FDA Workshop
Immune Globulins for Primary Immune Deficiency Diseases Antibody Specificity, Potency, and Testing
Center for Biologics Evaluation and Research, FDA
Immune Deficiency Foundation
Plasma Proteins Therapeutic Association
HHS Office of the Secretary/Office of Public Health and Science
Lister Hill Auditorium
Building 35A
National Institutes of Health
Bethesda, Maryland
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April 26, 2007
Welcoming remarks
Dorothy Scott, M.D., FDA
Introduction and history and rationale for measles antibody testing
Dorothy Scott, M.D., FDA
Toby Simon, M.D.
Measles antibody testing and decline in measles antibody Titers in Immune Globulins over time
Susette Audet, B.S., FDA
Measles antibody levels in PIDD patients
Othmarr Zenker, M.D., CSL Behring
Measles infections and estimated protective titers in PIDD, and potential reemergence of epidemic measles in vaccinated individuals
Diane Griffin, M.D., Ph.D., Johns Hopkins University School of Medicine
Panel Discussion
Moderator: D. Scott, M.D.,
Panelists: T. Simon, M.D., S. Audet, B.S., O. Zenker, M.D., D.Griffin, M.D., Ph.D.
Q2. How much anti-measles antibody is needed to attenuate or prevent measles in PIDD?
Q3. What is the potential clinical impact of diminishing Anti-measles titers in Immune Globulin products?
Q4. What are propose approaches to address decline of Anti-measles titers in Immune Globulins:
A. with respect to clinical efficacy to prevent measles infection?
B. With respect to utility as a test for lot-to-lot consistency?
P R O C E E D I N G S
Agenda Item: Welcoming remarks
DR. SCOTT: Good morning. I think we will get started. I would like to make a couple of announcements. First, if you need a taxi to the airport or anywhere else as a matter of fact see Rhonda Dawson at the desk. She will arrange a taxi or shuttle to the airport.
The second announcement is also housekeeping but important is that you have an evaluation form that is on the left hand side of your folder and please fill those out, and also leave them at the desk with Rhonda as you go out.
Agenda Item: Introduction and history and rationale for measles antibody testing
DR. SCOTT: I would like to introduce this session. We are here to address a concern which is the decrease in measles antibody potency in the immune globulins and as we have already heard yesterday it is a lot release test and I just wanted to point out that a product that failed its lot release testing for any reason has to be either discarded or reworked. So, this becomes a problem from the manufacturing point of view.
However, from the clinical point of view and regulatory point of view what we want is products with adequate potency and today we are going to be asking you to examine where we are and where you think we might go with this.
Measles antibody levels are a standard measure of potency in US immune globulins. It has historically been a very important specificity because measles obviously was not controlled as Dr. Seward showed you yesterday in the 1960's when this testing first came about.
The tests are easily available and they are correlated with protection. The levels are correlated with protection in normal subjects. In other words there are defined reasons for antibody levels that are going to prevent measles disease and levels that can prevent infection. You will be hearing more about that later.
For the immune globulin lot release test measles antibodies are measured by hemagglutination inhibition or neutralization. Those are considered accepted here and our problem is that declining antibody levels have been observed in products over the past several years.
There are some hypotheses about why this is the case. One is that natural, well, one hypothesis that seems likely at least is that natural infection results in higher antibody levels where the proportion of vaccinated as opposed to naturally infected donors is likely to be increasing. The vaccine was licensed in 1963 and was implemented over ensuing years and we heard that the schedule and regimen of vaccines has changed in order to provide greater efficacy and also the naturally infected population of donors is aging.
As we get older we apply some donor deferral criteria and fewer people are able to donate that have been naturally infected and they also drop out of donating for other reasons.
This is just a schematic Vaccines, Second Edition. There is data in the literature that backs this up and what it demonstrates is that natural measles infection does give you higher titers of antibody over a longer period of time and this goes all the way up to 16 years post-vaccination infections.
So, how did this all come about? I am going to repeat some of what was mentioned yesterday in the introduction but in 1944 the first publication about prophylaxis with immune serum came out and that was by Stokes in Journal of Clinical Investigation although prior to that immune serum globulin was already used purpose and around 1953 the minimum requirements for immune serum globulin published by the Department of Health, Education and Welfare stated that several lots should be effective in the prophylaxis of measles in order for a product to be licensed.
We came out, at that time it was the Division of Biological Standards, a predecessor of CBER with reference measles antiserum called lot 1. That was interestingly primate hyperimmune measles serum.
So, these were primates that originally had a natural infection with measles and then were boosted with measles antigens in IFA and then they were exsanguinated to make lot 1.
So, our first standard was not actually a human serum standard. In 1963, the first vaccine was licensed as I mentioned and in 1964 WHO came out with an international reference standard that was a human standard for measles antibody titers.
Somewhere between 1965 and 1968, the Code of Federal Regulations published a requirement for measles antibody potency testing for immune globulin and in 1981, the first intravenous immune globulin was licensed.
This gives you an overview of the history and this again is something I showed yesterday but the original requirements of the Department of Health, Education and Welfare were that specific antibody should be shown in immune serum globulins by titrations using tests and that some lots needed to be tested for their ability to prophylax measles.
In 1961 at the same time as this new standard was distributed for the testing of immune globulins a potency level was defined and this is determined by considering that all the licensed immune serum globulins were actually potent for measles prophylaxis based on the clinical trials and the cutoff titer was statistically arranged to permit 95 percent of these lots to pass specifications. That was based on a study done of 60 lots looking at the titers.
Very briefly for the standards lot 1 was replaced in 1971, 10 years later with what was called lot 175 which is a human immune globulin and also at that time we accepted either hemagglutination inhibition or neutralization methods as testing assays and those were prepared by CBER scientists.
In 1992, lot 175 was replaced with the current standard lot 176. However, it is a long time since 1992, and although this lot has been fairly stable in its current form we are looking at replacing lot 176 with a new lot 177 and this actually is connected to what we were talking about yesterday because we may also want to be testing this for new candidate specificities.
So, how much immune globulin is protective against measles? The intramuscular immune globulin still carries the indication for measles prophylaxis. It is recommended to give it within the first 6 days of exposure to measles in a susceptible host using a dose ranging from 41.2 to 82.5 milligrams per kilogram depending on the patient and this is a 16-1/2 percent solution that is at least .6 times the reference standard.
Now, for IGIV the lowest package insert recommended dose is 200 milligrams per kilogram and that also needs to meet the reference standard specifications that is when you have diluted everything appropriately so that the reference standard in immune globulin is the same concentration when you test them and this is what everybody does.
The typical IGIV dose exceeds the recommended measles prophylaxis dose for immune globulin and you will see some of these calculations in more detail later from Susette Audet.
However, IGIV is given every, approximately every 21 to 28 days sometimes more often and sometimes a little less often.
So, in our paper we calculated the trough anti-measles titer for IGIV using PK parameters that are average or typical and we calculated that for current lots it is likely that for most patients even at the trough level they would still achieve 120 million IUs per ml their serum, but this really is a calculated value, and I think that what Dr. Cunningham-Rundles said yesterday that it is important to understand what actually happens in these patients is highly relevant.
The 120 million IUs per ml is the estimated protective level against measles clinical disease. Actually you need a somewhat higher level as estimated for protection against even getting infected.
What are some options to address the declining measles titers in immune globulins? There are several but there is one that at least is possible through the regulations and in 21 CFR 640.104 which discusses potency of intramuscular immune globulin it states that the Director of the Center for Biologics can advise manufacturers of an appropriate antibody level and so of course that recommendation could be changed if it is clinically and scientifically justifiable to do so and that is where we want to focus today.
There are obviously other potential options like lot testing but that is something for the audience to discuss and really not for us to suggest.
So, the questions for the panel and the audience, and we will be providing you lots of information from our speakers to help you address these, is measles infection of current clinical concern for primary immune deficient patients and how much measles antibody is needed to attenuate or prevent measles in primary immune deficiency? What is the potential clinical impact of diminishing anti-measles titers in immune globulin products, and what are the possible approaches to address the decline of anti-measles antibodies in immune globulins with respect to clinical efficacy in prevention of measles infection and with respect to its utility as a test for lot-to-lot consistency which is the other purpose of having this titer tested in our immune globulin products.
We have a group of speakers and the design of this is to look first at the measles antibody titers in plasma donors, and Dr. Simon will be showing you data that has been collected on that to give you a sense of where it has been and where it is going.
Susettte Audet will be talking about the amount of measles antibodies in our current products. Dr. Zenker will be talking the amount of measles antibodies in patients with primary immune deficiency, and Dr. Diane Griffin will be talking about measles infections and estimated protection immune deficient patients.
I would like to thank Dr. Griffin for making the trip down here from Baltimore. She has a longstanding interest in antibodies and their ability to prevent and treat viral infectious diseases as well as in measles.
So, with that I am going to turn the podium over to Dr. Simon who will tell us about measles antibody titers in plasma collected from donors.
