FOOD AND DRUG ADMINISTRATION








This transcript has not been edited or corrected, but appears as received from the commercial transcribing service:  Accordingly the Food and Drug Administration makes no representation as to its accuracy.




                         Friday, July 23, 2004


                               8:00 a.m.




                        Gaithersburg Holiday Inn

                      2 Montgomery Village Avenue

                      Gaithersburg, Maryland 20877





         Kenrad E. Nelson, M.D., Chair

         Linda A. Smallwood, Ph.D., Executive Secretary

         Pearline K. Muckelvene, Scientific Advisors

            & Consultants Staff




         James R. Allen, M.D., M.P.H.

         Kenneth Davis, Jr., M.D.

         Donna M. DiMichele, M.D.

         Samuel H. Doppelt, M.D.

         Jonathan C. Goldsmith, M.D.

         Harvey G. Klein, M.D.

         Suman Laal, Ph.D.

         Katherine E. Knowles,

           Acting Consumer Representative

         D. Michael Strong,

           Non-Voting Industry Representative




         Liana Harvath, Ph.D.

         F. Blaine Hollinger, M.D.

         Katharine E. Knowles

         Matthew J. Kuehnert, M.D.

         Susan F. Leitman, M.D.

         Keith C. Quirolo, M.D.

         George B. Schreiber, Sc.D.

         Donna S. Whittaker, Ph.D.



                            C O N T E N T S



      Update on West Nile Virus, Hira Nakhasi, Ph.D.             6


            IV. Hepatitis B Virus Nucleic Acid Testing (NAT)

                         for Donors of Whole Blood:


                A. Introduction and Background,

                   Gerardo Kaplan, Ph.D., Laboratory

                   of Hepatitis and Related Emerging

                   Viruses, DETTD, OBRR, FDA                    28

                B. Serological Course of Hepatitis B,

                   F. Blaine Hollinger, M.D.,

                   Baylor College of Medicine                   32

                C. Preclinical and Clinical Data for

                   HBV MP NAT, Steven Herman, Ph.D.,

                   Roche Molecular Systems                      51


                   Allan Frank M.D., M.S.,

                   Roche Molecular Systems                      65


      Open Public Hearing:

                Michael Busch, Blood Centers

                  of the Pacific                               103

                William Andrew Heaton, Chiron                  121

                Sherrol McDonough, Gen-Probe                   129

                Richard Smith, NGI                             136

                Harvey Alter, AABB                             144


            IV. Hepatitis B Virus Nucleic Acid Testing (NAT)

                         for Donors of Whole Blood:


                E. Committee Discussion and

                   Recommendations                             154


           V. Current Trends in Plasma Product Manufacturing


                    A. Introduction and Background,

                       Mark Weinstein, Ph.D., Associate

                       Deputy Director, OBRR, FDA              223

                    B. Presentation, Jan M. Bult, CEO,

                       Plasma Protein Therapeutics

                       Association                             225


      Open Public Hearing:

                Patrick Schmidt, CEO, FFF Enterprises          258




  1                      P R O C E E D I N G S


  2             DR. SMALLWOOD:  May I ask all advisory


  3   committee members to, please, take your seats?


  4   Welcome to the second day of the Blood Products


  5   Advisory Committee meeting.  Yesterday I read the


  6   conflict of interest statement that applies to this


  7   meeting, however, we have a new process now and we


  8   will read a conflict of interest statement for each


  9   day.


 10             So, if you will indulge me, I will read


 11   that at this point.  This brief announcement is in


 12   addition to the conflict of interest statement read


 13   at the beginning of the meeting yesterday, and is


 14   part of the public record for the Blood Products


 15   Advisory Committee meeting on July 23, 2004.  This


 16   announcement addresses conflicts of interest for


 17   topic V.


 18             Drs. Liana Harvath, Blaine Hollinger,


 19   Matthew Kuehnert, Susan Leitman, Keith Quirolo,


 20   George Schreiber, Donna Whittaker and Ms. Katherine


 21   Knowles have been appointed as temporary voting


 22   members for this meeting




  1   Dr. Michael Strong is participating in this meeting


  2   as the non-voting industry representative, acting


  3   on behalf of regulated industry.  The Food and Drug


  4   Administration has prepared general matters waivers


  5   for the special government employees participating


  6   in this meeting who required a waiver under Title


  7   XVIII, United States Code 208.


  8             In addition, there are regulated industry


  9   and other outside organization speakers making


 10   presentations.  These speakers have financial


 11   interests associated with their employers and with


 12   other regulated firms.  They were not screened for


 13   these conflicts of interest.  I would just like to


 14   remind everyone participating to, please, make


 15   known, if you have not already done so, any


 16   affiliation you may have and your status with that


 17   affiliation prior to speaking.


 18             Our committee chairman, Dr. Kenrad Nelson


 19   has joined us this morning, and we also have Dr.


 20   Blaine Hollinger who will also be part of the


 21   committee this morning.


 22             I just wanted to announce to those who




  1   were not here yesterday that the next date, which


  2   is tentative however pretty much firm, for the next


  3   Blood Products Advisory Committee meeting will be


  4   October 21st and 22nd, 2004.


  5             At this time I will turn over the


  6   proceedings of the meeting to the chairman, Dr.


  7   Kenrad Nelson.


  8                    Update on West Nile Virus


  9             DR. NELSON:  Thank you, Dr. Smallwood.  I


 10   will try to keep awake after the 24-hour airplane


 11   ride.  I came in last night but I feel really


 12   pretty good and I am very interested in the topic


 13   today so I think that will help.


 14             The first topic is an update on West Nile


 15   virus by Hira Nakhasi.


 16             DR. NAKHASI:  Good morning.  I just want


 17   to give you an update, as Dr. Kenrad Nelson


 18   mentioned, on the West Nile epidemic and donor


 19   testing which is happening now, in 2004.  First I


 20   will try to wrap up last year's things and then


 21   come up to 2004.


 22             Next slide, please.  The topics which I




  1   will update you on are, as I said, last year's


  2   epidemiology and the investigational West Nile


  3   testing outcome of that, and some of the


  4   transfusion-transmitted cases, and then the trigger


  5   for the ID-NAT testing.  Then I will update you on


  6   the West Nile donor and product management


  7   recommendations with the recent revelations we have


  8   got.  Then I will update you on the 2004 epidemic


  9   and investigational West Nile testing, and also our


 10   efforts in-house on the panel development and other


 11   scientific issues--you know, the variation among


 12   the strains of viruses infectivity of these


 13   studies.


 14             Next slide, please.  If you summarize in


 15   one slide the last year's epidemic, it really


 16   basically sums up that we had approximately 1000


 17   [sic] cases or, to be precise, 9862 cases, human


 18   cases, and 264 deaths.  And, the proportion of the


 19   West Nile meningitis/encephalitis was 29 percent,


 20   whereas, the fever was 69 percent in the human


 21   cases.


 22             Forty-six states, including Washington,




  1   D.C., were endemic, and donor testing started, as


  2   all of you know, in July of 2003, using two


  3   investigational NAT testing.  In some cases, a


  4   small proportion started in the middle of June.


  5   Despite this testing, I think these two


  6   investigational NAT testing--these are minipool and


  7   the two tests were the Gen-Probe test and the Roche


  8   test, and Roche tested, as you know, in pools of 6


  9   and the Gen-Probe test involves a pool of 16.


 10             Despite testing, there were some


 11   transfusion-transmitted cases and CDC had


 12   investigated a total of 23 cases.  They were


 13   confirmed by NAT and IgM reactivity and also by


 14   follow-up of both the donor and the recipient.  Out


 15   of the 23, 6 were confirmed cases.  Only 4/6, you


 16   may recall, had very low viremia, around 0.1


 17   pfu/ml.  Eleven cases did not confirm; 3 were


 18   inconclusive because of the follow-up situation;


 19   and 3 were open investigations.


 20             Next slide, please.  As I said, since it


 21   started on July 1 of last year, screening using


 22   minipool NAT and IND, all geographic regions of the




  1   U.S. were screening at that time.  With that, what


  2   happened 1000 units of West Nile infected blood


  3   donors were interdicted after screening


  4   approximately 8 million donations.  So, I think it


  5   was a very, very vast improvement over the year


  6   before when there was no testing.  The last


  7   positive donation was reported in the middle of


  8   December in 2003.


  9             Despite this testing, as you see, the


 10   majority of cases were interdicted, more than 75


 11   percent, but there was a small percentage which


 12   went through because, as you know, this was done in


 13   minipool NAT.


 14             Next slide, please.  This slide is Mike


 15   Busch's slide where he showed why we were missing


 16   some of these cases, and we knew that minipool NAT


 17   sensitivity was such.  The areas, you know, where


 18   the wrap-up takes place when--you know, he calls it


 19   stage I, II, III, IV and V, and in stage I and II


 20   they are ID-NAT positive but minipool NAT negative,


 21   IgM negative.  So, it could be plus/minus.  So,


 22   during that stage they become IgM positive but they




  1   become minipool negative and they are still ID-NAT


  2   positive.  So, this region and this region were the


  3   ones where they went through.  But, you know, these


  4   were IgM negative and these were IgM positive so


  5   the question is what is the infection of these


  6   types of samples.


  7             Next slide, please.  So there was a


  8   potential for transmission of West Nile through


  9   minipool NAT negative blood of low viremia in some


 10   patients.  Therefore, what happened at that time is


 11   that limited prospective ID-NAT testing started in


 12   high incidence areas.  If you remember last year,


 13   Colorado, Kansas and certain other areas, and


 14   Nebraska were hot spots and ID-NAT was triggered at


 15   that time, and the trigger was based on if the


 16   preceding the rate of 1/200 minipool NAT positive


 17   rate of 1/250, then they would start testing with


 18   ID-NAT testing.  Also, what happened at that time


 19   is that there was voluntary withdrawal of the


 20   frozen transfusables in the high incidence areas


 21   before the ID-NAT was initiated by some blood


 22   establishments.




  1             Next slide, please.  There was also


  2   another initiative started at that time.  The


  3   initiative was to go back to do the retrospective


  4   study on the minipool NAT negative samples and test


  5   them by ID-NAT to find out how many we missed.  It


  6   would also let us know what was the low level of


  7   viremic high incidence samples in high incidence


  8   areas where minipool NAT did not pick them up.


  9             The other purpose of the study was also to


 10   identify samples which are like minipool NAT low


 11   titer, minipool NAT negative but ID-NAT positive


 12   for infectivity studies.  I told you that we do not


 13   know whether those samples are still infectious at


 14   low levels, and what is the level of infectivity.


 15   So, these samples would be tested in various animal


 16   models including non-human primates.  Also, the


 17   purpose of these samples is to really find out the


 18   relative clinical sensitivity of various West Nile


 19   investigational testing.  I will report in a minute


 20   what is happening with the infectivity state.


 21             Next slide, please.  Based on the


 22   observation that we had minipool testing and we




  1   missed some of the samples because the viremia was


  2   low, and also in the ID-NAT testing in the high


  3   incidence areas--based on those studies and based


  4   on the logistics issues, the question was what


  5   should be the trigger for ID-NAT, and also logistic


  6   issues such the availability of adequate resources,


  7   recruitment, reagents and trained technologists.


  8             So, the discussion about the trigger for


  9   ID-NAT was held in collaboration with the AABB task


 10   force.  By the way, we are very indebted to the


 11   AABB task force for the biweekly meetings almost


 12   throughout the year, and weekly meetings with the


 13   task force during the epidemic to update us and


 14   jointly discuss the strategies for how to go


 15   forward with the testing performance, as well as


 16   the epidemic.


 17             So, based on that discussion, which was


 18   held in February, the recommendations were the


 19   following for the ID-NAT trigger:  It was discussed


 20   that we should monitor reactive rates by zones


 21   daily, enrolled 7 days when the epidemic was


 22   starting, which was usually, you know, around the




  1   beginning of July and early June even and this year


  2   even May some cases were found.  The trigger was


  3   that if you have 2-4 cases in any geographic


  4   area--that is the blood collection, and the


  5   frequency of 1/1000.  This was based on the fact


  6   that every 1/4 would be missed by minipool NAT and


  7   require ID-NAT.  This was the study done by ARC and


  8   BSL and they found out that that would be the


  9   trigger.  And, you go back to minipool NAT only


 10   when you see ID-NAT reactivity and you don't find


 11   zero cases in a consecutive 3-4 day period or the


 12   rate is less than 1/1000.  So, that was the trigger


 13   because, you know, we wanted to be prepared this


 14   year because last year it was on an ad hoc basis to


 15   start ID-NAT testing in those hot areas.  So, we


 16   wanted to be prepared this year if these areas


 17   become hot so that we get the logistics present


 18   there so we can start without interruption of the


 19   ID-NAT testing.


 20             Next slide, please.  Now we come to 2004,


 21   where are we now?  As of July 20, which is a couple


 22   of days back--as you see, every week the numbers




  1   keep changing.  Last week there were 108.  This


  2   week it is 182 human cases out of which there were


  3   4 deaths.  There are 2 from Arizona, 1 from Texas


  4   and 1 from Iowa.  Out of total infections, 74


  5   percent of cases are neuroinvasive West Nile


  6   illness and 26 percent cases are West Nile fever.


  7   At the moment there are 35 states endemic for West


  8   Nile.  This slide has been kindly provided by Jen


  9   Brown, from CDC, and other slides which I will


 10   mention later.


 11             The total number of presumptive West Nile


 12   viremic donors reported to the CDC ArboNet--that is


 13   why I highlighted this, is 23.  There are more


 14   cases than that but, as you know, there is a delay


 15   in reporting to the ArboNet from the health


 16   departments.  So, using minipool NAT as well as


 17   ID-NAT in select areas, starting on May 4.  Out of


 18   these 23 presumptive West Nile viremic donors, 21


 19   are from Arizona.  The majority are from the


 20   Maricopa county near Phoenix, in Arizona; 1 from


 21   New Mexico and 1 from Iowa.  But this is the tip of


 22   the iceberg.




  1             Next slide, please.  This slide, again, is


  2   provided by Jen.  You can see the distribution of


  3   the West Nile, both the animal, avian and mosquito


  4   infection, which is in this color, and the blue


  5   color shows you the human cases.  You can see it is


  6   very high in Arizona and California.  I am telling


  7   Mike Strong that it is creeping up in Washington


  8   soon.  So, he has been telling me we don't see


  9   anything and I said, well, wait and watch!  As you


 10   remember, in 1999, how this started and how it is


 11   spreading and, you know, it just keeps on going.  I


 12   hope it will end up in the ocean sometime.


