + Turb., Lung 6 Ferret Turb. 1 ml 100 TCID50 IM + Lung, Turb. 7 it ii ii 0 Study 4: Experimental Infection in Pregnant Ferrets Inoculum: 0.5 ml 100 TCIDc;n RV Intramuscularly Animal Pregnancy Outcome Day 9 Day Sacrificed RV Recovery 1 Spontaneous delivery- cannabalized 21 ND 2 1! II ND 3 Spont. delivery- 7 live born 10 + Study 4-B: Recovery of Rubella Virus (RV) from Pregnant Ferret and offspring. Animal Day Sacrif. Anterior Turb. Boney Turb. Spleen Lung Liver Mother #3 10 + + + 0 0 Offspring #7 Age< 1 day CNS/0 Rest of carcass/0 rel6 29c Study 5: Experimental Rubella Infection in 2 week-Pregnant Ferrets. Sacrificed 10th Day Animal Route Inoculum i Amount RV Recoveries Uterine Contents Other 1 IN 0.3 ml 100 tcid50 0 + 2 IN 0.3 0 + 3 IM 1.0 0 + 4 IM 1.0 0 0 Summary: Recovery of RV in Experimental Infection of Ferrets Route No. Recovery of RV % Weanling IN 19 15 79 IM 4 2 50 Pregnant IN 2 2 100 IM 3 2 67 Total 28 21 75% rel6 29d Neutralizing Antibody Response of Ferrets in Experimental Rubella No. Route Pre-Inoc. 3 6 Days after 9 10 12 Inoc 14 21 Weanling 10 IN <1/4 <4 <4 <4 <4 <4 5 IN ii <4 1 IN ft 16 2 IM ii 16 32 Pregnant 2 IM f 1 8 16 Inactivated Ferret Serum vs o.5 log RV Study 2: Recovery of RV from Ferrets Inoculated with 0.5 ml of 100 Intranasally: Sac. Day 10. Animal No. Ant.Turb. Boney Turb. Spleen Lung Brain Serum 1 + + 0 + 0 Tox. 2 0 + 0 0 0 Tox. 3 + + +? + 0 Tox. 4 c C C + 0 Tox. 5 + + + + 0 Tox. 29e rel6 re 17 30 in maternal circulation which might have been present? DR. PARKMAN: Yes, this is certainly a possibility. DR* SEVER: Would you venture a guess also as to the reason for the higher titer being in the intestine? DR* PARKMAN: I really don’t know why the intestine was higher* There was some attached mesentery so it is not really intestine, It is a mixture of tissues. These were not pure specimens, certainly the extremities and the face specimens contained skin, muscles) and other tissues. DR* MEYER: We seem to be in that peculiar position today as compared to yesterday of being way ahead of time* I am not sure which is worse* I believe Dr* Schiff if you can get your slides, we will go ahead with some of the work on experimental animals and take our coffee break in about thirty minutes or so* REMARKS OF DR, SCHIFF DR, SCHIFF: The availability of an animal model system for experimental rubella would be of particular value in the studies of the mechanisms of pathogenesis,of the dis- ease, the development of vaccines for rubella, and the evalua- tion of newer anti-viral chemo-therapeutic agents* re 18 31 In our laboratory, attempts to produce disease in mice, guinea pigs, hamsters, rats, rabbits, A.G. monkeys and baboons have failed. Similar attempts in rhesus and squirrel monkeys have yielded some early encouraging results, but to date these have not been confirmed. Tbe ferret was incorporated into our rubella studies following the report by Coates and Chanock describing the use of this animal with respiratory syncytial virus. If I could have the first slide. (Slide) This is the ferret* The ferret is a member of the weasel family* It is amniverous, it eats fruit, berries, dog food and careless investigator’s fingers* It has been used for centuries to hunt rats and mice, to rid buildings of rats and mice, also small field rodents and rabbits* In addition, they have been used in laboratory research in connec - tion with canine distemper virus, influenza, rubeola, denta hygiene, reproductive cycles, and epilepsy. Slide off. For these studies six week old weanling and preg- nant ferrets were obtained from the Gilman Marshal Company. The inoculum used was our laboratory strain of RV rubella virus. This virus was initially isolated from the ferrets of a six year old boy within 24 hours of the appearance of rash. It*s been kept the last two years in tissue culture in our re!9 laboratory. The animals were pre-bled, inoculated, and then sacrificed at various intervals up to 14 days* At the time of sacrifice, the organs were removed, placed in a ten percent suspension, and tested for virus with the interference technique. Several ferrets were allowed to live up to 21 days, at which time they were bled for antibody levels. Again, the antibody titrations were done with the interference technique* All specimens were passaged three times before being considered negative. All isolates were identified with specific hyperimmune rubella antisera* Portions of the organs removed at the time of sacrifice were examined histologically* To date, five studies have been performed. In none of these have the animals appeared to be clinically ill* Although the studies are not complete to date, no pathological lesions have been seen on the histological sections* May I have the next slide, please? (Slide) Now, study 1, ten weanling ferrets were used* They were inoculated intranasally with our passage 7, seven times in monkey kidney virus* As you see#a total of half a milli- liter of ID50 intranasally, .25 ML’s in each nostril* Then at three-day intervals, except for the last interval two re20 animals were sacrificed. From these, on the third day we were unable to recover virus. Now the term anterior turbinate refers to the epithelial tissues overlying the turbinates; the bony turbinate was underneath, and also a little bit posterior to the epithelial tissue. On the sixth day with one passage we were able to \ recover virus from the lungs of both of these animals* On the ninth day we were able to recover a half log of virus from the anterior turbinate* An undiluted specimen yielded virus from the bony turbinate and again it took a passage in the lung. On day 12 we got virus out of turbinate; at 14, out of turbinates lAnj one lung. Could I have the next slide, please? (Slide) Some attempt to show growth of virus. The dotted line is the threshold of ability to recover from a specimen. Passage means it took one passage for the virus to come out. The anterior turbinate samples seem to yield the greatest amount of virus. On day 12 we got one and a half logs. The bony turbinate seemed to yield second most and then from the lung it would move up in amounts It also took passage to get material out of the lung. Next slide, please. re21 34 (Slide) We then got an additional group of five ferrets in. We gave them slightly higher inoculum intranasally; sacrificed all on day 10. Then we had recoveries from the turbinate; this time from the spleen from the lung; no virus was recovered from the brain. We were unable to recover virus from the serum. I might go back: In the first experiment we tried for serum and we were unable to do so. Unfortunately, this serum had been inactivated before we tried it. This time the serum was not inactivated. It proved quite toxic to our tissue culture despite steps taken to prevent the toxicity. This was a one to four solution. No virus from the brain. We have also tested liver kidney without any recovery of virus. Next slide, please. (Slide) Another group of weanling ferrets; these were all sacrificed on day 10• These received different inoculum. This inoculum is 6-A, the strain of virus which has been through nine passages in monkey kidneys and three passages in rabbit kidney. This titers six in rabbit kidney. We gave some of these animals intramuscular injec- tions* This also is a mistake, this also should be IM* From re22 these we recovered virus from the turbinates and supplies to the animals* We inoculated some animals intranasally with this inoculum and got isolations from turbinate, lungs and spleen. We also took a suspension from which we recovered virus from the turbinates of the ferrets in our first study, and inoculated intramuscularly and we were able to recover virus from the lung wi the turbinate. Now to date the titrations of these have not been completely finished, or not all specimens have been titered* There does not seem to be any increase in titer from these over — they are all in the range of 10*"1 and 10“2. Next slide, please* (Slide) The next two studies were gauged at trying to determine the effect of rubella virus on the ferret fetus* The gestational period of ferrets is 42 days* However, these are quite difficult to breed successfully, and are probably difficult to maintain in the early part of pregnancy. This is due to the temperament of the animal and also to the great increase in susceptibility to infection during pregnancy* Now the first pregnant study involved three animals. While