Agenda Item: Measles antibody titers in plasma donors
DR. SIMON: Thank you. I am grateful for the opportunity to present this data on behalf of CSL Behring. I am with ZLB Plasma which is the unit of CSL Behring that is responsible for recruiting and processing the donors in such a manner that the plasma that is obtained is unique to these
In the pre-vaccine era there were approximately 500,000 cases of measles per year in the United States. By contrast in 2005, the latest publication on this from the CDC showed 66 confirmed cases of which 34 were from a single outbreak associated with a traveler suggesting that this is an area of risk to our patients as well as for our donors. This was the largest outbreak in the United States since 1966. The current incidence is less than one case per million. The measles vaccine was introduced in 1963 and by the 1970s and 1980s in most states it was required for admission to school. The two-dose vaccine requirement was phased in in the beginning of 1989, and by 2001, 96 percent of states required the two doses for school entry and the median coverage was estimated at 97 percent and as has been shown in Dr. Scott's slides the issue that has impacted us in terms of our donors is seen in the data she showed as well as this quote from a different textbook from the section on the measles vaccine which said that the antibody titers elicited by vaccination do decline over time and as do those induced by natural infection and may become undetectable. Vaccine-induced antibody titers are typically lower than those induced by natural infection. The protection is there the way I like to think about it because the lymphocytes remember and the individual can still mount an immune response but we can't measure it in many cases in the antibodies in the circulation of the individual. For the data we are going to show we used a different method than was used for the product. Our measles antibody screening assay was an ELISA method for measles IgG using Eti-Max system and the kit from Diasorin. We developed an internal calibrator using the WHO 66/202 standard. Now, one of the issues with the assay is the reporting range of 0.5 to 10 international units per ml. So, this is the lowest value we can record. Values lower than that would have to be recorded as undetectable. The coefficient of variation ranges from 2.7 to 14.7 percent and is higher at the lower concentrations. We did run a correlation with an in-process assay used by one of our manufacturing groups which is the Dade-Behring ELISA and the correlation was very high. As you will see the data are similar to what was shown yesterday from products using the hemagglutination inhibition test. This is data based on a random sample of 4356 source plasma donors doing three snapshots in March 2006, December 2006 and March 2007 and then the data were aggregated. Here you see the birth year of the individuals, this being 1963 when the vaccine was licensed and the antibody titers in the older individuals born before that and the antibody titers in the younger individuals and you see it fall over time as the assay of the test, excuse me as the vaccine was put into general use it began to be required for school, eventually falling to levels such that when we averaged the zeros and the ones that we could detect antibody in the majority of the donors were actually the detectable level in these birth years. So, the younger donors would clearly have a marked difference in detectable antibody compared to the older donors and this we believe is the source of the problem in the lower potency levels being found in the product. We, also, have these two graphs that amplify on that data. This is from recovered plasma, plasma from whole blood donations and this is from source plasma from donations by source plasma donors and the selection year is shown and this was the curve that was anticipated based on the experience that we had with the declining titers up to 2006. However, based on our snapshot that we took it appears that there is now an accelerated fall occurring in these antibody titers and this is we think related to the managed growth. As we bring in new donors who are younger than the older donors the overall antibody titers are falling. The difference between these two curves reflects the different age profiles in normal source plasma donors and donors from recovered plasma. The blood banks tend to recruit older donors than we do. We tend to have a skewed donor population towards the younger ages. Now, these curves are theoretical curves and they would tend at some point probably to flatten out as the younger donors have replaced the older donors rather than continue to fall. This again shows the short-run deviation below the long-run line due to managed growth, bringing in the new donors and similarly the greater decline that we are seeing in some snapshots that we took at the same time in recovered plasma. These are the observed titers over the time plotted with the line above indicating what was anticipated and this line indicating the snapshot and in March 2006, we had a mean of 0.91. In December 2006 the mean was 0.66 and then in March 2007, we had a number of 0.57. This was 31 percent fall between the first two snapshots and 37 percent between the first and third and we believe the flattening of this line or the reduced fall in the titers is probably due to the holidays, the Christmas and New Year's holidays when there would have been a reduction of new donors being brought on line, but the actual reduction is above. It came out that we had anticipated reflecting growth in the donor population and the recruitment of donors who would be primarily in the younger age population and this is a histogram which tries to bring this together showing the senior donors here. That is the donors who were born before 1963, when the vaccine was licensed and the junior donors here who were the donors born after 1963, after licensure and this is the mean level of the donor population, the total donors and as you can see the total donor line is moving closer to the younger donors. If we looked at very high levels virtually all of the donors would have values below this level and if we go to the very low levels we already see in our junior donors over half of the donors as indicated on the first set of data points are below the detectable limits of the assay whereas in our senior donors about one-quarter are below the detectable levels. If we were to select another level like 2.0 international units per ml about 50 percent of the senior donors are below that level and already about 90 percent of the junior donors are below that level but a histogram makes the point that we are seeing a very high proportion of the donors below levels that we would want to achieve in the donors in order to have a product that meets the specifications. Of course we could immunize donors in order to get donors with higher titers,but there are a number of reasons why we believe this is not an acceptable solution that we would want to utilize. First I think there are ethical issues if we don't have a clear patient benefit to offset any risk to the donors who are being immunized. This would be a booster vaccine for most of our donors. I contrasted this with the rabies program. We do give rabies vaccine to donors with the risk related to that but the product is a live-saving product. So, if someone in this room were to be bitten by a rabid animal rabies immune globulin could mean the difference between life and death. I don't believe we have had that scenario with measles. Also, this vaccine is a live attenuated virus vaccine. It contains the active form of the live virus. The virus replicates in the body for 6 weeks, and we do not have any other vaccination programs with live attenuated virus and in addition we would have a 6-week from donation which is a problem for the donors since we generally recruit for immunization programs from committed donors that are donating regularly and in addition we wouldn't be able to capture any plasma during that 6-week deferral period. The side effects handling and management of this vaccine in terms of temperature, light exposure and those sorts of things are more complex and more difficult than the other vaccine programs we have in place, and research that has been done at one point by our company into the immunization possibility indicated that there are a number of uncertainties about the titers which would actually be obtained so that we would need to do a pilot program to determine if this was even feasible. So, for all those reasons we do not believe that this is a feasible solution to the issue of adequate potency in the final product. In summary falling measles titers are anticipated over time in normal donors. This has been verified by snapshot data in both recovered and source plasma donors and if you wonder why we just don't just use more recovered plasma this source only grows with the number of whole blood donations that are taken and many of the blood centers are moving to automated platforms for red cell donations and not obtaining plasma at the same time. So, more than 80 percent of the available plasma for product is from the source plasma donor. The reduction in plasma titers currently being seen will make it difficult in the coming years to achieve product specifications. Immunization programs pose significant issues and we believe that the measles antibody specifications for immune globulin products need to be reconsidered particularly based on the falling antibody levels in the donor population. (Applause.) DR. SCOTT: Do we have questions for the speaker? DR. EPSTEIN: Thank you very much. The snapshot data was really a 1-year interval and it is rather startling a change over simply 1 year. I am just wondering whether the age distribution of donation showed a comparably large difference. DR.SIMON: Certainly the age distribution is changing in that direction. We are getting more and more younger donors and the older donors are less inclined to return. So, it is certainly compatible with the data we have on the age distribution, changing age distribution PARTICIPANT: An alternative to changing recommendations would be to use plasma that has a high titer. That has been known for RSV immune globulin. I think they rejected 19 out of 20 to get a high titer RSV. DR. SIMON: You mean only use titers just all these -- PARTICIPANT: Measure the titers of your donors and just make a high-titered measles immune globulin. DR. SIMON: The problem is the specification for the total product and we wouldn't be able to get enough product because we have fewer and fewer donors with the higher titers. So, if we were making a measles immune globulin that would be an approach but I think for the final product that is not an approach that I believe would work. PARTICIPANT: Are all your donors from North America? DR. SIMON: Yes. PARTICIPANT: Because measles has been controlled here for a much longer period of time than it has in Europe. DR. SIMON: Yes, I should have specified this is all US. DR. SEWARD: I wonder about some potential populations that might be receiving MMR doses in adulthood. Health care workers come to mind and they are routinely tested and if they are antibody negative get a booster dose. The military are another population and women after pregnancy. You know anybody who is rubella seronegative gets an MMR dose. I know it is hard. It may be too difficult to search out those adult populations but have you thought of that? DR. SIMON: I think that is an interesting thought. We hadn't actually considered it. Military donors are a problem because of CJD travel restrictions. So, that is a portion that probably would not be available and postpartum women tend to delay coming back in if they were donors and they generally don't start donating when their children are young even after the 6 weeks, and I guess health care workers I don't know how widespread that is. They are tested for their levels and then boosted? DR. SEWARD: I think more often than other adult populations they are tested or have to show a vaccination history and if they can't show it then they usually get a dose. So, I think they are more often getting doses in adulthood. DR. SIMON: That would be an interesting point. I don't know how feasible it would be. I know in the past that has been done with health care workers who got hepatitis B vaccine. I guess that would be a possibility. I would like to ask an ignorant question. Maybe Judy here can help. I mean we know from neutralizing antibody studies that there are levels of immunity about the protective level in people that are vaccinated 10 to 15 years out. So, this WHO standard, is there any need to reset that, but this is an ELISA? DR. BEELER: There haven't been good comparisons. The protective level of antibody, a level of 120 international units is relative to WHO standard 66.202 and that was demonstrated in a small clinical study and I am not aware of a similar study that has verified that in an ELISA titer. May I just make a reassuring comment that when you do compare titers by ELISA and neutralization frequently the ELISA tests are set for a very high cutoff. So, even though you are detecting, seeing that in a number of individual donors who are negative in their ELISA test we do know that most of those, virtually all of those individual have neutralizing antibody against measles. They have a negative reading in the ELISA test. So, individuals once they are exposed to measles virus or measles vaccine retain that antibody essentially. So, they don't seroconvert. PARTICIPANT: I would just like to confirm that because we use actually as volunteers for a number of our studies health care workers because they get identified by ELISA as being negative, and they have to re-immunized and then we recruit them to study and when we look at their original titers they have all had histories of having been immunized. They may be relatively low titers but they are not negative. DR. SIMON: Right, that is consistent with what I tried to show but I think using the IgG assay we are able to show a marked difference between the older donors and the younger. Obviously we are at a much lower level. PARTICIPANT: It has been about 17 years now since the recommendation for second immunization. So, those donors are going to be now in their early twenties. Is there any indication that this new wave of donors that had two immunizations will have higher titers than the ones that had just a single immunization at 12 to 15 months? DR. SIMON: It hasn't shown up in our data. As of this time we do recruit students as donors. So, one might expect to see it if it occurred. It is something we could look for but so far at least with this assay we have not been able to find it. DR. BEELER: Charlie Lebaron at the CDC has a long-term 10-year follow-up study that was recently published that looked at that question and two doses of measles vaccine. The second dose was either given to children at 4 to 5 years of age or 11 to 12 years of age and were followed for 10 years and we see a very small rise in measles antibody titers but by 6 months of age immune titers essentially went to baseline but the titers in the children who received a second dose are much higher. DR. SIMON: So, what you are saying is this is consistent with the data we showed. DR. BEELER: Exactly. DR. SEWARD: I couldn't see these birth years on your slide that showed over time, could you just tell us what year the titers got down to .5? It must have been in the seventies or so, but I would like to know. DR. SIMON: About 1969. DR SCOTT: Just a minor point for the military donor deferrals that deferral really gone down in 1996. So the current military cohort I guess have been revaccinated probably would not defer on that basis. DR SIMON: The new recruits would not be. I mean I think what has happened is we generally have moved away from donor centers at the military bases. DR.SCOTT: Okay, I think we will move forward and Susette Audet from Judy Beeler's group is going to be speaking about measles antibody titers in products. This is the results of a study that we did with CBER.
