587
MODERN VACCINES From Jenner to
genes—the new vaccines
Starting with Jenner’s vaccine against smallpox in the last decade of the eighteenth century, we have now advanced to the point where we are able to protect man and his domestic animals against most of the important infectious diseases. However, some diseases remain for which no vaccine exists-notably, malaria, parasitic diseases, and of course AIDS. Moreover, even with the currently available vaccines there are pressing reasons why we should try to improve their quality and method of preparation. The existing vaccines are, with the exception of that for hepatitis B, prepared by one of two methods. For inactivated vaccines the causal agent is grown in large amounts and then inactivated, usually with formalin, p-propiolactone, or an imine, under conditions that ensure the retention of the immunogenic activity of the protective antigens. For the preparation of attenuated vaccines, the virulent organism obtained from the infected host is weakened by growth in an unnatural host or under conditions such that the product will then proliferate in the natural host without causing disease. For smallpox, Jenner used a naturally occurring attenuated strain of the virus—cowpox virus. Despite their excellent success record, the current vaccines have disadvantages. With inactivated vaccines there is always the need to ensure that the product no longer contains any live organisms. Moreover, the handling of large volumes of virulent organisms is a hazard to the personnel involved and to the immediate environment. With attenuated vaccines the disadvantages include the possible presence of adventitious agents in the cells or medium used for production, the need for storage at refrigerator temperatures, and the hazard of reversion to virulence (even the highly effective Sabin poliovaccine occasionally causes paralytic poliomyelitis). The aim, clearly, must be to produce vaccines by methods that will allow greater control of their biological properties and eliminate, as far as possible, their side-effects. Such goals have now been brought within reach by advances in our understanding of the structure of the genomes of many different organisms, to add to our knowledge of the structure and function of protective antigens. These advances are best understood by reference to viral diseases because the structures of many of the agents causing them have been characterised in great detail. Consequently this paper will concentrate on these agents. The advances resulted largely from the techniques which became available to grow viruses in quantities sufficient to
allow purification and chemical characterisation. Moreover, by growing them in the presence of radioactive precursors of nucleic acids and proteins it was possible not only to monitor the purification procedures but also to label the individual
Fig 1-Schematic representation of rabies virus. (Reproduced by permission from J Virol 1972,10: 256--60).
constituents. These analyses, on which the classification of viruses is now based,1 quickly revealed that there are many different groups of these agents. For example, the viruses causing poliomyelitis, the common cold, and foot-andmouth disease fall into a single family, with each member consisting of a spherical particle with icosahedral symmetry, 30 nm in diameter, and with one molecule of RNA and 60 molecules of each of four proteins. In contrast, the genome of influenza virus consists of eight segments, each of which codes for a different protein, encapsulated in a lipid envelope. Despite the differences in the basic structure of the viruses, however, there are unifying principles regarding their function as immunogens. Thus it was shown more than 20 years ago that protective immune responses could be elicited by individual proteins isolated from viruses such as those causing influenza, measles, and rabies that are easily dissected by dissolving their lipid envelope.2 In each instance the immune response was associated with the surface projections that protrude through the lipid envelope (fig 1). Even with foot-and-mouth disease virus, which is ADDRESS Department of Virology R & D, Wellcome Biotech, Langley Court, Beckenham BR3 3BS, UK (Prof F Brown, PhD,
FRS)
588
difficult to dissect under conditions that allow retention of immunogenic activity, one of the four capsid proteins is immunodominant. However, the level of neutralising antibody elicited by the separated protein is much lower than that elicited by the intact virus particle. more
Identification and expression of the genes coding for immunogenic proteins The first step in the design of a new vaccine is to identify the gene coding for the immunogenic protein. Most of the information on the genomic organisation of viruses has come from work on their replication. The genomes of most viruses code for several proteins in addition to the structural proteins involved in the immune response. Thus in the picornavirus family, of which foot-and-mouth disease virus is a member, only about one-third of the viral genome codes for the structural proteins. By a series of mapping experiments that had been devised for poliovirus and other picornaviruses, it was shown that the part of the genome coding for the structural proteins was located near the 5’ end
(fig 2). step in the procedure is to transcribe the appropriate segment of RNA into DNA so that this can be ligated into a suitable vector and expressed. There are many expression systems available but essentially only two different ways in which these systems can be used for vaccines. In the first the vector is cultivated in vitro to produce a large amount of the protein for use as inactivated vaccine. In the second the foreign DNA is inserted into a vector that can replicate in the host species in the same way as an attenuated vaccine. Virus proteins have been expressed in bacteria, in mammalian and yeast cells, and in viruses.3 Escherichia coli cells were the first to be used for this purpose but the expressed proteins are not glycosylated-a drawback since many of the immunogenic proteins of viruses, such as the surface projections of lipid-containing viruses, are glycosylated. Nevertheless the yield of the non-glycosylated immunogenic protein of foot-and-mouth disease virus was very high and accounted for as much as 20% of the total The
Fig 3-Construction of vaccinia virus recombinants.