we had asked for animals early in pregnancy, as it turned out these were quite late in pregnancy. We inoculated these intramuscularly with this passage 7, seven times in monkey kidney strain. These delivered, much to our surprise, re23 much earlier than we had thought* They delivered sometime during the evening on the 9th day after inoculation* The mothers apparently were hungry and ate what they delivered* These were not sacrificed but were allowed to go on and were used for antibody studies* The third mother — if I could have the next slide (Slide) was sacrificed the next morning or day 10. From her we were able to recover virus from turbinates in the spleen. Also from here seven offspring which were sacrificed on the follow-| ing morning, on the morning of day 10, we tested the brains and the rest of the carcass minus the skin. These have been through three passages in tissue culture and have been negativ|e. The next slide, please* (Slide) The final study, which has not been completed yet, from the isolation point of view, involved four pregnant animals* They were again sacrificed on day 10. Two received intranasal inoculations of our passage 7 material in two IM inoculations* This time the pregnancy was interrupted on the tenth day. They appeared to be in their early pregnancy: the uterine contents were carefully removed and washed and ground up and tested for virus* This appeared through one tissue passage and has been negative* From two of the mothers we have been able to recover r©24 virus — excuse me — from three of the mothers virus from the turbinate the lung or spleen* Next slide, please* (Slide) This is a summary of the recovery of RV from our experimental rubella in ferrets to date* This includes the two which we attempted to recover virus from on the third day* Overall we have 75 percent batting average* Next slide, please* (Slide) This is a study of the antibody studies done to datej. From the weanlings, those which were sacrificed, they were at the time of sacrifice also tested for antibody levels. They all were less than one to four on the pre-bled, but none of these which we sacrificed up to date — 14 — were we able to detect any antibody. The five which were allowed to go 21 days all showed at least a significant fourfold rise, This is 21 days after inoculation. This is done in monkey kidney and rabbit kidney. This one goes up to 128. Any questions? DH. MEYER: Any questions regarding ferret infection? DR# SEVER: I would like to add that in our studies with rabbits we have tested the products of conception after the intramuscular inoculation of rubella virus to the pregnant re25 rabbits approximately on the 6th day of pregnancy and in the one large series that we have completed tests we have not recovered the rubella virus from the material obtained at the terminus. Of course these rabbits at that point, that extended the current study by injections directly in the uterus to the fetus* DR. MEYER: Dr. Phillips? DR. PHILLIPS: Have you doneany studies so far as contamination to find out whether this is transmissible from ferret to ferret? DR. SCHIFF: No, we have not. DR. MEYER: Since rabbits were mentioned again — they were mentioned yesterday — let me ask you: Do you have evidence that you can recover virus, ignoring the fetal rabbit,from the mother, can you get virus types from rabbit tissues, rabbit blood, or other types of material? In other words, what evidence is there, for Mow good the rabbit is as an experimental animal? DR. SCHIFF: The studies to date using intranasal infection have not revealed as good results as ferrets, from the tentative findings which we have just received. Rabbits may be of some use; however, this needs to be explored in detail. We don’t have the final Information on that. DR. MEYER: Dr. Buescher? re26 39 DR* BUESCHER: May I ask the strain of rabbit that is being used? DR. SCHIFF: White rabbits. VOICE: NIH rabbits. (Laughter) DR. MURRAY: If these are NIH rabbits this can be \ established. DR. SEVER: No, they are not, it's Norwegian rabbits or something like that. DR. MEYER; Let me ask Dr. schiff a question, again turning to rabbits. The reason we keep pushing this is I think the experimental host is something we are all very interested in, since this has been a real need in the rubella field, and there seems to be evidence that rabbits can be infected —< how useful it may prove to be remains to be seen. Has anyone had any evidence of communicability in rabbits? Has there been any work done on cage contacts or anything of this sort? DR. schiff: From our own studies we have had no evidence of communicability of rabbits. We have not seen the development of neutralizing antibodies in rabbits held in the same general area as infected rabbits. We have not done the very elaborate study that we havecbne with ferrets and other animals, and cages specifically set up to promote this type of investigation. re26 39a TABLE 1. GERMAN MEASLES VIRUS. LYMPHADENOPATHY SCORES & SEROLOGIC RESPONSES OF CERCOPITHECUS MONKEYS INOCULATED WITH STRAIN M33 & OF THEIR CAGE CONTACTS INOCULATION MONKEY LYMPHADENOPATHY SCORE TITER VOLUME STATUS ING. AXIL. ON DAY ON DAY & ROUTE INOO. CONT. SEX NO. L R L R 30 50 1.0 ml. IP ♦ 0.5 ml. IN X X M 561 + + + 10,11* 50 20 X F 567 + + + + 10,11 13 10 • X M ')*>/ - - - + 21,22 5 <4 X F 56H - - <4 II 1.0 ml. SC X M V- 1 - - 20 5 X F 5t> y To TA/re<£T. i/v SfAutn A '.*)S/(/-¥■) C^uL€“A/(SF^ No. MiTH |No. WjiTHOUT Ru££l/-* ftu6£*4i./f t',\C - t /« 7~ 5~Q- ' £ T’Atss/vr 2 7 //7?6Lje £o6a£CTS CLw/caC 1 SofiC. lf*c. :- /■ F. C/F ■1 ■ -' - t'ftt't jit -!' ' SPr.a- /-‘2 • zz zz O 1 /o * 3 n *■ * // 5“ , vO C ; . . _ Bf. i e r i i . . s / 1 0 i 1 .i CLirttCAL CAStS l ft/** AKb MTt&Qbj RUr U)iT¥ OR UJiT-tfouT v/iftO-S l’SolRr/0/ £ f&RRSOt 60T MTiflflfc/ M)*T* **uATfcouf v<*>* LaT'«* 49b PREVALENCE OF RUBELLA VIRUS ANTIBODY ACCORDING TO AGE % WITH NEUTRALIZING ANTIBODY (SERUM DILUTION 1 -4) AGE IN YEARS NUMBERS WITHIN COLUMNS INDICATE NUMBERS Of SUBJECTS *ALMOST ENTIRELY FEMAlS IN THE FIRST TRIMESTER Of pregnancy EXPERIMENTALLY TRANSMITTED RUBELLA 32 CASES TIME OF ONSET OF LYMPHADENOPATHY AND RASH LYMPHADENOPATHY RASH % OF 32 CASES OF RUBELLA DAYS AFTER I.M. INJECTION OF RUBELLA SERUM 49c EXPERIMENTALLY TRANSMITTED RUBELLA IN FIFTY SUBJECTS RELATIONSHIP OF PRESENCE OF VIRUS IN BLOOD. PHARYNX AND STOOL TO TIME OF APPEARANCE OF RASH ■ RuBE~_A VIRUS ISOLATED '' Y:T SO.ATEC NUMBER OF SPECIMENS NUMBER OF DAYS BEFORE —* RASH «— NUMBER OF DAYS AFTER EXPERIMENTALLY TRANSMITTED RUBELLA NEUTRALIZING ANTIBODY TITERS IN 9 SUBJECTS SERUM DILUTION DAYS 8EF0RE RASH DAYS AFTER RASH MONTHS 50 virus. That represents two of the five. DR. PHILLIPS: The other three had been housed here for a long time. Excuse me. DR. MEYER: The question of communicability is of course of interest both in man and in monkeys. Especially in man as it relates to any tissue culture passage material. We deliberately have not given any data on this, because our data at the moment are inconclusive. I think, Doctor Cabasso, you were using 17 passage material? DR. CABASSO: 25. DR. MEYER: You were using relatively high passage material and in the experiment you reported on there didn’t seem to be too much evidence of communicability. We hope to have a definitive answer to that shortly. It is possible earlier passage material, such as we are using, might be communicable and when the higher might not. Maybe the New York people can tell us something about man which would be even better than what we are finding out about monkeys. Thank you, Doctor Phillips. Dr. Green? EXPERIMENTAL INFECTION OF MAN WITH RUBELLA VIRUS Experimental infection of children. DR. GREEN: This report concerns the experimental transmission of rubella to children which our group has been conducting during the past two years. 