Continuous Cell Lines as Substrates for Biologicals

Conferencer Reports syncytmm m i n b m o n assays A Osterhaus showed data m the FeLV system, winch proved that both monoclonal and polyelonal antibodies rinsed against either monoclonal or polyclonal antiviral antlbo&es, reduced anlaviral antlbo&es m the absence of viral antigen m nuce D Davies presented a paper on the use of synthetic peptldes as lmmunogens, focussing special attention on the recently developed criteria for the sectaon of B- and T-cell epltopes In the session on antigen preparation, A Osterhaus gave an overview of the &fferent systems available at present for the enhancement of the immunogemcity of viral antigens Speoal attenuon was g~ven to multamenc presentation forms of viral proteins hke mlcelles, hposomes and ISCOMs Data obtmned m collaboratmn with B Morem and O Jarrett m the FeLV system, showed that FeLV ISCOMs also mduced protective ]mmumty m cats The same amounts of viral anlagen presented with a conventional adjuvant system clearly gave inferior responses In the session on assessment of tmmune responses, P Mortimer and H Hmsman presented their extensive experiences w~th the evaluation of the first and second generatmns of antibody assays and the tests used to confirm the results of these serological tests It was concluded that the second generation of serological tests was supenor to the first generatmn and that, as a routine, more than one confirmatory test should be used So-called false posmve reactmns, winch showed mmnly antlbo&es reactive with p24 of HIV1, are hkely to be the express]on of other related retroviruses occurnng m man Two papers were presented on the subject of cell-medmted lmmumty to HIV m man A McMlchael speculated on the role of cytotorac T-lymphocytes and their role m controlhng or contnbutmg to the pathogenesls of the disease He showed that cytotoxlc T-lymphocytes with specificity for the gag protein are present m seroposmve donors, using an autologous lymphoblastold cell line mfected with a vacclnia-gag recombinant virus, as a target Based upon expenences in the influenza system, he speculated on the posslbihty of predicting certain T-cell ep~topes on the bas~s of the respectwe HLA structures and on the possible prognosUc value of the presence of cytotoxac T-lymphocytes in the circulation In addition, M Plata reported on a system permitting the quantitative analysis of HIV-specific cytotox~c Tlymphocytes using bronco-alveolar lavage cells from the lungs of seroposltIve patients with lymphocytic alveohtls These cells were able to kdl autologous HIV-mfected cells and mouse p815 cells transfected with both HIV and HLA

class I genes These cells were consldered classsc cytotoxsc T-lymphocytes since they recogmze and kill HIVinfected or transfected cells m a class I H L A restricted manner and express the CD3 and CD8 surface markers In the last session winch dealt with chnlcal tnals, P Minor gave a paper on essentml prechmcal tests and mentioned a number of points to consider m the development and the manufacture of a future AIDS vaccine He stressed the importance of the implementation of GLP and GMP standards from very early stages of development onward and advised research workers to consult w~th national control authontaes at an early stage E Miller gave an introduction to the design of phase I, II and III clinical trials, in winch ethical and statistical considerations were reviewed The selection criteria for subjects m the respectwe trials and the evalualaon of chmcal reaclaons and of the results of laboratory mvest]gatlons were discussed and it was estimated that it would not be possible to conduct more than one phase I trial m the U K Therefore the careful selection of a potential vaccine will be crucml It was also concluded that for postexposure vacclnatmn trials, the methodology would be much simpler J Smith also pointed to the range of problems to be expected in organizing chmcal trials The potentaal vaccines should, from the very beginning, be developed and produced in hcensed premises, according to internationally accepted GMP standards Standardization reqmrements and the need for an independent assessment by national control anthontles hke NIBSC were considered essential and all chmcal trials should be conducted with a chmcal

tnal certafice and etincal comrmttee approval For tins reason a group has been formed under the auspices of the MRC in the U K to develop gmdehnes for tins work Since only a limited number of phase III trials can be conducted, only the most pronuslng preparatsons should be considered and a central apprmsal or approval orgamzatlon should be involved To ensure optimal international benefit, centralized coorchnation centres, field umts and expert laboratones should participate m all cases G Scinld presented data on the role of the W H O and the U K &rected programme on AIDS The MRC programme, winch has a £15 nulhon budget for the next 3 years and may be expected to expand further d u n n g the cormng 10 years, is p n m a n l y &rected towards the development of vaccines and novel antwlral drugs All the different approaches winch might eventually be expected to be successful wdl be sponsored in tins programme A collaborative network of informal worlong groups has been established, winch operates apart from the mmn committee, on vanous scientific subjects and to winch collaborators from other countries can be invited The W H O programme, in winch p n m a n l y the InsUtut Pasteur (France), the MRC (UK) and the NIAD (USA) participate, mainly focusses on the estabhshment of collections of well documented panels of virus ~solates, infectious molecular clones, mono- and polyclonal antlbo&es and cell hnes These will be made freely available to all research restitutions, winch m turn will be encouraged to participate in the programme