next
protein. expressed in mammalian and yeast cells are glycosylated and so resemble more closely the naturally occurring surface projections. The yeast cell system has proved to be particularly attractive on the industrial scale and the surface antigen of hepatitis B virus produced in this way forms the basis of a highly successful vaccine. The virus expression system that has received most attention is the Proteins
The DNA sequence coding for the foreign gene IS inserted into the with a vaccinia virus promoter and vaccinia is sequences The resultant recombination vector then introduced into cells infected with vaccinia virus to generate a virus that expresses the foreign gene.
plasmid vector along thymidine kinase (TK)
nuclear polyhedrosis virus of Autographa californica. The yields of the foreign protein obtained with this virus are in most instances good enough to warrant serious consideration for industrial application. In 1982, Paoletti and Moss and their co-workers4 put forward the idea of adapting a virus that was itself used as an attenuated vaccine. These workers showed that vaccinia virus, which had been so successful in the control and eventual eradication of smallpox, could be used for the expression of foreign antigens, thus inducing immunity of laboratory animals against the pathogens in question (fig 3). Because of concern about their safety, application of the vaccinia recombinants has not proceeded beyond the laboratory so far, but field trials in animals are planned for vaccines against rabies and rinderpest. (Laboratory experiments showed that these two vaccines generate solid immunity in the natural hosts.) Other live vectors are being studied intensively. The most recent is the attenuated strain of poliovirus. The principle is the same as that used with vaccinia virus but there is the important difference that poliovirus is an RNA virus. However, the viral RNA can be transcribed into a complementary DNA which is infectious and can thus be manipulated in the same way as other DNA molecules. Moreover, poliovirus has the additional advantage that its three-dimensional structure is known at close to atomic resolution, so that the insertions can be made at positions corresponding to the loop regions of the parent virus in the expectation that these will lead to negligible distortion of its structure.
Fig 2-Genetic organisation of the genome of foot-and-mouth disease virus showing the position of the structural proteins and the immunogenic peptides. (Reproduced by permission from Nature 1986; 319: 549-50)
One of the safest and most effective of the attenuated viral vaccines is the 17D strain of yellow fever virus. A single immunisation results in persistence of neutralising antibody for as long as 40 years and complications are extremely rare. Now that an infectious complementary DNA has been produced, the way is open to develop the 17D strain as a recombinant vaccine vector.
589
Two attenuated bacterial species are also being used for the expression of foreign genes-namely, Salmonella typhimurium6 and Bacillus Calmette-Guerin (BCG).7 The great advantage of salmonella recombinant vectors would be that they can be given orally, thus providing the mucosal immunity that is clearly required for many infectious diseases. BCG is the attenuated vaccine most widely used in the past four decades and more than one billion doses have been given with a very low frequency of complications. Only a single inoculation is required to induce cell-mediated immunity for long periods, it has adjuvant activity, and it can be given repeatedly. The exploration of this system is only in its early stages but it holds out great promise, particularly because the vaccines would be very cheap to
produce.
Peptides as vaccines Reductionism has been taken a stage beyond the identification of individual proteins carrying immunogenic activity: epitopic sites on proteins can elicit neutralising antibodies in amounts sufficient to protect against infection. The concept is not new. As long ago as 1963, Anderer showed in a historic paper that short fragments of the protein of tobacco mosaic virus would elicit antibody that neutralised virus infectivity; and this observation was followed by the demonstration that a synthetic peptide with the same aminoacid sequence as the C terminal fragment would also elicit neutralising antibody. At that time the work could not be taken much further because few aminoacid sequences of proteins of microbiological interest were available; it was only when DNA sequencing methods were described by Maxam and Gilbert and by Sanger, Nicklen, and Coulson (thus allowing aminoacid sequences to be derived from the sequences of the genes coding for immunogenic proteins) that this approach could be
pursued. The development of synthetic peptides that might be useful as vaccines depends on identification of immunogenic sites. Several methods have been used and protection against challenge infection was obtained with a linear sequence of 20 aminoacids corresponding to a segment of one of the four proteins of foot-and-mouth disease virus.8 What has emerged from this and other work is that successful presentation of the peptides to the immune system depends on (a) the presence of a helper T cell epitope in addition to the B cell epitope9 and (b) the physical structure of the antigen." These are considered in turn.