51 The titration originally described by Dr. Buescher and Weller and their associates on the African Green Monkey kidney system has been used exclusively for our virus isolations and in the antibody determinations. First slide. Now this slide is a composite of a number of experiments in which attempts were made to transmit| rubella to subjects, some of whom we didn't know were immune — well, we didn't know their immunity status. And in some instances virus was inoculated and in others I suppose it was by contact. This will come out in the next slide. But I think you can see at once that individuals who had base line serum antibody of less than 1-4 were by and large sus- ceptible. Forty-six out of 54, or about 85 percent contracted the disease in marked contrast to those who did possess such antibody; 37 in this group all uniformly resisted infection. Next slide, please. Now this slide illustrates the efficacy of several methods of exposure in transmitting the experimental disease. When virus in the form of infectious serum was injected intra - muscularly, all of the 22 subjects developed classical rubelli. When similar serum was sprayed into the nasal pharynx, it wasn't nearly so successful. Seven out of 10 developed rubella and three of these were sub-clinical cases. In other| words, the diagnosis was a laboratory one, but they did not have a rash. Two types of contact exposure had been tried. 52 The idea here was to try to simulate the conditions that occur in nature, first to have prolonged or repeated contact that happens when the child or children of a pregnant mother contracts rubella and she is with them day in and day out for weeks, and here we took children infected with rubella and put them with groups of susceptible subjects and allowed them to mingle freely for a period of days. This was pretty effective, as you can see; 16 of the 17 susceptible subjects developed disease, but interestingly enough, five, or about one-third had sub-clinical disease. Finally, the brief single type of contact that might occur when a pregnant woman meets a friend and chats with her briefly and then the next day her friend calls up and says ”1 have got a rash.” Here this obviously is not very effective as a method of transmitting the disease. Only one of five such subjects developed rubella. Next slide, please. We have attempted to determine the prevalence of rubella virus antibody according to age. I should say at the outset that these are highly selected groups; virtually all of the children were institutionalized children, many of them had been there for sometime, although a fair number had come in from the outside. In the greater than 15 year group practically all were young pregnant females who did not have a history of 53 rubella. But I think our figures here are somewhat higher than those that were shown yesterday. It seems to be some increase up to, oh, the 5 to 10 year group, then it seems to level off and there is not much difference after that. Next slide, please. x This slide illustrates some of the clinical manifestations of the experimentally transmitted disease. The rash and lymphadenopathy were in no way different from that seen in the naturally occurring disease. These children by and large didn't have fever, they seldom did, and did not appear ill. After injecting the virus in the form of infectious serum, most of them got lymphadenopathy within about eight days and the rashes occurred about two weeks later, about a week later, or two weeks afterinoculation. Now in the disease that was acquired by contact, this was pushed over about a week. In other words, the lymphadenopathy and rash actually occurred about a week later, which corresponds very nicely with the usual period. Next slide please. Now as you have heard today and yesterday, it is apparently pretty easy to isolate virus from the pharynx, seijum and stool of infected persons. Here we have compiled all of the data on 50-some subjects using the day of rash as a 54 focal point. One can see at once that virus is almost always present in the pharnyx on the day of rash and for a few days before and after. In one instance, I guess in two or three instances we found virus as early as seven days beforethe appearance of rash and in one instance as late as two weeks after the appearance of rash. In contrast to this, virus is found most commonly in the serum for a few days preceding rash and on the day of rash there is a rather sharp drop, and after rash, after the day of the appearance of the rash, we have isolated virus in only two instances, one on the first day after rash and once on the second day after rash. Now as you will see in the next slide, the disappearance of viremia corresponds very nicely to the appearance of antibody. Also virus has beenisolated from rectal swabs a number of days before and after rash. But as you can see this is pretty irregular. Next slide please. Here we are trying to demonstrate the pattern of development of antibody in the experimentally transmitted disease. These are nine subjects in whom we had serial bleedings with some interruptions. This antibody is almost always absent before rash and in a few instances it gets a low titer on the day of rash, but by and large it makes its 55 appearance two to five days after the appearance of rash. It then seems to reach a peak within a month and from what little data we have it would seem that the peak levels are maintained for at least a period of six to 12 months. That is the last slide. To go back to your question about degree of we have not done a great deal in this regard, but in some instances we have found, and Doctor Balsamo may want to elaborate on this, but we have found titers as high as 10 to minus 3. DR. MEYER: Are there questions? DR. ROBBINS: I think the question that Doctor Meyer raised was the relative susceptibility of the child as opposed to the tissue culture. Don't you have data on this? VOICE: I think what the question is is how far the titration of infectious serum in children went as opposed to infectious serum in tissue culture and in childrenj we had infection out to 10 to minus 3, whereas in tissue culture the virus was present only on second passage, it was not present in first passage. DR. GREEN: Yes, there is quite a gap. VOICE; Therefore the child was much more sensitive than the tissue culture system. DR. GREEN: Yes. 56 DR. MEYER: This is working with infectious human serum as the source. You have no information on tissue culture material as the infecting source? VOICE: No. DR. WARREN: In the individual patient, when you compared antibody titer and carriage of virus in the throat, was there any correlation, because your slides suggest that you can have lots of antibody and still have lots of infections virus persisting in the throat for sometime. DR. GREEN: We have not actually analyzed our data carefully for that point, but I have the impression that that is true. DR. HENDERSON: Henderson, Atlanta. I wonder if you could elaborate a bit as to what you mean by prolonged contact and whatyou mean by brief contact? What sort of prolonged contact was it? DR. GREEN: We have actually two isolation wards at Willow Brook, and in the prolonged experiments, consist- ing of prolonged contact, we infected children by the intramuscular inoculation of known infectious serum and then we just turned them loose on a ward with a number of suscept- ible subjects and they stayed there during the entire experi- ment. In one experiment I think we didn’t turn them loose on the ward, so to speak, until their rash had developed. Now the brief contact, two subjects on the day of 57 appearance of rash, after they had been infected by intra- muscular injection of serum, were allowed to kiss the sus- ceptible subjects and that was it. They were just brought into the room and they kissed each other. VOICE: Just once? DR. RUEGSEGGER: Were any of your nurses \ (?).....antibody before and after? t DR. GREEN: I couldn’t hear you. DR. RUEGSEGGER: Were any of your nurses or ward attendants tested for antibody before or after this? DR. GREEN: No, we have not done this. DR. RUEGSEGGER: Did you try to screen them for a history of it? DR. GREEN: No, we haven’t done it. DR. KRUGMAN: In answer to this question, the same attendants and the same nurses and the same physicians have been present in this unit for the past year and a half and have been intimately exposed time and time again to natural rubella. I think this is an important point. And none have ever acquired any evidence of disease. DR. SCHIFF: I can answer Doctor Warren’s question partially with a slide here. Before I do that, about our own volunteer studies with adults, we had two patients who were antibody free, two volunteers antibody free in the same room with those who had the disease for a period of eight days and these did not show any evidence of disease nor could virus be recovered or antibody conversion. VOICE: They didn't kiss each other? DR. SCHIFF: They might have. DR. SEVER: I think it should be pointed out in those