A. Osterhaus

Continuous Cell Lines as Substrates for Biologicals Joint Meeting of WHO, lABS (Cell Culture Committee Subsect=on) and ESACT, 26-29 May, 1988, Wash)ngton, USA The meeting was attended by about 200 delegates, mainly from the USA and Europe Both the regulatory agencies and Industry were well represented in association with a sampling from the universities After a day of plenary lectures, which covered basic aspects, the second day consisted of four 'workshops', which were formal presentations of research reports or papers covering adventitious contaminants, tumorigenicity, cell characterization and banking, and experiences with biological products The final day received reports of the workshop session chairmen, with

their views on how the meeting sensed the &rectlon of future thlnkmg with regard to the regulation of blologlcals made from anmaal cells m culture The following outstandmg points emerged for further cons~deratlon Should it be shown that the most efficient producuon system for a specified biological reqmred the use of a continuous cell line (CCL) and that CCL was known to be a tumongemc agent, then it would not be necessary to provide further evidence for such tumorlgenlcaty Where other things are equal, a CCL

Vaccine, Vol 6, October 1988 461

Conference Reports winch is known not to be tumongemc is to be preferred, in which case tests would be required to confirm such nontumongemoty CCLs of lower populataon doubhng levels, (specifically not passage levels), are less hkely to be tumorigenic and are to be preferred It was reco£mzed that manufacturers of biologleals would present to a regulatory agency data derived from all the conceivable test systems available to characterize the cell substrate, the production system and the product, (primarily to obviate the need for a protracted, time-consurmng correspondence with the agency) Such overkall was, however, probably unnecessary and there should be clear reqmrements to remove older less reliable or demonstratlve tests with newer more sensmve and robust descriptors of the process and its materials ATCC, E C A C C and W H O collaborate on the comprehenswe characterization and storage of the super-master cell bank of CCLs for distribution at or below cost to those who have a need to use such cells for the production of btologlcals, the cells to be so stored are Vero, BHK-21, (already m process of such storage under the aegis of WHO), MRC5 and WI38 The D N A fingerpnntmg techmque should be used for cell characterization work in apposition to karyology winch yields relatively meamngless data wath CCL preparations Tests for endogenous retroviruses should be multifaceted, as any one test is inadequate to characterize such viruses

Cells winch house endogenous retroviruses can be used in bioproduct generation providing it can be shown that, with 'spiked virus' dummy runs, the concentration and purification processes remove adequate amounts of virus It should be noted that although any individual operataon is capable of decreasing virus tItre by a measured amount it is not necessanly valid to conclude that such processes used together will decrease the virus tltre by the sum of the individual decrements It should also be noted that whereas ~20% of m u n n e hybrldomas contain retrovlruses which can be shown to be infectious in cell culture, the CHO cell lines contain retrovirus-hke particles, but all efforts to recover infectious virus from such cells have, so far, failed Putative cell substrates should be examined for adventitious v~ruses and these should be ehminated during the production process by either physical or chemical means It should be noted that such viruses, while not necessarily present in the cell substrate, may well be introduced, particularly via fetal calf serum, which often contains both mycoplasma and the virus responsible

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for bovine viral &arrhoea This latter has been shown to be present, although it and the mycoplasma can be ehrmnated by gamma irradiation (2 53 5 Mrad cerafied by dosimetry) It is important to consider that cellculture-based systems should be used m place of whole animal expenments wherever possible, not only for reasons of cost and convenience but also for etincal reasons With regard to cells with engineered genes it is important to check the final product for the presence of the vector, vector-associated proteins and the possibihty that the lnsertaon of the vector into the host chromosome could have activated some host gene or endogenous varus sequence There was a general consensus that cellular D N A posed a vamshmgly small risk of causing abreactlons, whereas the clear threat was from those v~ruses winch could be present m the final product and winch have been shown to be tumorlgemc It should also be noted that such viruses, to be tumongemc, have to be present in the form of a complete genome (m one or many pieces) to be effective In terms of the quality of the final product, there should not only be <100 pg D N A present but also the matenal should not have antigemc properties when inJected into the body of the target ammal Tins is particularly Important for those matenals whose efficacy depends on repeated apphcation The challenge to the industry is to manufacture and market a cost-effective product which, when all the testing has been taken into account, can sUll stimulate a demand for its use This could be quite difficult in the area of live or other virus vaccines Europe, in formulating its regulations governing the use of drugs and blologlcals, should not reinvent the wheel but rather should adopt existing and acceptable guidelines and where necessary make the modifications to smt the local conditions Tins, it is hoped, would improve conditions for international trade It was generally held to be remarkable that the opinion of many people in the field was that the CCL route to bioproducts would meet such resistance by the regulatory agencies that it would be foolhardy to embark on a project which involved such materials It would now appear that, after the licensing of the OKT3 monoclonal antibody, the CHOdenved tPA and the EB-vlrus-transformed lymphocyte (Namalwa) for umterferons, the way is open for the more general use of CCLs in the manufacture of products for both prophylactic and therapeutic application as and when such cell substrates are characterlzed and controlled in a manner which is