Importance of T cell epitopes generally assumed that, because of their small size, peptides would behave like haptens and would therefore require coupling to a protein carrier to enhance their immunogenicity. We now know that synthetic peptides can be highly immunogenic in their free form provided they contain, in addition to the B cell epitope, sites which can elicit help for antibody production (T cell epitopes). Such T cell sites can be provided by carrier protein molecules but the addition of T cell epitopes, either from a foreign antigen or from the same molecule as the B cell epitope, can provide help. With the peptide from foot-and-mouth disease virus, a normally unresponsive mouse haplotype will respond when a suitable T cell epitope is added. It seems, therefore, that we should be seeking universal T cell epitopes for each species, which could be added to the B cell epitopes of interest. It was
immunogenic proteins and peptides to the immune system
Presentation of
Proteins separated from virus particles are generally much less immunogenic than the intact particles. This difference in activity is usually attributed to the change in configuration of a protein when it is released from the constraints imposed by the structural requirements of the virus particle. Many attempts have been made to enhance the immunogenic activity of the separated proteins: the results would be of direct application for genetically engineered proteins that are produced in what is essentially a "foreign" environment. In the most successful procedure a mixture of the plant glycoside saponin, cholesterol, and phosphatidylcholine provides a vehicle for presentation of several copies of the protein on a cage-like structure. Such a multimeric presentation mimics the natural situation of antigens on microorganisms. These immunostimulating complexes have activities equivalent to those of the virus particles from which the proteins are derived, thus holding out great promise for the presentation of genetically
engineered proteins. Similar considerations apply to the presentation of peptides. Tam, working at the Rockefeller University, has shown that, by building the peptide of interest onto a framework of lysine residues so that eight copies instead of one are present, the product elicits a much greater response than the monomeric form. Clarke and colleagues have described an approach that allows the presentation of the peptide in a polymeric form combined with T cell epitopes. The sequence coding for the foot-and-mouth disease virus peptide was expressed as part of a fusion with the gene coding for the hepatitis B core protein. The hybrid protein, which forms into spherical particles about 22 nm in diameter, elicited levels of neutralising antibodies against foot-and-mouth disease virus that were at least a hundred times greater than those produced by the monomeric peptide. Gentle disruption of the particle into the monomeric protein considerably reduced the response to the added peptide; thus, presentation in a multimeric form was crucial for effective responses. Milich and his group have studied in great detail the immunological characteristics of the hepatitis B core particle and have identified several of the T cell epitopes on the particles that are responsible for the help provided by the core particles in eliciting high levels of neutralising antibody. This approach to the design of completely synthetic vaccines, in which the appropriate B cell epitope is linked to a T cell epitope and presented as a particle containing many copies of the immunogenic site, is being pursued with several systems and will give us information about the basic requirements for an antigen to stimulate a protective immune response.
Conclusions Amidst all the euphoria generated by much elegant molecular biology it is important to remember, when protection against infection is the goal, that the type of immunity required is not the same for all diseases. Thus a vaccine that prevents or modifies a systemic infection may not be effective against localised mucosal infection. For infections involving mucosal surfaces, an attenuated salmonella vector is likely to be a suitable way of presenting foreign antigen, whereas for systemic infections recombinant vaccinia viruses are likely to be more useful.
590
The realisation that it was not sufficient merely to produce large amounts of immunogenic proteins-that these proteins also had to be in a configuration which mimics that on the intact organism-led to the development of immunostimulating complexes and research on other methods of presentation. The presentation of immunogenic peptides is likewise important to the immune response. Another advance has been the combination of T cell sites with the specific B cell sites as a means of overcoming genetic
2. Brown F. Synthetic viral vaccines. Annu Rev Microbiol 1984; 38: 221-35. 3. Murray K. Polypeptide production by recombinant DNA techniques. In: Bell R, Torrigiani G, eds. New approaches to vaccine development. Basel: Schwabe, 1984. 4. Mackett M, Smith GL. Vaccine virus expression vectors. J Gen Virol 1986; 67: 2067-82. 5. Burke KL, Dunn G, Ferguson M, Minor PD, Almond JW. Antigenic chimaeras of poliovirus as potential new vaccines. Nature 1988; 332: 81-82. 6. Dougan G, Hormaeche CE, Maskell DJ. Live oral salmonella vaccines:
restriction. The information
7.
gained by this molecular approach to vaccination has greatly advanced our understanding of what is required for effective immunisation. It has already led to one product, a vaccine against hepatitis B, and the future will doubtless
see
many others.