studies, tissue-grown virus in fifth or seventh passage was used. 58 DR. SCHIFF: Yes. This was fifth passage materialj This is one of our volunteers from whom we recovered virus from the throat as early as the fourth day after inter-nasal administration, rash developed on the 12th day and virus was recovered up to the 21st day. On the 13th day we got an antibody level of 1 to 8 and then on the 16th day it was the same and about three weeks later it was up to 1 to 32. But this is the virus from the pharnyx, trying to quantitate it somewhat. As you can see at the time of the rash is where virus is at its peak and then it drops off. DR. MEYER: Doctor Feldman? DR. FELDMAN: These are children whose personal hygiene may not be of the best? DR. GREEN: Exactly. DR. FELDMAN: And you got virus out of rectal swabs? DR. GREEN: In a small percentage of cases. DR. FELDMAN: Could this conceivably be a source of 59 infection for prolonged contact? DR. GREEN: It might be. I suppose it could. DR. FELDMAN: This would be quite different. DR. MEYER: For those of you on the back row who couldn’t hear Doctor Feldman, he is interested in the question of whether there is or there is not poor hygiene in the children and this might be a question of spread of virus from rectal material, if I got that correctly. Perhaps you gave this information, but I didn’t catch it. In your contact cases, the children who contacted either inapparent or apparent infection from your inoculated children, what was the ratio of clinically apparent infection in the contacts to inapparent infection in the contacts, getting back to this question we were talking about yesterday, if a child got contact infection, how frequently did he show clinical symptoms? DR. GREEN: Well, I think it was in that slide, the second slide, I believe. With prolonged contact, there were 17 susceptible subjects, 16 developed rubella and five of the 16 had sub-clinical disease. It was almost one-third. DR. MEYER: The vast majority had clinical disease Is this clinical disease as defined by rash or by lympha- adenopathy? DR. GREEN: Rash. 59a SUMMARY OF STUDIES OF THE PROPHYLACTIC USE OF GAMMA GLOBULIN TYPE OF EXPOSURE GAMMA GLOBULIN TOTAL NO. SUSCEP SUBJECTS NO. WITH RUBELLA NO. WITHOUT RUBELLA PRIMARY 1 CASES SECONDARY 2 CASES VIRUS INOCULATED 0.12cc / lb 9 7 (3 2 fo 0 NONE * 13 10 p 3 F 0 PROLONGED (REPEATED) CONTACT 0.15 CC TO 0 20cc / lb 11 8 [5 2 Jo 1 NONE 11 2 p» 2 BRIEF (SINGLE) CONTACT 0.20cc / lb 4 o r— fo * 3 2 NONE 6 2 H" 3 1 INCUBATION PERIOD 12 TO 24 DAYS 2 INCUBATION PERIOD= 25 TO 53 DAYS □ NUMBER OF SUBCLINICAL CASES VtREMIA IN contact Rubella JIN EVAUMTiow of gamma GLOBULIN OF soejf^rs' GAtAHA GlOtUUlN SoB JEcTS j NUMBER OF SEtfoM SPEC/MEVS1 i.l iTU i \am l n | lT£S~£D IPOSIT. Jt ! V.- i ' 1 5 rioME H- / 3 / o 7 7 J<; 5 L 0.iS*<-J IL A n 7 37 % i ' ALL SUBJECTS had CLlMiCAL c:iszasZ % * ALL fiETwEEN DAY Of tfASH A NO F/vE DAYS' BEFORE RASH 60 DR. MEYER; Doctor Henderson? DR. HENDERSON: I wondered one other point I may have missed, what passage material was this you were using in the inoculating the children? DR. GREEN; Well, it was serum obtained from a patient on the day of appearance of rash. We have done some tissue culture passage material. Doctor Balsam© may present some of that later. DR. MEYER: Any other questions for Doctor Green? Thank you, Dr. Green. Doctor Balsamo. GAMMA GLOBULIN PROPHYLAXIS OF RUBELLA DR. BALSAMO: The data I am about to present repre-j- sents the result of six separate gamma globulin experiements carried out at the Willow Brook State School. In order to inform some of the people here about the Willow Brook State School, some of the procedures utilized there, I will briefly| mention that there is a full-time pediatrician, primarily interested in infectious diseases, who examines each child in our study daily. Daily throat swabs are taken on each patient, as well as daily rectal swabs. Daily serum specimens are obtained at the expected critical period, critical period * that we have learned to expect from previous studies. Serum specimens are obtained at frequent intervals in other than expected critical periods. 61 We have performed actually three separate categories of gamma globulin experiments. The first category was a virus inoculation experiment in which infectious rubella serum was inoculated into a number of children. Half of these children| received gamma globulin; one-half did not. In the next inoculation experiment, infectious serum was sprayed into the nasal pharnyx of a group of children. One half hour afte:r the nasal pharnyx was sprayed, one-half of the group received gamma globulin and the other half served as controls. The next category of experiments were the contact type experiments alluded to earlier by Doctor Green. In two of these experiments children were inoculated and exposed, in two separate experiments, children were inoculated and exposed to a number of subjects, test subjects, so to speak. Twenty-four hoursafter the development of rash in the inoculated patients, one-half of the test subjects received gamma globulin and one-half of the test subjects served as controls. This contact rubella or continuous contact rubella encompassed three separate experiments. In the third experiment the children inoculated with, the so-called pilot cases were isolated until the day of rash. When they developed rash they were then exposed to the subjects who were going to serve as the test subjects. Twenty-four hours after exposure one-half of the children received gamma globulin, one-half did not. The last experime it 62 was the brief contact experiment mentioned by Doctor Green, and I don’t think it is necessary for me to repeat his des- cription any further. Before I go into the details of our findings, I should mention that we have thus far tested 11 separate gamma globulin specimens and three gamma globulin pools. Using the interference technique for determining neutralizing antibody, four of these gamma globulin specimens have had a titer of one to 32, five have had a titer of one to 64, two have had a titer of 1 to 128. I should mention that one specimen was obtained from Captain Miller. This gamma globulin was collected after a mild outbreak of rubella at the Great Lakes Naval Station in the fall of 1962. The titer on this material was 1 to 64. We have also obtained specimens of gamma globulin from the National Institutes of Health and the titer on that material was also 1 to 64. In the gamma globulin experiments I will mention now, gamma globulin with a titer of 1 to 64 was used in all experiments but one and in that first experiment gamma globulin with a titer of 1 to 32 was used. May I have the first slide, please? Just to refresh your memory about the three categories of gamma globulin experiments, in the first column here we have the virus inoculated group, which includes the inoculated with virus intermuscularly and the virus sprayed 63 into the nasal pharnyx. The middle column combines the three contact experiments, since there was essentially no difference in the results between the three separate experi- ments. In the last horizontal column, the results from a brief contact are summarized. In the inoculated subjects who receive .12 cc’s of gamma globulin per pound, there were 9 susceptible Seven developed primary rubella as designated by a rash — excuse me, as designated by an incubation period of 12 to 24 days. This was confirmed by virus isolation studies or antibody studies. In the seven primary cases there were three sub- clinical cases. In the two secondary cases there were no sub-clinical cases and no child escaped. I should mention that the small numbers i rthe boxes indicate the number of sub-clinical cases as is apparent from the slide. In the control subjects in the virus inoculated group, there were 13 susceptible patients, 10 developed primary rubella and six of the sub-clinical variety. The three secondary cases were of the clinical nature. In the so-called repeated or continuous contact experiment/ the amount of gamma globulin administered varied from test to test, from .15 cc’s in the first test to .20 cc's 64 per pound in the last two test groups. There were 11 sus- ceptible subjects in the gamma globulin group and 11 in the non-gamma globulin group. Eight of the susceptible subjects received gamma globulin, developed primary rubella; five of the eight had sub-clinical disease. The two patients in this