REVIEW Folia Microbiol. 42 (2), 105-112 (1997)

Multivalent Vaccines: Prospects and Challenges* R.E. SPIER University of Surrey, Guildfor~ Surrey, UK Received August 12, 1996 ABSTRACT. T h e advantages and disadvantages of combination vaccines are set out with a clear indication that, although there are m a n y (described and discussed) influences on in the immunogenicity of a vaccine it would still be of considerable value to be able to deliver such materials with the m i n i m u m n u m b e r of needle-stick injections. Such a situation m a y become facilitated in the future as vaccines based on plasmid D N A become available; a prospect which is examined in outline in the final section.

CONTENTS 1 Background 105 2 T h e vaccines and their advantages 105 3 Issues to be resolved 106 4 Nucleic acid vaccines on the threshold 111 5 Conclusions 112 References 112

1 Background There are two driving forces which lead to the promotion of multivalent (a term which will be used synonymouslywith polyvalent) and combined vaccines. The first is the Children's Vaccine Initiative (CVI) which, under the aegis of the WHO, seeks to immunize all children against 6 major infectious diseases (polio, diphtheria, tetanus, pertussis, measles, tuberculosis). This requires a minimum of 5 injections (Ellis 1996) and 5 visits to the medical center. So an ideal vaccine becomes an immunopropylactic which is effective, will cover a wide range of diseases, administered (preferably by mouth) in a single or small number of doses, will provide lifelong immunity, is stable, can be transported easily and is affordable. The second factor which impels the development of multivalent vaccines is the development and licensing of newly available vaccines which it would be desirable to administer to the same children who are to be covered by the current initiative. Such already licenced vaccines could be those for hepatitis B and A, Hwmophilus influenzae type b, mumps, varicella and rubella. While the prospects for the licensing of additional vaccines which would be protective against such diseases as meningitis, cholera, other diarrhceal diseases, malaria, respiratory disease and diseases caused by parasitic nematodes and helminths are promising, it is to be hoped that such vaccines would be included in the vaccination programmes when the vaccines become available. The inclusion of these additional and prospective vaccines into a large-scale programme of immunization would require many more visits to the medical center and a corresponding increase in the number of needle stick events which child vaccinees have to suffer. Development of multivalent vaccines for the coadministration of many vaccines simultaneously is therefore an objective which is worth attaining.

2 The vaccines and their advantages It is clear that the simple way to make a multivalent vaccine is to make a mixture of the individual vaccines and inject them simultaneously. Such a system does work for many of the vaccines used in the CVI (Parkman 1995). When the combination of the inactivated polio vaccine was made with the normal diphtheria, tetanus, pertussis (DTP) the thimerosal preservative used for the latter destroyed the polio vaccine. The use of 2-phenoxyethanol as a way round this difficulty is under investigation (Sutter et al. 1995). There are a number of alternative ways of combining immunogens so they may be

*Given at a FEMS International Symposium on Novel Methods and Standardization in Microbiology, at Ko~ice (Slovakia), July 1-4, 1996.

106 tLE.SPIER

Vol. 42

administered simultaneously. One such way is to link a number of immunogenic haptens to a common carrier such as hepatits B core protein or tetanus toxin or the cytolysin of Bordetella pertussis (~ebo 1996). An alternative method is to use a bacterial or viral vector. Amongst the bacteria, Salmonella Ty21a and BCG can be considered candidates, while such viruses as the vaccinia derivative NYVAC and the modified avian pox virus ALVAC may be engineered to carry multiple immunogenic epitopes (protectopes). Recently it has become possible to consider the use of naked deoxyribonucleic acid in the form of a bacterial plasmid as an immunogen. Clearly many immunogens may be engineered into a single plasmid or a mixture of plasmids and they may be made with relative ease since they are all similar in chemical composition and structure. The advantages which may be expected from combining vaccines into single delivery compositions are that fewer visits to the medical centre will be required. (This is a major benefit in those countries where most people find transportation difficult; it could be a disbenefit to those health care personnel who rely on the fees paid for vaccinating children to augment their incomes.) There would be an improvement in the degree of vaccine coverage obtained which will have epidemiological benefits and also a lowering of the unit cost of the vaccine may be expected as there would be decreases in the costs of separate vials, labels, packages, cold chain storage, needles and syringes, the time of medical personnel and the size of inventories. However, there may be come difficulties which need to be overcome before ideal vaccines are in the market place.