REFERENCES 1. Matthews REF. Viral taxonomy for the Microbiol 1985; 39: 451-74.
non
virologist.
Annu Rev
potential use as carriers of heterologous antigens to the immune system. Parasite Immunol 1987; 9: 151-60.
Jacobs WR, Tuckman M, Bloom BR. Introduction of foreign DNA into mycobacteria using a shuttle plasmid. Nature 1987; 327: 532-36. 8. Bittle JL, Houghten RA, Alexander H, et al. Protection against foot-and-mouth disease with a chemically synthesised peptide predicted from the viral nucleotide sequence. Nature 1882; 298: 30-33. 9. Milich DR. Synthetic T & B cell recognition sites: implications for vaccine development. Adv Immunol 1989; 45: 195-282. 10. Clarke BE, Newton SE, Carroll AR, et al. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature 1987; 330: 381-84.
VIEWPOINT What
causes
diabetic renal failure?
do some diabetic patients develop renal failure while others do not? Two Lancet editorials, in 19721 and 1988,22 have pointed out the discrepancies between histological changes in the kidney and the occurrence of proteinuria or renal failure yet, despite the low correlation between nephropathy and histological changes, glomerular disease is still widely believed to be the primary cause of diabetic nephropathy. In a necropsy study of 17 matched diabetic patients with and without nephropathy, Thomsen and colleagues,3as reported in the later editorial,2found that "the prevalence and severity of Kimmelstiel-Wilson nodules, exudative lesions (fibrinoid caps and capsular drops), and arteriolar lesions were indistinguishable between the two groups. Morphological features which identified patients with uraemia were a reduction in the area of open capillaries and an increase in glomerular mesangial tissue". However, Thomsen et al also found an increased interstitial space in patients with nephropathy-an increase that correlated with serum creatinine levels, as previously observed by Bader and colleagues.’ Although tubule dropout (disappearance of tubules whose glomeruli became totally ischaemic) may contribute to the increased interstitial space, experimental evidence indicates a separate and partly independent mechanism that contributes to increased interstitial pressure and volume in the diabetic kidney. Could these interstitial changes contribute to the aetiology of renal failure in diabetic nephropathy? The microvascular complications of chronic diabetes mellitus affect both glomerular and peritubular capillaries .5,6 The total surface area of peritubular capillaries is up to 40 times that of glomerular capillaries.7 Normally 99% of the glomerular filtered fluid volume is reabsorbed through the
Why
walls of peritubular capillaries because of hydrostatic and colloid osmotic pressure differences across the capillary walls, but in chronic diabetes mellitus the permeability of peritubular capillaries to plasma proteins increases:5 this altered permeability may affect fluid entry into peritubular capillaries in two ways. First, the normally low interstitial protein concentration tends to increase, with a corresponding fall in the colloid osmotic pressure gradient across the capillary wall; as a result, interstitial fluid tends to accumulate, with increased interstitial volume and hydrostatic pressure. However, the increased interstitial pressure augments the hydrostatic force that moves fluid into the peritubular capillary, so that tubular fluid reabsorption may be largely unaltered-at the price of increased interstitial volume and pressure. Secondly, peritubular microangiopathy may change the reflexion coefficient to macromolecules of the capillary wall resorptive surface. Normally, the reflexion coefficient is close to 1 -0, its limiting valuer but if it were to become severely depressed by microangiopathy the colloid osmotic driving force would be further diminished. How does the kidney respond to increased leakage of proteins from the peritubular capillaries? Most importantly, the extravasated plasma proteins do not accumulate in the ADDRESSES Departments of Physiology, University of Maryland School of Medicine, Baltimore, USA, and Biomedical Sciences Division, King’s College, London, UK (Prof G. G. Pinter, MD), and Department of Nephrology, Walter Reed Army Institute of Research, Washington DC, USA (J. L. Atkins, MD) Correspondence to Prof G. G. Pinter, Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
MODERN VACCINES From Jenner to
genes—the new vaccines
Starting with Jenner’s vaccine against smallpox in the last decade of the eighteenth century, we have now advanced to the point where we are able to protect man and his domestic animals against most of the important infectious diseases. However, some diseases remain for which no vaccine exists-notably, malaria, parasitic diseases, and of course AIDS. Moreover, even with the currently available vaccines there are pressing reasons why we should try to improve their quality and method of preparation. The existing vaccines are, with the exception of that for hepatitis B, prepared by one of two methods. For inactivated vaccines the causal agent is grown in large amounts and then inactivated, usually with formalin, p-propiolactone, or an imine, under conditions that ensure the retention of the immunogenic activity of the protective antigens. For the preparation of attenuated vaccines, the virulent organism obtained from the infected host is weakened by growth