group who developed secondary rubella had clinical \ disease. One patient escaped as evidenced by no change in antibody titer, base line, or at time of discharge from the experiment isolation wards. In the non-gamma globulin group, seven children developed primary rubella; two of these cases were sub-clinical. The two secondary cases were both of the clinical variety. Two children likewise escaped in this non-gamma globulin group. The last column demonstrates the brief contact experiment mentioned by Doctor Green. There were four sus- ceptible subjects who received0.20 cc's gamma globulin per pound; no child developed primary rubella, two children developed secondary rubella of the clinical variety, and two escaped. In the six susceptible children who did not receive gamma globulin, there was one primary case and two secondary cases. One of the secondary cases was of the sub-clinical variety. Three children escaped. I would like to draw your attention to the fact that 65 in our prolonged or repeated contact experiments, five of the eight primary cases of rubella were sub-clinical, whereas only two out of the seven non-gamma globulin primary cases were sub-clinical. This may be of some significance when related to the epidemilogical data presented yesterday. In the last horizontal column, I think this type of experiment is of vital importance in order to evaluate thej place of gamma globulin in the prevention of rubella and congenital malformations. It is obvious from this chart however that our attack rate was so low that we can make no conclusions about this experiment, and further work is obviously needed. One question that arises frequently in gamma globulin experiments of this kind is the effect of gamma globulin on the development of viremia. Next slide. We have five patients who received gamma globulin and had extensive studies for the development of viremia. Five patients did not receive gamma globulin and were studied in a similar manner. I should mention these ten patients had clinical disease, throat swab isolations of the rubella virus and an antibody rise. All serum specimens enumerated on this chart were taken from five days before the day of rash up to and including the day of rash. There are two points of importance on this chart. The first point is that 66 we were able to isolate virus from at least one serum specimen in four out of five patients in each group. The next significant point is that in 10 out of 13 serum specimens tested, in the non-gamma globulin group, virus was isolated for a percentage of 77 percent of the specimens tested. In the gamma globulin group, only 7 out of 19, or 37 percent were positive. The significance of this data is not clear. I think the demonstration of viremia after gamma globulin given early is of some importance. To summarize our conclusions about the gamma globulin data, it is I believe the consensus of opinion in our group that the gamma globulin that we have used in our studies has failed to be of prophylatic value in preventing the development of rubella in inoculated or in continuous contact exposure. The brief contact exposure is obviously of importance, but we have no definitive information in that regard. And the viremia chart stands for itself I believe. That is all. (Applause.) DR. MEYER: Captain Miller? CAPT. MILLER: I just wanted to make clear, because this has been misunderstood in the past, that the material from Great Lakes was not collected from, was not selected necessarily from rubella patients, it was a general over-all | 67 collection of about four percent of the donors that had rubella. And then one other point. I think it is going to be very important to see how well gamma globulin, how effective gamma globulin is in the preventionof viremia in the young adult, because we did not look for viremia, of course, in our gamma globulin prophylaxis on recruits and it is very possible, although we were preventing clinical apparent rubella, we were not preventing viremia. DR. SEVER: I think these findings are most important for the problem of prevention of rubella in pregnancy and prevention of fetal defects relating to rubella. I am sure we are all aware of the extensive studies reported by Lundstrom and more recently the studies in the Britieh Medical Journal on 6,000 pregnant women, both of these indi- cating that gamma globulin was effective in reducing the incidence of malformations. The studies presented today though certainly warrant extensive consideration and further investigation, I am sure, as you are probably planning to do in the study of natural infection in pregnant women with the availability of the techniques that are now at hand, I would imagine that an obvious extension of your studies would be to compare antibody status prior, and subsequent to the administration of gamma globulin to pregnant women and to correlate this information with the outcome of their pregnancy. DR. BALSAMO: An epidemiological survey is now underway. DR. CABASSO: This points up at least in my mind the necessity for those interested in gamma globulin to compare titers they get in the gamma gloculin and a necessity for perhaps in the early days to have a reference that people can test against, because the 1 to 64 titer mentioned here differs from the others and we don’t know whether it is the same or a different level. If it were a true level, as compared to the levels obtained by others, it may be the reason for the lack of difference between the two groups. So it is essential to have, to know whether your titer is comparable to the titers obtained by others. DR. BALSAMO: I believe some investigators have titered the same gamma globulin. Does anyone care to comment on that? DR. MEYER: Just one comment about the reference. There is an obvious need for a serologic reference, I mean just in the course of comparing notes between various investij- gators, and there is a DBS reference volume available which can be sent to people on request. We were planning on mentioning that this afternoon. We can talk further about it) then. (VOICE)— What is the titer of that gamma globulin in DBS? 69 DR. MEYER: Here again it depends upon your test system. With the test system Doctor Parkman described, again depending on the virus dose, about 1 to 100. It runs higher and lower, depending on the test. DR. KEMPE: Doctor Balsamo, do you know the titer of Doctor Lundstrom's rubella gamma globulin? DR. BALSAMO: He uses different units than we do apparently. I have never spoken to him about it. I don't know anything about the comparative — DR. SCHIFF: We have compared his lot with other lots and his is higher than anything we have. Again figures don’t mean much here, but his was 4,096 whereas our lowest was 236. This was a sample of material which he had used successfully clinically. DR. KEMPE: I raise this point because this would be an ideal time, which may not happen again in ten years, for us to collect an experimental and perhaps clinical lot for clinical reference of rubella immune gamma globulin. In one small college in Colorado we have 200 young adult females who would be potential donors of a convalescent pool and I think between the people here it might be quite to get the 500 or 1,000 units that might be required to make a lot that would pass DBS standards and be used for clinical research. And this is something we might decide today, because the opportunity won’t present itself again. 