Agenda Item: Measles antibody testing and decline in measles antibody titers in Immune Globulins over time MS. AUDET: I want to thank the organizers and the sponsors for giving me this opportunity to talk about a study that was published in 2006 in the Journal of Infectious Diseases entitled “Measles Neutralizing Antibodies in Intravenous Immunoglobulins.” Now, Dot gave a nice summary of this, and I just want to reiterate that all immunoglobulins must meet a minimum potency of measles antibodies assayed in the CFR. Although not specifically stated in the CFR, IGIV must also meet a minimum potency for measles antibodies. With this in mind, we wanted to study any factors that could potentially influence the potency of this product, and because we knew that with universal use of measles vaccine, this would more likely have an impact on the product. On this slide I just briefly highlighted some interesting facts regarding measles epidemiology. Some of this you have heard before. Individuals born before 1957 are presumed to have measles immunity due to natural infection. Therefore, individuals born in or after 1957 are more likely to have the vaccine-induced measles immunity. Measles antibody titers after vaccination do tend to be lower than natural infection. However, irrespective of the type of immunity, antibody titers do dwindle over time. As of 2000, there is no endemic measles virus circling in the United States. The cases that do occur are due to importation of the virus from other countries. Therefore, in the United States, there are fewer opportunities for immunological boosting. We had four main study goals for this project. One was to evaluate the potency of measles antibodies in the IGIV products over successive manufacturing years, and also to evaluate changes in donor demographics and to evaluate factors related to manufacturing that could influence the potency of the product. Finally, we wanted to estimate the serum levels of measles antibodies achievable with various IGIV products that would provide sufficient antibodies to maintain levels that have been known to be associated with protection in the vaccines. Summarized on this table are our seven IGIV products that we used in the study. Included are the type of plasma used to produce these products and a brief synopsis of the manufacturing procedures for each product. Finally, we have also listed the number of lots we tested for each manufacturing year, starting from 1998 to 2003, for a total of 166 lots. Summarized on this table are the plasma donor demographics for source plasma donors, as shown in the red, and the recovered plasma donors, as shown in the blue. We also subdivided each of the donor groups into two birth cohorts: the percentage of individuals born before 1957, as shown in the dashed line, and the percentage of individuals born in or after 1957, as shown in the solid line. If we look in 1987, about 42 percent of the source plasma donors were born before 1957. Therefore, about 58 percent of these donors were born in or after 1957. The difference between these two birth cohorts dramatically changes by 2003. In 2003, we only had 10 percent of the source plasma donors born before 1957 and 90 percent of the donors born in or after 1957. We saw a similar trend with the recovered plasma donors, but the difference between the birth cohorts wasn’t quite as dramatic. In 1995, there were about 50 percent recovered plasma donors born before 1957 and after 1957. By 2003, about 43 percent of the recovered plasma donors were born before 1957 and 57 percent of the recovered plasma donors were born in or after 1957. What this data suggested to us was that the source plasma donors do tend to be younger than recovered plasma donors and were more likely to have vaccine-induced immunity. The next two slides actually show the measles antibody titers for the products we tested in the study. Listed on this slide are the measles antibody titers for IGIV products produced from source plasma, as shown in the red bar, or recovered plasma, as shown in the blue bar. We did see this declining trend for products produced with source plasma starting from 1998, as shown in red, to 2002. We did see a similar trend for the recovered plasma, in that lots produced from recovered plasma in 2000 were slightly lower than lots produced in 1998. Perhaps this is not a big surprise that we saw this trend, because, as I mentioned in my previous slide, source plasma donors do tend to be younger, and therefore were more likely to have vaccine-induced immunity. Summarized on this slide are the measles antibody titers for the individual IGIV products. Each of the bars represents a given manufacturing year, starting from 1998 to 2003. The bold, dashed line going across the bars represents the overall mean titer for that product. In this slide, we do see this declining trend of measles antibodies for products A, B, and C. Overall, product D had the lowest level of measles antibody titers of any of the other products. Product E had the highest level of measles antibody titers for a majority of the manufacturing year. This product also had the overall highest measles antibody titers compared to the other products. As I have shown you in my previous slide, the type of plasma can influence the potency of the product. However, this could not be the only determinant. As seen in product D, this product has the overall lowest level of measles antibody titer, yet this product is produced from a similar starting plasma as products A, B, C, and F. So we investigated to see if there were manufacturing factors that could influence the potency of our products. We investigated the contribution of each IgG subclass to measles-neutralizing activity. In addition, we wanted to see the effect fragmentation would have on the measles activity. This slide shows the measles-neutralizing antibody activity for the different IgG subclasses. The IgG subclasses were separated from one IGIV blot using FPLC, and they were tested at the same IgG concentration in our neutralization assay. As you can see from this graph, the IgG1 had the highest overall measles antibody activity, followed by IgG3, and the IgG2 had the lowest level of measles activity. In order to determine if this loss in IgG1, or perhaps IgG3, would affect the potency of our products, we measured the percentage of each IgG subclass in our seven products. Listed on this slide is a summary of that work. Here is the distribution of the IgG subclasses and the percentage for IgG1, 2, 3, and 4. In addition, we also have listed the potency for these lots that we have tested, in this column. If you exclude product D, overall the other six products had very similar IgG profiles. IgG1 was the highest, followed by 2, 3, and, finally, 4. In contrast, product D had the lowest level of IgG1 and also the lowest level of IgG3. Interestingly, this product also had the lowest level of measles antibody titer. Next we looked at the effect fragmentation would have on the measles activity. In order to evaluate the neutralizing activity of the fragments, one lot of IGIV was digested using pepsin or papain agarose, and both the monomer and the fragments were tested on an equimolar basis in our neutralization assay. You can see from this slide that antibodies that were valent had the highest percent measles activity compared to the FABs. This suggested to use that fragmentation can have a significant impact on the functional activity of antibodies when evaluated by neutralization assay. The next thing we wanted to do was to measure the serum level, followed by IGIV infusion, in order to determine if a product would have sufficient neutralizing antibody presence to maintain levels that have been associated with protection in vaccines. That level was designated as greater than 120 mIU/mL. On this slide are the theoretical measles antibody serum levels after one dose of IGIV. Listed here is the code for products, the mean measles antibody titer for that particular product. Here are the total measles antibodies when given one dose of IGIV at 400 mg/kg for a 10-kg recipient. Listed in the last three columns are the neutralizing antibody levels at time of administration, equilibrium, and trough level. We also performed a back-calculation to determine the theoretical minimum potency needed in order to obtain a trough level of 120 mIU/mL. In our calculation, as seen in red, a theoretical minimum potency of 600 mIU/mL would be able to maintain a trough level of 120 mIU/mL, which has been shown to be associated with protection from disease in vaccinees. As you can also see from this table, all of our seven products would exceed this theoretical minimum potency. In addition, all of our products would be able to maintain a trough level of 120 mIU/mL. In summary, we have shown that the percentage of plasma donors born after 1957 is increasing. The products made from recovered plasma do tend to be higher than products made from source plasma. We did see a decreasing trend of potency for the IGIV products produced from 1998 to 2002. In addition, the IGIV products with the lowest level of IgG1 and IgG3 have the lowest measles antibody potency. We also showed that IgG1 was the predominant subclass with measles neutralizing activity, and bivalent antibodies had higher functional activity than FABs. Finally, all of the IGIV products would be expected to achieve a level of 120 mIU/mL after one month, although I do want to reemphasize that the level of measles antibody needed for measles prophylaxis in an immunodeficient individual for protection is not known. In conclusion, the decrease in measles potency of IGIV may be reflective in the donor demographic and can also be affected by the type of starting plasma. Perhaps other manufacturing procedures could affect the potency of the product. Also pharmacokinetic studies -- Also, finally I want to thank a lot of collaborators on this project, Dr. Judy Beeler, my supervisor has been a tremendous support and mentor all these years. Paul Albrecht actually developed the plaque reduction neutralization assay that we used throughout this entire study and all our wonderful collaborators in the Office of Blood and finally I would also like to thank Mary Gustafson and Ed Notari for providing all the demographics. Thank you. (Applause.) DR. SCOTT: Questions? PARTICIPANT: The minimum level of gamma globulin for normal children is less than that given to immunosuppressed children. Now, was this calculation of 400 milligrams per kilogram based on the level that is thought to be appropriate for immunosuppressed children or for normal children? MS. AUDET: I think I am going to refer that to Dot. DR. SCOTT: I think the 400 milligrams per kilogram was chosen with the idea in mind of thinking about primary immune-deficient patients. I would also point out that there is a difference in terms of when one might measure the trough or when it becomes important because for measles prophylaxis you want to give IG within 6 days of exposure. So, you are really only looking at that time period for efficacy of day 0 to 6 post-exposure whereas for primary immune deficient patients obviously you want them covered in a prophylactic fashion all the time. Does that answer your question? PARTICIPANT: What did you make of the trend in your data after 2002 where it appears that the titers are rising? MS. AUDET: Right, we did see that and obviously we don't have any kind of vaccination history and on these donors we really don't have a history of their measles exposure. The only thing that we could think of as sort of an unknown to us and we have mentioned this in the paper that perhaps if you remember from yesterday's talk in late and mid-1980's there was this huge outbreak of measles in the United States and we thought perhaps those individuals that were exposed during that time are now eligible for donation and perhaps they sort of gave a quick rise in the donor plasma but basically we do not know why there was this sudden increase in the measles antibody titers for the lots produced in 2003. We just don't have an answer. DR. OCHS: Dr. Ochs. I wonder this preparation D I think it was with the low level and your referral to digestion, was this a preparation that perhaps had pepsin or some enzyme that -- MS. AUDET: Audit. OCHS: -- in a breakdown? MS. AUDET: Right, this preparation I believe has a hydrolase step, this particular product. So, we did think that perhaps that procedure did alter antibodies in some way. So, it was purposely done to create fragments to see what would happen although I think Dr. Finlayson did point out that fragmentation does not occur very often because of other manufactured procedures that will prevent that from occurring but if it did it would have an effect on the antibody. So, yes, there is a specific step for that particular product that has a sort of hydrolase procedure. DR. OCHS; There was one product for many years that had pepsin, a small amount of pepsin in it and I was wondering if it was that preparation? MS. AUDET: No. DR. EPSTEIN: Could you come back to the slide in which you did the calculated plasma levels? MS. AUDET: Sure. DR. EPSTEIN: I am puzzled by the bottom two lines. I don't understand how 600, this is on the left-hand column can be the minimum needed potency because if you just look at the reference standard at four times that level you achieved less trough than on the theoretical calculations. So, how do you reconcile those two? MS. AUDET: Right, from my understanding, perhaps Dr. Finlayson can help with this, there is a theoretical minimum of potency for an IGIV product and the calculation and IG product was slightly different, and, Dr. Finlayson I don't want to put you on the spot but perhaps you can elaborate.. DR. FINLAYSON: I knew Jay would see this. Yes, t he US reference is a 16.5 percent intramuscular preparation and the trough level was based on the assumption that it would be given at the package insert recommended level for an intramuscular preparation. DR.SCOTT: Just a reminder that this product isn't indicated to achieve a trough level at 3 or 4 weeks of greater than or equal to 120. It is really used within the first 6 days. So, even though the trough is low for the reference IG which is a 16-1/2 percent standard that isn't when the patients that are at risk for measles would need that level if that makes some sense. DR. FINLAYSON: If I can intrude if one goes back one half life, in other word 22 days before the average of 27 to 32 you are just about back where you wanted to be 6 days afterwards and so you would double that 105 figure at that point. DR. SCOTT: Any more questions? I think we will proceed then to the next talk where we will hear about trough levels in recipients. Thank you, Dr. Zenker.