3 Issues to be resolved There is a wide range of problems which require solution before the way is cleared for the widespread use of the combination vaccines. Some of these issues are theoretical and, while of considerable interest, should not be allowed to stand in the way of the creditable progress which has already been made in using combination vaccines (Bloom 1995). Such vaccines were made by empirical methods and when assayed in the laboratory, small animal unit and field were found to be viable immunoprophylactics. Nevertheless, it behoves us to examine the new vaccines from all viewpoints which will include manufacturing and social issues as well as the biological phenomenologies involved.

3.1 The issue of immunological interference Perhaps the most salient problem which has to be addressed is that of immunogen interference. There are two areas where such interactions may militate against a combination vaccine. The first involves the prospect that an immunogen in one vaccine may suppress the immunogenicity of another vaccine while the second issue posits that the mixing of materials may create a combination which will increase the number and severity of deleterious side effects. 3.1.1 Immune interference There are two aspects to an immune response; the first is quantitative while the second is qualitative. Both of these arms are important for, while it is possible to generate the most effective kind of immune response, the amount of antibody or number of activated cells may not be quantitatively sufticient to be protective. By contrast it is possible to have a quantitatively massive immune response which is of a type which does not provide immunity. An example of the latter is the response of cattle to peptide vaccines which generates much IgG1 antibody but does not lead to a protective response (Dimarchi et al. 1986). A similar situation pertains in the case of the human immunodefficiency virus (HIV) where the presence of antibody to the gpl20 or gpl60 is also not necessarily protective. The qualitative aspects of the immune response may be divided into those responses which are T-cell independent and those which depend on T-cells. Antigens which provoke an immune response independent of T-cells are in the categories of polysaccharides, polymerized flagellin, L-amino acid polypeptides and some hepatitis B-core antigen conjugates. Most other immunogens operate through the T-cell system and have amongst their members all viruses and bacteria as well as toxoids, particulate antigens and most soluble proteins. The T-cell response is itself tricameral (Fig. 1). When a virus or bacterium penetrates into a cell, it is degraded by a complex of hydrolytic proteins called the proteosome. This process generates polypeptides which contain 8 - 1 2 amino acids some of which will fit into the groove in the bimolecular major histocompatibility complex (MHC) of the type I variety. The length of the peptide is critical and there are at least two key amino acids which act as the anchoring residues. When such an MHC I molecule is loaded with a polypeptide it presents

1997

MULTIVALENT

VACCINES:

PROSPECTS

AND

CHALLENGES

--

rev/zw 107

this molecular complex for interaction with the bimolecular T-cell receptor, presented by a cell of the CD8 lineage, after which activation, often requiring the presence of the cytokine interleukin-12 (IL-12), the T-cell becomes transformed to a cytotoxic lymphocyte (CTL) or natural killer cell (NKC). Such cells will interact with other non lymphocytic cells which express the protein from which the MHC I peptide has been derived, on the cell surface and destroy them. MHC DEPENDENT IMMUNE RESPONSES

virus and intracellular pathogen

MHC[

+ peptide (8-12 aa) - --.~ (protoesome treatment)

CD8+ (CTL)

antigen IL-12 MHCII + peptide (12-24

~

'

r

aa)-~--.1~ CD4+

Fc-receptor

antigen-antibody

INF-y " " 11.-2,TNF-I], IgG2a / Inhibltl~ DTH, CTL CMI

IL-4, IL-IO IL-5, IL-6 ",~ 11-9 9 IgG1, IgE

antibodies TH1 RESPONSE TH2 RESPONSE Fig. 1. The three principal reactions of the T-cell response.