70 DR. BALSAMO: We have started or planned to do this On a very small scale, only in patients from whom we have isolated rubella virus from the blood or pharnyx. But this is a very small scale. DR. MEYER: I would like to make one comment on this question of immune gamma globulin, which I think Doctor Parkman made some passing allusion to previously and others have commented on. One problem on convalescent gamma globuliji or convalescent serum to make a gamma globulin pool is by far and large your convalescent titers in rubella are not icantly higher than the titers of many individuals you see in the ordinary population. We ran into this ourselves in trying to get convalescent serum pools for use in labor- atory tests as compared to let's say ordinary serum pools. Doctor Parkman has as high a titer and he hasn't had rubella in years as the average convalescent person we run across. DR. KEMPE: But Doctor Lundstrom’s data are so much better than anybody else’s and there is this difference in the titer. DR. BALSAMO: This is what we found. The titer really doesn’t change. You can’t tell after the first two weeks of infection between a recent and late infection. One of the reasons we are interested in collecting gamma globulin, collecting this material from the patients who had a very recent infection is that perhaps there is some factor 71 other than neutralizing antibody that may be important. DR. KEMPE: This may be the point. Maybe we are not measuring the right substance. DR. MIRICk: I think one question was asked a short while ago, which I don't believe has been answered, and that is how the titers obtained by Doctor Belsame compared with titers on the same specimens obtained elsewhere, And there was one which was checked I believe by you, Doctor Parkman, where it was the same, 1 to 64. DR. KRUGMAN: I think in this discussion it is important to think not only of titers of gamma globulin, but to re-emphasize a point made by Doctor Green earlier, when he presented the slide which showed that the virus that is present in the pharnyx as long as seven days before onset of rash. And this epidemilogical fact plus the obser- vations made and reported by Doctor Balsamo, of no protection at all following prolonged intimate contact, I think is very clear, at least to me, that given this type of intimate household type of contact, the gamma globulin, no matter what the titer is, is given too late. It is given in all probability after infection has been established. As far as the brief contact is concerned, we need / more data about that. But it would be very — we have had a number of instances in clinical practice, under controlled situations, where mothers have been exposed to their own 72 children and gamma globulin in one situation was given in a dose of 40 milliliters by a pediatrician to his wife and 19 daj|s later this woman had classical rubella. There have been a number of instances like that. And this is not because of a titer of antibody, I think it is because of establishment of infection prior to the administration of the gamma globulin. As far as Dr. Lundstrom's studies are concerned, to the best of my knowledge these are not controlled studies. Docto| Lundstrom has given gamma globulin to large groups of pregnant women, but he does not have a comparable control group. I think it is very difficult to determine whether he really observed a reduced incidence of congenital malformatio^i; unless you have information to the contrary I don't believe his studies are controlled, DR. MURRAY: Apart from the possible use of gamma globulin prophylatically, which of course presents problems, I think Dr. Kempe's suggestion has considerable merit, if we are going to rely on preparing a pool of gamma which will have laboratory value, this may be the time to do it. The epidemic seems to be migrating westward. I don't know how far it has gotten yet. But by contacting the various blood collecting agencies, it may be possible to get a large number of pools, donations of blood from persons who have had rubella within the past few weeks. The average pool of gamma globulin prepared commercially represents an average of 73 approximately 2,000 donations and if you could include in thijs 2,000 only those who recently had rubella, you might have material which has a somewhat elevated titer, which could have some scientific value. DR. ROBBINS: I think the point also made by Docto| Kempe should be emphasized again and that is that we are going entirely on the basis of data with one kind of test, namely a neutralization test, and it may well be that when we get better, a different and diverse techniques, we will find that recent convalescent gamma globulin has antibodies --- -’*■*,/ that are quite significant. Also I would agree with what Dr. Krugman said about the studies from Sweden. Although they certainly look j suggestive as you go over the data of Dr. Lundstrom, if you talk to him you will talk to an evangelist who is firmly con- vinced himself, but certainly the data are far from conclus- ive in my humble opinion. The data that Doctor Miller showed from Great Lakes, I think it is well worth rememberingj that this was before exposure in a significant number of your recruits, so that this is not comparable to giving gamma globulin to exposed women. Is that correct, Doctor Miller? DR. MILLER: I think that is right. DR. ROBBINS: This is truly prophylaxis of disease rather than — 74 DR. MILLER: It was given about ten days after arrival and I am sure some of the exposures had occurred prior to that time but the bulk were not. DR. BALSA WO: We had one patient, a medical studeny, in which the gamma globulin was given seven to ten days before exposure and it didn’t prevent any of the manifestations \ of the disease. DR. WELLER: I would just like to second Doctor Kempe’s suggestion that the time has come now to prepare somej sort of a reference standard, I don’t care whether it is gamma globulin or high titer convalescent serum, I think in terms not only of standardization of future preparations but in terms of asking each laboratory that works in the to somewhere put down their titer of antibody that they achieve against this reference standard, thereby giving some reference for us all to cross check. Now I would doubt a little bit from the informatioji at hand that we need to make a convalescent pool. I would agree titers seem to stay up very well. We have impressive results with the Sindbis interference neutralization test, using 25 TCID50 of rubella virus, in which four lots of gamma globulin were prepared and given to us by 4 Laboratory in Massachusetts, lots 1 and 2 were prepared in 1962 from 1500 to 1800 liter plasma pools, lot 3 was prepared at 54 from an 850 titer plasma pool and lot 4, and 75 Dr. Feldman may know sometmng about the source of this, was prepared from bleedings of 137 patients in Ithaca and New York who had rubella in 1952, 7 to 18 weeks prior tc bleeding. Now the titers on these four lots were really all of the order of 1:1024. , in replica determinations it is true that lot 3 and 4 occasionally went as high as 1:4096. But the range in all of them was on the order of 10 to 24, I think it would be extremely helpful if we could at this session decide to set up a common pool of material that we could all then use as a standard for antibody deter- minations, because we are going to want hopefully in the future to be studying antibody responses and being able to compare each other's results. DR. BALSAMO: If I may make one comment in reference to the reference pool, we have tested at NIH lot 174, which I believe is the measles reference pool, and I think Paul has tested it and I would not be surprised if the NIH group has tested the same lot. So maybe we have comparative data already. DR. KRUGMAN: I think the lot of gamma globulin, Tom, which you refer to, as being collected from Ith4ca or Syracuse, is a lot which was prepared some years ago as convalescent rubella gamma globulin by Doctor Korns and members of the New York State — is that the one? 76 DR. WELLER: That is the one. DR. KRUGMAN: This is convalescent rubella gamma globulin. We used this material at Willow Brook State School in 1952, in a trial where it was mixed with serum known to contain rubella virus, and it was completely neutralized. This was also used in Taiwan during an epidemic of rubella. But it was used there and the quantity used in this trial was less than that of a commercially available lot of gamma globulin and from what you suggest this indicates I believe that the titer is no different from ordinary gamma globulin. DR. WELLER: Of course we don’t know anything about its storage history over the past 12 years and the stability of the material. So one might have to take this with a little bit of question. But as far as we can tell, these titers are high and regular pools have good high titers, within the sensitivity of the test. What this means one doesn’t know. DR. SEVER: I think to assist in resolving this question of having this material available, we prepared rubella from convalescent gamma globulin provided by recruits at Chanute Air Force Base last year. The total of approximately 150 units were included in this preparation and the gamma globulin made from that we have distributed to a number of laboratories on request. We would be very happy to provide the bulk of this material to DBS for further 77 distribution as one