Agenda Item: Measles antibody levels in PIDD patients DR. ZENKER: Good morning, everybody. First of all I would like to thank the organizers to give us the opportunity to present our data on measles antibodies for patients with primary immune deficiency disorder disease. As already announced yesterday I will speak about titers in the blood and not in the bottle. Some repetition at the beginning. Measles infection but also vaccination induces a lifelong immunity in almost all persons. Individuals with wild-type infection tend to have a higher antibody titer than by vaccination and after introduction of the vaccination in the sixties the antibody titers in donors are falling and as a consequence also in the immunoglobulin preparations. From a medical perspective it is more important to talk, to discuss the protective titer especially in PID patients. From outbreak and vaccination studies it is expected that this protective titer is in the area of 0.1 to 0.2 units per milliliter. For PID patients not much is known. Calculations from Susette Audet as already presented suggests that 0.21 units per milliliter provide at least as much measles antibody as recommended for measles prophylaxis. The measurement of measles antibodies was not foreseen in the study protocol of several IVIG and subcutaneous immunoglobulin studies performed by us in the past years. Nevertheless we have sufficient frozen retention samples from one subcutaneous study to perform this pilot study on measles antibodies. This is a very complex study design of the US Pyrota(?) study for viviglobulin(?). I will not go into detail here but just mention that the samples we have analyzed in our study are all coming from the efficacy phase here, after the individualized subcutaneous dose adjustment. The samples were analyzed with a commercially available ELISA test but also with a neutralizing assay for comparison reasons. Some words to the patient characteristics. Currently we have data from 20 patients. Six of them have XLA and the remaining 14 CVID. The age range was between 9 years and 62 years as expected for the study population. The median IgG trough level was 9.1 with a wide range from 5.7 to 19.2 grams per liter. The weekly subcutaneous dose was 158 milligrams per kilogram body weight. These data do not differ significantly from the total patient population in the study. Now to the results of the ELISA test. The range of measles antibodies goes from 0.5 in this patient to approximately 4.9 in this patient. We have excluded one patient with an anti-measles of more than 14. Further details of these patients have to be evaluated but we think that this patient has a natural immunity due to a wild-type infection in childhood. You can further see on the chart that all patient samples were well above the cutoff level of 0.12 units per milliliter. There is some correlation between the trough IgG levels and the measles antibody level but this correlation is not very strong as mentioned here in this correlation coefficient. We,also, looked at the correlation between the dose given for these patients and the anti-measles titer which showed similar results. Now, to the functional assay. The anti-measles titer ranges between 1.3 in this patient and 6.3 in this patient. Also, here the titer is well above the protective titer of 0.12 units per milliliter. The correlation between the trough level and the anti-measles titer is again moderate. Finally, we have analyzed the correlation between the two assays. Here we found a quite good correlation with a coefficient of 0.94 but mainly based on the patient with the very high titer here. This patient had the titer of more than 14 in the ELISA and around 12 in the functional assay. When you look at the data points in the range where most of the samples were here in this area you might see also a correlation but definitely more data are needed to confirm these results. Now, I come to the conclusion. First of all and most importantly all clinical samples analyzed were far above the protective titer for normal subjects of the vaccination. We could show a good correlation between our two assays which has to be confirmed in further studies. As expected due to the variability of the past and the variability within a patient only a moderate correlation between the anti-measles titer and the trough IgG levels could be observed. There is a lot more to do. The current presented data were only preliminary. Further analyses are in preparation. These are additional measurements in the same but also in further patients depending on the availability of the retention samples but also from the statistical perspective further work is needed. We have started to match clinical data to the anti-measles data like diagnosis, age groups and dose but also the lot-to-lot variability will be taken into consideration as several lots with different anti-measles antibody levels have been used in the study. Last but not least we will include pharmacokinetics of anti-measles IgG in further clinical studies to have more data available in the future. These are my colleagues who did most of the lab nd the clinical work. Thank you very much. (Applause.) DR. SCOTT: I have a question. Here you are looking at the subcutaneous immune globulin dose, and my recollection is that the trough levels for total IgG also in that study were quite high. I wanted to ask how you can relate this to regular IGIV and whether or not you are, also, doing studies to look at the trough levels in patients who received intravenous product. DR. ZENKER: Yes, as I have mentioned we have started a program also to look in patients that have been treated with IVIG. This will be looked at in the upcoming studies. Results will be available probably in a year. Regarding the trough levels, the trough levels after subcutaneous treatment are higher than after intravenous treatment and this will be also reflected probably in the anti-measles titer. DR. SCOTT: Right, and they were higher by approximately how much if you could just state what the average trough level was. DR. ZENKER; The average trough level in the viviglobulin study was higher, approximately 1 gram per liter. DR. GOLDING: I think it is important maybe to clarify that. The trough levels you are saying in the subcutaneous was higher than the intravenous, but the other PK parameters may be equally or more important such as area under the curve, C max, etc. So, am I right in saying that if you look at the total PK data that the two are similar and that the reason why you have higher trough levels is because you are just giving it more frequently, and you are actually giving a higher dose or over the month you end up giving about 37 percent more of your product than the IV? DR. ZENKER: Right, the study was designed to match the area under the curve of the previous intravenous immunoglobulin. So, overall the area under the curve also falls. For the measles titers should be the same. DR.SCOTT: I think what I am trying to do is perform a crude calculation to figure out what it might be for a typical intravenous product. So, for example, if you have an average trough level of total IgG of 1 gram per liter and people are really aiming for half of that let us say in a typical trough and the clinicians can help us with that even your lowest neutralization titer that you got here if you divide that by two still puts you at 650 milli IUs per ml. I mean that is not clinical data but that may be a ballpark estimate that tells us it is still quite well above what is considered the protective level against disease. DR. BERGER: In fact, I think the trough levels of the patients in the study if you go to the North American study and the European study for the trough levels on IV were in fact in the eight hundreds. I think Paul Ingersoll has made the observation and reported it at the last meeting that over the years many clinicians have been increasing the doses of IV given to PID patients and the trough levels have been drifting up. DR. SCOTT: Any further questions. Thank you very much and we look forward to even more information in the future. Now, we will hear from Dr. Griffin and she is going to tell us a lot about measles and antibody.
Agenda Item: Measles infections and estimated protective titers in PIDD, and potential reemergence of epidemic measles in vaccinated individuals DR. GRIFFIN: I was hoping to be able to use my computer but maybe that is not going to be possible. Is there a way to plug it in? Success. Right, well, thanks for inviting me to this interesting workshop and as previously suggested I worked on measles for a while and I have a lot of interest in antibodies and the role of antibodies both in protection and clearance and I am going to just give you the data that we have or that I have collected from the literature that I think is relevant. Some of it has already been mentioned. I will try not to be too repetitious, but I will note that even though measles is at an extraordinarily low level in the United States and there has been no endemic transmission of measles since 2000 really since the institution of the two-dose strategy for immunization that is not necessarily true in the rest of the world and as already mentioned most of the cases, essentially all the cases we see in the US are due to importations but they may then spread in the community with one outbreak of 34 cases having been mentioned. It does remain one of the 10 most important causes of death due to infectious disease in the world. So, if you have patients that are traveling there are still risks even though in the United States the likelihood of being exposed is quite low and not surprisingly these are the parts of the world where measles is most common and there are major efforts to try to control measles and particularly efforts are ongoing in Africa. I, also, just want to point out that it is not only developing countries. At the time this map was made which was 2003 and these shift around a little bit, there are two countries that we would consider well developed and that appear on this particular year's map, Italy, Japan are both countries and Europe has only had a major effort to control measles within the last few years. So, you can travel to many parts of the world and be exposed to measles even though in the Americas we are essentially measles free, and measles remains the second most common cause of deaths due to infectious disease in children under 5 that can be prevented by immunization. So, for vaccine preventable disease, measles is still the second most common cause, second only to pneumococcus. So, I am just going to talk a little bit about the pathogenesis of measles because I think it is necessary in order to be able to try to understand where antibody comes in and where it may play an important role and the basics, basically after infection there is a lot of spread of virus with relatively high titers about 10 days after infection has occurred. That is the reason you can intervene at this point with prophylactic immunoglobulin and have an effect and between 10 and 14 days the rash appears and that is the stage at which measles is diagnosed and recognized clinically and that is coincident with the appearance of the immune response and so this appearance of immune response which is a T cell response as well as an antibody response which is clearly recognizable at the time of the rash most individuals will have within 4 or 5 days of the onset of the rash detectable IgM which is the way the diagnosis is usually confirmed and then subsequently large amounts of IgG are produced. Then coincident with that you have clearance of virus that is occurring thanks to this immune response. So, one point to make is that the rash is a manifestation of the immune response. Therefore one of the problems you can have with immunocompromised individuals who are exposed to measles is they might not develop a classic presentation and the diagnosis can be very difficult. The rash itself is a manifestation primarily of the cellular immune response. So, that is the skin rashes during the eighty-nine, ninety-one outbreak of patients admitted to Hopkins with measles and if you stain for the types of cells that are present there is a lot of lymphocytic infiltration into the area of the rash and as I say it is primarily a manifestation of the cellular immune response. If you don't have a good cellular immune response you are not going to get a rash. So,the roles of the immune response are really two, first of all for clearance as I just mentioned and then also for protection from reinfection which is what we are most interested in in this particular group but just to point out that these two are not the same necessarily. So, clearance is going to require elimination of infected cells as well as well as circulating virus. Individuals who have difficulty with clearing the infection can have a different clinical manifestation of measles. First of all we see now in a number of individuals and it has also been reported in the literature on the correlation and hasn't become so clear until lately that a desquamating rash actually is seen in individuals who often have very slow clearance or they eventually do clear; they eventually do recover but they have this different type of a rash, and then what I have already mentioned is you can get progressive disease often without manifestation of a rash and particularly in a situation of deficiency of the cellular immune response. Now, for protection you are really trying to prevent this initial infection so that you don't have to go through any of this clearance. You just are protected from being affected at all and many studies, many of which have been mentioned already this morning the best correlate that we have for whether an individual is or is not going to be protected from measles in an outbreak situation is the level of neutralizing antibody. Now, measles is a human disease, doesn't have an animal reservoir and animal models for measles are hard to come by but the Rhesus macaque is a good animal model and monkeys in general are susceptible to measles. In fact, measles could be a major problem in primate colonies and we have used the Rhesus macaque model to look at a variety of aspects of the pathogenesis of measles and particularly what the protective immune response needs to be in order to protect animals from a wild-type infection and the characteristics of the infection are very similar if not identical to what happens in humans when they are infected and you get viremia, rash and you get clearance of this evident virus replication coincident with the rash and then you get a lot of changes in the immune system that are consistent with measles in humans and I will just point out that we are using a wild-type strain of measles isolated from an outbreak in the Netherlands. So, one of the things using a macaque model allows you to do is you can look at immunodeficiency and what the outcome is and you can selectively look at various components of the immune response and what is