in an unnatural host or under conditions such that the product will then proliferate in the natural host without causing disease. For smallpox, Jenner used a naturally occurring attenuated strain of the virus—cowpox virus. Despite their excellent success record, the current vaccines have disadvantages. With inactivated vaccines there is always the need to ensure that the product no longer contains any live organisms. Moreover, the handling of large volumes of virulent organisms is a hazard to the personnel involved and to the immediate environment. With attenuated vaccines the disadvantages include the possible presence of adventitious agents in the cells or medium used for production, the need for storage at refrigerator temperatures, and the hazard of reversion to virulence (even the highly effective Sabin poliovaccine occasionally causes paralytic poliomyelitis). The aim, clearly, must be to produce vaccines by methods that will allow greater control of their biological properties and eliminate, as far as possible, their side-effects. Such goals have now been brought within reach by advances in our understanding of the structure of the genomes of many different organisms, to add to our knowledge of the structure and function of protective antigens. These advances are best understood by reference to viral diseases because the structures of many of the agents causing them have been characterised in great detail. Consequently this paper will concentrate on these agents. The advances resulted largely from the techniques which became available to grow viruses in quantities sufficient to
allow purification and chemical characterisation. Moreover, by growing them in the presence of radioactive precursors of nucleic acids and proteins it was possible not only to monitor the purification procedures but also to label the individual
Fig 1-Schematic representation of rabies virus. (Reproduced by permission from J Virol 1972,10: 256--60).
constituents. These analyses, on which the classification of viruses is now based,1 quickly revealed that there are many different groups of these agents. For example, the viruses causing poliomyelitis, the common cold, and foot-andmouth disease fall into a single family, with each member consisting of a spherical particle with icosahedral symmetry, 30 nm in diameter, and with one molecule of RNA and 60 molecules of each of four proteins. In contrast, the genome of influenza virus consists of eight segments, each of which codes for a different protein, encapsulated in a lipid envelope. Despite the differences in the basic structure of the viruses, however, there are unifying principles regarding their function as immunogens. Thus it was shown more than 20 years ago that protective immune responses could be elicited by individual proteins isolated from viruses such as those causing influenza, measles, and rabies that are easily dissected by dissolving their lipid envelope.2 In each instance the immune response was associated with the surface projections that protrude through the lipid envelope (fig 1). Even with foot-and-mouth disease virus, which is ADDRESS Department of Virology R & D, Wellcome Biotech, Langley Court, Beckenham BR3 3BS, UK (Prof F Brown, PhD,
FRS)
588
difficult to dissect under conditions that allow retention of immunogenic activity, one of the four capsid proteins is immunodominant. However, the level of neutralising antibody elicited by the separated protein is much lower than that elicited by the intact virus particle. more
Identification and expression of the genes coding for immunogenic proteins The first step in the design of a new vaccine is to identify the gene coding for the immunogenic protein. Most of the information on the genomic organisation of viruses has come from work on their replication. The genomes of most viruses code for several proteins in addition to the structural proteins involved in the immune response. Thus in the picornavirus family, of which foot-and-mouth disease virus is a member, only about one-third of the viral genome codes for the structural proteins. By a series of mapping experiments that had been devised for poliovirus and other picornaviruses, it was shown that the part of the genome coding for the structural proteins was located near the 5’ end
(fig 2). step in the procedure is to transcribe the appropriate segment of RNA into DNA so that this can be ligated into a suitable vector and expressed. There are many expression systems available but essentially only two different ways in which these systems can be used for vaccines. In the first the vector is cultivated in vitro to produce a large amount of the protein for use as inactivated vaccine. In the second the foreign DNA is inserted into a vector that can replicate in the host species in the same way as an attenuated vaccine. Virus proteins have been expressed in bacteria, in mammalian and yeast cells, and in viruses.3 Escherichia coli cells were the first to be used for this purpose but the expressed proteins are not glycosylated-a drawback since many of the immunogenic proteins of viruses, such as the surface projections of lipid-containing viruses, are glycosylated. Nevertheless the yield of the non-glycosylated immunogenic protein of foot-and-mouth disease virus was very high and accounted for as much as 20% of the total The
Fig 3-Construction of vaccinia virus recombinants.