form of convalescent gamma globulin if this would be of assistance and if you wish to in that way have it available for comparative studies by different laboratories. DR. BUESCHER: To go back to Doctor Kempe's original suggestion of a dual purpose, if there is question that processed gamma globulin can indeed be effective in prevention, I would like to raise the question as to whether or not part of this shouldn't be retained as whole serum. The question basically is would it be justified to evaluate the gamma globulin in the whole serum, the whole serum particularly, in such a circumstance as the New York group has. DR. MEYER: Can I say one more thing? Before going further on this subject, this question pertains a bit I believe to some of the things we wish to discuss this afternoon, which basically is the reference standards. Perhaps we could be thinking about it between now and then and enlarge on the discussion of what might be feasible in the way of gamma globulin standards and collections in the afternoon session. I would like to ask one more question before we move on to the next topic. Vie have had no data presented regarding the use in man of tissue culture passed rubella with the exception of one slide Doctor Shiff showed. Does 77a I SpKay Of T"'Ss*« Cvltm* P ? >« j 78 anybody have any data concerning use of tissue culture passed rubella virus in man? DR, BALSAMO: Yes, I think I have a slide on that. This material was first passage material in the patas monkey kidney. It was sprayed into the nasal pharnyx of 11 susceptible children. The titer of this material, it caused interference in the green monkey kidney tissue culture at a dilution of 1 to 10. I am sorry, it was sprayed into eight children, sprayed into the nasal pharnyx undiluted in four susceptible children, diluted 1 to 10, and four sus- ceptible children were kept as controls. In the four sus- ceptibles that received undiluted material, four of them developed rubella, and an incubation period of 17, 22 and 29 days. In all cases it was the clinical variety. In the four who received tissue culture material, diluted one to 10, three developed rubella at an incubation period of 33 and 35 days. One was of the sub-clinical variety. It is apparent that these patients most probably escaped who developed contact disease, but working with tissue culture material, we can’t be sure about that. There was one escape in this group. In the control children who did not receive the tissue culture preparation by nasal spary, one developed typical clinical rubella on day 63 and two escaped. It should be mentioned that two of the escapes in the control 79 children represent crib patients and it is possible their contact was not as good as the contact in the other patients. We have since this time administered 25th passage tissue culture material with a titer of 4 and a half to a number of children at Willow Brook. But unfortunately three days after the experiment was started an outbreak of wild rubella occurred and the results are not interpretable. DR. MEYER: Any questions on this last slide? DR. WARREN: This is — before we leave the matter of experimental infection, has anybody any information on whether the immune adult, in intimate contact with a sick child, carries live virus? Can you isolate live virus in the nose and throat? DR. BALSAMO: I can’t comment about adults. I can only comment about children. Immune children, we have not been able to isolate the virus from immune children, after close intimate contact with infected children, after nasal sp'ay or intramuscular injection DR. PLOTKIN: In doing a fairly large amount of diagnostic virology, which we have been forced tcdo recently, we have tested a number of pregnant women who have been in contact with their own children or in some cases with other children, who had frank clinical rubella and on no occasion have we isolated virus from such a woman when there has been a neutralizing antibody titer in the first serum specimen we had, which has usually been early in expoetnre,. DR. BALSAMO: I take back vtot I wid earlier. I forgot we also have been running a viral diagnostic laboratory for pregnant females in New York and we have exact 1]| the same results. DR. MEYER: And these are the sane result# Doctor Buescher reported yesterday. I have forgotten the number involved. But the recruits that got antibody to start with, didnot have a shift in antibody, they did not excrete virus. DR. BUESCHER: That is correct. They didn't have virus intheir throats by the schedule that was used for sampling. DR. BALSAMO: One otherword about sub-clinical patients. Isolation from the throat swab in sub-clinical patients are prolonged, even though no manifestation of the disease is present. We have isolated virus in as long as 14 consecutive days, without clinical manifestation of the disease. DR. SHELOKOV: Was there a rise in the antibody content of the serum of thesis pregnant females? VOICE: We have not identified any rise in titer in someone with a titer in the first specimen. We have of course had rise from infected people. DR. SHELOKOV: But not the ones with pre-existing antibody, no rise? 80 (a) neutralization of Rubella Virus (KV) by Paired Serums of Rubella and Control Patients Fort 1. Ord, Calif., Patients Neutralization Acute Convalescent rubella (recruits) * ▼ 2. 3- _ - 4. - ♦ 5- 4- + 6. - ♦ 7- . •4- 8. - ♦ 9- - ♦ Virginia, rubella (pediatric) 1. - 4- 2. - 4- 3- - + Fort Ord, Calif., , normal controls (recruits) 4 2. ♦ 4 3- ♦ 4- 4. ♦ 4- 5- 4- 4- 6. ♦ 4- 7- - - •tt. 4- 4- 9- 4- 4- 10. 4- ♦ 11. * 4- 12. * + Fort . ord, Calif. , poet rubella (recruits) 1. ♦ 2. 4- 3- 4 4. 4 6. 4- 7- 4 8. 4 9- _ 10. 4- • Screen neutralization with 50 and 500 TCH 50 FV. T neutralization, no neutralization. 80b HUBELLA VIRUS (RV) HEUTRAUZnC ARTIBODT I. Response to Administration of Rubella Virus (KV) and Control Fluid to Human Volunteers With and Without Pre-existing Antibody A. Rubella Virus* Neutralizing Antibody Clinical Volunteer Acute Convalescent Rubella 1 - + 4- 2 - 4- + 3 - 4- + 4 - 4- 4- 5 4- 4- - 6 4- 4- - T + 4- - 8 + 4- - 9 + 4- - 10 + + * - * Intranasal, 100 TCID50 of virus (Primary African green monkey- kidney-tissue cultures) B. Control' Tissue Culture Fluid* Neutralizing Antibody Clinical Volunteer Acute Convalescent Rubella 1 - - 2 - * Primary African green monkey-kidney-tissue cultures ' Titers of Reference Convalescence Gamma Globulin and Reference Human Serum Using Various Concentrations of RV Strain Rubella Virus a @ a Reference Convalescence Rubella Gomma Globulin(16.5%) • • • Reference Human Serum LOG OF RUBELLA VIRUS RECIPROCAL OF GAMMA GLOBULIN OR SERUM DILUTION 80c Table 2. Rubella neutralizing antibody in young adult males with natural and experimental infection. No. of Neutralizing Cases Antibody Titer* NatuAal a* faction* 3 16 2 32 1 64 4 128 txpe•umcntai me of our findings with formalin - treated rubella virus vaccine. Thesa studies represent a continuing effort, some of which was reported at the meeting of the Society last year and some of which is still in progress at the present time. Briefly the techniques involved in this series of investigations have been reported previously in our original observations with volunteer administered rubella virus which was reported in the Journal of American Medical Association and the techniques with gamma globulin and serum titers have been described in detail in the publication of Dr. Shiff’s, in Science. To summarize just something of the information going prior to the main part of this study, I would like to have the first slide please. This data was obtained several year? ago in studies of serum specimens from recruits at Fort Ord , California, children with rubellar, normal controls at Fort Ord , and 82 recruits at Fort Ord who were sampled only in a convalescent I state, in which the history of rubells three weeks earlier had been given. These initial determinations then using the virus interference method, shewed the conversion of serum specimens from negative to positive in almost all of these patients who had clinical rubella. At this time of course we were just working with new techniques and in additional studies reported at the same time, the serum from a number of different outbreaks was tested under a code. These included outbreaks in island populations as well as in this country, and the reproducabili ;y of the test run at 1 to 4 for the screening