important for clearance is really what we have looked at here and so these are monkeys that have been depleted of CD8 T cells and then infected with measles and then just look at the clearance and those are the control monkeys and these are the ones that have been CD8 depleted and there are two things to notice. The main thing is that the titers are higher during the viremia and complete clearance of the viremia and this is detection of infectious virus take longer perhaps partly because of the initial levels of virus replication. So, CD8 T cells are clearly important for containing that measles virus replication and for the clearance process but diseased animals did eventually clear. Now, the mechanism for depleting CD8 T cells is an antibody that really only depletes the T cells for 2 to 3 weeks, and we also then used the same kind of a model system but now to look at depletion of B cells so to try to take a look at the independent role of antibody and in these cases this is looking at amounts of viremia by two different methods. One was the same that I just showed you on the previous graph which was actually amounts of infectious virus in peripheral blood mononuclear cells. This is an RT PCR assay for measles virus RNA in the same kinds of samples. So, these are most comparable to what you just saw and these are the control monkeys. These are monkeys that have been depleted only of B cells and these monkeys were deleted of both B cells and CD8 T cells and so you can see that the ones that are depleted just of B cells actually had viremias that were very similar as measured by either of the two parameters as the control monkeys but if you depleted both then it took longer to clear virus and although titers were somewhat higher but not really substantially higher in this particular group, nd one of the things that we did see or Sally Permar and Norm Leckman who really did these studies was that these animals developed a desquamating skin rash. So, there is something to do with either the CD4 response which we do have that may lead to this or the lack of either antibody or CD8 response that may lead to this particular clinical manifestation of a desquamating rash and this is a picture of this rash probably best seen here but the regular histology in these monkeys and if I could just go back you can also see giant cells that are present in this, a characteristic pathologic feature of measles which are also present in this desquamating rash. So, it suggests there is more measles virus replication in the skin at least and probably elsewhere because this is just a visible aspect of the disease and so we have also seen in our studies in Africa where we have been conducting studies in Zambia for a while that a desquamating rash is also associated with both malnutrition and possible with HIV, underlying HIV infection and we are currently looking at this population in a little more depth to try to understand the immune response in this group of children. So, there are lots of studies and many of them have been mentioned that suggest that antibody alone can be important for -- go back a minute. This slide is just to counter the monkey studies and say that there are studies out there that would suggest that antibody can be important for clearance or may contribute to clearance even though we did not see that with the viremias and these are just listing those studies that levels of ADCC antibody correlate with cessation of viremia but may correlate with other things that are going on with the immune response at the same time. There are a number of studies from Africa that show low antibody responses to infection, predict poor outcomes but again cellular immune responses weren't usually measured and these individuals may be deficient in more than one thing and then there are some in vitro studies that were published quite a while ago that show that antibody can have an effect on virus replication intracellularly, independent in fact in vitro. So,I think we really still don't have a total picture of the contribution of antibodies to clearance itself and to look at the kinds of diseases then that one might see in an immunodeficient individual who has been exposed to measles but now gets a progressive measles disease, the two classic presentations of progressive measles infection are giant cell pneumonia and measles inclusion body encephalitis and there are many case reports in the literature of both of these clinical pictures occurring in children with a wide variety of,mostly children but also some adults with a wide variety of immunodeficiency diseases mostly involving both C and B cells. The clinical picture as I mentioned before is often there is no rash at the time of the measles infection. So, there is usually quite a substantial delay in the diagnosis because what the individuals present with is a progressive pulmonary or CNS disease usually of unknown etiology and this usually is occurring weeks to months after this initial exposure, so, getting a history of an exposure to a sibling or something like that with measles but many of these diagnoses weren't made until autopsy basically. Measles wasn't suspected as a cause of this disease. This is a picture of a giant cell pneumonia in such an immunodeficient child and a little more emphasis on the time course of manifestation of clinical disease in individuals who develop measles and there are three different central nervous system diseases which I will talk about only with measles inclusion bodies encephalitis because this is the disease that occurs in immunocompromised individuals, central nervous system disease. The earliest manifestations are usually somewhere around 3 or 4 months after the exposure to measles and these children as I say frequently do not have rash and many of them may have both pulmonary and CNS disease and one or the other may predominate with the presentation. There is one mention of this other disease, subacute sclerosing panencephalitis. This occurs in individuals who as far as we can tell are immunologically normal at the time that they, immunologically normal period. They have often acquired measles at a young age, under the age of 2, most children with SSPE and those children also may have been reported either not recognized they have measles or reported to have a very mild disease. So, it is probably some problem clearly with control and it ends up with progressive measles virus infection and in the nervous system again internuclear inclusion bodies you don't see giant cells in the nervous system but you do see inclusion bodies and you can stain lots of cells for measles virus antigen. So, there are really abundant amounts of the virus that are present either in the lung or the nervous system of individuals at the time the present with such disease. So, as I mentioned there are lots of case reports of progressive measles infection associated with immunodeficiency. Usually these are occurring in individuals that have combined B and T cell immunodeficiencies. I can find no and I would be very interested if anybody in the audience has such information, good reports on or even reports at all on individuals that have pure B cell deficiencies who are really only lacking antibodies. The dogma is that those individual recover normally but if you actually look for the documentation it is a little slim and then many of the cases are in secondary immunodeficiencies due to leukemia, lymphomas, various tumors, chemotherapy, other immunosuppressive therapy and quite a number reported with HIV infection and with HIV infection as well as some of these other diseases this progressive disease has been reported with a vaccine virus as well as with the wild-type virus, much more common with the wild-type virus but the vaccine virus can do that if an individual is inadvertently immunized because as mentioned before this is a live virus vaccine. So, what is the role of antibody in protection from infection which is really the heart of what people in this audience are interested in? So, there are really three sources of information to say that antibody is protective and it is the main determinant of protection and one is that we know that infants are protected by passively acquired maternal antibody. So, we don't have a T cell issue in infants who are protected from measles. There is passive administration although infants are now having the same problem that you are talking about with immune globulin. Mothers have lower titers. Infants therefore get lower titers. They become susceptible to measles at a younger age than used to be the case a couple of decades ago. As has already been mentioned and I won't go into it any more detail passive administration of immune globulin can protect after exposure and has been used and probably is still being use for that reason and then what we really relied on primarily is that the best correlate for vaccine induced immunity, protective immunity is the level of neutralizing antibody. So just to remind you not to go into a lot of virology but the measles virus has a number of proteins and eight of them, six that are present in the virion so that in addition to surface glycoproteins there is a nuclear capsid protein which is most abundant and made most abundantly as well as a couple of polymerase proteins. So, when you are looking at antiviral antibody particularly in ELISA test you are looking at antibody to all of these proteins that are present on the ELISA plate and this is just documenting the kind of immune response to these various proteins that are made. This is during natural measles virus infection but I think it would be a very similar picture if you looked after immunization that this is the percent of individuals that have responded to each of these proteins. This is the relative amounts of antibody that is made to each of the different proteins. So, if you look at the nuclear capsid protein not only has essentially everybody responded most promptly with antibody to nuclear capsid but that is also the most abundant antibody that is present after infection and then coming up somewhat more gradually is the antibody to the hemagglutinin and then much lower levels in general of antibody to the other proteins. So, I have already talked about a couple of, three methods basically of measuring measles antibody but just to reiterate that each of them is measuring antibody to something different and so the neutralization assay is measured by the biologic test that is the inhibition of plaque formation, of a tissue culture adapted strain of measles on Vero cells that Albrecht developed the test that all of us use to measure neutralizing antibody that is standardized across the world and then there is the anti-immunoassay,the ELISA which is usually used as lysate of MV-infected Vero cells or sometimes a recombinant nuclear protein as antigen but commercial tests are whole measles virus lysate primarily. I will just point out that for the neutralization test neutralizing the two surface glycoproteins the hemagglutination and effusion both contribute to neutralization. So, you are e usually measuring antibody too both of those but by far and away the biggest contributor to the neutralizing antibody titer is the antibody against the hemagglutinin protein as the most potent neutralizing antibody. Therefore the hemagglutination inhibition assay which is measuring antibody just to H is able to correlate very well with the neutralization titer. As was mentioned I think or alluded to at least the ELISA titer which is measuring antibody to all of these proteins correlates well with the neutralizing titer at high titers, but there are a number of studies and we certainly confirmed that in our own lab that at low titers there is not a very good correlation but we use as the cutoff for the ELISA that correlates are not particularly good at those low titers and it is probably because of the mix of what kinds of antibodies you actually have. So, the evidence that antibody can be protective, first that infants are protected by passively-acquired antibody, I am not going to go into a lot of the primary data. I will just give you the basic picture. There is a number of studies. Most of these studies have looked at response to immunization as a measure of whether the infant is protected from measles or not because most of the studies have done trying to figure out what the proper age for immunization is and maternal antibody, residual maternal antibody is an important determinant of whether you get seroconversion with the vaccine or not. These are data from Haiti that actually looked at whether children were getting measles and at what age they were getting measles and they also were then looking at the age at which you could begin to immunize. So, the main point of the study was to determine the optimum age of immunization but this is just basically 6 to 12 months, the decline of maternal antibodies. These are data from Haiti but could be mimicked in a number of developing countries. So, there is a steady decline and so the routine age of immunization in the US is between 12 and 15 months and that is true for most developed countries and that is because at that age most children have lost essentially all of their maternal antibody and you have a high rate of seroconversion to the vaccine. However, in developing countries this was the percentage, almost 40 percent of children had already acquired measles by the age of 12 months in this setting of high measles transmission. So, in developing countries 9 months is the compromise time. You have a lower rate of seroconversion but still you prevent a large number of infections that may be occurring even just in that window of susceptibility between 9 and 12 months. So, these kinds of data say that antibody alone can not only prevent seroconversion to the vaccine but also prevent measles. So, there is a couple of studies that have really formed a lot of the basis in addition to these maternal antibody studies, the basis for understanding what the magic titer is or how much antibody you need. This is just one other maternal antibody titer where basically children were determined whether they were positive or negative. So, these are pretty low titers and then looked at seroconversion to vaccine and again there is a very strong correlation between the level of antibody and whether an infant at the same age will or will not seroconvert to the vaccine. So, lastly let us talk about the correlates of protection with respect to vaccine-induced protection and as has already been mentioned we don't have data on the critical you know if you passively transfer antibody, what do you need to protect from natural disease. So, we really only have the vaccine-induced data. Now, these kinds of data could be acquired from monkeys but they haven't been as far as I know. There are some data but it all looks at response to immunization not response to wild-type infection. So, the study that I think is most often quoted is from Chen at the CDC where they had during these outbreaks