next
protein. expressed in mammalian and yeast cells are glycosylated and so resemble more closely the naturally occurring surface projections. The yeast cell system has proved to be particularly attractive on the industrial scale and the surface antigen of hepatitis B virus produced in this way forms the basis of a highly successful vaccine. The virus expression system that has received most attention is the Proteins
The DNA sequence coding for the foreign gene IS inserted into the with a vaccinia virus promoter and vaccinia is sequences The resultant recombination vector then introduced into cells infected with vaccinia virus to generate a virus that expresses the foreign gene.
plasmid vector along thymidine kinase (TK)
nuclear polyhedrosis virus of Autographa californica. The yields of the foreign protein obtained with this virus are in most instances good enough to warrant serious consideration for industrial application. In 1982, Paoletti and Moss and their co-workers4 put forward the idea of adapting a virus that was itself used as an attenuated vaccine. These workers showed that vaccinia virus, which had been so successful in the control and eventual eradication of smallpox, could be used for the expression of foreign antigens, thus inducing immunity of laboratory animals against the pathogens in question (fig 3). Because of concern about their safety, application of the vaccinia recombinants has not proceeded beyond the laboratory so far, but field trials in animals are planned for vaccines against rabies and rinderpest. (Laboratory experiments showed that these two vaccines generate solid immunity in the natural hosts.) Other live vectors are being studied intensively. The most recent is the attenuated strain of poliovirus. The principle is the same as that used with vaccinia virus but there is the important difference that poliovirus is an RNA virus. However, the viral RNA can be transcribed into a complementary DNA which is infectious and can thus be manipulated in the same way as other DNA molecules. Moreover, poliovirus has the additional advantage that its three-dimensional structure is known at close to atomic resolution, so that the insertions can be made at positions corresponding to the loop regions of the parent virus in the expectation that these will lead to negligible distortion of its structure.
Fig 2-Genetic organisation of the genome of foot-and-mouth disease virus showing the position of the structural proteins and the immunogenic peptides. (Reproduced by permission from Nature 1986; 319: 549-50)
One of the safest and most effective of the attenuated viral vaccines is the 17D strain of yellow fever virus. A single immunisation results in persistence of neutralising antibody for as long as 40 years and complications are extremely rare. Now that an infectious complementary DNA has been produced, the way is open to develop the 17D strain as a recombinant vaccine vector.
589
Two attenuated bacterial species are also being used for the expression of foreign genes-namely, Salmonella typhimurium6 and Bacillus Calmette-Guerin (BCG).7 The great advantage of salmonella recombinant vectors would be that they can be given orally, thus providing the mucosal immunity that is clearly required for many infectious diseases. BCG is the attenuated vaccine most widely used in the past four decades and more than one billion doses have been given with a very low frequency of complications. Only a single inoculation is required to induce cell-mediated immunity for long periods, it has adjuvant activity, and it can be given repeatedly. The exploration of this system is only in its early stages but it holds out great promise, particularly because the vaccines would be very cheap to
produce.
Peptides as vaccines Reductionism has been taken a stage beyond the identification of individual proteins carrying immunogenic activity: epitopic sites on proteins can elicit neutralising antibodies in amounts sufficient to protect against infection. The concept is not new. As long ago as 1963, Anderer showed in a historic paper that short fragments of the protein of tobacco mosaic virus would elicit antibody that neutralised virus infectivity; and this observation was followed by the demonstration that a synthetic peptide with the same aminoacid sequence as the C terminal fragment would also elicit neutralising antibody. At that time the work could not be taken much further because few aminoacid sequences of proteins of microbiological interest were available; it was only when DNA sequencing methods were described by Maxam and Gilbert and by Sanger, Nicklen, and Coulson (thus allowing aminoacid sequences to be derived from the sequences of the genes coding for immunogenic proteins) that this approach could be
pursued. The development of synthetic peptides that might be useful as vaccines depends on identification of immunogenic sites. Several methods have been used and protection against challenge infection was obtained with a linear sequence of 20 aminoacids corresponding to a segment of one of the four proteins of foot-and-mouth disease virus.8 What has emerged from this and other work is that successful presentation of the peptides to the immune system depends on (a) the presence of a helper T cell epitope in addition to the B cell epitope9 and (b) the physical structure of the antigen." These are considered in turn.