of antibody titers like this was a little over 90 percent. The cross testing of course also demonstrated no serological difference betweenj the various isolates of rubella virus. Now subsequent to this, after extensive work in monkeys failed to demonstrate the clinical findings which we had hoped to be able to use for these studies, the first volunteer studies were initiated after review by the medical board here at NIH and the Director of NIH and these studies were done in conjunction With Dr. Shiff and Dr. Huebner, and the Federal Reformatory System. Next slide. In these studies volunteers did not have the technical antibody, four of these men, and in a combination of two studies, for divergent purposes individuals 83 with pre-existing antibody were challenged with 100 TCID 50 cj fifth passage primary African green monkey tissue cultures. This was given in a one cc inocculation internasally. A13 of the men negative for antibody developed clinical, rubella. Clinical rubella did not occur in any of the individuals with pre-existing antibody. And the antibody conversion occurred in the volunteers who were susceptible. The virus could be re-isolated from the nasal pharnyx of course of these individuals, clinical rubella in this way occurs on the 11th to 12th day with the internasal administration of tms quantity of virus, Virus isolations from the na,6al pharnx can be made for a period of approximately a week prior to the appearance of clinical rubella and virus persists in the nasal pharnyx of these young adults for at least a week in many instances. The period of viremia is for a week prior to the occurrence of clinical symptoms and then disappears very shortly thereafter. This information was summarized on the slide Dr, Rhiff showed earlier. Viruria occurs for approximately the same duration as viremia, namely, for roughly a week prior to the occurence of clinical symptoms, and disappears shortly after the appearance of clinical symptoms. Two volunteers included in these studies did not have neutralizing antibody in their first specimen. They were 84 given the control tissue culture material, which was tested in the same way as the initial material, and there was no conversion and no evidence of clinical rubella. This is a control of course simply on the presence of possible undetectj- able agents in the tissue fluids which might conceivable produce serological changes and clinical symptoms indistingu- ishable from rubella. This of course did not happen. The titrations of antibody which I am going to refer to involved primarily determinations with the inter- fering method, which has been described previously, and secondly, we extended these and reworked all of the previous titrations and added on a number of other studies, using the RK-13 system. Next slide please. In this data, this is a slide of the paper of Dr. Shiff’s of course, the amount of virus used in these titrations is 0.5 log, and the reason for this selection is the reproducibility of the tests handled at that level and the fact that it is possible to make a good separ- ation of titers between low and high titered gamma globulin and low and high titered human serums, so the test has value for separating it out; when one runs into a sensitive range you can block out and dampen this effect by adding more virus to the system. Another advantage is that serum specimens screened at one to 4 have consistently been useful, using this amount 85 of virus, since individuals without antibody have been resis- tant to either natural or experimental disease, and individuals with antibody, I am sorry, individuals without antibody have been all susceptible and individuals with detectable antibody at the 1 to 4 level, using this amount of virus, with anti- body, theyhave been resistant, without antibody they have been susceptible. The titers obtained with this system, if we can hav|e the next slide please, here again from the same paper with natural infection the titers are in the range of 16 to 128. With experimental infection, titers of 16 tc 128. With natural infection, 10 to 15 years earlier, again the titers seem to be persisting quite unchanged, at least during this time interval, These of course were identified as natural infection by history, since obviously the techniques have not been available to document the occurence of rubella that long ago. Interestingly, the titers obtained with this method then, when run on RK 13, as reported previously, using 100 TCID 50, are quite comparable, and data between the two tests can be compared quite readily, the titers being withinj usually a two-fold variation of the titers determined in the African Green system. Now the determination of inactivation of rubella virus with formalin is given in the next slide. 86 These tests were done with rubella material which initially haji approximately, threelogs of rubella virus. The time in hours is given across the bottom. At 37 degrees, with formalin 1 to 4,000, there was no detectable virus at four hours; detectable virus was still present, one log, at three hours. But after that period it was not possible and of course the time has been extended here past 24 hours, there was no detectable virujs. There is some thermal inactivation seen by the other line and this occurs with virus stored at 37 degrees centigrade. This has been found by a number of other investigators. Our interest in this material, of course we were wondering if it would be possible to study this for its possible use as an antigen, formalin treated material for usej for rubella. Our studies were first conducted in a series of experimental animals. Next slide please. Now in these studies with rabbits, two separate preparations of the formalin inactivatecj material of the fifth passage in primary African Green monkej kideny tissue cultures is given. The material had been in- activated for £ period of six days at 37 degrees centigrade. The initial titers obtained with one injection — this is two weeks after the first injection and then the second injection was given at two weeks, just after the bleeding was obtained from these animals, and a final bleeding was obtained then three weeks after the second injection. Therefore the titers| 87 are after one injection, taken two weeks after, and taken three weeks after two injections. In the rabbits, the untreated material gave titers in excess of 512. The loss or decrease ii antigenicity occurred primarily then during the intitial six hours of treatment until the titers got down in the range of 16, 32 and 128, even 256, in these instances. It would appear then that after the initial six hours that there is also a slow decrease in antigenicity through the 8th day as indicated particularly by the specimens taken after one injection of this treated material, and yet this decrease is considerably less rapid than the initial decrease, which is first seen. Antigenicity in these two preparations persisted in the material through eight days of formalin treatment. Of course bisulfite was added at various periods, the materiaj was stored and refrigerated during the period of treatment for subsequent studies, Further studies were done with the material, par- ticularly that at six days, simply because this was well after the last detectable virus was present. Studies were done in monkeys and a number of other species of animals. Titrations were done initially of course in the interference system and also thensubsequently in the RK-13 system. These titers ' presented here incidentally are specifically those from the RK-13 determinations; one could superimpose the other titers for the most part within two-fold when determined in the African 88 Green system. The animal titers were quite similar to those, the findings were quite similar to those found here in In monkeys, antigenicity was demonstrated and the titers were in the range of generally 8to 32. These titers are a little less than, in the monkey situation,than titers which we get frequently with untreated tissue cultures. With the availability then of antigenic material, which demonstrated its antigenicity, all studies were done with an intermuscular inoculation of one mililiter of preparation, with a demon- stration of anitpenicity, and the lack of any detectable and after the thorough safety tests paralleling those very closely that are stipulated for measles vaccines, it was decided to inititate studies in a small group