that were occurring in the US in the late eighties and early nineties and it happened that as one of the schools where there was an outbreak there had been a blood donor program that had occurred shortly before the outbreak. So, they had plasma from the individuals and they could determine what their titers were before the outbreak and then who was protected and who was not protected during the outbreak then at that school, and so these data then if you look at those that had antibody levels under 120 milli international units per ml there were nine of those and eight of them developed measles during the outbreak. Those that had higher titers that were this high or higher there were 71 of them and none of them developed measles during the outbreak. So, this kind of data is what we have solidified at 120 as a protective level for immunity and I just want to say that what we are looking for here was measles and so that means the characteristic disease with a rash, etc. -- in that same study. If you look more carefully at viremia rather than just rash, then you find that actually there is another group that can develop viremias without rash and have not been protected from infections, though they have been protected from disease. This is a group of monkeys that we used in a vaccine study. It was a DNA vaccine. It gave very variable levels of neutralizing antibody to these different vaccines. These are all individual monkeys. In red are the clinical outcomes after these monkeys were challenged. These were the titers just before challenge. What you can see is that there were some monkeys that developed both rash and viremia; they got clinical disease. They, in each case, had the lowest levels of neutralizing antibody induced. There was another subset of monkeys that developed viremias, but didn’t develop rashes. In general, they also had low titers, but they were somewhat higher and, in every case, were over the 120 cutoff, the commonly accepted cutoff. So there are different levels of protection. The same kind of thing was observed, then, in the Chen study. If we look at these schoolchildren that had intermediate titers, some of them developed an antibody response. So the other thing that one would observe in these monkeys that developed a viremia is that you would get an IgM response and a boosted antibody response that would occur simultaneously with that infection. What they measured there were the individuals that developed an antibody response, a booster response, to measles, as evidence of having been infected, even though they weren’t -- sometimes they had symptoms, but they didn’t have a rash and they didn’t something that was clinically diagnosed as measles. If they had titers over 1052, then they didn’t have any evidence of even having been infected. So they were a fully protected group of individuals. So those are basically the data we have. In conclusion, I think that after infection, clearance of measles is dependent primarily on cellular immunity. There are a lot of data that suggest that. Defects in clearance are associated with unusual manifestations of measles, such as not developing a rash, progressive infection, and now we think desquamating rash also is another feature. The protection from infection is best correlated with a level of neutralizing antibody greater than or equal to 120 for protection from disease, a rash, but a higher level for protection from infection and viremia. The people that have been involved in both the monkey and the human studies that we have done over the years have been Fernando Polack, Sallie Permar, and Bill Moss. The depletion studies Sallie, who was a Ph.D. student in my lab, did, in collaboration with Norm Letvin at Harvard. Thanks. I would be glad to answer any questions. PARTICIPANT: I am still concerned that the level achieved in normal children to protect against infection may be different than for an immunocompromised. I wonder if the protective antibody levels differ in children that are normal versus the immunocompromised child. DR. GRIFFIN: Absolutely. I just don’t know where the data are for immunocompromised children receiving IVIG, the levels they have, and then exposure in an outbreak situation, which is basically what you need -- those data and then whether they were or were not protected. Those are not studies that we have done anything like, and I can’t find them in the literature. You people are more likely to know that, if there are case reports, et cetera -- anything that gives us a hint in that regard. PARTICIPANT: Do you know where the data came -- the recommendation for preventing measles in an immunocompromised child is double, I think, that for a normal child. Is there data for that, or was that just sort of out of the blue? DR. GRIFFIN: I don’t know. I didn’t know there was a different recommendation for immunocompromised children and normal children for protection, if the normal child was totally naïve. DR. BEELER: Maybe Dr. Finlayson -- I think he is the best one to answer that question. DR. FINLAYSON: In the old days, when we wrote to equipment manufacturers -- in the days of snail-mail -- we always would write at the bottom of the letter, “This is an inquiry and not a purchase order.” So this is a guess, and not data. I strongly suspect that Dr. Charles Janeway, Sr., just said that was the way it was, and then everybody said that was the way it was. I base that on the fact that, for many, many years, when there was no product available except the product for intramuscular use, a circulating level of 200 mg/mL was considered protective. There were, I think, minimal data, if any, on that figure, but that was sort of what you could achieve. That is just my speculation. DR. GOLDING: Dr. Griffin, you did these elegant monkey experiments. Did you get any clues from that? For example, if you took a B-depleted monkey and you wanted to protect, or a TNB-depleted monkey and you wanted to protect, did you need a higher dose? DR. GRIFFIN: We didn’t do that study. We depleted them, then we challenged them and said, can they recover or not, and what are their viremia levels? You could take those same monkeys and now passively transfer antibody to them at different levels and then challenge them and get an answer to that question. Unfortunately, it just wasn’t the study we did. We were asking a different question. DR. OCHS: I may have missed it. How did you infect the monkeys? DR. GRIFFIN: They are intratracheal. It’s a respiratory route of infection. DR. OCHS: What is the normal, in humans -- how do humans get exposed to the virus? DR. GRIFFIN: It’s a respiratory route of infection. It’s transmitted through the respiratory route. DR. OCHS: If it’s respiratory or through the mucosal membrane, what role does IgA play in the protection from normal exposure? DR. GRIFFIN: I don’t think we really know the answer to that question. The vaccine is given subcutaneously or intramuscularly. It does induce some IgA response, but I am not sure how carefully people have looked at the respiratory tract. Again, Judy may know more about the IgA levels. DR. OCHS: The polio story was always -- we were taught that the natural infection and the oral polio would provide protection through the membrane. The Salk was given the other way, and you would not get surface protection. Is there a similar thing between -- DR. GRIFFIN: It could be. There is a fair amount of IgG that actually gets into the respiratory tract. But we don’t know that the individuals who get exposed may not have some level of epithelial infection. Measles is really a systemic infection. There is very little in the way of respiratory symptoms initially, although there is some cough and that sort of thing early, and there is this prodrome. But I don’t think that we know for sure that you haven’t just prevented spread, that it just never gets out of the respiratory tract. I think the thought is that it’s really a solid protection and that you are probably getting some IgA, as well as IgG. T cell responses may also be contributing to that solid protection that you get from the current vaccine. DR. GOLDING: Can I say something about IgA? It often comes up at these meetings. Maybe I have a bias. But I think there is an experiment of nature where you have quite a common condition of people being selectively IgA-deficient. The treaters here know better than I do, but I don’t think those people run into particular problems with measles or other infections. DR. GRIFFIN: They have more infections with otitis and sinus infections and that sort of thing, I think. DR. GOLDING: Yes. The other fact that to me is quite interesting is that there have been studies -- particularly I remember Ruth Frubeck’s [phonetic] with HIV, where she gave IGIV passively to monkeys against HIV and found neutralizing titers in the mucosal lumen. The immune globulin, the IgG component, does diffuse, as you were saying, into the mucosal surface. We are not sure, in that situation, how important IgA is. DR. GRIFFIN: I would agree with you. PARTICIPANT: If you give immune globulin to a patient late in the incubation period, you can modify that. What is modified measles? DR. GRIFFIN: People who have been previously vaccinated and are not completely protected -- that occurs. In the outbreaks that were occurring in the late 1980s, early 1990s, a high percentage of those individuals had a history of having been immunized. They may not have responded to the vaccine. We didn’t have a lot of data on that, although in a couple of outbreaks we had some We have seen modified measles in our monkeys. They get a much milder rash. They get lower titered viremias. I think they are just inducing an anamnestic response. They have enough of an immune response to sort of curtail things a little earlier than a totally naïve individual or a totally naïve monkey. People where there are vaccine failures often have milder disease. I think they have been primed. They may not have gotten enough of an immune response to be fully protected. They still get infected and they get some rash, but it’s much milder disease. DR. SCOTT: I have a question to the treaters in the audience. About a year and a half or maybe even two years ago, we made an inquiry through the Immune Deficiency Foundation about the level of concern for measles in your primary immune-deficient patients. I think about 10 treaters responded. The responses were that they had not seen any measles infections. I wanted to ask now, what is your level of concern? The second question I have is, is it possible that you could be missing a very atypical case of measles that is affecting the central nervous system or the lungs that just simply isn’t diagnosed because there was never a history of a rash or measles infection? DR. BUCKLEY: In terms of measles being a problem in our primary immune-deficient patients, I haven’t really seen that in recent years. Back before we could do bone-marrow transplants in SCID babies, measles giant cell pneumonia was an important problem. But since we have been doing transplants, we really haven’t seen that as so much of a problem as, for example, varicella or polio. Many of these babies did get vaccine-induced poliomyelitis. But for some reason, we have not seen it as a problem. Dr. Stiehm, you wrote a paper about neurologic disease in immunocompromised people. I just wondered, did you find any evidence of measles in any of those patients? DR. STIEHM: The patients were from all over the world. We did not identify any microorganisms or viruses or anything like that. Careful studies were really done, including viruses and immunofluorescent studies on the brain. There was no evidence that they had measles. DR. BERGER: We don’t particularly worry about it nor see it in antibody-deficient patients treated with IVIG. First of all, I think, better than previously, the word has gotten out to the pediatricians to withhold live-virus vaccines from patients with immune deficiency. There is a population of DiGeorge patients with partial T cell defects. Actually, the CDC is launching a study, through the Clinical Immunization Safety Assessment Network, to try to understand how many patients are getting live-virus vaccines without having problems versus in how many patients the live-virus vaccines are withheld and then they get a vaccine-preventable disease. But, in general, the incidence of cases of measles in schools and daycares is so low that it’s not a practical problem. That is my clinical feeling. DR. BLAESE: When we did the survey of the treaters for the IDF, the universal experience was that no one was seeing measles in their community, and so they had no way of knowing whether there was exposure, but they doubted it. We were also asked to ask about the last 10 or 15 years. So it didn’t go back three decades, where it might have had more natural measles. DR. BEELER: You alluded to the DNA vaccine, your DNA vaccine program in the monkeys. I just wondered if you could comment on the potential for using those vaccines in certain immune-deficient populations, as an alternative. DR. GRIFFIN: Right. We have continued to work on developing vaccines with the main purpose of being able to give them much earlier. But they are also all non-live-virus vaccines. We currently have a DNA vaccine, actually, that looks quite promising in monkeys and provides solid protection, expressing both the H and the F protein. I guess some of that would be a vaccine that is not a live-virus vaccine and induces both the T cell and a B cell immunity. Whether it could be used in the immunodeficient population, it certainly would be a safer vaccine, at least, than the current live-virus measles vaccine. PARTICIPANT: It might be of interest to see if measles was a threat by going to some semi-developed countries that have both immunodeficient subjects and endemic measles. DR. GRIFFIN: We didn’t try that, but we have tried to identify some of those kinds of issues in our studies in Africa, where there has been and probably will be a lot of circulating measles. We tried to look at neurologic disease -- SSPE, for instance. The problem is that there is very little specialized medical care. I don’t think these children get diagnosed. There is high infant mortality, high childhood mortality. For the most part, people don’t know what the children die of. I think if you are an immunodeficient child born into this medical atmosphere, unless you are born to a rich family, you are highly unlikely to get a diagnosis. I don’t know if that is better in some areas in India, perhaps. They still have lots of measles. There may be more sophisticated medical care. DR. EPSTEIN: We also showed that there is a significant outbreak in Italy, and you have the SID registry. I was wondering whether there might be any data available in that registry to comment on vaccination history, in particular, and/or occurrence of disease before diagnosis. DR. GRIFFIN: Both Europe and Italy that year had a lot of measles. Other countries in Europe have as well, although control is now better. Nevertheless, there is a fairly long history. Japan had, two years ago, 30,000 cases of measles. That is another country where, I think, these kinds of diagnoses would be made. Those might be the places where you could get the answers to some of these questions, where there is still circulating endemic measles at a fairly high rate. DR. SCOTT: It’s time for our break. If people could return in 15 minutes, that would be terrific. We will have our panel discussion to address the questions that you see in your agenda. (Brief recess)