 13             Next slide, please.  This is just to give


 14   you how early the human cases can be detected.  As


 15   you see from the slide, the earliest one was in


 16   April.  So, you know, there is an expansion of this


 17   epidemic, it looks like.  We were told in the


 18   textbooks it is mostly in August and September or


 19   late July but you can see it as early as April now,


 20   and last year we saw it as late as December, in the


 21   middle of December.  So, you know, it is almost a


 22   year-round activity now.




  1             Next slide, please.  Thanks to all the


  2   blood establishments and testing establishments, I


  3   got these data from several folks and I will


  4   acknowledge them as I speak.  The total number,


  5   according to my calculations but this may not be


  6   right, is 61 presumptive viremic donors reported,


  7   starting in May, 2004.  As I said, some of them are


  8   reported to ArboNet and some of them are not.  So,


  9   it is not in addition to that; it is inclusive of


 10   the ArboNet reports.  ARC has told me--Sue Stramer


 11   gave the data from June 16 to July 20, 7 hard


 12   cases.  Again, this is also in the Arizona area.


 13   But she says no region has their ID-NAT trigger.


 14             Mike Strong gave me this data from Roche.


 15   There are 2 positive confirmed by ID-NAT--around


 16   300,000 donations screened.


 17             BSL, Sally Caglioti and Mike Busch told me


 18   that there are 23 confirmed, out of which 16 came


 19   from minipool NAT and 7 came from ID-NAT, confirmed


 20   positives.  There are 14 pending and he was saying


 21   that some of them are ID-NAT and would have been


 22   missed by minipool NAT.  Also, some of them are low




  1   viremic and also there are some which are IgM


  2   positive.  The denominator is around 400,000.


  3             Gen-Probe, Leanne Kiviharju, gave the


  4   data.  These are non-ARC data but I am not sure--I


  5   sent an email to Leanne--whether this is also


  6   non-BSL but I am not sure; maybe we can find out


  7   from here, but 21 confirmed positive and 7 are


  8   pending.  I am glad that you guys sent me several


  9   slides.  I was basically trying to summarize what


 10   the presumptive donors are and, you know, I really


 11   appreciate your sending extra slides.


 12             The Department of Defense, Ron Hagey sent


 13   me the data which has 8 confirmed out of 62,774


 14   since January of 2004.


 15             So, you know, this is the majority of the


 16   screening going on at this time and there may be a


 17   few cases which have not been reported yet, but


 18   this is where we stand as of today.


 19             Next slide, please.  I just wanted to sort


 20   of briefly remind you that FDA is still continuing


 21   to work closely with the test kit manufacturers and


 22   we would like to facilitate implementation of these




  1   tests and expediter test licensure.  I just want to


  2   remind you that we issued two guidances in October,


  3   2002 and May, 2003.  There are 3 INDs for West Nile


  4   minipool-NAT.  One is from Roche, one from


  5   Gen-Probe and one from ARC.  This is public


  6   information.  FDA is continuing to work with the


  7   AABB task force.  I think that has been a


  8   wonderful, wonderful collaboration with the AABB


  9   task force and the people on the task force are


 10   really helpful in doing this project together, and


 11   with the CDC, NIH help, and to monitor the epidemic


 12   and monitor the testing.


 13             Next slide, please.  Both ARC and BSL did


 14   a study, which is unpublished observation.  We had


 15   a small discussion at the task force on what they


 16   found out in some of the viremic donors when they


 17   followed up.  They wanted to find out what is the


 18   rate of the disappearance of RNA when they convert


 19   IgM and IgG.  As you remember, in the last years


 20   before the testing started the literature was that


 21   it can go as long as 28 days of viremia.  But from


 22   their studies, and I don't want to go into detail




  1   here because these are unpublished and, you know, I


  2   don't want to divulge information--the gist of that


  3   was that what they found out in both cases is that


  4   the viremia may last up to 49 days in one case and


  5   39 days in the ARC study, and in the BSL 49 days,


  6   and West Nile RNA may go coexist with IgM.


  7   Therefore, this sort of started us thinking.  In


  8   the guidance document we put 28-day donor deferral


  9   and so we may have to rethink the deferral for


 10   that.


 11             Next slide, please.  We have not discussed


 12   it with the AABB task force but we will be


 13   discussing with the task force that, you know, the


 14   integration of West Nile testing information.  We


 15   are thinking about maybe 56-day deferral for West


 16   Nile diagnosis of symptoms, including headache and


 17   fever, or 14 days after symptom resolution if it is


 18   more than 56 days.  Potential reinstatement of


 19   donor deferral for West Nile symptoms only


 20   following 30 days without symptoms, and negative by


 21   West Nile IgM or ID-NAT.  Again, this is current


 22   thinking.  We have nothing in the works yet but we




  1   have internal discussions, and we will discuss it


  2   at our next regular AABB task force before we come


  3   up with a recommendation.  Dr. Alan Williams is


  4   spearheading this initiative.


  5             Next slide, please.  With regard to our


  6   activities in-house, as I mentioned last year also,


  7   we are still working on the panel development.  The


  8   purpose is to monitor sensitivity of assays to


  9   detect viral nucleic acid antibodies, and also


 10   trying to isolate and characterize West Nile


 11   strains from human samples during 2003 and 2002


 12   epidemics. The purpose of this study, which is done


 13   by Dr. Maria Rios in our group--and all these


 14   studies actually really are done by Dr. Maria Rios'


 15   group--is the genetic variation of viral strains;


 16   detection by currently available West Nile assays.


 17   The purpose is to really see if there is any


 18   genetic variation and also infectivity studies


 19   using animal models.  Currently, the samples have


 20   been identified which could be used for infectivity


 21   studies.  However, there are logistic issues about


 22   the animals, baboons, which are being worked out




  1   with the Southeast Medical Center.  I guess the


  2   task force is working on that.  Hopefully, we will


  3   get some information by fall and we will be set to


  4   do those studies.


  5             Next slide, please.  Briefly, they have


  6   two isolates, NY99 in 2002, which have been


  7   characterized by genetic sequencing which I can


  8   show you in a minute.  The viral infectivity is


  9   determined by in vitro studies using cell lines and


 10   primary human blood cell cultures.  Final panel


 11   specifications are being established through the


 12   collaborative studies, and the range of


 13   concentration ranges between 1000-5 copies/ml.


 14             Next slide, please.  Just a piece of


 15   information here that Maria was kind enough to


 16   provide to me.  You know, she did the comparison of


 17   the human 2002 strain and the NY99 flamingo isolate


 18   and then passed through the Vero cells.  She found


 19   there were 20 nucleotide mutations and one


 20   insertion.  The mutations are distributed all


 21   across the region which result in 5 amino acid


 22   substitutions.  She is characterizing more isolates




  1   and she already has 6 from 2002, 11 from 2003 and 6


  2   from 2004.  So, the purpose is to really compare


  3   and to see what the differences are and how those


  4   differences impact on our tests.


  5             Next slide please.  The outcome of the


  6   panel testing--six laboratories participated in


  7   that.  She tells me there were no false-positive


  8   results reported.  More variability in detection


  9   was found towards the lower end of the viral


 10   concentration, i.e., 80 percent of the time


 11   detected 100 copies/ml member but all laboratories


 12   detected 100 percent of the time the panel members


 13   of 500-1000 copies/ml.  Further testing is going to


 14   define the consensus copy number.


 15             Next slide, please.  This is the important


 16   slide.  I would like to thank all the people who


 17   really helped to make this talk possible.  Jennifer


 18   Brown, whom I have always been bugging to provide


 19   the slides.  Thank you, Jennifer.  Dr. Sue Stramer,


 20   Dr. Mike Busch and Sally Caglioti, Dr. Mike Strong,


 21   Leanne Kiviharju, Roland, Maria and all these


 22   people--whoever I send an email they are kind




  1   enough to respond quickly.  Also my colleagues at


  2   the FDA, Maria Rios, Alan Williams, Dr. Epstein,


  3   Martin Ruta, Indira Hewlett--always helping in this


  4   whole project and, last but not the least, the AABB


  5   task force.  I am really, really grateful to them


  6   for providing all the information and helpful


  7   discussion.  Thank you very much.


  8             DR. NELSON:  Thank you.  Any questions or


  9   comments?  Yes?


 10             DR. GOLDSMITH:  Do you have additional


 11   data on the level of viremia in these samples that


 12   you have been studying?  What is the maximum level


 13   of viremia?


 14             DR. NAKHASI:  Which samples are you


 15   talking about?


 16             DR. GOLDSMITH:  The ones that you


 17   recovered from the viremic donors.


 18             DR. NAKHASI:  From the viremic donors, I


 19   don't know.  Maria, do you know what the levels


 20   are?


 21             DR. RIOS:  Between 10                                          

                                       5 and 106 is the high


 22   level of viremia that we have found.  Are you




  1   asking for the range of viremia or the high level


  2   of viremia?


  3             DR. GOLDSMITH:  I was just curious about


  4   the high but it is fine to give the range.


  5             DR. RIOS:  It varies.  It varies.  The


  6   assays, in general, that use lower volumes do not


  7   detect them.  Assays that have higher volume and


  8   high throughput detect, but do not give accurate


  9   quantitation, to 10                                                      

       6 copies/ml.


 10             DR. NELSON:  One of your slides had 23


 11   positives with 16 by minipool and 7 by ID.  Were


 12   those 7 not detectable by minipool or was it just


 13   that ID screening was triggered and they weren't


 14   tested by minipool?


 15             DR. NAKHASI:  I think they came for the


 16   ID-NAT testing.  Is that true?  Yes.  You know, in


 17   BSL they had already started ID-NAT testing in


 18   Maricopa County.  The trigger had started earlier.


 19             DR. NELSON:  So, they were negative by


 20   minipool?


 21             DR. STRONG:  No, the trigger was activated


 22   and they started doing ID screening so they haven't




  1   gone back yet, I think, to see if those would have


  2   been picked up by minipool.


  3             DR. BUSCH:  Actually, 7/12 that were


  4   picked up in the region that had been converted to


  5   ID-NAT, 7 of them had been fully worked up and 5 of


  6   those 7 are negative at 1:16 dilutions so they


  7   would have been missed by minipool.  Of those 5, 1


  8   of them is antibody negative and 4 have IgM and


  9   IgG.


 10             DR. NELSON:  Has anybody looked at the


 11   characteristics of the donors that have low levels?


 12   Are there host factors that might influence whether


 13   somebody has high level or low level?  I know one


 14   feature may be antibody but in those that are


 15   antibody negative, I wonder if there are any donor


 16   characteristics that influence the level of


 17   viremia.


 18             DR. BUSCH:  Sue has looked at that I think


 19   more formally and there wasn't any correlation.


 20   These are representative donors of the donor pool


 21   in terms of the viremics, non-viremics and low


 22   viremics.  I think it is just by chance.  This




  1   phase of early viremia is completely asymptomatic.


  2             DR. RIOS:  It may have some inherited


  3   characteristics that limit the viral replication.


  4   The reason why we think that is because we have


  5   performed some in vitro studies with human primary


  6   macrophages and there is a great variability not


  7   only in the day of the viral peak, but some


  8   individuals can have a very steady and low titer


  9   that doesn't progress to peak.  So, that indicates


 10   that some inheritance variability may interfere


 11   with replication.


 12             DR. NELSON:  That is interesting.  Other


 13   questions?


 14             DR. LAAL:  Unless I misunderstood, I


 15   noticed that in 2003 we had a majority of your


 16   isolates from people who had fever, and about


 17   one-quarter were from neuroinvasive cases.  In 2004


 18   it is reversed.


 19             DR. NAKHASI:  Yes, that is an important


 20   point.  I discussed it with the CDC folks and they


 21   said, you know, don't pay attention to that because


 22   the fever cases were--you know, this year they are




  1   paying more attention so some of the fever cases


  2   were not real fever cases.  You are right, you saw


  3   the switch.


  4             DR. LAAL:  But then in the isolates that


  5   you are picking up now for the genetic studies, are


  6   you carefully making sure that you look at both


  7   types?


  8             DR. NAKHASI:  Maybe Maria can say; I don't


  9   know.


 10             DR. RIOS:  The isolates that have been


 11   studied so far don't come from patients.  Actually,


 12   that is the effort we are going to move towards


 13   now.  They are identified through the blood


 14   screening.  So, in order to evaluate if there is


 15   any isolate that may not be picked up by the blood


 16   screening we need to acquire samples from cases


 17   that are non-blood donors to investigate this


 18   possibility.


 19             DR. NELSON:  Yes, Mike?


 20             DR. STRONG:  Just a quick comment on the


 21   donors.  In the studies that were done last year,


 22   many of the donors that were interviewed, in fact,




  1   were symptomatic either shortly before or shortly


  2   after their donations but the screening questions


  3   just didn't pick them up.


  4         IV. Hepatitis B Virus Nucleic Acid Testing (NAT)


  5                    for Donors of Whole Blood


  6             DR. NELSON:  Thanks.  The next topic is


  7   hepatitis B virus nucleic acid testing for donors


  8   of whole blood.  Dr. Gerardo Kaplan will introduce


  9   this and give us background.


 10                  A. Introduction and Background


 11             DR. KAPLAN:  Good morning.


 12             [Slide]


 13             I am Gerardo Kaplan, Chief of the Lab of


 14   Hepatitis and Related Virus Emerging Agents.  I am


 15   with the Office of Blood, and I will introduce for


 16   you the hepatitis B virus n nucleic acid testing


 17   for donors of whole blood.


 18             [Slide]


 19             The general agenda for this meeting is


 20   that after the introduction and background, Dr.


 21   Blaine Hollinger will give us an update on the


 22   serology of hepatitis G.  This will be followed by




  1   two presentations from the Roche Molecular Systems


  2   and their preclinical and clinical data in support


  3   of their application.  Finally, I will come back to


  4   give you the FDA perspective on hepatitis B MP-NAT


  5   and present the questions for the committee.  I


  6   understand that there will be a break and then a


  7   public hearing.