When an antigen enters a cell, having been previously attached to an antibody, it goes through the Fc receptor on the antigen presenting cell (APC) external membrane. Such antigens are also degraded but this time the products of the hydrolysis are peptides of between 12-24 amino acids long. Such polypeptides are accepted into the groove found in the bimolecular MHC II and are held in the maw of this molecule by two or three anchoring residues. This complex is presented to the T-cell receptor (CD4) of a T-cell of the CD4 lineage and the resulting activation of that cell can take one of two pathways. On the one hand it can generate a TH1 response which involves the production of interferon (INF)-y, IL-2, tissue necrosis factor (TNF)-~, IgG2a along with delayed type hypersensitivity (DTH) and a CTL. Alternatively the cell may effect a TH2 response and secrete the ILs 4, 10, 5, 6, 9 and cause B-cells to make the IgG1 and IgE forms of antibodies. These two responses are generally mutually exclusive in that INF-y inhibits the TH2 response while IL-4 and IL-10 inhibit the TH1 response. Nevertheless, in practice both responses do occur often with a clear predominance of one type of response over the other. Factors which predispose the immune system to respond with either a TH1 or TH2 response are the nature and relative amounts of the immunogen, adjuvant, carrier, cytokine and route of injection. Table I shows the way different materials promote differences in the quality of the immune response. This quality is of crucial importance in some cases. For example, the mounting of a TH1 response to Mycobacterium leprae will lead to a stable tuberculoid lesion whereas the inculcation of a TH2 response will lead to lepromatous leprosy and death. It may therefore be of significant value to measure the relative amounts of IgG1 and IgG2 as a function of each and every immune response as this will give additional information about the T-cell engagement without effecting difficult and irreproducible T-cell assays. Of course the improvement of T-cell function assays is also a key necessity in monitoring, controlling and understanding the efficacy of a vaccination activity.

108

1LE.SPIER

Vol. 42

Table I. Materials and factors affecting the nature of the immune response Material type

Material

Response 1

Response 2

Response 3

Immune response

Leishmania M. leprw

IL-12 IL-2

INF-y INF-y

TH1 TH1

IL-4

IL-5

TH2

Immunogen

(tuberculoid) M. leprce

(lepromatous) M. (various) (intracellular) L/steda toxoplasma

IL-12

TH1

IL-12

TH1

Adjuvant cholera toxin B. penuss/s alum (AI salts) CFA, (FCA) BCG/MDP MPA, LPS,

IgG1

IgE

poor DTH

TH2

IgG1 (some)

IgG2a

DTH CMI

TH1 + (TH2) TH1

IgG2a

TH1

IgG2a IgG2a

TH1 & TH2 TH2 > TH1

Brucella abortus

liposome + MPL or MDP quail A, block copolymers

IgG1 IgG1 >

proteins, KLH

IgG1 all lgGs

Carriers B. abortus-TNP

LPS-TNP

TH2 TH1 > > TH2

lgG2a premodominates

Cytokines IL-12,2, INe-y, TNF@ IL-4.5,6?,9,10 B7-1 B7-2

IgG1

IgG2a lgE

DTH, CTL CMI

TH1 TH2 TH 1 TH2

Route of injection intraperitoneal with SAF-1 adjuvant subcutaneous, intramuscular subcutaneous/ gene gun (NAV) intramuscular (NAV)

lgG2a

TH1

IgG1

TH2

IgG1

TH2 lgO2a

CrL

TH1

In addition to the quality of the T-cell response changing, there is also the possibility of changes to the isotype profile, V-region usage, functional spectrum, virus neutralization, anamnestie response and the priming and memory of the antibody generating system to take into account. Actual interference in immunogenic effects resulting from the mixing of viruses has focused on the immunogenicity of pertussis vaccines when mixed in with either inactivated polio vaccine (IPV) or oral polio vaccine (OPV) or Haemophilus influenzw type b conjugate vaccine (HibOC). In the ease of polio the Canadian data generated between 1970-1992 are difficult to interpret for, while it would seem that both IPV and OPV did not affect the number of pertussis eases between 1970-1974, between 1975-1987 it would seem that in the presence of IPV more cases of pertussis were recorded while in the presence of OPV fewer cases were reported. Between 1987 and 1992 this trend was reversed (Halperin et al. 1995). On the other hand, when an acellular pertussis vaccine was examined in the context of a DTP vaccine with additional HibOC, then there were virtually no significant differences observed when the antigens were administered singly or as a combination (Paradiso 1995).