Agenda Item: Panel Discussion and Summary DR. SCOTT: It looks like nearly everyone is back and engaged in fruitful conversation, but I think we will get started with our panel. Dr. Seward has, also, kindly agreed to join us and she was able to stay over today and what I would like to do is begin with a bit of a summary of today's session and what we have seen so far. One is that the measles antibody titers seem to be declining in plasma of blood and source plasma donors recovered in source plasma, and we have seen a couple of lines evidence for that, and we have also seen that over the years there has been a decline as well in overall the titers in the products for this potency lot release test which is measles neutralization. What we haven't show in our FDA data is what has happened to the products in 2004 through 2006, and I think that is a gap that we would like to address in a broader sense. We have seen trough titers in a single study with subcutaneous IG. and those have appeared to be what might be considered sufficient to protect a normal person from measles clinical infection and actually many cases from measles infection at all. However, we have identified I think some data gaps there. It would be very useful to us I think and to the community to have more trough antibody titers for measles as assayed by the hemagglutination inhibition or neutralization test in people who have received, primary immune deficient patients who have received intravenous immune globulin and to be able to relate that to the actual titers in the product that they received. We have also heard that measles still could be a potential problem. The question is how much of a problem is it for patients now and that people with T cell defects as well as Cell defects certainly seem to be more likely to develop severe clinical disease if exposed to measles. We have also learned that the level of antibody needed in immune compromised people is somewhat uncertain but I think that perhaps there are ways to address that and I would like to focus the discussion in part on that question and that is my summary of what has happened so far and the first question we have which I think was answered before this discussion by our clinical people here but I would like to pose it again is measles infection of current clinical concern for primary immune deficient patients in the US. . I would put that in the context of the possibility of travel of outbreaks elsewhere and again what has been observed in by treaters over the years. So, do we have any particular additional comments from the panel or from the people in the audience about the current clinical concern for measles infections in people with PID? DR. SEWARD: I think the epidemiology of measles in the US now as I showed yesterday and is your experience, I mean there is a very low likelihood now of being exposed to measles in this country. There is the risk of travel but I for one would hate to see antibody levels in IVIG decline. I think this is one disease that we really want to maintain protection for. It is highly infectious. We don't know what might happen in the future although if there is a change in epidemiology we would be looking at change in vaccine policy if needed, but I think we do need to be cautious. We weren't expecting the big resurgence of measles in 1989 and 1990 and we may see more measles in the future if we don't maintain a high vaccine coverage. DR. SIMON: It did seem, however that from the treaters in the audience that the specific issue related to the patients with primary immune deficiency was not of great significance so that it looked like for this particular product and the patients who need it that the clinical significance was not there. DR. OCHS: I would add to this that it is much of greater concern to us so-called "treaters" not "traitors" that we run out of gamma globulin if lots would be rejected based on an arbitrary titer of antibody. So, that would be throwing the baby out with the bath water, and I think we have to keep that in mind if we try to maintain or change our criteria. What you suggested is that we monitor our patients and I think we could set up a program where we constantly monitor trough levels on patients who are either on IVIG every 4 weeks or every 3 weeks. I have patients who inject themselves intravenously every 2 weeks and then we have of course a group a patients who get subcutaneous once a week and have a very consistent, not a trough level but a maintenance of he level and this would give us some idea to be prepared once the titer sinks lower and lower to see if our patients are protected and if once every month a huge dose gives us trough levels that are too low maybe then we rather than rejecting gamma globulin preparations maybe those patients would have to be put on a different scheduled and maybe we have to treat them every week with subcutaneous. So, if we could work out something with the FDA or with CDC to monitor our patients and have select centers and select patients where we use them as canaries for the population with PIDD and see what happens to their trough levels. DR. BAKER: You know I do want to make sure that we understand and this is coming back to Toby Simon's presentation that we are approaching a demographic cliff with respect to the measles antibody. For our current source donors for our company about 20 percent fall into that age cohort that was naturally infected but this is approaching a pretty steep demographic cliff and the reality in front of us, folks, is we have got to do something. Either we reset the measles specification or we won't have enough product or we have got to enroll AARP and start collecting in retirement communities. I mean we can't do nothing. We have got to do something and so the question in front of us of the various options that we have, what are we going to do? DR. GRIFFIN: What is the possibility of having a measles immune globulin as a different product that would be used particularly at, well, certainly in the event of any outbreaks that were occurring in the US but also would be used for travel and that sort of thing, that what you got routinely was different than what you got if you were on your way to wherever, actually almost anyplace outside the United States except for Canada and might be exposed as another approach? DR. SIMON: I think it would be very difficult as some of the other people in the industry could amplify because as I discussed usually we create an immune globulin, a specialty immune globulin product through an immunization program and it does not appear that we are well set up to do that in the case of measles and we have this declining number of donors with natural high levels of immunity .So, I think that would be a difficult product to achieve and nobody feels differently in the industry. DR. SCOTT: Historically we have had measles immune globulin and actually the original standards and CFR specified a titer for the measles immune globulin which was about double what it was for regular immune serum globulin. Right now immune globulin intramuscular preparation is indicated for that post-exposure prophylaxis but it is not a case where you would get a shot and go out for a month to another country, but what we did show is that the levels of measles antibodies in the product are predicted to be protective based on what we know about protection if you give enough. So, it is a good question though. Would that be a stopgap for people who might need an extra boost? Now, the question of whether you can collect plasmas with those high titers I think with selection that probably such a product could be made. DR. GRIFFIN: It is my understand that is what is done for RSV is immune globulin or was done. I mean that was not an immunization program. They were selecting high titer dose. PARTICIPANT: In the discussion this morning someone said that you really can't get much of a boost in measles titers by giving second vaccination. So, is it even possible to develop a hyperimmune population for measles if we are basing it strictly on vaccination and not natural disease? DR. GRIFFIN: Certainly people get very varied levels of antibody in response to vaccine. I mean there is fairly wide biologic variability in those titers. So, there will certainly be a subgroup of people who will have high titers independent of being re-immunized. DR. BLAESE: I want to just comment that I think it is important to consider this population. I have a lot of immune deficient patients that have been in college and going off to third world countries and right now they are probably covered but in 5 or 6 years with that population will there still be sufficient antibody titers to cover those kids, and I think we have to be concerned about the long range. PARTICIPANT: There is the issue of cellular immunity and that probably persists and would give you an anamnestic response in most of those adults anyway who have either seen a natural infection or a vaccine. We don't know the answer to that. DR. SCOTT: I think the people with combined T and B cell deficiencies are the ones you would most worry about which is a substantial proportion of the primary immune deficient population. DR. GOLDING: Am I misrembering(?) the slide that had all the levels and trough levels and they all seemed to be twice what you really needed? I know there is a caveat that people who are immunodeficient may need more of the product. So, isn't there a big difference between what the levels are for lot release and the levels that you would require to get protection and doesn't that give us some room here for maneuvering without doing anything drastic? DR SEWARD: I think antibody data tell you that we have got protection in the levels that we achieve and so it is more an issue of that test, isn't it? DR. SCOTT: The required specification. DR. SEWARD: The required specification I mean. DR. SCOTT: Right. I would also like to say with respect to the decline in titers in the donors that the data we saw was all ELISA data and I think that it would be extremely useful to see the neutralization data for that because the slope of the decline could be different and the absolute amount of antibody that is useful in those donors might be different. DR. SIMON: What I would think and we discussed a little bit of this at the break is that the ELISA of course included the neutralizing. The issue would be I think the sensitivity of the assay at the lower level. So, I think as Dr. Griffin said, you would get, we would see detectable levels that you could measure but they would presumably still be at that low level. DR. SEWARD: Judith mentioned the paper this morning that she is a co-author on with Charles Lebaron from CDC and 10 years after second dose I think we have reached a steady state now with as you saw in the presentation that with vaccinees that is going to stay the same now. So, people at 20 will have had two doses of vaccine and you know looking at neutralizing antibodies 10 years out they are all protected . Now, 30 years out, I mean 20 years out or 30 years out they may not be. So, we have got to keep looking but right now there is nobody who is below 120 10 years out from their second dose according to the longitudinally collected data that Judith mentioned this morning that was published in April 2007, but projections are if decline continues and this was just modeled data based on these data and so we acknowledge that the modeling may not be correct but we do predict that there will be people susceptible in another 10 or 20 years. PARTICIPANT: One point from the agency might be bottom line seeing as the titers are going to fall unless we get new populations and can boost them or whatever and increase the titer in the product it seems that the titers are going to fall. We will be forced to set a lower specification or else deny the product to the people who need it and that being the case what sort of rationale could we use to support a reduction in the titers? Under what circumstances would you guys bless our rationale for lowering the specifications? What kind of data do we need? DR. SCOTT: Actually you are leading right into the second question of how much anti-measles antibody is needed to attenuate or prevent measles in primary immune deficiency and the fact is we don't know that but what I would like to hear is some educated guesses and some thoughts on how we could find out more about that. PARTICIPANT: I think that the statement that you heard from a number of individuals about the relative protection in the general population today is that the antibody levels that are present in the general population appear to be protective at least of those individuals. Now, whether or not that translates immediately to the PID community is a little bit of a stretch but it seems as if we are going to have to stretch that way. My biggest personal concern is that I think that a preparation that is drawn from US donors that has an anti-measles titer consistent with titers that are present in US donors is a good preparation but a preparation that came from a plasma pool in an area that still has endemic measles that only had the titer that is present in the US would suggest a preparation in which large portions of the functional antibodies might have been destroyed and that would not be a preparation that I would like to see used in PID patients. You know, I think that what you have heard from the Individuals who are the current users and practitioners is that they are not providing PID subjects with measles prophylaxis. They are providing subjects with general prophylaxis and in fact the measles titers are being used as a surrogate for the activity of all of the different specificities that are present in the preparation. DR. SEWARD: If you go for a safety factor of doubling for example, I mean we don't know, but if they are 120, say, that immune deficient people need 240, then almost all the preparations cover that except for D is close to not being and you know maybe manufacturers based on this work might change the antibody, relative antibodies in the product to increase. So, you know that is just a guess but allowing a little bit of margin of safety above 120 might be -- PARTICIPANT: Can't one come to an estimate just by looking at the transplacental antibody cutoffs for protection over time and you would get some educated guess from that? That is all IgG. DR. SCOTT: That might be a little tough if you are thinking about the primary immune deficient population but it probably is useful data. I