  8             [Slide]


  9             So, the issue is that the FDA is seeking


 10   the opinion of the committee on the performance of


 11   the Roche COBAS AmpliScreen HBV test in minipools


 12   of 24 samples to screen blood for transfusion by


 13   nucleic acid testing, and its proposed intended use


 14   as an alternative to hepatitis B surface antigen


 15   testing in conjunction with testing for antibodies


 16   to hepatitis B core antigen.


 17             [Slide]


 18             In support of their claims, Roche


 19   Molecular Systems performed a clinical trial.  The


 20   study objectives of the clinical trial were to


 21   determine whether the COBAS AmpliScreen test, which


 22   is a minipool of 24 samples of plasma from




  1   volunteer blood donors, can detect hepatitis by DNA


  2   in hepatitis B surface antigen-anti-core negative


  3   window period cases.  This is the primary objective


  4   of the study.  In hepatitis B surface


  5   antigen-positive donors, who are acutely infected


  6   or chronic carries, and in persons previously


  7   exposed to hepatitis B as the secondary objective.


  8             [Slide]


  9             During the clinical trial, Roche Molecular


 10   Systems identified two window period cases in about


 11   600,000 volunteer whole because donations screened


 12   by hepatitis B NAT using their minipools of 24


 13   samples.


 14             RMS, Roche Molecular Systems, claims that


 15   the use of the COBAS AmpliScreen HBV test in


 16   conjunction with the anti-core test would reduced


 17   the residual risk of transfusion-transmitted


 18   hepatitis B, and also that this test, the COBAS


 19   AmpliScreen, could be used as an alternative to the


 20   commonly used hepatitis B surface antigen donor


 21   screening test.  I would like to point out that


 22   blood in the U.S. is currently being tested by




  1   surface antigen and core antibodies.


  2             [Slide]


  3             So, we would like to request comment of


  4   the committee on three questions.  Basically, the


  5   firs would be do the sensitivity and specificity of


  6   the Roche COBAS AmpliScreen hepatitis B test in


  7   minipools of 24 samples support licensing of the


  8   assay as a donor screen?


  9             [Slide]


 10             If so, assuming continued use of screening


 11   tests for anti-hepatitis B-core, do the data


 12   support use of the Roche COBAS AmpliScreen


 13   hepatitis B test in minipools of 24 samples to


 14   screen blood for transfusion as an equivalent


 15   alternative to the surface test?  If not, which


 16   studies would be required to validated such a new


 17   test?


 18             The third question is do the data support


 19   use of the Roche COBAS AmpliScreen hepatitis B test


 20   on minipools of 24 samples to screen blood for


 21   transfusion as an added test in conjunction with


 22   licensed donor screening tests for hepatitis B




  1   surface and anti-core?


  2             Now I would like to introduce Dr. Blaine


  3   Hollinger.  He will give us an update on hepatitis


  4   B serology.


  5               B. Serological Course of Hepatitis B


  6             DR. HOLLINGER:  Thank you, Gerardo.  It is


  7   always a pleasure to come to these meetings and I


  8   always tend to learn more than I think anyone here


  9   because there is always so much information.


 10             [Slide]


 11             The talk today is about serology, and


 12   serology can be confusing.  It is confusing to our


 13   GI residents and fellows in trying to determine


 14   what happens after hepatitis B infection.  The


 15   clinical and serologic changes that occur following


 16   infection represent a complex interaction between


 17   the host, the virus and specific antigens.  If you


 18   have an understanding of these items, I think it


 19   becomes very easy to understand what happens.


 20             There are certain changes that occur after


 21   infection with all viruses that seem to be very


 22   similar.  Viruses are either enveloped or




  1   non-enveloped.  Hepatitis B virus happens outcome


  2   have a lipoprotein envelope that contains hepatitis


  3   B surface antigen as the envelope protein.  There


  4   are also some other particles in this serum which


  5   are seen in excess of the infectious virion, and


  6   these are the small particles here, and these


  7   tubular forms which actually come off, in many


  8   cases, from the hepatitis B virion.


  9             The inside, or the nucleocapsid of the


 10   virion is comprised of the hepatitis B core


 11   antigen, and this encloses a relaxed, circular,


 12   partially double-stranded DNA molecule.  Another


 13   antigen which is seen also is the hepatitis B


 14   antigen, but this is not part of the virion in any


 15   way.  It is a secretory protein, about 16-17 kd in


 16   size, that is secreted during active infection, and


 17   is representative usually of high concentrations of


 18   virus in the bloodstream.  We don't really know


 19   what is the reason for this particular antigen.


 20             [Slide]


 21             This is the HBe genome.  It has four open


 22   reading frames.  One of them is responsible for the




  1   hepatitis B-surface antigen and is comprised of a


  2   small, a medium and a large protein.  A second one


  3   is the core gene which expresses the core-antigen,


  4   the hepatitis B core-antigen and also the e-antigen


  5   as well.  There is a DNA polymerase that is


  6   important for the replication of this virus, and


  7   there is an x-gene here which is very important for


  8   initiation and probably maintenance of the


  9   infection.  It is probably also weakly oncogenic as


 10   well.  Now, once one understands these things, you


 11   can then start to see what happens after an


 12   infection.


 13             Next slide, please.  This slide shows what


 14   happens in the typical course--now, I am going to


 15   talk about typical things here.  Certainly all of


 16   us know about the exceptions and we have all seen


 17   them at one time or another, but I think it is


 18   important to deal right now with the typical


 19   changes that can occur.  Again what happens, you


 20   have a hepatocyte that is infected.  There is a


 21   very short eclipse phase in which we would not see


 22   virus in the hepatocyte.  Then viruses start to be




  1   produced and are transported out of the cell.


  2   Then, one sees initially HBV DNA in the


  3   bloodstream.  It precedes the expression of


  4   hepatitis B-surface antigen which then starts to


  5   occur here very early in the course of the disease


  6   and peaks usually during the acute phase of a


  7   clinical illness.


  8             What happens then in this non-cytolytic


  9   infection is that there are usually some holes that


 10   are punched in the plasma membrane of a hepatocyte.


 11   ALT, which is in the cytosol, aminotransferase


 12   enzyme which is in the cytosol of hepatocytes, are


 13   released into the bloodstream.  So, the ALT is


 14   elevated in these patients and then follows the


 15   clinical symptoms of anorexia, fatigue, jaundice,


 16   etc.


 17             Usually about the time the ALT begins to


 18   appear, HBeAg and IgM anti-HBc are found.  IgM anti


 19   HBc appears in high titer or high concentration in


 20   individuals as a response to the synthesis of


 21   nucleocapsids and, therefore, represents active


 22   viral replication.  The IgM rises during the early




  1   courses of infection and then starts to disappear.


  2   Usually by 4-6 months or so, or 6-9 months, the IgM


  3   anti-HBc disappears, although it may be present for


  4   up to 2 years in individuals, if you use very


  5   sophisticated research tools.


  6             Usually the HBV DNA disappears in the


  7   acute infection at the time that a hepatitis


  8   B-surface antigen disappears.  So, you have a


  9   pattern here.  HBV DNA first; HB surface antigen,


 10   and the HBsAg disappears and usually about that


 11   time HBV DNA is gone, or at least is


 12   non-detectable.


 13             Again, another pattern appears.  When


 14   HBsAg disappears it is replaced by its antibody,


 15   anti-HBs.  Now, we know that probably anti-HBs is


 16   produced very early in the course of the infection,


 17   even probably back in this area here, which results


 18   in serum sickness, vasculitis or other things, but


 19   you don't detect it because there is so much


 20   hepatitis B surface antigen available so it is


 21   non-detectable.  But eventually the surface antigen


 22   goes; anti-HBs then is detected.  When HBeAg




  1   disappears in the active infection, then anti-HBe


  2   replaces it.  Anti-HBe lasts in most patients for a


  3   long period of time but in about a third of the


  4   patients disappears after about 6 months of


  5   follow-up.


  6             Of course, the IgM anti-HBc--there is a


  7   switch-over between the IgM anti-HBc and IgG


  8   anti-HBc.  What is important to recognize is that


  9   the assay, the total anti-HBc assay, detects not


 10   only IgM but IgG antibodies.  It is not an IgG


 11   test; it detects both IgG and IgM.  So, whenever


 12   IgM is present you ought to see the total anti-HBc


 13   test positive.


 14             Next slide, please.  This is an older


 15   slide I have but I think it helps us a little bit


 16   understand the relationship of all these assays


 17   that we are looking at.  The EIA assay and those


 18   that are being developed can detect down to about a


 19   tenth of a nanogram of HBsAg.  HBsAg circulates up


 20   to 200 mcg or more per ml of blood.  These tests


 21   really have an upper level of sensitivity of about


 22   1 mcg/ml.  So, usually they are at their upper




  1   limits of detection for most infections.


  2             The second test that came along them were


  3   the hybridization assays, the liquid phase and the


  4   filter hybridization assays.  These could detect


  5   down to a tenth to 1 pg of genomic equivalence in


  6   the blood.  At least, at 24 hours if you have the


  7   autoradiographs stay for maybe up to 5 days, you


  8   can detect anywhere from 2- to 10-fold more virus


  9   in the blood.  So, they are detecting about this


 10   level here, somewhere between here and here, and 1


 11   pg is equivalent to about 300,000 copies/ml.  You


 12   will see 280,000, 330,000 but let's just talk about


 13   300,000 copies/ml.  So, 1 pg is here, 300,000,


 14   30,000, 3,000 and 300 down at the femptogram


 15   levels.


 16             The PCR assays, which are somewhere


 17   between 700-7,000 times more sensitive than


 18   hybridization assays, can detect down to 50 copies


 19   or less per ml.  So, they are down in this range,


 20   here.  As I said, this is 300,000, 3,000 and 300


 21   copies/ml.  This would be 30 copies/ml.  These are


 22   usually with nested PCRs or other assays, nucleic




  1   acid--the tests that are available today.


  2             So, you see the differences between these


  3   assays.  One might ask why is it then that the EIA


  4   tests are detecting HBsAg very closely to the HBV


  5   DNA.  Part of the reason for that is that there are


  6   anywhere from 1,000 to 10,000 times more--at least


  7   10,000 times more hepatitis B surface antigen


  8   particles per infectious virion, and there may be


  9   even more naked HBV particles.  So, you have a lot


 10   more HBsAg produced initially than you do


 11   infectious virions.  That is the reason I think


 12   that there is not a great deal of difference, at


 13   least initially, in the detection of these


 14   particles.


 15             Next slide, please.  So, during the course


 16   of infection HBV DNA is detected from 2-5 weeks


 17   after infection and up to 40 days before the HBsAg


 18   is detected.  However, there is a wide coefficient


 19   here of variation but the mean of that is around


 20   6-15 days, and there are some exceptions to this.


 21   It rises slowly during the course of infection at a


 22   relatively low level, say 10                                             

                               2 to 104 copies/ml




  1   during the seronegative period, that is, before the


  2   HBsAg appears.


  3             Next slide, please.  HBsAg, on the other


  4   hand, appears 1-3 weeks before the ALT becomes


  5   abnormal or 3-5 weeks before the onset of symptoms


  6   or jaundice.  It reaches a peak during the acute


  7   stage of the disease and then it declines to


  8   undetectable levels within 4-6 months.


  9             Next slide.  IgM anti-HBc is indicative,


 10   as I said, of ongoing viral replication and appears


 11   at the onset of ALT abnormality at high


 12   concentration.  It is present but undetectable in


 13   some chronic infections and may reappear,


 14   therefore, during reactivation of HBV during


 15   chronic infection.


 16             Now, one of the questions that often


 17   arises is when you have acute hepatitis how do you


 18   determine that this is not a reactivation of


 19   chronic disease compared to an acute disease?  That


 20   has always been an issue.  For all practical


 21   purposes, the IgM anti-HBc test can be utilized to


 22   determine that.  It is at high concentrations. 




  1   Whereas, patients who have reactivation of their


  2   hepatitis B virus infection from chronic disease


  3   often have a relatively low concentration of IgM


  4   anti-HBc in their test.  We use a sample to cut-off


  5   ratio and it is usually somewhere between 1-3 in


  6   those cases, whereas in acute disease it is at its


  7   maximum.


  8             In addition, the other way, if you have


  9   the means to do this, is that the IgM has a 19S


 10   sedimentation coefficient during acute infection.


 11   It has a 7S sedimentation coefficient during


 12   chronic infection.  That is another way that one


 13   could use to determine acute from chronic.


 14             Next slide, please.  The anti-HBs is a


 15   neutralizing antibody occurring during recovery and


 16   after infection, and in most cases indicates


 17   protection against reinfection.  However, it may


 18   become undetectable in up to 20 percent of patients


 19   after several years.


 20             Next slide.  This slide sort of shows what


 21   happens in a patient who gets acute disease and


 22   gets over it.  The anti-core is a very strong




  1   antibody, high concentration of antibody compared


  2   to anti-HBs.  Over time, as I said, in about 20


  3   percent of patients this antibody will disappear.


  4   Then you are left with a person who has only an


  5   anti-HBc response.


  6             Next slide.  One of the ways that one can


  7   tell that that is present is you can do an HBsAg


  8   vaccine challenge.  You can give them a vaccine.


  9   About 2-4 weeks later you have them come back in


 10   for a quantitative anti-HBs test and if the


 11   response is greater than 10 milli-international


 12   units/ml, that is indicative that this person


 13   probably had a prior infection and was immune.  It


 14   is unusual for a susceptible person or a person


 15   with a low-level infection to respond to the


 16   hepatitis B surface antigen by 2-4 weeks.  Probably


 17   less than 3 percent in 2 weeks will respond to the


 18   vaccine at this particular level.


 19             Next slide.  I have talked a little bit


 20   about chronic infection.  The difference is that


 21   arbitrarily, say, if the HBsAg is still positive


 22   after 6 months or the person has ALT abnormalities




  1   for a period of time, then they probably have


  2   chronic infection.  In addition, the HBsAg is at


  3   very high concentrations through that period of


  4   time.  It doesn't start to decline.  So, if you


  5   could dilute these samples as a patient is being


  6   followed along, you will find that the HBsAg


  7   concentration is declining.  Just remember that the


  8   upper level of their reference is about 1 mcg/ml so


  9   you do have to do some dilutions in order to see


 10   this happening.  The IgM does disappear but remains


 11   usually positive at very low levels.  With research


 12   tools you can detect it in most patients.  It just


 13   isn't positive in the way the test is done, which


 14   is to dilute the sample about 1:1000 or 1:2000.