1997

M U L T I V A L E N T VACCINES: P R O S P E C T S A N D C H A L L E N G E S -- rev/ew

109

3.1.2 Safety It is easy to suggest that when a mixture is made from materials whose individual safety was deemed to have been acceptable there could occur such interactions between the components that an unsafe mixture results. Indeed when one examines the list (Wassilak et al. 1995) of potential problems it would be hardly surprising if something did not turn up. Notwithstanding such theoretical musings, in practice little has emerged to engender a sense that mixing already well tested and much used vaccines will lead to products which are inherently less safe than the progenitor materials. One such examination did reveal small differences in that the combined material was somewhat more reactive than the individual vaccines given separately, but the differences were not great (Paradiso 1995). 3.2 Manufacturing/documentation issues

In making a combination vaccine it may be necessary to mix immunogens made by different manufacturers who are in competition. Such operations will have to strike agreements about transfer prices, delivery dates, product specifications, communication systems and, possibly, the most difficult of all, a name for the new mixed product and the design of the logo which will carry it into the world. A more complex problem is presented by the in-package data sheet. For the quadrivalent Eli Lilly vaccine, Tetraimmune, this amounts to some 7000 words. Much of such verbiage is legalese seemingly designed to decrease the exposure of the manufacturing company to the possibility of legal actions being taken out against the company (McLintock 1995). Other documentation issues are uncovered when the forms recording the vaccination history of an individual need to be completed. In view of the increase in the possible number of vaccines and, with that, the consistency with which succeeding doses relate to previous vaccinations, it is difficult to preorganize the forms to cater to such expected variation. 3.3 Product licence issues

There is little doubt that revisions in the licencing procedures will be required if the potential of combination vaccines is not be to stifled at birth. It is clear 4 vaccines may be presented in 15 combinations. For each such combination there are, say, 4 possible dose regimens. This leads to some 154 schedules for testing or 50625 test schedules. Assuming a median cost of $ 200000 per schedule, then the overall bill could be as high as $10 billion (Halsey 1995). This is a financial burden which is both unnecessary and unwise to levy from the vaccinees who may not always be in a position to afford such unnecessary expenditure. In testing mixed vaccines it is clear that the placebo control cannot be used, for such people would not have the protection against infectious diseases they would have had were they vaccinated normally. Therefore the appropriate control has to be the existing vaccines given separately. The key issue in testing the new vaccines either in combination or even separately is that of determining the 'correlates of protection'. Here the possibilities proliferate almost without limit: antibodies and their types, T-cells and their lineages, the different arms of the immune system TH1, TH2 as well as the performance of the immunogeus in model systems based on laboratory animals, in vitro cen culture systems and in the final analysis, the target species itself. It may also be of interest to determine the rise in the total amount of immunoglobulin as well as the increase in the total lymphocyte count as gross indicators of immune system performance. All such reactivities could be related back to the physicochemical composition of the immunogen as made. Were is possible to forego the animal and cellular assays in favor of the physico-chemical, then it is possible that much effort and expenditure would be saved. But at the time of writing it would seem that such correlates of protection are few and far between. Indeed, the most prevalent assay is that for humoral IgG (isotype not specified) and assays for antitoxins to the D and T components of DTP. In terms of testing the new multivalent vaccines it has been proposed that a provisional licence be issued contigent on the performance of the vaccine in the post-market field situation. Special centres would be established to refer any cases of vaccine malfunction and such foci would cover the world in which vaccines are used. The objective of the exercise would not be to determine whether all the components of the vaccine were working in a manner equivalent to their separate administration, but rather a satisfactory mixture would be defined by the similarity of its effects to what would have occurred through separate application. In brief it is expected that malfunction events would occur with low frequency. Therefore it is necessary to use the preparations on a massive scale to observe any problem

110

R.E. SPIER

Vol. 42

areas either in safety or immunogenicity. Experience from the field will then generate the information from which improvements to existing capability can be construed.