Importance of T cell epitopes generally assumed that, because of their small size, peptides would behave like haptens and would therefore require coupling to a protein carrier to enhance their immunogenicity. We now know that synthetic peptides can be highly immunogenic in their free form provided they contain, in addition to the B cell epitope, sites which can elicit help for antibody production (T cell epitopes). Such T cell sites can be provided by carrier protein molecules but the addition of T cell epitopes, either from a foreign antigen or from the same molecule as the B cell epitope, can provide help. With the peptide from foot-and-mouth disease virus, a normally unresponsive mouse haplotype will respond when a suitable T cell epitope is added. It seems, therefore, that we should be seeking universal T cell epitopes for each species, which could be added to the B cell epitopes of interest. It was
immunogenic proteins and peptides to the immune system
Presentation of
Proteins separated from virus particles are generally much less immunogenic than the intact particles. This difference in activity is usually attributed to the change in configuration of a protein when it is released from the constraints imposed by the structural requirements of the virus particle. Many attempts have been made to enhance the immunogenic activity of the separated proteins: the results would be of direct application for genetically engineered proteins that are produced in what is essentially a "foreign" environment. In the most successful procedure a mixture of the plant glycoside saponin, cholesterol, and phosphatidylcholine provides a vehicle for presentation of several copies of the protein on a cage-like structure. Such a multimeric presentation mimics the natural situation of antigens on microorganisms. These immunostimulating complexes have activities equivalent to those of the virus particles from which the proteins are derived, thus holding out great promise for the presentation of genetically
engineered proteins. Similar considerations apply to the presentation of peptides. Tam, working at the Rockefeller University, has shown that, by building the peptide of interest onto a framework of lysine residues so that eight copies instead of one are present, the product elicits a much greater response than the monomeric form. Clarke and colleagues have described an approach that allows the presentation of the peptide in a polymeric form combined with T cell epitopes. The sequence coding for the foot-and-mouth disease virus peptide was expressed as part of a fusion with the gene coding for the hepatitis B core protein. The hybrid protein, which forms into spherical particles about 22 nm in diameter, elicited levels of neutralising antibodies against foot-and-mouth disease virus that were at least a hundred times greater than those produced by the monomeric peptide. Gentle disruption of the particle into the monomeric protein considerably reduced the response to the added peptide; thus, presentation in a multimeric form was crucial for effective responses. Milich and his group have studied in great detail the immunological characteristics of the hepatitis B core particle and have identified several of the T cell epitopes on the particles that are responsible for the help provided by the core particles in eliciting high levels of neutralising antibody. This approach to the design of completely synthetic vaccines, in which the appropriate B cell epitope is linked to a T cell epitope and presented as a particle containing many copies of the immunogenic site, is being pursued with several systems and will give us information about the basic requirements for an antigen to stimulate a protective immune response.
Conclusions Amidst all the euphoria generated by much elegant molecular biology it is important to remember, when protection against infection is the goal, that the type of immunity required is not the same for all diseases. Thus a vaccine that prevents or modifies a systemic infection may not be effective against localised mucosal infection. For infections involving mucosal surfaces, an attenuated salmonella vector is likely to be a suitable way of presenting foreign antigen, whereas for systemic infections recombinant vaccinia viruses are likely to be more useful.
590
The realisation that it was not sufficient merely to produce large amounts of immunogenic proteins-that these proteins also had to be in a configuration which mimics that on the intact organism-led to the development of immunostimulating complexes and research on other methods of presentation. The presentation of immunogenic peptides is likewise important to the immune response. Another advance has been the combination of T cell sites with the specific B cell sites as a means of overcoming genetic
2. Brown F. Synthetic viral vaccines. Annu Rev Microbiol 1984; 38: 221-35. 3. Murray K. Polypeptide production by recombinant DNA techniques. In: Bell R, Torrigiani G, eds. New approaches to vaccine development. Basel: Schwabe, 1984. 4. Mackett M, Smith GL. Vaccine virus expression vectors. J Gen Virol 1986; 67: 2067-82. 5. Burke KL, Dunn G, Ferguson M, Minor PD, Almond JW. Antigenic chimaeras of poliovirus as potential new vaccines. Nature 1988; 332: 81-82. 6. Dougan G, Hormaeche CE, Maskell DJ. Live oral salmonella vaccines:
restriction. The information
7.
gained by this molecular approach to vaccination has greatly advanced our understanding of what is required for effective immunisation. It has already led to one product, a vaccine against hepatitis B, and the future will doubtless
see
many others.