 15   That is why you don't see it in these individuals.


 16   That is how the test is configured.  Part of that


 17   is because of prozone effects and other things


 18   which could make a positive test negative if you


 19   did not dilute.


 20             E antigen is usually present initially,


 21   but over time about 5-10 percent of patients will


 22   seroconvert from HBeAg positivity to anti-HBe




  1   positive.  During that period of time they usually


  2   show reactivation of their disease.  Often it is


  3   just an elevation of the liver enzymes but they may


  4   become jaundiced and they may develop symptoms that


  5   look just like acute disease.  Until we understood


  6   this, this was one of the reasons we used to think


  7   that 10 percent of patients who had acute disease


  8   would become chronically infected.  Now that we


  9   know that many of these patients were chronically


 10   infected to start with, we now feel that probably


 11   less than 3 percent of the immunocompetent patients


 12   who develop acute disease will actually become


 13   chronically infected.  Of course, the anti-core and


 14   HBsAg remain positive in most patients.


 15             Next slide, please.  Let me just go


 16   through, finally, some of the findings that we just


 17   talked about.  This is HBVd and a surface antigen,


 18   anti-HBc and anti-HBs.  You can see in the


 19   pre-seroconversion window period--we have a couple


 20   of windows here because we used to talk about a


 21   window period between the end of the HBsAg phase


 22   and the development of anti-HBs in which the IgM




  1   anti-core is present during acute disease.  But


  2   here I am talking about the pre-seroconversion of


  3   antibodies or the seronegative, if you will, window


  4   period.


  5             Then, in the early acute infection both


  6   the HBVd and a and HBsAg are positive.  The


  7   anti-core and the anti-HBs are negative.  The


  8   presence of HBsAg is indicative of a hepatitis B


  9   infection, either acute or chronic.  Just the HBsAg


 10   alone--you can't tell.  But the presence of all


 11   three of these together in the absence of anti-HBs


 12   indicates an HBV infection, either acute or


 13   chronic.  Eventually you will lose these markers


 14   and you are left with anti-HBc and anti-HBs, which


 15   means a previous infection with immunity.


 16             Next slide.  The other findings are in


 17   individuals who have a solitary anti-HBc test


 18   present.  They have no anti-HBs and no HBsAg.  Some


 19   of them may contain HBV DNA and these represent


 20   low-level carriers which do not have enough surface


 21   antigen present in the blood to be detectable by


 22   the current assays.




  1             The other thing is that if this is


  2   negative, this could also indicate an early


  3   convalescent period in which this anti-HBc will be


  4   IgM.  It could represent an HBV infection in the


  5   remote past, as we talked about, in which the


  6   anti-HBs has disappeared and you could do a vaccine


  7   challenge, or it could be in many cases a


  8   false-positive reaction.  Most of these reactions


  9   show anti-core at a very low level, again, a sample


 10   to cut-off ratio somewhere between 1-3.  The


 11   anti-HBe is usually absent in these individuals as


 12   well, and they don't respond to the vaccine


 13   challenge.


 14             Then you have a group that have no


 15   markers.  This usually excludes an HBV infection in


 16   this case.  Then you have individuals with anti-HBs


 17   only.  This represents a vaccine type response.  It


 18   is really unusual to see something else happening,


 19   or you shouldn't see anything other than anti-HBs


 20   in a patient who gets the vaccine because it only


 21   contains HBsAg.


 22             Next slide.  This is my final slide.  It




  1   looks at discordant or unusual hepatitis B


  2   serologic profiles requiring further evaluation.  I


  3   have probably seen all of these at some time or


  4   another in my career.  For example, I have a


  5   patient right now who has HBsAg positive only, who


  6   is anti-core negative, with 900 million copies or


  7   IU of virus per ml of blood.  He has been like that


  8   most of his life.  It happens in some young


  9   children who get hepatitis B and become chronically


 10   infected.  It is seen in other adults, particularly


 11   in Asians.  But it is a really unusual phenomenon.


 12   So, that is a possibility but really rare.


 13             Finding of all these three markers in one


 14   patient does occur in about 5-10 percent of


 15   patients.  It is mostly in drug users or people who


 16   have been exposed to different genotypes of the


 17   virus.  It could be found in people who have been


 18   vaccinated and have made antibodies to a different


 19   genotype, and it usually is in patients who have


 20   more serious liver disease.


 21             Anti-HBs or anti-HBc positive only--we


 22   have talked about those phenomena.  The are not so




  1   uncommon.  HBsAg negative individuals who are HBeAg


  2   positive--you shouldn't really see that but I have


  3   seen at least two cases that have been HBeAg


  4   positive, HBV DNA negative but do not have the


  5   presence of surface antigen present.


  6             You could have patients who are positive


  7   for both e antigen and anti-e at the same time.


  8   That has to do with the nuances of the test and


  9   probably nothing else.  And, you can find patients


 10   who are anti-core negative--I mean, you should not


 11   find a patient who is anti-HBc negative but IgM


 12   anti-HBc positive.


 13             Well, I hope now you are a little bit more


 14   familiar with the serology of this disease.


 15   Antigens are followed by antibodies.  HBV DNA


 16   precedes the development of antigens very early in


 17   the course and usually remains throughout the


 18   disease entity itself.  Thank you very much.


 19             DR. NELSON:  Thanks, Blaine.  Are there


 20   any questions?  Harvey?


 21             DR. KLEIN:  Blaine, there have been a


 22   number of reports of people who have low levels of




  1   HBV DNA and have anti-HBs.  Do you know if any of


  2   those are infectious?  Have you ever seen one?


  3             DR. HOLLINGER:  Well, it is not whether we


  4   have ever seen one.  The issue I guess is that we


  5   have this question all the time about occult


  6   infection in HBV DNA even though there are no


  7   markers, usually in people who are very sick and


  8   would not be donors, for example, liver cancer


  9   patients and immunocompromised individuals.  So,


 10   you wouldn't find these as donors anyway.  It is my


 11   belief that most of these issues of finding nucleic


 12   acid in the presence of antibodies probably means


 13   it is neutralized and it is not infectious.  You


 14   see that with RNA found very late in the course of


 15   the disease.  In the presence of antibody it


 16   doesn't appear to be infectious biologically,


 17   clinically or in trials.


 18             So, I think that finding anti-HBs at a


 19   reasonable concentration and HBV DNA would probably


 20   not indicate somebody is infectious.  But you just


 21   have to do these studies, and no one has really,


 22   say, taken a chimpanzee and looked at these.  I




  1   think Prince did so a few years ago and the data


  2   was that they were not infectious.


  3             DR. NELSON:  You didn't mention the


  4   genetic variations in the surface antigen.  Could


  5   those lead to a false-negative surface antigen test


  6   in people who were actually infectious?


  7             DR. HOLLINGER:  Yes, Kenrad, it is a good


  8   question.  There are about six or seven genotypes


  9   of this disease, from a to h, but because they all


 10   have a group a antigen present, they all seem to be


 11   detectable by the usual tests which are available


 12   today.  Now, could it happen?  Sure, I think it is


 13   possible but I think it would be unusual.


 14             DR. NELSON:  One other comment too, we


 15   have studied hepatitis B in injection drug users


 16   and it is extremely common for them to have


 17   hepatitis B core antibody only, without surface, at


 18   very high levels.  They are actually truly infected


 19   and it is up to 20 percent of injection drug users.


 20   It is possible that co-infection with hepatitis C


 21   may play a part in that because they are all


 22   infected with hepatitis C as well.  It is a very




  1   complex interaction.  But seeing hepatitis B core


  2   antibody only in somebody who actually is a drug


  3   user and comparing drug users with others without a


  4   history, it is like 20 percent of the drug users,


  5   maybe 1 or 2 percent in gay men who have had


  6   hepatitis B infection.


  7             DR. HOLLINGER:  Yes, you see it not only


  8   in drug users but you see it in volunteer donors


  9   who are found to be anti-HCV positive and then you


 10   go do some other tests and they are found to be


 11   anti-HBc positive only.  They don't respond well to


 12   the vaccine.  And, we know that HCV interferes with


 13   the replication of hepatitis B virus so that is a


 14   possibility.


 15             DR. NELSON:  Other comments?


 16             [No response]


 17             Thanks.  Dr. Herman, from Roche?


 18         C. Preclinical and Clinical Data for HBV MP NAT


 19             DR. HERMAN:  Thank you very much.  It is a


 20   pleasure to be here to describe to you the results


 21   of our non-clinical and clinical performance


 22   studies on our COBAS AmpliScreen HBV test.




  1             [Slide]


  2             I am going to begin the talk with a


  3   description of our non-clinical performance


  4   studies, and Dr. Frank will conclude with a


  5   description of our clinical study results.


  6             [Slide]


  7             Here is an outline of the talk.  I am


  8   going to give an overview of the COBAS AmpliScreen


  9   system and then I am going to describe the


 10   different portions of the non-clinical performance


 11   studies and give you their results.


 12             [Slide]


 13             The COBAS AmpliScreen system is designed


 14   for screening of minipool samples or individual


 15   donor samples.  The samples are put onto the


 16   Hamilton microlab pipetting robot which prepares


 17   the pools.  Then the pooled or individual samples


 18   are processed to extract nucleic acid with the


 19   generic sample processing method.  There are two


 20   variations of the method, the multiprep method for


 21   the pooled samples and the standard method for


 22   individual donor samples, and I will describe them




  1   in the next slide.


  2             Aliquots of the extracted material can be


  3   processed in parallel with our three different


  4   COBAS AmpliScreen tests, the test for HBV and the


  5   licensed tests for HCV and HIV.  The analysis is


  6   done on the COBAS Amplicor analyzer which automates


  7   the nucleic acid amplification and detection


  8   processes.  Then, the software for the analyzer and


  9   the software for the pipetting machine interact


 10   with the data output management system.


 11             [Slide]


 12             This slide describes the two sample


 13   preparation methods, the multiprep method which is


 14   used for the minipools, and the standard method


 15   that is used for individual donor samples.  For the


 16   minipool method, 1 ml of the pool is centrifuged at


 17   23,500 g to enrich for the viral targets, HCV, HIV


 18   and HBV.  After the centrifugation, 900 mcL of the


 19   supernatant is discarded, leaving behind the pellet


 20   and 100 mcL of the supernatant.  The nucleic acids


 21   are extracted by adding a k-atropic lysis reagent


 22   which contains the internal control.  After a brief




  1   incubation, isopropanol is added to precipitate the


  2   nucleic acids.  The nucleic acids are collected by


  3   centrifugation.  The pellet is washed with ethanol


  4   and resuspended in a specimen diluent, and then an


  5   aliquot of the specimen diluent can be used in each


  6   of the AmpliScreen tests.


  7             The standard specimen processing method


  8   for individual samples is almost identical.  All


  9   the green steps are identical and the differences


 10   are highlighted in red.  So, the starting specimen


 11   volume is 200 mcL instead of 1 ml, and there is no


 12   concentration step.  The k-atropic lysis reagent


 13   with the internal control is added directly to the


 14   200 mcL specimen and then the remaining steps are


 15   the same.


 16             [Slide]


 17             Now I am going to describe the analytical


 18   sensitivity studies that were performed.  These are


 19   the results using the multiprep procedure.  We used


 20   the World Health Organization HBV DNA international


 21   standard and we prepared dilutions of it between


 22   100 and 3 IU/ml and analyzed 120 replicates of each




  1   dilution, at Roche, using 2 different kit lots.


  2   This shows the results.  So, we had 100 percent hit


  3   rate at 100, 30 and 10 IU/ml and a 95.8 percent hit


  4   rate at 5 IU/ml.  The predicted 95 percent limit of


  5   detection, using the PROBIT statistical method, is


  6   4.4 IU/ml.


  7             [Slide]


  8             This slide shows the results using the


  9   standard specimen processing procedure.  In this


 10   study we analyzed dilutions of the World Health


 11   Organization standard from 300 to 10 IU/ml.  There


 12   was 100 percent hit rate on 100 replicates at 300


 13   and 100 IU/ml, and 99 percent on 30 IU/ml, and 97


 14   percent at 20, and 95.8 percent at 15 IU/ml.  The


 15   predicted 95 percent limit of detection, using the


 16   PROBIT method, is 16 IU/ml for the standard


 17   specimen processing method.


 18             [Slide]


 19             This slide describes the results on a


 20   panel prepared by CBER.  The original panel


 21   contained 3 members at 100, 10 and 0 copies/ml, and


 22   at Roche we prepared 4 additional dilutions from




  1   the 100 copy/ml member at 50, 25 5 and 2.5


  2   copies/ml.


  3             This slide shows the results using the


  4   multiprep specimen processing method and on 6


  5   replicates, 100 percent hit rate at 100 copies and


  6   at 10 copies/ml, then 67 percent and 42 percent


  7   hits at 5 and 2.5 copies/ml.


  8             Using the standard specimen preparation


  9   method, 100 percent hits at 100 copies/ml with 6


 10   replicates and 92 percent and 75 percent at 50 and


 11   25 copies/ml.


 12             [Slide]


 13             This slide shows the results of our


 14   analysis of the performance of the test on HBV


 15   genotypes A through H.  These samples were obtained


 16   from commercial sources, and we analyzed up to 25


 17   individual isolates for each genotype.  The samples


 18   were quantified using the COBAS Amplicor HBV


 19   monitor test or, in the case of genotypes G and H,


 20   monitor tests that we have in development.  Then


 21   the samples were diluted to approximately 2-3 times


 22   the limit of detection for the multiprep method and




  1   the standard prep method, and then they were


  2   analyzed using the COBAS AmpliScreen test.  So,


  3   with the multiprep method and the standard method


  4   all the isolates yielded positive results.  So, the


  5   test detected HBV genotypes A through H.


  6             [Slide]


  7             This slide summarizes the results on 40


  8   commercially obtained seroconversion panels using


  9   the multiprep method and the standard prep method.


 10   The slide shows the number of days that DNA was


 11   detected prior to detection of surface antigen


 12   using a licensed surface antigen test, the Ortho


 13   surface antigen test system 3.  The orange bars


 14   show the results with the multiprep test, and for


 15   that analysis the samples were diluted 24-fold to


 16   simulate minipool testing.  The blue bars show the


 17   results with the standard prep on undiluted


 18   samples.  The panels are sorted by the number of


 19   days prior to antigen that DNA is detected with the


 20   multiprep method.  So, using the multiprep method,


 21   in 38/40 panels HBV DNA was detected prior to


 22   surface antigen, and in 2/40 panels HBV DNA was




  1   detected in the same bleed as surface antigen.