3.4 The complexity issue As more vaccines become available and there are more components which can be added to the vaccines, the complexity of the vaccinators' work increases. Such a development can lead to increases in waste and mistakes, but perhaps most importantly it will tempt those who are engaged in vaccination programmes to 'mix and match' vaccines which have not been subject to thorough examination prior to reaching the field situation. For example there are several HibOC vaccines (based on differences in the number of repeat units in the linker, the particular saccharide which is coupled to the carrier and the concentration of the saccharide relative to the carrier material). Additionally, with pertussis, there are many possible acellular vaccines and vaccine components. At some time it will be necessary to consolidate such variety into a 'suite of consensus' vaccines irrespective of free-market philosophies which emphasize choice in all things.

3.5 Ethical issues People are becoming increasingly sensitized to the way they, and other people, behave. In a mass media multicultural society individuals see and hear more in a month than an inveterate explorer of the middle-ages saw in a lifetime. Styles and manners are becoming universal; and there is a corresponding demand for fairness and propriety in the interactions between people, particularly when there is a disparity between the resource availability of two nation states. This relates keenly to vaccines as such materials are often made and tested in relatively wealthy states for use in relatively poor states. One example of an issue under current discussion is that of the appropriate way in which a vaccine which is targeted to the disease problem of a lesser developed or developing country (LDC or DC), comprising some 80 % of the world population, should be tested in such a country. There are several questions which can be asked. Firstly: would the results generated in the L D C / D C be applicable in a resource abundant country (RAC)? Secondly, if the risk of damage as a result of testing the vaccine rests within the LDC/DC can benefit be accrued in a RAC? Thirdly, who will pay compensation for any vaccine induced damage which may occur and at what level of payment? And, after having been exposed to some risk and having provided the resource which has demonstrated the safety and efficacy of a given vaccine, would the vaccine be affordable by the rest of the population of the country in which the test was done? These are not insoluble issues but people in all countries who could benefit from vaccines tested in one country should contribute to a more equitable distribution of cost and benefit. Cultural differences need to be respected. Some prople regard the extraction of blood samples and biopsy materials as being contrary to their traditional culture. Should such people be denied the right to engage in tests because of such reluctances? Are there ways round such problems by the use of non-invasive techniques? Or should one approach a large number of individuals with appropriate bribes at hand and 'persuade' people to participate? A related issue to the extraction of biological specimens is the ownership of the specimen material. If such a material became involved in a genetic engineering experiment which went on to provide cellular materials from which a biotechnology company made a profit, it is possible to ask the question as to whether any benefit should revert to the source of the original specimen? In general, ethics committees have taken the view that once a biopsy material has been removed, it ceases to be the property of the donor and becomes the property of the institution which generates the added value from what would otherwise have been discarded (Human Tissue 1995). This draws us into the issue of informed consent. It may not be possible for a massive trial in the field, involving thousands of people, many of whom could be illiterate and uneducated, to adopt the techniques for establishing informed consent such as that practised by D. Sack at lohns Hopkins Clinical Research Centre. The measures they apply include a training session for would-be volunteers followed by an examination (multiple choice questions) to satisfy the examiners that the candidates for the vaccine test understand the risks they will experience and, notwithstanding such knowledge, yet consent to entering into the trial. Being safe is an absolute state; to be sought but never reached. The appropriate question becomes, what level of safety is appropriate under which particular set of circumstances? Troops going to war might accept a higher risk of vaccine damage than citizens at peace (particularly as military history relates that many more servicemen and women died of infectious disease than the bullets of the

1997

MULTIVALENT VACCINES: PROSPECTS AND CHALLENGES -- rev/ew 111

enemy). Again when disease is prevalent as in the LDC/DC areas of the world the safety standards of the RAC may not be applicable. So what standards should be regarded as appropriate? Clearly there will have to be a balancing of cost and benefit and, on the basis of this information, decisions can be made and updated as necessary. 4 N u c l e i c acid vaccines on the threshold