think that has been considered for the normal population, that kind of data. I think one of the questions that comes up is there any way to come to aa more educated guess using animals models to have an idea, let us say B and T cell deficient animal models of what might be protective titers, and we talked a little bit about this during the break but I would like to hear some thoughts about what might be a useful type of experiment. DR. GOLDING: I think Dr. Griffin probably is the expert but I think you would want to think about what is the worst case scenario. So, if you took a monkey for example and you T and B cell depleted I think that would be the worst case that you could use in that model and then determine what is the dose, the minimum dose required to achieve sterilizing immunity. DR. GRIFFIN: Right, I think that monkeys would be sort of a most definitive way to look at this and you can certainly immunosuppress them in a variety of different ways so that they wouldn't have an either B or T cell response t the vaccine. One of the other model systems that we were talking about at the break is cotton(?) rats. Cotton rats can get infected with measles. They don't get measles. You know, they don't get a rash even if you shave them and look not just because they are furry but you know that they can get infected and it would be a situation in which you could give passive antibody of various varieties. You could immunosuppress them in different ways with an anti-T cell product or an anti-B cell product or vice versa. It would just be a smaller, cheaper model system in which you could get some preliminary data at least that will give you some ideas that would maybe bracket a more definitive study in a macaque model. DR. OCHS: I would just make one comment or argument and that is if we assume that the product reflects what at the end of the product reflects what comes in and if we assume that we have normal population to tell us when the antibody titer goes below protection we try to treat our patients to have them at the trough level as high as the general population. So, we would, if the general population isn't sensitive to measles then I think our patients are protected as well. If the product is not like product D where in some way the antibody to measles has been reduced and so we have sentinels out there. So, if the CDC recognizes that our population gets susceptible to measles then we have to worry about our patients but otherwise unless you know they need more or less no evidence that patients with actual gamma globulin anemia need more protection in terms of antibodies then healthy human beings then there should be no problem. We have the sentinels out there and if the population starts to get measles then we have to worry about those who get the product from the general population. DR. SIMON: I think that is a good lead in. I think from the point of view of those of us who produce the plasma the standard logically would be based on the antibody levels that we are now finding in the younger donors and as long as what we are hearing from the treaters like following up on what you said that that should meet the needs of the patients I think that would be the way to go because there is no particular way to enrich this population with higher antibody levels. Some of the suggestions of places we could recruit would just be small increments into the general donors .So, I think I would echo the urgency of this as we recruit younger donors and the need to get at least standards that reflect where our donor population is which is after all protected against measles. DR. SCOTT: I think we have more or less covered the third question, what is the potential clinical impact of diminishing anti-measles titers in immune globulin products but if people have additional comments it would be useful to hear them. I think one of the interesting comments is that these very healthy primary immune deficient patients now are going overseas and potentially being exposed to measles but that we don't seem to have a problem in this country with those patients with respect to measles and finally what are the proposed approaches to address decline of anti-measles titers in immune globulins? I think first we do need to look again at the plasma donors to find out in terms of neutralization where they are because actually the lot release titers that are there for most products would suggest a lack of this decline and I have to add the caveat that we have not looked at the more recent products ourselves and that needs to be done. We also are very interested in understanding what the trough level titers are in the primary immune deficient patients and I think Dr. Ochs has mentioned the possibility of having some access t that from communities but it does occur to me and I want to ask our industry folks who are here that there very often are retained or leftover samples from clinical studies of new immune globulins and that that those trough levels could be assayed for measles antibody neutralization titers and those titers could be related back to the amount of immune globulin in the titer in that that they received during the clinical study and that might be a method to use too be able to have a better assessment of the real trough titers in these patients. I would like to hear from our industry colleagues about the possibility of doing studies like that. You already have the assays with which to do them. DR. ZENKER: At CSL we have started a program with that. We will continue and analyze also our samples that we obtained after intravenous treatment, but it takes time. You know that the neutralization assay takes more time and is more expensive than the ELISA. DR. GOLDING: Could I make a point about the assay? It seems to me what I learned today is that the assay is a lysate usually and the neutralizing component on the envelope of the virus is the hemagglutinin. So, I think one could improve that binding assay significantly if it was based on a hemagglutinin so a recombinant hemagglutinin being used as the binding protein on the ELISA plate because I would assume that the correlation between that and the neutralizing antibody would be much tighter than using a viral lysate. PARTICIPANT: There is one other difficulty we have anytime we want to explore measles titers through our process and even in our incoming plasma and that is the limited supply of reference 176. We really have only enough to support lot release not to do any exploration. So, I was wondering where are we as far as developing the next reference standard and what would it be derived from? DR. SCOTT: I would like to call on my colleague Dr. Yu to respond. DR. YU: We have been thinking about lot 177 for quite a while. Of course what you said is correct. Lot 176 the supply is very limited and recently there is a new WHO standard but that is actually anti-measles is in serum not in IgG format and we already have some reports that maybe Lot 176 compared with that may be a little bit less than what they determined when they used Lot 176 anyway. We have some candidate materials for manufacturers and actually Dr. Don Baker has provided us some candidate material. So, we need the characterizing and the preparation is not going to be like 16-1/2 percent. It probably will be 5 percent or 10 percent IGIV preparation but again we are waiting for this particular IGIV workshop, I mean this immune globulin workshop to make sure what kind of surrogate markers that we need to put in and what kind of ratio. Now, in the past for one last point, 176 when compared with Lot 175 we did put some surrogate markers which are anti-HIV and also anti-HBV, you known anti-HBS. So, this time it is going to be different probably. We don't know but again it is in the thinking and we have got to do it quickly but I have to carefully plan and so forth. So, in the meantime there is still some Lot 176 but very limited,. So, we would like to carefully distribute that particular product. I hope I have answered the question. DR. SCOTT: So, I think what we need to do from our viewpoint is determine from manufacturers who are willing to look at these trough levels by a neutralization assay an estimate of how much of Lot 176 they would actually need to do these studies while we are in the process of developing Lot 177 and we will have to parse out how doable that is going to be. PARTICIPANT: While you are doing this for measles wouldn't it be a good idea to do hepatitis A antibody and hepatitis B antibody, too, because these titers are also falling in the products and so that maybe the difference between what is in the product and what is a protective level are different magnitude than for measles but I am just wondering that maybe we can reinvestigate this all next year and we are worrying about hepatitis A titers. DR. YU: Thank you. We will do that probably because actually anti-hepatitis A and anti-HBS are very, it is by ELISA assay and that already through the vaccine study we know they are neutralizing. They can correlate. The EIA titer can correlate with neutralizing titers. It is easier to do and that is what we did with Lot 175 when compared with Lot 176 and John, you would like to comment about that I am sure. That is before my time. So, we need to do that. DR. GOLDING: I think that people use, maybe use different differently the IM as prophylaxis for HIV but for the HB my understanding, Dr. Yu is that we really have it in there because it is an extra protection because if there is any virus surrounding the product that would neutralize the virus and it is not really at concentrations that would be useful in treating with either HB hyperimmune. So, do we really think that IG preparations or IGIV could be useful in treatment of hepatitis B? PARTICIPANT: It is necessary to prevent patients with primary immunodeficiency against hepatitis B and you are not going to achieve that by vaccine. DR. GOLDING: So, what you are saying is that the lot release titer that we have now for those products is sufficient too protect PID patients? DR. YU: I would like to just add one more information about anti-HBS. I know some of the manufacturers if not all, they all make sure their plasma pool has certainly titer of anti-HBS before they make all these immune globulin products. So, that is just to be sure. So, that is why we haven't, maybe that is why we haven't seen any hepatitis episodes. DR. HOLLEY: Hi, I am Preston Holley from Nabi. I would like to pick up on something that was mentioned yesterday by Dr. Ochs and others and that is characterizing certain lots that maybe manufacturers voluntarily characterized their product from a central lab or something like that to look at various antibody levels in particular lots. This would be maybe another way of having that available, say, high-titered lots that have a lot of measles antibody for example that would be characterizing in case there was an outbreak and then maybe those lots could be selected for use in that type of an area or if someone was going to an endemic area and needed extra protection to be sure then they could use those lots. DR. SCOTT: As it is now since everybody does this as a lot release test it would be possible even to call manufacturers and ask them to suggest a high-titer lot and that is the kind of thing that has been done in the past for these sorts of special needs and it gets back to some of the things that we were discussing yesterday about finding products with a particularly high titer that happens to be know for certain circumstances. In this case it would be a lot easier because these tests are already being done for each lot and it would be not too difficult for a manufacturer to go and look at their most recent lot titers. PARTICIPANT: Dr. Scott, I wanted to go back to what you mentioned a minute ago about donors. You suggested that there needed to be further study of that observed decline in antibody titer using neutralization assays as opposed to the ELISA. Would you expand on that for me? DR. SCOTT: I think that the particularly precipitous looking decline really needs to be confirmed in the context of looking at neutralizing antibody which we know is the most important specificity to look at to ascertain the real degree of that decline in that donor population. PARTICIPANT: Yes, I think I understand the concept but I just wondered in terms of proposing an actual study to do that how large a study would you think? Neutralizing is a little more difficult to do mass screening with neutralizing antibody obviously than with ELISA. I guess that is something we could discuss off line. DR.SIMON: I would assume that we could do a smaller number than what was done before. So, hopefully we could do this but it would still probably take in the hundreds of donors randomly selected. DR. SCOTT: Can I call for any other comments? DR. GRIFFIN: This is just an information question. I assume that there is some sort of network or reporting mechanism or something so that if individuals who are receiving these products do become exposed or get any of the diseases that are supposed to be being prophylaxed against that there is some mechanism for identifying that as a problem. True? I mean if all of a sudden there were some cases of measles or hepatitis A or whatever disease occurring in populations that are getting prophylaxed and they may just be just, and they would be just individuals you know at various places in the country but some way, early warning system that there was a problem. DR. SEWARD: I think unofficially at CDC we have all confirmed measles cases reported to us. Undoubtedly some are missed but we don't miss any outbreaks I am sure and if we had a case reported in such a patient I think we would let FDA know just as a courtesy and because of the interest. We certainly haven't seen any. DR. BLAESE: And certainly the patient registries that we are setting up in Europe and the US I think could be but it is going to be a long time before we have enough coverage of the whole population that it would really be a sort of sentinel detector. DR. SCOTT: What is very useful is that you are also looking at whether they have been vaccinated and received live vaccines and you will know if they have been on immune globulin at the time and that might give us some additional information that would be useful and you will know what kind of immune deficiency they have. So, it may be that some of the data you collect in the aggregate can be put together in a way that can help us with some of these questions. PARTICIPANT: Just one other comment if I could just to thank you for all the attention that is going to trying to do this carefully and come up with the right measures. I think it is important to remind people that given the trend lines we are seeing from the plasma side that eventually it does impact on our ability ultimately to have that IVIG available. So, I just think that has to be kept in mind as well as a counterpoint to all the discussions around this and then the realistic time lines around studies, etc., b