  2             Using the standard prep method on neat


  3   samples in 39/40 panels HBV DNA was detected prior


  4   to surface antigen, and in 1 panel HBV DNA was


  5   detected on the same day as surface antigen.  I am


  6   going to show you the results on one of the panels,


  7   this panel, 39.


  8             Before that, with the multiprep method, on


  9   the 38 panels on which DNA was detected prior to


 10   surface antigen, DNA was detected an average of 17


 11   days earlier, and with the standard method DNA was


 12   detected an average of 22 days earlier.


 13             [Slide]


 14             Here are the results on panel 39.  This


 15   panel had 14 bleeds.  This column shows the results


 16   with the Ortho Surface Antigen Test System 3, and


 17   you can see that this turned positive on bleed 12


 18   at day 143.  This column shows the results with the


 19   minipool test, and you can see that it was positive


 20   on the first and third bleed and then positive


 21   again at the 11th bleed on day 113.


 22             On the previous slide, on this panel it




  1   was described as having DNA detected prior to


  2   antigen 30 days in advance this one bleed.  Even


  3   though DNA was detected intermittently earlier than


  4   that, we made a conservative interpretation and


  5   only counted the number of bleeds that DNA was


  6   continuously detected prior to surface antigen.


  7             In this column, the results with the


  8   standard preparation method on undiluted samples is


  9   shown.  There were 11 bleeds prior to detection by


 10   surface antigen and the HBV DNA was positive in 7


 11   of them but did not yield a positive result on


 12   bleed 11.  So, in the previous graph the standard


 13   prep method was described for this panel as being


 14   detected on the same day as surface antigen.


 15             [Slide]


 16             This just illustrates that even though DNA


 17   was detected intermittently earlier, on multiprep


 18   method we only counted 1 bleed, which was 30 days


 19   in this case.


 20             [Slide]


 21             This slide summarizes the results on the


 22   40 seroconversion panels.  Using the multiprep




  1   method on samples diluted 24-fold to stimulate


  2   pooling, DNA was detected prior to surface antigen


  3   in 38/40 panels and on the same bleed in 2/40


  4   panels an average of 17 days prior to surface


  5   antigen.  Using the standard procedure on undiluted


  6   samples, DNA was detected prior to antigen in 39/40


  7   panels an average of 22 days earlier, and on the


  8   same bleed on the 40th panel.


  9             [Slide]


 10             This slide summarizes the results of the


 11   analytical specificity testing and the testing of


 12   potentially interfering substances.  For the


 13   analytical specificity we looked at 38


 14   microorganisms and cell lines, and all these


 15   samples yielded negative results with the


 16   AmpliScreen and valid internal control results.


 17   There was no cross-reactivity observed.


 18             For the potentially interfering


 19   substances, we looked first at 95 clinical


 20   specimens with different diseases in patients


 21   infected with these viruses or with autoimmune


 22   disease or with yeast infections, and multiple




  1   specimens of each.  These specimens were tested


  2   both with the addition of HBV target and without.


  3   So, the testing with the addition of HBV target was


  4   looking for interference.  No interference was


  5   observed.  Testing without HBV target was looking


  6   for cross-reactivity and no cross-reactivity was


  7   observed.


  8             Then we looked at these various


  9   potentially interfering substances.  Again, they


 10   were tested with and without the addition of HBV


 11   target.  On the tests with HBV target there was no


 12   inhibition and on the tests without HBV target


 13   there was no cross-reactivity.


 14             [Slide]


 15             This slide and the next look at our


 16   reproducibility study.  This slide shows the


 17   results of the multiprep procedure.  The study was


 18   conducted at 3 clinical sites using 3 lots of


 19   reagent.  The study was conducted over 5 days with


 20   2 operators at each site.  Each day the operators


 21   analyzed the 6-member panel, blinded 6-member panel


 22   consisting of 2 negative samples and 4 samples at




  1   low and moderate target concentrations.


  2   Essentially the same results were obtained with the


  3   3 kit lots and at the 3 sites.  So, there was 1


  4   false-positive result with lot 3 and that happened


  5   at site 3.  At the low copy samples the percent


  6   hits were nearly identical among the 3 lots and


  7   among the 3 sites.


  8             [Slide]


  9             This slide shows the reproducibility study


 10   with the standard procedure and the same outcome.


 11   Essentially the same results between the 3 lots and


 12   between the 3 lights on the negative and the


 13   positive samples.


 14             [Slide]


 15             Finally, this slide describes the


 16   performance of the AmpliScreen test alone or in


 17   combination with the licensed anti-core test on 918


 18   antigen-positive clinical specimens.  I am showing


 19   the results with the multiprep test on samples


 20   diluted 1:14 to simulate pool testing and with the


 21   standard method on undiluted samples.  So, looking


 22   at the results of the AmpliScreen test alone with




  1   the minipool method, 871 of the 918 samples were


  2   DNA positive for sensitivity of 94.9 percent.


  3   Using the standard method on undiluted samples, 898


  4   samples were positive for sensitivity of 97.8


  5   percent.  These 20 samples that were not detected


  6   by the standard test were among the 47 that were


  7   not detected by the minipool test.  All the NAT


  8   negative samples were sent out for anti-core


  9   testing and all of them were positive by the


 10   anti-core test.  So, using the multiprep method, 1


 11   sample had insufficient volume for the testing, a


 12   sample that was positive with the standard sample


 13   prep and negative with multiprep so we couldn't get


 14   a result on that so it was excluded from the


 15   sensitivity analysis.  So, for the 917 samples with


 16   NAT positive results and anti-core results on the


 17   NAT negative samples the sensitivity was 100


 18   percent for NAT and core combined relative to


 19   antigen.  On the 1900 samples with evaluable


 20   results using the standard method, again, the


 21   sensitivity was 100 percent.


 22             [Slide]




  1             To summarize the non-clinical performance


  2   studies, the analytical sensitivity was determined


  3   on the World Health Organization standard.  With


  4   the multiprep method the limit of detection at 95


  5   percent probability is 5 IU/ml and with the


  6   standard method the limit of detection at 95


  7   percent probability is 15 IU/ml.  The performance


  8   on the CBER panel with the multiprep method, 100


  9   percent hits at 10 copies/ml and with the standard


 10   method 92 percent hits at 50 copies/ml.  Genotypes


 11   A through H were detected by the test.


 12             [Slide]


 13             On the 40 seroconversion panels using the


 14   multiprep method on samples diluted 24-fold, HBV


 15   DNA was detected prior to antigen in 38/40 panels


 16   an average of 17 days or earlier, and with the


 17   standard method on undiluted samples DNA was


 18   detected in 39/40 panels prior to antigen an


 19   average of 22 days earlier, and there were no


 20   panels in which antigen was detected prior to DNA.


 21   There was no cross-reactivity or interference


 22   observed in the analytical specificity and




  1   potential interfering substances studies.  And, the


  2   sensitivity on antigen-positive specimens, in


  3   combination with the anti-core assay, was 100


  4   percent.


  5             [Slide]


  6             I would like to acknowledge all the people


  7   who did this work, Yuanfeng Yang and his group at


  8   Roche Molecular Systems did an excellent job


  9   developing the assay and performing these studies.


 10   Larry Pietrelli coordinated the clinical studies


 11   and, in this part of the talk, he coordinated the


 12   reproducibility studies.  They were conducted at


 13   the Community Blood Center of Greater Kansas City,


 14   Royal Blood Centers, Minnesota and the Gulf Coast


 15   Regional Blood Center.


 16             Now I think I will turn the microphone


 17   over to Dr. Frank who will describe the results of


 18   the clinical performance study.


 19             DR. FRANK:  Thank you, Dr. Herman.


 20             [Slide]


 21             It is my pleasure to be here today to


 22   describe to you a prospective study to evaluate the




  1   screening of plasma pools from volunteer blood


  2   donations for the presence of HBV DNA.


  3             [Slide]


  4             A clinical trial summary will focus today


  5   on two areas of primary focus of improved safety of


  6   blood donations by HBV minipool-NAT.  The key focus


  7   in that particular area will be looking at the


  8   potential window period or pre-seroconversion areas


  9   of HBV.  A secondary focus will be replacement of


 10   surface antigen by HBV minipool-NAT.  To focus on


 11   that, we will look primarily at the areas of


 12   samples that were surface antigen positive that


 13   were picked up by minipool-NAT and surface antigen


 14   positive picked up by anti-core.


 15             This clinical study was initiated in


 16   August of 2002 at 5 U.S. sites, and was completed


 17   in April of 2003.  A total of 704,902 specimens


 18   were tested with HBV minipool-NAT, of which 581,790


 19   were included in the sensitivity and specificity


 20   analysis.  The differential between the two numbers


 21   occurred because specimens were excluded if no


 22   electronic results for surface antigen and/or




  1   anti-core were available from the site.


  2             [Slide]


  3             If you look at this chart, which is a


  4   schematic of the results listing the analytes


  5   anti-core, surface antigen and DNA--if you can't


  6   see in the back of the room, the red means they


  7   were HBV negative for that particular analyte; the


  8   green means they were positive for that particular


  9   analyte.  The totals are on the far right-hand


 10   column, the numbers that had that criteria.  As you


 11   can see, the majority of the donors were, as


 12   expected, negative for all three.


 13             [Slide]


 14             We were primarily interested in looking at


 15   this group down here, and those were enrolled in


 16   our follow-up study, the protocol for which I will


 17   be describing briefly.


 18             [Slide]


 19             Of these 4 eligible for the follow-up


 20   study, the 2 areas we wanted to look at are


 21   primarily the last row, which is the potential


 22   window cases, that is, those cases which were HBV




  1   minipool DNA positive but negative for core and


  2   surface antigen.  This represents the type of


  3   pattern one would expect in pre-seroconversion


  4   window cases, and then the potential surface


  5   antigen false-positive results, that is, surface


  6   antigen positive but core negative and HBV minipool


  7   negative as well.


  8             [Slide]


  9             I mentioned the follow-up protocol that


 10   was utilized and this is the follow-up protocol for


 11   those columns that we followed up, the test index


 12   donation was tested by alternate NAT, provided by


 13   the National Genetics Institute.  If alternative


 14   NAT was positive, the result was quantitated and


 15   then the subject was offered enrollment in a


 16   6-month follow-up study for weekly draws times 4


 17   then monthly draws to complete 6 months of


 18   follow-up.  The analytes tested during follow-up


 19   are as listed on your screen.


 20             [Slide]


 21             The primary objective was to improve the


 22   safety by use of HBV minipool-NAT for detection of




  1   window period cases.


  2             [Slide]


  3             As stated previously there were 23 donors


  4   that were HBV DNA positive, surface antigen


  5   negative and core negative.  Of these 23, 14 were


  6   enrolled in the follow-up study.  It should be


  7   noted that 9 donors declined follow-up and for the


  8   purposes of our calculations for sensitivity and


  9   specificity these were presumed to be false


 10   positive and were negative on additional index


 11   testing as well.


 12             Of the 14 enrolled donors, 2 were


 13   confirmed window period cases, the details of which


 14   I will be showing next, and 12 were shown to be


 15   valse positive on HBV due to persistently negative


 16   anti-core, persistently negative surface antigen


 17   and persistently negative HBV DNA, and negative by


 18   alternate NAT index specimens.


 19             [Slide]


 20             This window period case is a 26 year-old


 21   male repeat blood donor with no known risk factors.


 22   His HBV DNA was positive for a quantitative level




  1   of 2000 on index donation.  On day 17 post index


  2   donation the surface antigen was positive, and on


  3   day 48 anti-core was positive.


  4             [Slide]


  5             The next window case is a subject who is a


  6   49 year-old female repeat blood donor, healthcare


  7   worker, with a history of HBV vaccination.  This is


  8   an interesting case because she had a negative


  9   anti-surface antibody result 2 months prior to her


 10   index donation when she donated blood previously.


 11   What is interesting is between that time, where


 12   this result 2 months prior was negative, and here


 13   index donation was 2,340--now, this normally


 14   wouldn't be done during her blood screening but she


 15   was HBV DNA positive and she quantitated out at 200


 16   copies/ml, which would be an infectious dose.  So,


 17   this is a window period case of an individual who,


 18   on follow-up, never did convert for surface antigen


 19   and did, on day 22, convert for anti-core.  What


 20   this likely represents is that somewhere between


 21   the 2 weeks prior to her index donation when this


 22   was negative and the result of her index donation,




  1   she became infected with HBV and had a nice immune


  2   system response to her previous HBV vaccination.


  3             [Slide]


  4             There are additional window cases I would


  5   like to show to you today, and these were detected


  6   by sites continuing to use HBV NAT following


  7   conclusion of our clinical study.  The dates for


  8   those 3 sites were April, 2003 to the present, and


  9   there are 3 additional window period cases that


 10   were detected in the one million donations


 11   screened.


 12             [Slide]


 13             The first case is a 27 year-old male


 14   repeat blood donor who reported high risk male


 15   sexual partners.  He had HBV DNA positive on


 16   donation with a quantitative level of 61,000.  On


 17   day 7 his surface antigen turned positive and on


 18   day 36 his core was repeat reactive.


 19             [Slide]


 20             This case is a window period case of a 29


 21   year-old male repeat donor who has no appreciable


 22   risk factors for hepatitis B, other than a possible




  1   acupuncture treatment for 8 weeks prior to his


  2   donations, weekly treatments.  His HBV index was


  3   positive at a quantitative level of 2,300.  His


  4   surface antigen turned positive 7 days later and


  5   his core turned positive 28 days later.


  6             [Slide]


  7             This last case is still on follow-up but


  8   this is a 50 year-old male repeat blood donor,


  9   positive on index donation with 37,000 copies/ml.


 10   On follow-up he remains positive on HBV DNA and the


 11   traditional blood banking serologies are still


 12   pending.