REFERENCES 1. Matthews REF. Viral taxonomy for the Microbiol 1985; 39: 451-74.
non
virologist.
Annu Rev
potential use as carriers of heterologous antigens to the immune system. Parasite Immunol 1987; 9: 151-60.
Jacobs WR, Tuckman M, Bloom BR. Introduction of foreign DNA into mycobacteria using a shuttle plasmid. Nature 1987; 327: 532-36. 8. Bittle JL, Houghten RA, Alexander H, et al. Protection against foot-and-mouth disease with a chemically synthesised peptide predicted from the viral nucleotide sequence. Nature 1882; 298: 30-33. 9. Milich DR. Synthetic T & B cell recognition sites: implications for vaccine development. Adv Immunol 1989; 45: 195-282. 10. Clarke BE, Newton SE, Carroll AR, et al. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature 1987; 330: 381-84.
VIEWPOINT What
causes
diabetic renal failure?
do some diabetic patients develop renal failure while others do not? Two Lancet editorials, in 19721 and 1988,22 have pointed out the discrepancies between histological changes in the kidney and the occurrence of proteinuria or renal failure yet, despite the low correlation between nephropathy and histological changes, glomerular disease is still widely believed to be the primary cause of diabetic nephropathy. In a necropsy study of 17 matched diabetic patients with and without nephropathy, Thomsen and colleagues,3as reported in the later editorial,2found that "the prevalence and severity of Kimmelstiel-Wilson nodules, exudative lesions (fibrinoid caps and capsular drops), and arteriolar lesions were indistinguishable between the two groups. Morphological features which identified patients with uraemia were a reduction in the area of open capillaries and an increase in glomerular mesangial tissue". However, Thomsen et al also found an increased interstitial space in patients with nephropathy-an increase that correlated with serum creatinine levels, as previously observed by Bader and colleagues.’ Although tubule dropout (disappearance of tubules whose glomeruli became totally ischaemic) may contribute to the increased interstitial space, experimental evidence indicates a separate and partly independent mechanism that contributes to increased interstitial pressure and volume in the diabetic kidney. Could these interstitial changes contribute to the aetiology of renal failure in diabetic nephropathy? The microvascular complications of chronic diabetes mellitus affect both glomerular and peritubular capillaries .5,6 The total surface area of peritubular capillaries is up to 40 times that of glomerular capillaries.7 Normally 99% of the glomerular filtered fluid volume is reabsorbed through the
Why
walls of peritubular capillaries because of hydrostatic and colloid osmotic pressure differences across the capillary walls, but in chronic diabetes mellitus the permeability of peritubular capillaries to plasma proteins increases:5 this altered permeability may affect fluid entry into peritubular capillaries in two ways. First, the normally low interstitial protein concentration tends to increase, with a corresponding fall in the colloid osmotic pressure gradient across the capillary wall; as a result, interstitial fluid tends to accumulate, with increased interstitial volume and hydrostatic pressure. However, the increased interstitial pressure augments the hydrostatic force that moves fluid into the peritubular capillary, so that tubular fluid reabsorption may be largely unaltered-at the price of increased interstitial volume and pressure. Secondly, peritubular microangiopathy may change the reflexion coefficient to macromolecules of the capillary wall resorptive surface. Normally, the reflexion coefficient is close to 1 -0, its limiting valuer but if it were to become severely depressed by microangiopathy the colloid osmotic driving force would be further diminished. How does the kidney respond to increased leakage of proteins from the peritubular capillaries? Most importantly, the extravasated plasma proteins do not accumulate in the ADDRESSES Departments of Physiology, University of Maryland School of Medicine, Baltimore, USA, and Biomedical Sciences Division, King’s College, London, UK (Prof G. G. Pinter, MD), and Department of Nephrology, Walter Reed Army Institute of Research, Washington DC, USA (J. L. Atkins, MD) Correspondence to Prof G. G. Pinter, Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