suffioent for the purpose for which they are intended The reviewer was unable to attend all four of the workang groups and so can only report on those papers which he was able to attend, four of winch were of particular interest The paper by Dr Moyer showed clearly that it IS not possible to produce turnouts m her rodent test systems with parts of the SV40 virus genome, the whole genome or a mLXture of all the chfferent parts of the genome is needed for tumongemclty Dr Wurm demonstrated the products of his method to detect the site of the insertion of a gene, (amphfied or not), into the chromosome Tins was acineved by using a blolanylated D N A probe winch was made evident by sequential layers of fluorescem-conlugated axadln and antabody coupled to fluoresceln-conlugated avldln Dr Albert used flow fluorocytometry to show the health of the cells, by interpretlng Ins data to ln&cate the number of cells in S phase at any gwen state of the culture Dr Doyle demonstrated that the D N A fingerpnntlng techmque (of paternity suit fame) would provide a very powerful techmque for the characterization of cells for both cell bankers and manufacturers A number of other issues were aired and need to be more thoroughly addressed in order to make progress in this difficult area The case by case evaluation by the agencies creates a situation which defines an area where mdlvaduals can operate and all other areas are out of hmits A n alternative approach would be to define a set of pnnclples deternumng the minimum requirements which, once met, leave other possibihtles open for action The present system is hke that of the insect exoskeleton and the other is more akm to the endoskeleton It would be pertinent to operate on the restrictive basis untal there has been such an accumulation of knowledge and practice that both the agencies and socaety are confident and comfortable with the practaces and products of the manufacturer of Inologlcals, and then change to the less restrictive approach based on p n n o p l e s winch can be culled from the practaces winch have proved their value to socaety A second and fundamental issue winch was not addressed was the way m winch the balance between costs versus benefits is to be evaluated Should the regulatory agencies be the sole repositories of wisdom in tins regard 9 Clearly the manufacturers cannot stand in tins position Is it the job of responsible individuals drawn from or representing society 9 On those occasions when a drug or biological has caused damage to those who have been treated, sooety has responded by urging those who have responsibility for the affairs of socaety to

Conference Reports

ughten their grasp of the regulatory process winch, in pracUce, has promoted yet more restncUve measures Those m the Scientific and Techmcal commumty (lncludmg umverslUes, manufactunng mdustry, and research workers and offictals m the regulatory agencies), who have the knowledge and expenence, should bring to the attentmn of socmty both the costs and the benefits of the new materials, so that soaety can carefully adopt those new matenals m a way m winch the benefit can be clearly appreciated and the cost accommodated. In a m v m g at an acceptable process it m often necessary to define the mater-

mls mvolved Such a reqmrement for defimUon presupposes that we know mstmcUvely when we have used those verbal expressions winch adequately describe that winch we seek to dehneate But it is always possible to rinse those borderline cases winch reqmre a revamon of the original detimtlon At some point it becomes counterproductive to continue thts relterauve refinement process What ts the pomt~ How far should we go m cell charactenzaUon or defimUon~ When should we lump and when should we spht~ U t l h t a n a n guidelines winch would help m tins area could well provade some tmprovement m negotiating the many requtrements

winch the manufacturer of btolog~cals has to overcome to brmg a product to the market place The meeting provided much on winch the future can be bruit There was a resoluUon to have a further meeting m 1990, for m tim area progress ts mewtable, w~th some 300 monoclonal antibody matenals seeking hcence and with a corresponchng number of recominnant products, the pace m ever acceleratang It is hoped that the conference will facthtate progress m b n n g m g to the market place the latest and the most advanced of the btolog~cal products from the manufacturers

R.E. Spler

/

Structure and Function of Domestic Animals Dr W Bruce Currie Associate Professor of A n i m a l Physiology, C o m e l l University, USA

One of the hrst structure and function books focusing on domestic animals to be written by a physiologist rather than an anatomJst * Provides a comprehensive introduction for courses m anatomy and physiology or general animal science * Thorough explanations are provided for all technical terminology and are easily located from the index * Each chapter is complemented by exercises and test questions *

C o n t e n t s : T h e structural n a t u r e of a n i m a l s • The basis of a n i m a l production • The excitable tissues - nerve and m u s c l e . C h e m i c a l c o m m u n i c a t i o n • Vital life support systems • Digestive m e c h a n i s m s • M e t a b o l i s m . G r o w t h and d e v e l o p m e n t • Stress and defence m e c h a n i s m s • Reproductive m e c h a n i s m s • Lactatlonal biology • Appendices • Glossary • Index September 1988

254

x

179 mm

Hardcover 464 pages approx

0 409 90044 3 Illustrated £32 95 approx

For details of other Butterworth Soentff~c t~tles please contact the appropriate office Orders should be sent to the appropnate office hsted below The UK headquarters serves all UK and overseas markets except where there is a local Butter~,orths office United Kingdom Butterworlhs Borough Green Sevenoaks Kent TN 15 8PH England

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Vaccine, Vol 6, October 1988 463