 13             [Slide]


 14             In summary, I thought it was interesting


 15   to look at the 5 cases that HBV minipool-NAT picked


 16   up.  If you look at the left-hand column, and as I


 17   have noted in the footnotes of your slides, all of


 18   these are repeat blood donors who could normally be


 19   expected to be donating blood regularly to the


 20   blood pool, and all donated blood units that would


 21   have been missed by the current blood banking


 22   algorithms.  All of them had infectious dose




  1   concentrations on their index donations.  In fact,


  2   even the lowest dose at 200 copies/ml, if the unit


  3   were divided appropriately, would be sufficient to


  4   infect everyone in this room.


  5             [Slide]


  6             Well, what is the yield for HBV


  7   minipool-NAT?  If you look at the clinical study


  8   yield alone we had 2 cases that I showed you out of


  9   approximately 0.7 million donations for a yield of


 10   1/350,000.  If we look at the continuing site data,


 11   which I also showed you for the 3 cases in


 12   approximately 1 million donations, the yield is


 13   similar, 1/330,000.


 14             Well, what does this mean in perspective


 15   of what we are already doing in blood banking to


 16   protect the nation's safety of the blood supply?


 17   The yield for HBV minipool-NAT is approximately


 18   equal to the yield for hepatitis C minipool-NAT and


 19   the yield for HBV minipool-NAT is far greater than


 20   the yield for HIV minipool-NAT.


 21             [Slide]


 22             If we look at this data that was presented




  1   by Mike Busch in Paris of this year, this


  2   emphasizes what I have just stated, that for HBV


  3   the yield is approximately 1/340,000, based upon


  4   his presentation.  For hepatitis C, 1/230,000, but


  5   for HIV, 1/3.1 million.  Although these aren't


  6   quite comparable, West Nile Virus was included for


  7   because there aren't a plethora of antibody and


  8   antigen tests available yet implemented for West


  9   Nile Virus but that yield is 1/5,000.


 10             [Slide]


 11             This brings us to a sensitivity and


 12   specificity calculation.  You can see how we


 13   assigned HBV status, on the screen here, for


 14   positives, negatives, the HBV status unknown for


 15   those that were surface antigen negative but core


 16   positive.


 17             [Slide]


 18             So, again, in summary, here are the totals


 19   and the results of the initial testing.  These are


 20   the potential surface antigen false-positive


 21   results.  These are the potential window cases.


 22   This last row I have gone into detail on, the row




  1   above, the 4 and the 3 will be going into the


  2   secondary objective.


  3             [Slide]


  4             The final status determination is here, on


  5   the fir right-hand column.  The ones that could not


  6   be determined are yellow, for those of you in the


  7   back of the room.  The green is HBV positive.  The


  8   red is HBV negative.  The window cases that were


  9   proven in the clinical study are shown here as 2.


 10   The remaining 21, which will be accounted for in


 11   sensitivity and specificity calculations, were


 12   counted as HBV positive but final status HBV


 13   disease negative.


 14             [Slide]


 15             To that point, our specificity


 16   calculations, if you look at the total number of


 17   subjects for which we have data--the difference


 18   between the numerator and the denominator is the 21


 19   cases that I just spoke about a second ago to yield


 20   a specificity calculation of 99.9964 percent, with


 21   the ranges noted on your screen.  For sensitivity


 22   we looked at the positive AmpliScreen minipool HBV




  1   results in specimens with positive HBV status and


  2   divided by the total number of specimens with


  3   positive HBV status.  Again, these calculations are


  4   for HBV minipool-NAT alone and the sensitivity here


  5   is 89 out of 105 or 84.7 percent, with a range of


  6   74.4 to 91 percent.


  7             [Slide]


  8             So, the primary objective conclusion is


  9   that COBAS AmpliScreen HBV test has identified


 10   individuals in  pre-seroconversion window period


 11   that would otherwise have been undetected by the


 12   blood banking system.  The COBAS AmpliScreen HBV


 13   test is suitable for blood screening with minipool


 14   strategies presented today, and these data indicate


 15   that HBV minipool-NAT will increase the blood


 16   safety.


 17             [Slide]


 18             Let's turn now to the secondary objective,


 19   and that is consideration for the HBV minipool-NAT


 20   and anti-core for replacing surface antigen and


 21   anti-core.


 22             [Slide]




  1             The index donations of interest for this


  2   discussion then would be those that were anti-core


  3   negative/ surface antigen positive and we will


  4   discuss those first.  Those donors fitting this


  5   category were 7 out of the total number for which


  6   we have data, or 0.0012 percent.  Those that had


  7   core-negative/surface antigen positive HBV DNA


  8   negative were 3.  Of those 3, 1/3 enrolled and


  9   converted to anti-core.  The other 2 remaining


 10   donors declined the follow-up study.  These were


 11   determined to be HBV final status positives.  For


 12   DNA negatives, that is DNA negative, core negative,


 13   surface antigen positive donors, 2 out of these 4


 14   enrolled in follow-up.  Two of the remaining


 15   declined to enroll in follow-up but we do have


 16   additional data that has been available and I will


 17   make available and show to you today.  All 4 of


 18   these were assessed to be HBV negative.  Let's look


 19   at some of those details.


 20             [Slide]


 21             This first subject was HBV DNA negative,


 22   surface antigen positive, core non-reactive.  On




  1   follow-up this subject remained core non-reactive


  2   throughout the follow-up study.  On surface antigen


  3   the subject has non-reactivity on follow-up, and


  4   HBV DNA remained negative, as did the other


  5   follow-up indicators.


  6             [Slide]


  7             This would indicate a case of an initial


  8   surface antigen test which did not repeat on


  9   follow-up, and all other negative serology results


 10   indicate this patient had an initial surface


 11   antigen positive that is consistent with


 12   contamination or carryover.


 13             [Slide]


 14             The next case is a little bit more


 15   interesting.  This case is surface antigen positive


 16   again on index donation, HBV negative, core


 17   non-reactive.  It should be noted that the surface


 18   antigen was positive and repeat reactive throughout


 19   follow-up but that the surface antigen was negative


 20   on neutralization for all of these testing time


 21   points.  The donor also states a history of HBV


 22   vaccination.  Of note, the core never did react




  1   throughout the course of follow-up and HBV DNA was


  2   negative throughout the course of follow-up.  The


  3   anti-surface antibody was positive and that we


  4   attribute to his vaccination status, and the repeat


  5   reactive, again, was negative on neutralization


  6   throughout.


  7             In summary for this case, the anti-surface


  8   antibody is explained by the donor's vaccination


  9   history.  And, the persistent surface antigen EIA


 10   repeat reactivity, which is negative on


 11   neutralization, combined with all the other


 12   negative results such as anti-core, is consistent


 13   with a false-positive surface antigen result due to


 14   cross-reactivity.


 15             [Slide]


 16             We have additional data for the 2 donors


 17   who declined the follow-up protocol.  Of the


 18   remaining 2 donors, 1 donor retested 1 month


 19   following his index donation and was negative for


 20   surface antigen, anti-core and anti-surface


 21   antibody.  The other donor was retested a year and


 22   a half post index and was negative for surface




  1   antigen and for core antibody.


  2             [Slide]


  3             This slide may look somewhat familiar.


  4   This is a look at the total HBV infected, HBV


  5   surface antigen positive specimens.  In our study


  6   there were 103 specimens fitting that


  7   categorization.  If we look at sensitivity from


  8   that perspective alone, HBV detected 87 of the 103


  9   specimens and was negative on 16, for a sensitivity


 10   as a stand-alone of 84.5 percent in this clinical


 11   study.  However, the proposal of HBV minipool-NAT


 12   plus anti-core antibody yielded positive in all 103


 13   of 103 HBV positive cases and missed none of the


 14   cases, for a sensitivity of 100 percent.  This


 15   parallels the non-clinical data shown to you


 16   earlier today by Dr. Herman.


 17             [Slide]


 18             What are the secondary objective


 19   conclusions?  All 105 HBV positive donors were


 20   identified by anti-core and HBV minipool-NAT


 21   combined.  These 105 include 2 window case donors


 22   that would have been missed by anti-core and




  1   surface antigen combined, and 103 surface antigen


  2   positive cases that were identified by the HBV


  3   minipool-NAT and anti-core as an alternative.


  4   These data then suggest that HBV minipool-NAT


  5   combined with anti-core provide an alternative to


  6   combined surface antigen and anti-core screening.


  7             [Slide]


  8             In summary conclusion today, I offer that


  9   the COBAS AmpliScreen HBV test improves the safety


 10   of blood transfusion.  I offer that COBAS


 11   AmpliScreen HBV test combined with anti-core may be


 12   considered as replacement for surface antigen


 13   combined with anti-core.  This is supported by the


 14   clinical trial data, and also supported by the


 15   non-clinical trial data, specifically


 16   seroconversion panel data shown here today and the


 17   other HBV positive clinical specimens shown here


 18   today by Dr. Herman.


 19             [Slide]


 20             With that, I would like to acknowledge the


 21   blood centers that participated in this large


 22   study, as well as the Roche Molecular personnel. 




  1   Thank you.


  2             DR. NELSON:  Thank you very much.


  3   Questions or comments?  Yes. Harvey?


  4             DR. KLEIN:  On the two window period cases


  5   that you detected with HBV alone, what was the


  6   antigen assay that was used for those?


  7             DR. FRANK:  In the two in the clinical


  8   study, the first window period case was Ortho Test


  9   System 2, and all remaining four window cases that


 10   were presented were Abbott Auzyme.


 11             DR. KLEIN:  And, do you by any change have


 12   any data with head-to-head comparisons with PRISM?


 13             DR. FRANK:  I don't believe we ran this


 14   test against any test that was not approved by the


 15   FDA.


 16             DR. KLEIN:  Even in Europe?


 17             DR. HERMAN:  We analyzed the


 18   seroconversion panels against the PRISM and the


 19   Ortho and I can show that data if you would like to


 20   see it.  That was the European version of the PRISM


 21   test that has the Paul Ehrlich Institute approval.


 22   Do you want to see that data?




  1             DR. NELSON:  Yes, I think so.


  2             [Slide]


  3             DR. HERMAN:  These are the same 40


  4   seroconversion panels that I showed you previously


  5   and the same NAT results but compared to the Paul


  6   Ehrlich Institute licensed Abbott PRISM test, and I


  7   guess this is the lot number.  Again, these are


  8   sorted by the results with the multiprep test on


  9   samples that were diluted 1:24-fold.  Those are the


 10   orange bars.  The blue bars represent the results


 11   with the standard test.


 12             [Slide]


 13             With the multiprep method DNA was detected


 14   an average of 14 days prior to antigen, and with


 15   the standard method DNA was detected 19 days prior


 16   to antigen.


 17             [Slide]


 18             This slide has the summary.  Compared to


 19   the European PRISM test, with the multiprep method


 20   DNA was detected prior to antigen in 34 of the 40


 21   panels, and with the Ortho DNA was detected prior


 22   to antigen in 38 of the 40 panels.  On the other 6




  1   panels DNA and antigen were detected in the same


  2   bleed.  Using the standard method compared to the


  3   PRISM test, DNA was detected prior to antigen in 38


  4   of the 40 panels, and on the same bleed in the


  5   other 2 panels.


  6             DR. NELSON:  Thank you.


  7             DR. HOLLINGER:  I just have a question.


  8   We talk about the mean and the median between the


  9   appearance of one antigen for nucleic acid before


 10   the other one.  But these are not tests that are


 11   done every day so the panels do not include--and to


 12   say 14 days or 19 days is a little misleading I


 13   think in some cases because theoretically if you


 14   draw blood today and you drew one 200 days from


 15   now, and that is all you had, you would say, well,


 16   it would detect things 200 days before one was


 17   positive and one was negative.  So, you know,


 18   unless samples are collected every single day for a


 19   period of time, this is somewhat misleading.  So,


 20   give me some feeling about the days between each


 21   one of these sample collections.


 22             DR. HERMAN:  It varies between the panels.




  1   So, the only data that I have available on the


  2   slides here is the one panel I showed you where the


  3   interval between bleeds I think was as few as a few


  4   days and as large as 30 or 40 days.


  5             DR. HOLLINGER:  It is not your fault in


  6   doing this; it is just that, unfortunately, that is


  7   how panels are set up.  But I think all of us have


  8   to understand that it may be much less than that.


  9   It won't be more than that but it certainly could


 10   me much less than that.


 11             DR. HERMAN:  That is absolutely correct,


 12   and that is a limitation of these studies so one


 13   shouldn't interpret this to conclude that if


 14   testing was done every day these would be the


 15   numbers.  This is an approximation, subject to the


 16   limitations of the interval between the bleed dates


 17   and the different panels.


 18             DR. NELSON:  Actually, often


 19   epidemiologists would assume a median--a mid-point


 20   seroconversion between the two days.  So, if you


 21   had a sample that was taken on day zero and day 14,


 22   you would assume that the one that the one that was




  1   positive on day 14 seroconverted on day 7.  That


  2   would be one way, a different analysis of the data


  3   than probably what you have done.


  4             DR. EPSTEIN:  Could I get you to clarify


  5   whether the apparent window period samples


  6   themselves were tested with HBsAg assays with known


  7   sensitivities of 0.1 ng/ml?


  8             DR. HERMAN:  Let me defer to my


  9   colleagues.  The window period cases from the


 10   clinical study?


 11             DR. EPSTEIN:  Correct, and you say you had


 12   five of them.  Right?  Two from the trials that


 13   analyzed sensitivity and specificity and the


 14   additional three--you have five available samples


 15   that could have been retested with the most


 16   sensitive available HBsAg assays.


 17             DR. STRONG:  I have to declare my conflict


 18   here because I am one of the clinical test sites,


 19   but we have been able to test four of the five.


 20   The most recent one hasn't been tested, but the


 21   four have been tested on the Ortho system 3, the


 22   new licensed test.  It picked up one of the four




  1   but missed three.


  2             DR. KUEHNERT:  Could you clarify what


  3   those are?  You said five.


  4             DR. STRONG:  The five window cases that


  5   are described here.  The first four were tested and


  6   it picked up the one that you would expect it to


  7   pick up, which is the high copy number sample.  The


  8   fifth sample, which hasn't been tested, also has a


  9   relatively high copy number and you might expect it


 10   to be picked up as well, with 37,000 copies.


 11             DR. NELSON:  Are there blood donor


 12   policies or recommendations for the interval


 13   between receiving hepatitis B vaccine and donation?


 14   Because that is a source of a false-positive


 15   infection for the surface antigen.  Are there any


 16   policies on that?