Much progress has been achieved since that startling discovery by J.A. Wolf et al. (1990) that the [3-galactosidase gene can be injected into muscle cells and will express the enzyme in situ. A number of symposia have been held on this subject (reviewed by this author, Spier 1995, 1996; Liu et al. 1995). It is clear that plasmid DNA synthesized in Eschetichia coli containing the genes whose expression would result in immunogenic materials can be used as vaccines. Such materials when injected by a dermis penetrating gun generate a TH2 type of response while the same material (perhaps in larger amounts) when made up in a sallne solution and injected into muscle generates a TH1 response. In test situations such preparations have been shown to be immunogenic for influenza and possibly for HIV. Human clinical trials are in progress for both of these latter materials at this time. Were it possible to present all the necessary immunogens for the CVI and future vaccine campalgns as plasmid DNA (with either all the immunogens coded for on the same plasmid or a multiplicity of plasmids), many of the technical problems for the production of multivaleut vaccines would disappear. Also it may be expected that the regulatory agencies might learn how to become comfortable with such materials, and once they had been shown to be safe, efficacious and to be made by a consistent process then many of the awkward and difficult situations with such agencies might be eliminated. However, the agencies express some reservations about the new nucleic acid immunogens (Smith 1996). The agency reservations in relation to these vaccines stem from the following considerations: * the injected plasmids might integrate into the host chromosomal DNA and 'turn on' cancer genes, 9 integrated DNA might find its way into gametic cell chromosomes and produce inherited changes, 9 endotoxins from the Escherichia coli producing organism could contaminate the final product, 9 the plasmid DNA could elicit antibodies which would react with self DNA and lead to a disease like lupus erythematosus, and 9 the low level of immunogen expression on a continuing basis could lead to a situation akin to low dose tolerance and the generation of an unprotectable person. In answer to these reservations it is possible to assert the following: 9 we live in a plasmid rich environment and nobody has raised the suggestion that having been infected by a bacterial plasmid has caused a human disease, 9 many of the vaccines currently in use or on trial may have (or generate) segments of DNA which integrate with the human chromosome (pox, herpes, HIV), 9 were any plasmid DNA to integrate then it will integrate in one position in one cell and a totally different position in another cell; it would be unlikely to integrate in the same position in a large number of cells and trigger the expression of a cancer causing gene, 9 our recent experience in attempting to achieve the integration of vectors carrying genes to correct genetic defects has been salutary; it has been found to be extremely difficult to obtain an integration which can generate a lasting response, 9 to obtain cellular transformation in vitro using the SV40 virus it is necessary to use the whole virus; genomic parts thereof do not effect in vitro transformation; and the contamination of millions of doses of polio vaccine with the SV40 virus has not led to reported cases of cancers which can be attributed to the polio vaccinations, 9 plasmid DNA is unstable in the serum, 9 methods (based on enhanced PCR techniques) have been developed to detect plasmid DNA molecules which have integrated in 1 in 150 000 or 1 in 500 000 examined cells; to date integrated plasmid DNA has not been found, 9 recombinant materials made from Escherichia coli have been in use for some 10-15 years (insulin 1982, u-interferon 1986) without hitting the problem of the endotoxin toxicity;