 17             DR. EPSTEIN:  FDA has no policy for donor


 18   deferral after HBV vaccination.  I am not certain


 19   whether AABB has a voluntary policy, but I think


 20   for non-live vaccines they would also not recommend


 21   deferring.  Someone else would need to clarify


 22   that.




  1             DR. KLEINMAN:  I think that is right, Jay,


  2   except that there have been some instances where


  3   people have had false-positive surface antigen so


  4   it is sort of unspoken that you might want to defer


  5   people for a week or so, but there is no absolute


  6   AABB requirement to do that.


  7             DR. LEITMAN:  Can I ask a different


  8   question.  I am not sure I am following what is


  9   going on in the 16 donors who were HBsAg positive,


 10   anti-core positive but multipool-NAT, COBAS


 11   negative.  Were those individuals individual donor


 12   NAT-negative?  In other words, are they infectious


 13   or are they not infectious?  If they are not


 14   infectious, do they just happen to have a lot of


 15   HBsAg particles circulating but not infectious


 16   virion?


 17             DR. FRANK:  I have to apologize, I can


 18   only hear parts of the question because of where I


 19   am standing.


 20             DR. LEITMAN:  I don't think I can do that


 21   again.  The 16 donors who were HBsAg positive,


 22   anti-core positive but NAT-multipool negative, were




  1   they retested in individual donor NAT to see if


  2   they have low level viremia or DNA nucleic acid, or


  3   are they, in fact, true HBsAg positive but not


  4   infectious because they don't have intact virion


  5   but just have particles circulating?


  6             [Slide]


  7             DR. FRANK:  You are talking about these


  8   16?


  9             DR. LEITMAN:  Those 16, yes.


 10             DR. FRANK:  The final status here is that


 11   they were HBV positive.  The question you are


 12   posing is are these 16 patients infectious.  They


 13   have surface antigen positivity and core


 14   positivity.  I have to defer to my colleague as to


 15   whether or not we tried to quantitate with NGI the


 16   HBV minipool NATs.


 17             DR. PIETRELLI:  I am Larry Pietrelli, from


 18   Roche Molecular Systems.  Yes, on those 16 donors a


 19   sample was supposed to be tested by alternate NAT


 20   and also by individual donation.  One donor was not


 21   tested by either assay.  Of the 15 remaining, 10


 22   were positive by ID-NAT, of which 7 sere also




  1   positive by alternate NAT.  Two were negative.  One


  2   was not done.  Of the 5 that were negative by


  3   ID-NAT, 2 were positive by alternate NAT; 2 were


  4   negative; and 1 was not tested.  The viral


  5   concentration was low, as expected.


  6             DR. LEITMAN:  Thank you.


  7             DR. KUEHNERT:  I have a follow-up question


  8   about those 16.  They were core antibody positive.


  9   Is there any way to quantitate that level of core


 10   antibody positivity?  Were any of these at an


 11   equivocal level?  I am just sort of getting at, you


 12   know, whether you had greater numbers whether some


 13   of these might have been close to a threshold for


 14   missing them by core antibody positivity.


 15             DR. FRANK:  I don't have that data.  I


 16   don't know whether my colleagues have access to


 17   that data today.


 18             DR. PIETRELLI:  No, we did not collect


 19   that information but we could go back to the sites


 20   to see what the S to CO was.


 21             DR. HOLLINGER:  I have a technical


 22   question.  Is there are a reason you used 24,000




  1   times g for pelleting?  I know others have used


  2   40,000 times g for pelleting for an hour at the


  3   enhancement stage, so to speak.  Also, why did you


  4   use 24 samples in your minipool or multipool?


  5             DR. HERMAN:  I can answer the first


  6   question about the centrifugation.  It is a


  7   limitation of the instrument that is widely


  8   available, which is a table-top centrifuge.  It is


  9   not quite an ultracentrifuge so it is more


 10   convenient and easier to use.  That was a choice of


 11   what equipment would be the most suitable for the


 12   multiprep procedure for all three virus targets.


 13             DR. HOLLINGER:  And the other question?


 14             DR. HERMAN:  The choice of pool size, I


 15   will defer to Mike.


 16             DR. STRONG:  The pool size is one that was


 17   selected for the HIV, HCV trials which were


 18   concluded a couple of years ago and were licensed.


 19   So, it was just to continue with the logistical way


 20   in which laboratories operate to be consistent with


 21   the other testing, and it is what the software does


 22   for us.




  1             DR. NELSON:  It is interesting that there


  2   are many places, in the Orient particularly, where


  3   core antibody testing is not done because of very


  4   high rates of positivity.  So, that would cast some


  5   limitation.  Even though the NAT assay would be


  6   useful under those circumstances, it wouldn't


  7   perhaps be as useful as continuing using the core


  8   antibodies as is done in the U.S. and Europe.  You


  9   had a question, Donna?


 10             DR. DIMICHELE:  Thank you.  I was just


 11   wondering if anybody can estimate, based on these


 12   data or based on your data, what the residual


 13   risk--in using your assay, if your assay was to be


 14   implemented with core antibody testing, what do you


 15   estimate the residual risk of transmission of HBV


 16   would be through transfusion?


 17             DR. FRANK:  That is a good question, and I


 18   can say that we have shown the yield today but in


 19   terms of discussion of residual risk, Dr. Kleinman,


 20   would you like to comment?


 21             DR. KLEINMAN:  Yes, Steve Kleinman.  I am


 22   a consultant to Roche Molecular Systems on this




  1   project.  The residual risk estimate is dependent


  2   upon what you think the risk is now prior to


  3   initiating HBV NAT.  We have estimated that risk


  4   through the REDS study on two occasions, one that


  5   was published in 1996 and then we kind of confirmed


  6   the same numbers about six years later as about


  7   1/60,000.  The risk estimate is based on the


  8   incidence window period model and the data for that


  9   model with HBV are not as strong as they are for


 10   HIV and HCV.  So, other people have estimated the


 11   residual risk now prior to NAT as being lower than


 12   1/60,000, being about 1/200,000.


 13             At any rate, if you accept the 1/60,000


 14   risk estimate, and then you take a look--the


 15   question is how much does this new assay decrease


 16   the window period.  If you draw on data that was


 17   published by Robin Biswas and colleagues on their


 18   panel testing of decrease of the window period, you


 19   get about a 10-day decrease of the window period in


 20   that study, and in this study it looks like the


 21   window period decreases by 17 to 20 days based on


 22   the panel.  But, as Dr. Hollinger mentioned, that




  1   is a maximum case because you don't have closely


  2   spaced samples.


  3             At any rate, if you take a 10-day


  4   lessening of the window period and you apply it to


  5   what we think the current window period is, about


  6   59 days, and you work this through with the window


  7   period and the incidence of HBV, you would probably


  8   get that the residual risk is still 1/200,000 to


  9   300,000 even with this test being implemented.


 10   Actually, it may even be 1/100,000.  So, you are


 11   removing some of the risk but because you are only


 12   cutting the window period back 10 days out of a


 13   potential 59 days, the likelihood is that most of


 14   the risk still remains even with minipool-NAT and


 15   that in order to decrease that risk further you


 16   have to go to either smaller minipools or


 17   individual donation NAT, which would buy you more


 18   in terms of lowering the window period.  So, you


 19   are making an incremental step in decreasing the


 20   risk; you are not decreasing the majority of what


 21   we think is the residual risk.


 22             DR. NELSON:  These data are heavily




  1   dependent on what the window period is, which may


  2   vary in different populations.


  3             DR. STRONG:  Yes, they are dependent on


  4   the incidence and what you think the current window


  5   period is, and that is something that there hasn't


  6   really been good data on.  We have estimated the


  7   current window period as 59 days based on some


  8   older work from the TTVS study and then, through


  9   REDS, we made a revised estimate of the window


 10   period through mathematical modeling to be about 45


 11   days now, the infectious window period.  So, these


 12   are estimates.  We don't really know what the


 13   current window period is so it is a little bit


 14   difficult.  We don't know exactly how much we


 15   shortened it so that is why I think you have to say


 16   that we can't make the kinds of precise estimates


 17   that we have made about HCV and HIV.


 18             DR. DIMICHELE:  Can I just ask you a


 19   follow-up question then?  What is the


 20   estimate--let's say given that residual risk, what


 21   is the estimate of the impact of vaccination,


 22   widespread vaccinations now going on in the younger




  1   population with respect to when they become blood


  2   donors?  Does anybody know how the risk of


  3   transmission will be decreased just by vaccination,


  4   widespread vaccination alone?


  5             DR. KLEINMAN:  I don't know the answer to


  6   that.  I will answer kind of a related question,


  7   which is what is the risk to recipients?  As more


  8   recipients get vaccinated, then they should be


  9   immune to a challenge, you would think


 10   theoretically even if they were exposed.  However,


 11   most of our recipients are in the older age group,


 12   60 and above, and some have compromised immune


 13   systems.  So, I don't think that vaccination is


 14   going to protect much of the recipient population.


 15   Your question is different, whether it makes donors


 16   less likely to acquire new HBV infection and I


 17   don't know that anybody has worked that through


 18   mathematically.


 19             DR. ALTER:  Harvey Alter, NIH.  The


 20   mathematical models have been extremely useful but


 21   there has always been a little discomfort with HBV


 22   in that you just don't see the cases as frequently




  1   as you would have predicted from the model.


  2             It seems to me now that if you make the


  3   assumption that the real risk would be coming from


  4   HBV DNA positive donors, you could actually


  5   determine what the actual risk is now by taking the


  6   best or maybe all the different HBV DNA assays and


  7   testing these samples as single donations.  If you


  8   are picking up 1/350,000 now in a minipool format


  9   you will pick up even more in an individual donor


 10   format.  That theoretically would be the risk.  If


 11   we can exclude those donors, that risk would be


 12   removed.  It seems too simplistic to be true but I


 13   would like to hear a counter argument.


 14             DR. BUSCH:  Mike Busch.  I wanted to


 15   comment on the vaccine issue.  In this study, I


 16   think of the yield cases one of the yields that is


 17   in the original clinical trial, clearly by history


 18   vaccinated person, had an anamnestic anti-surface


 19   response, low viremia in the setting of the index


 20   sample.  I think one of the additional yield cases


 21   also had anti-surface on index.  It is interesting


 22   that the data from Taiwan is showing that when they




  1   have applied NAT to donor screening--this is a


  2   massively vaccinated population--they are seeing as


  3   these young people, you know, enter adolescence and


  4   in their 20s, they are seeing a fairly high rate of


  5   the same kind of phenomenon.  These are essentially


  6   vaccine breakthrough infections.  So the vaccine,


  7   rather than being sterilizing--still people are


  8   getting exposed and infected and, in a sense,


  9   boosted from a wild exposure.  In their setting the


 10   vast majority of their NAT yield are people who


 11   have been vaccinated and have anti-surface and


 12   never develop surface antigen, never develop


 13   chronic infection.  So, the critical question of


 14   whether these could transmit I think is still on


 15   the table.


 16             DR. STRONG:  Another point to follow-up on


 17   what you were commenting on, on the value of


 18   anti-core, I think this clinical trial also


 19   demonstrated, as has been published, a small yield


 20   of DNA positives from the anti-core positive group,


 21   roughly in the same area of 5.5 percent or


 22   thereabouts.  Also, I think it demonstrated a




  1   difference in specificity with the anti-core assays


  2   between the two licensed tests.  So, there is


  3   clearly a large number of false-positive anti-core


  4   donors that we have excluded on the basis of


  5   false-positive reaction.  I think those of us who


  6   have been involved in this trial, we have been


  7   anxious to try to push the sensitivity of the assay


  8   to allow us to reenter a lot of those donors


  9   because it is a very large number.


 10             DR. HOLLINGER:  I just want to follow-up


 11   briefly on what Mike said.  I mean, this shouldn't


 12   really surprise us very much, if you look at most


 13   of the vaccine trials and follow patients for ten


 14   years or so you will see anti-core appearing in


 15   these patients.  I just mentioned that anti-core is


 16   a marker of viral replication.  So, somewhere along


 17   the line these patients will probably have DNA in


 18   the bloodstream.  The issue is whether it is


 19   infectious or not and that is, of course, I think a


 20   very critical issue with it.


 21             Then I just wanted to ask a question


 22   either of Harvey or Steve about how many cases are




  1   recorded now of transfusion-transmitted hepatitis


  2   B?  I don't see very many.  I don't want to get


  3   back to this argument we had about 20 or 30 years


  4   ago when we said there was no hepatitis C or


  5   non-A/non-B hepatitis when really there was a lot.


  6             DR. KLEINMAN:  No, I think that is exactly


  7   right, Blaine.  There are very few post-transfusion


  8   hepatitis B cases reported.  As you know, it is a


  9   question about whether those people become


 10   symptomatic.  So, there may be asymptomatic cases.


 11   Some may go on to be chronic carriers.  That is


 12   certainly possible.  Secondarily, even if a case


 13   occurs, is it recognized by the clinician?


 14             It is certainly a correct statement that


 15   we don't see the number of HBV clinical cases


 16   reported as post-transfusion cases as the


 17   mathematical models would predict.  So, then you


 18   have two potential explanations.  One, the


 19   mathematical models are wrong and these cases don't


 20   occur or, two, the cases occur and they are not


 21   recognized and reported.  I don't think we have a


 22   way to sort out which one of those hypotheses is




  1   correct.  Harvey's point is that we would have to


  2   take a different approach to estimate risk, that


  3   is, we would have to use the most sensitive DNA


  4   assay available, hoping we could pick up one


  5   copy/ml or even less.  By that, we could basically


  6   make the equivalence that if we have a DNA-emic


  7   specimen that equates to an infectious specimen,


  8   and we could do that kind of study that was done 15


  9   years ago or I guess 17 or 18 years ago for HIV,


 10   but that would be an extremely laborious and


 11   expensive study to do.


 12             DR. NELSON:  I think we will take a break


 13   at the moment and then come back for the open


 14   public hearing.  Maybe 20 minutes.


 15             [Brief recess]


 16             DR. NELSON:  The meeting is now in session


 17   again and we will start with an open public hearing


 18   on the topic presented of HBV DNA testing.  Before


 19   we start I will read this statement.


 20             Both the FDA and the public believe in a


 21   transparent process for information gathering and


 22   decision making.  To ensure such transparency at




  1   the open public hearing session of the advisory


  2   committee meeting, FDA believes that it is


  3   important to understand the context of an


  4   individual's presentation.


  5             For this reason, FDA encourages you, the


  6   open public hearing speaker, at the beginning of


  7   your written or oral statement to advise the


  8   committee of any financial relationship that you


  9   may have with the sponsor, its product and, if


 10   known, its direct competitors.  For example, this


 11   financial information may include the sponsor's


 12   payment of your travel, lodging or other expenses


 13   in connection with your attendance at the meeting.


 14   Likewise, the FDA encourages you at the beginning


 15   of your statement to advise the committee if you do


 16   not have any such financial relationships.  If you


 17   choose not to address this issue of financial


 18   relationships at the beginning of your statement,


 19   it will not preclude you from speaking.


 20             With that, Mike Busch?


 21                       Open Public Hearing


 22             DR. BUSCH:  Thank you.




  1             I wanted to just briefly touch on three


  2   items.  One is just one slide sort of summarizing


  3   the different context of some of the issues that


  4   were raised in the questions as to the value of HBV


  5   minipool-NAT both relative to the other NAT systems


  6   and ID-NAT.  Then in most of my comments I want to


  7   focus on the issue of can we drop either anti-core


  8   surface antigen and, if not or if the data doesn't


  9   support it at this point, what further studies


 10   would be useful to address that.  I have Roche's


 11   computer here--


 12             DR. NELSON:  That sounds like a conflict


 13   of interest!


 14             [Slide]


 15             DR. BUSCH:  The committee does have this.


 16   This is a published analysis of cost-effectiveness


 17   and I am not going to talk at all about sort of


 18   cost-effectiveness but I just wanted to show one


 19   slide that sort of emphasizes the relevant clinical


 20   impact of these three viruses.  We tend to talk


 21   numbers as if each of these viruses is equivalent


 22   and the NAT yield is equivalent, but what this is




  1   showing is the number of quality life years lost


  2   per transmission.


  3             What you can see is that for HIV it is


  4   about 7 quality life years to the average 60 years


  5   old transfusion recipient.  Transmission of HIV


  6   would cost that person 7 quality life years; HCV,


  7   0.6 and HBV, only 1.6.  This is because most


  8   recipients are older people who naturally resolve


  9   the acute infection of HBV, and even if chronic it


 10   rarely evolves to significant morbidity/mortality


 11   over the persistent life span of the average


 12   recipient.  So, you can see that essentially HBV is


 13   one-quarter as clinically important as HCV and 44


 14   times less important than HIV.


 15             The other point I wanted to make has to do


 16   with this issue of going from current serologic


 17   testing to minipool versus serologic testing to


 18   ID-NAT, and how much of the yield that would be


 19   accomplished by going to ID-NAT is detected by


 20   minipool-NAT.  For HIV we had very sensitive


 21   antibody tests in place so NAT by minipool closes


 22   the window by about 5 days and ID closes it by




  1   another 5.  So, we picked up about 50 percent of


  2   what could be picked up but the risk is very low.


  3             With HCV, because of the long plateau


  4   phase, 95 percent of the window period is detected


  5   by minipool-NAT.  With HBV, because of the slow


  6   ramp-up, at least the model estimates and some of


  7   the empiric data like in the Biswas paper would


  8   suggest that minipool is only picking up about 23


  9   percent or a quarter of what could be detected by


 10   ID.


 11             Next slide.  The issue of anti core and


 12   could we get rid of anti core--there are three


 13   studies that have been either fully published or in


 14   abstract form, actually the Roche data, that have


 15   looked at high numbers of donations that were


 16   anti-core only reactive--so surface antigen


 17   negative, anti-core positive--and subjected then


 18   HBV NAT.  Each of these studies, as you can see,


 19   has given almost identical rates both on a per


 20   anti-core and, if you extrapolate, on a


 21   per-transfused unit basis of detecting DNA in


 22   donations that were surface antigen negative.  So,




  1   about a quarter to a half percent in all these


  2   studies.


  3             What is interesting is that in each of


  4   these studies there was quantitation done in one


  5   context or another, and all of these viremic


  6   anti-core reactive units, surface antigen negative


  7   have very low viral loads.  In Roche's data, for


  8   example, 92 percent based on previously presented


  9   work of their viremic anti-core only were negative


 10   by minipool but only detected by ID-NAT.  So, these


 11   are very low viral loads that really would require


 12   ID-NAT to detect.


 13             [Slide]


 14             This is just a summary and I am not going


 15   to walk through this but with respect to low


 16   viremic anti-core reactive donations or anti-core


 17   only's there are a number of studies that have


 18   documented transmissions, rare but there are


 19   transmissions from these low viremic units.  Again,


 20   I don't have time to go into these but we also know


 21   that in liver transplantation from anti-core


 22   reactive, surface antigen negative donors very




  1   frequently transmits the virus.  So, these people


  2   who have low viremia do harbor infectious virus


  3   and, obviously, the anti-surface status is a


  4   factor.  But I personally don't think there is any


  5   prospect for dropping anti core.


  6             [Slide]


  7             Next slide.  This was alluded to earlier.


  8   There was one study where purposeful transfusion of


  9   these anti-core reactive, DNA positive, surface


 10   antigen negative units into chimps was done.  This


 11   was by Fred Prince and was published.  These did


 12   not transmit.  Three patients who had remote


 13   hepatitis B, persistent anti-core, negative


 14   antigen, low-level DNA.  However, the caveat is


 15   that all three of these donors had anti-surface and


 16   the volume inoculated into chimps was quite small,


 17   1 ml.  So, I think larger studies with larger


 18   volumes may be indicated to potentially explore the


 19   infectivity of these units.


 20             [Slide]


 21             Next slide.  In terms of dropping antigen,


 22   which I think is the more likely and I believe




  1   optimistically we will get there, I think clearly


  2   for these viruses like HIV, HCV, HBV where there is


  3   not only the acute phase but there are chronic


  4   carriers and some of these chronic carriers have


  5   low-level infectious viremia the optimal paradigm


  6   really is the combination of a front-end sensitive


  7   direct assay and a serologic test for antibody.


  8   For HBV we have both antigen and NAT which could be


  9   viewed as redundant, analogous to P24 antigen in


 10   HIV NAT.  Clearly, on the front end we know, and


 11   saw from Roche and other studies, that HBV NAT is


 12   clearly more sensitive than antigen during the


 13   acute phase.


 14             Next slide.  But the problem is, as Blaine


 15   summarized, HBV is really an unusual virus in that


 16   it produces in chronic infections amazingly high


 17   levels of circulating antigen in the absence of


 18   infectious particles.  Therefore, this excess


 19   antigen can occasionally be detected in the absence


 20   of detectable DNA by minipool or even ID-NAT.  I


 21   want to present a recent study on that.  Therefore,


 22   to really be sure that we are not taking a step




  1   back and dropping antigen when we have NAT, we need


  2   to do large studies and I think Roche's data is


  3   very impressive but whether it is enough is for the


  4   committee to decide.  But these studies need to


  5   look at these issues in a different context,


  6   obviously, the front-end window period anti-core


  7   negative as well as chronic carriers.  But also I


  8   think they need to reflect populations with


  9   different endemnicity and different routes of


 10   transmission; populations that have been vaccinated


 11   because we are, as I mentioned earlier, seeing


 12   vaccine breakthrough infections, and how would they


 13   play out in terms of antigen versus DNA; and also


 14   genotypes and mutants.


 15             Next slide.  Just to contrast, one of the


 16   big differences in acute phase versus chronic is


 17   the relationship between the DNA load and the


 18   antigen reactivity.  This is one of a number of


 19   studies.  This is a Japanese study.  During this


 20   acute phase these were all anti-core negative


 21   front-end infections.  They were actually all


 22   detected, 181 units detected as negative by the




  1   Japanese particle agglutination antigen test.


  2   Actually, 105 of these were reactive by the PRISM


  3   assay.  So, only a subset, about 40 percent of


  4   these remained negative.  But what you are seeing


  5   here is a very nice linear regression relationship


  6   between the reactivity in the antigen test and DNA


  7   load.  That is typical of the front-end acute


  8   viremic phase.


  9             Next slide.  I want to summarize now a


 10   paper that is in press in collaboration with Mary


 11   Kuhns and Steve Kleinman from the REDS group, where


 12   we took 200 antigen positive, anti-core positive


 13   donations in REDS.  We first subjected them to


 14   quantitative HBV DNA using the Amplicor Roche


 15   assay.  This assay has a sensitivity of 400 copies,


 16   and 64 percent of the antigenic units had DNA above


 17   400 copies.  Those samples that were negative, the


 18   72, were taken through a more sensitive assay that


 19   had a sensitivity of about 65 copies, which is


 20   probably comparable to minipool type NAT, and 12


 21   remained negative even at that level.  Those 12


 22   samples were taken to a very high sensitivity,




  1   essentially 1 copy/ml assay, high volume, and still


  2   there were 6 that were negative.  So, we ended up


  3   with still 3 percent negative even after taking it


  4   through sequential increasing sensitivity assays.


  5   So, these are the chronic carriers in whom DNA is


  6   undetectable with at least one time point and one


  7   high sensitivity assay.


  8             Next slide.  This is showing the


  9   relationship between antigen reactivity and viral


 10   load.  Unlike that nice linear relationship, here


 11   there it is essentially a scatter plot.  These are


 12   the 3 that were negative by all NAT.  These are the


 13   ones that were detected only by the high


 14   sensitivity.  These are the Amplicor negative but


 15   single input, not as sensitive, positive.  And,


 16   these are the quantifiable.  You can see that


 17   relationship is not observed in chronic carriers.


 18             Next slide.  That is people who have


 19   anti-core.  I think Roche did a nice job of sort of


 20   saying that if we keep anti-core the issue is not


 21   that problem with anti-core positives, antigen


 22   positives having absence of DNA.  It is really are




  1   there donations that are antigen positive that lack


  2   anti-core that are truly infectious and not


  3   detectable by NAT?


  4             In the REDS program and in other studies


  5   varying 1-5 percent of antigen positive donations


  6   lack anti-core.  Now, when these are worked up most


  7   of these are window phase seroconverters so they


  8   have high-level DNA very consistent with acute


  9   infection.  But in a proportion of DNA negative,


 10   and we saw those cases in Roche, the issue is


 11   studying enough of those cases to sort out are they


 12   true HBV infections, that either there is a


 13   mutation not detected by NAT or individuals who


 14   have failed to form anti-core, chronic carriers who


 15   have low-level DNA and have failed to form


 16   anti-core.  Do they lack contamination, which I


 17   think most of them are?  Are they persistent,


 18   non-specific antigen reactivity, and Roche showed


 19   us an example of that?  Or, are they possibly


 20   recent vaccinees?


 21             Next slide.  This is three studies, two


 22   published papers and some unpublished data from




  1   REDS that have looked for these people who are


  2   antigen positive, anti-core negative and either


  3   lack or have very low-level DNA.  I am not going to


  4   go through these in detail but in Japan one study


  5   identified three such donors who had no history of


  6   vaccination and who remained low-level PCR-reactive


  7   with only a very high sensitivity enhanced assay;


  8   remained anti-core non-reactive, so never


  9   converted; and, again, remained antigen positive


 10   and very low-level PCR-reactivity for over a year.


 11             The French group, Couroce's group reported


 12   two donors from endemic countries as well, with 0.1


 13   percent of their antigen-positive donors who had


 14   this pattern of persistent antigen without


 15   anti-core and very low-level HBV DNA, and no


 16   history of vaccination or immunosuppression and no


 17   evidence of mutations to explain the failure of


 18   this person to form anti-core.


 19             In REDS--this is a cross-sectional study


 20   so we weren't able to do follow-up, but we had 20


 21   antigen-positive donors who lacked anti-core.


 22   Sixteen of these were positive for DNA and were




  1   probably typical acute phase, but 4 were negative.


  2   Three of these were probably contaminations.  They


  3   had no other serologic markers, probably represent


  4   surface antigen carryover.  But one had anti-e and


  5   could represent an atypical carrier.


  6             So, just a caution that there are some


  7   studies, particularly from endemic settings where


  8   there may be people who have antigen in the absence


  9   of anti-core and low-level or absent DNA, and I


 10   think we just need to study these further.


 11             Next slide.  Just in conclusion, I do


 12   think that we would see a small incremental yield


 13   by minipool-NAT but the clinical impact of that I


 14   think needs to be considered as well as the yield,


 15   and we also need to view the context of what would


 16   we get were we to get all the way to ID-NAT.


 17             I am optimistic that we will eventually be


 18   able, with ID-NAT or very small pools or high


 19   sensitivity HBV DNA, to get rid of antigen,


 20   however, I think this is going to require further


 21   studies.  Particularly, we are capturing units


 22   daily that could be worked up to accrue large




  1   numbers with high volume of the plasma components


  2   from these various sort of atypical patterns.


  3   Particularly relevant is that I do think the


  4   combination of NAT anti-core is the ultimate goal.


  5   Particularly relevant are these samples that are


  6   antigen positive, anti-core negative, and really


  7   both studying these samples and particularly


  8   enrolling and following the donors, as Roche did,


  9   to determine whether these are false positives or


 10   contamination.  Thank you.


 11             DR. NELSON:  Thank you.  Comments?  Yes,


 12   Harvey?


 13             DR. KLEIN:  Mike, we heard from Dr. Alter


 14   earlier that, with the problems in trying to


 15   calculate what the residual risk is, it might be


 16   reasonable as a start to do a study simply by


 17   taking 400,000, 600,000 specimens and using a


 18   single unit detection system along with the


 19   serology.  I would just like to know what your


 20   opinion on doing that kind of a study might be.


 21             DR. BUSCH:  Yes, I think that is a good


 22   idea.  I mean, we have the Biswas paper included




  1   comparing assays on window period panels neat


  2   versus minipool.  That is kind of these model


  3   estimates that minipool picks up only a quarter of


  4   what might be detected neat.  But, yes, I think


  5   such a large-scale study either with neat--you


  6   know, one of the issues with Roche's system is


  7   their multiprep high extraction volume assay--we


  8   saw the data that was comparing the multiprep pool


  9   of 24 versus the standard prep neat, but had they


 10   applied their high sensitivity multiprep neat,


 11   which is what was done in the Biswas paper, they


 12   would have seen a greater incremental closure than