112

R.E. SPIER

Vol. 42

methods are available to reduce the level of plasmid contaminants to the undetectable even using the most sensitive of modern methods. It would therefore seem that the reservations of the regulatory agencies may be swiftly overcome and we may move into an era of DNA vaccines more quickly than we think. Were this to be the case then the advantage of being able to make combinations vaccines will surely not be overlooked. 5 Conclusions The need to make multivalent vaccines is evident. Manufacturers, regulatory agency personnel, scientists, engineers and members of the health-care calling will all work together to bring into being new generations of multivalent prophylactics. Some such materials may be based on the novel plasmid DNA vaccines, while others will rely on mixtures of conventional licensed vaccines. We may estimate that each year 1/60th of the world's population dies (some 100 million people); of these 17 million die from infectious disease caught in the early years of life. Many of such deaths will in due course be prevented by vaccination. While recognizing the benefit resulting from requited efforts incurred while child-bearing there are also powerful intangible benefits derived from a more optimistic view of life; a view which can plan for the future; an attitude which leads to investment and progress. Vaccines, and their delivery as combinations, will contribute to this outcome. We could perhaps be standing on the doorstep of an age of progress like that which our ancestors of 200 years ago crossed when they adopted the smallpox vaccine of Jenner; a fitting bicentennial anniversary for one of humanity's greatest benefactors. REFERENCES BLOOM B.R.: A perspective on issues relating to future vaccines; in Combined Vaccines and Simultaneous Administration: Current Issues and Perspectives (J.C. Williams, ILL. Goldenthal, D.L. Bums, B.P. Lewis Jr., Eds). Ann.New York Acad.Sci. 754, 388-395 (1995). DIMARCm R., BROOKEG., GALE C.: Protection of cattle against foot-and-mouth disease by a synthetic peptide. Science 232, 639 (1986). ELLIS R.: Challenges and development of combination vaccines, pp. 127-132 in Novel Strategies in the Design and Production of Vaccines (A. Cohen, A. Shafferman, Eds). Plenum Press, London 1996. HALPERINS.A., EASTWOODB.J., LANGLEYJ.M.: Immune responses to pertussis vaccines concurrently administered with viral vaccines; in Combined Vaccines and Simultaneous Administration: Current Issues and Perspectives (J.C. Williams, ILL Goldenthal, D.L. Bums, B.P. Lewis Jr., Eds).Ann.New YorkAcad.Sci. 754, 89-96 (1995). HALSEYN.A.: Practical considerations regarding the impact on immunization schedules of the introduction of new combined vaccines; in Combined Vaccines and Simultaneous Administration: Current Issues and Perspectives (J.C. Williams, ILL. Goldenthal, D.L. Bums, B.P. Lewis Jr., Eds).Ann.New YorkAcad.Sci. 754, 250-254 (1995). Human Tissue: Ethical and Legal Issues, p. 153. Nuffield Council on Biocthics, London 1995. Lio M.A., HILLEMA~M.R., KORTH17,.(Eds): DNA Vaccines: A New Era in Vaccinology. New York Acad. Sci., Vol. 772 (1995). McLIwrOCK D.IL: Combination vaccines: regulatory issues; in Combined Vaccines and Simultaneous Administration: Current Issues and Perspectives (J.C. Williams, ILL. Goldenthal, D.L. Bums, B.P. Lewis Jr., Eds). Ann.New York Acad.Sci. 754, 27-34 (1995). PARADISO P.R.: Combination vaccines for diphtheria, tetanus, pertussis and Hwmophilus influenzw type b; in Combined Vaccines and Simultaneous Administration: Current Issues and Perspectives (J.C. Williams, ILL. Goldenthal, D.L. Bums, B.P. Lewis Jr., Eds).Ann.New YorkAcad.Sci. 754, 108-113 (1995). PAR~IAN P.D.: Combined Vaccines and Simultaneous Administration: Current Issues and Perspectives (J.C. Williams, ILL. Goldenthal, D.L. Bums, B.P. Lewis Jr., Eds).Ann.New YorkAcad.Sci. 754, 1-9 (1995). ~EBO P.: paper presented at a FEMS Internat. Symp. Novel Methods and Standardization in Microbiology, Ko~ice (Slovakia) 1996. SMITHH.: Report in vaccine, inpress (1996). SPIER R.E.: Report of a meeting on vaccines; new technologies and applications. Vaccine 13, 1038-1039 (1995). SI'IER R.E.: Meeting report for vaccine, inpress (1996). SUTrER R.W., PALLANSCHM.A., SAWYERL.A., CO,HI S.L., HADLERS.C.: Defining surrogate serological tests with respect of predicting protective vaccine efficacy:, polio vaccination; in Combined Vaccines and Simultaneous Administration: Current Issues and Perspectives (J.C. Williams, ILL. Goldenthal, D.L. Bums, B.P. Lewis Jr., Eds). Ann.New York Acad.Sci. 754, 389-299 (1995). WASSII~KS.G., GLASSERJ.W., CHEN R.T., HADLER S.C. and the vaccine safety data link investigators: Utility of large-linked databases in vaccine safety, particularly in distinguishing independent and synergistic effects; in Combined Vaccines and Simultaneous Administration: Current Issues and Perspectives (J.C. Williams, ILL. Goldenthal, D.L. Bums, B.P. Lewis Jr., Eds).Ann.New YorkAcad.Sci. 754, 377-382 (1995). WOLFJ.A., MALONER.W., WILLIAMSP., CHO~4GW., ACSADIG., JANiA., FELGNERP.L.: Direct gene transfer into mouse muscle in vivo. Science 247, 1465-1468 (1990).