Live-Virus Vaccines 219
Inactivated Virus and Virus Subunit Vaccines 223
Synthetic Vaccines 225
DNA Vaccines 226
Anti-Idiotypic Antibodies 226
Methods for Enhancing Immunogenicity 226
Comparison of Different Classes of Vaccines 227
Immunization is the most generally applicable way of preventing infectious disease. The control of so many important viral diseases by immunization is arguably the outstanding medical achievement of the twentieth century, recognized by the award of several Nobel prizes.
Traditionally, there have been two major strategies for the production of viral vaccines, one employing live avirulent virus and the other employing chemically inactivated virus. Recombinant DNA technology has now opened up an exciting range of additional options (Table 13-1).
The most successful viral vaccines are live avirulent mutants They have been instrumental in dramatically reducing the incidence of several important dis eases of childhood (see Fig. 15-1) and for eradicating smallpox (see Chapter 15). The key to their success is the fact that the live virus multiplies in the recipient, eliciting a lasting immune response but causing little or no disease. In effect, a live vaccine produces a subclinical infection, nature's own way of immunizing.
Approaches to Designing Viral Vaccines
Live-virus vaccines Virus attenuated in virulence by sciial passage in cultured cells Cold-adapted mutants and reassortants
Virus attenuated by gene deletion or site-directed mutagenesis Antigens expressed via recombinant live viral or bacterial vectors Nonreplicaling antigens Inactivated virions Purified proteins
Proteins from genes cloned in prokaryotic or eukaryolic cells Synthetic peptides Others Anti-idiolypic antibodies Viral DNA
Most of the live-virus vaccines in common usage today have been derived empirically by serial passage in cultured cells. Adaptation of virus to more vigorous growth in cultured cells is fortuitously accompanied by progressive loss of virulence for the natural host. Avirulence is demonstrated initially in a convenient laboratory model, often a mouse, then a primate, before being confirmed by clinical trials in human volunteers. Because of the requirement that the vaccine must not be so attenuated that it fails to replicate satisfactorily m vivo, it is sometimes necessary to compromise with a strain that does in fact induce trivial symptoms in a few of the recipients.
During dozens of passages in cultured cells these host range mutants accumulate numerous point mutations; it is generally not known which of these are responsible for attenuation. For most viruses, several genes contribute to virulence in different ways. Moreover, the avirulence of attenuated vaccines has generally not yet been characterized in terms of their pathogenesis in the vaccinee. In the case of some experimental vaccines administered by the respiratory route, multiplication of the attenuated virus is severely restricted and confined to the upper respiratory tract. On the other hand, the oral poliovaccine replicates in intestinal cells but has lost the capacity to infect the critical target cells in the spinal cord.
Despite the outstanding success of empirically derived live vaccines, a vigorous program of research is aimed at replacing what some see as "genetic roulette" with a more calculated approach to the design of live viruses of reduced virulence. These approaches, which have already yielded several promising experimental vaccines, are discussed below
The observation that temperature-sensitive (Is) mutants (unable to replicate satisfactorily at temperatures much higher than normal, e.g., 40°C) generally display reduced virulence suggested that they might make satisfactory live vaccines. Unfortunately, even vaccines containing more than one fs mutation displayed a disturbing tendency to revert toward virulence during replication in humans.
Attention then moved to cold-adapted (ca) mutants, derived by adaptation of virus to grow at suboptimal temperatures The rationale is that such a mutant might provide a safer vaccine for intranasal administration, in that it would replicate well at the lower temperature of the nose (33°C) but not at the temperature of the more vulnerable lung. Cold-adapted influenza vaccines containing mutations in almost every gene do not revert to virulence. They are used as "master strains" into whicb genes for novel hemagglutinin (HA) and/or neuraminidase (NA) proteins can be introduced by reassortment, but they are not as immunogenic as had been hoped.
The problem of back mutation could be circumvented by deleting nonessential genes that contribute to virulence. The large DNA viruses, in particular, carry a certain amount of genetic information that is not absolutely essential, at least for replication in cultured cells. Genetic surgery has been used to construct deletion mutants of certain herpesviruses, one of which (pseu-dorabies of swine) is now in use in veterinary praclice.
Site-directed mutagenesis now permits the introduction of prescribed nucleotide substitutions at will (see Chapter 4). When more becomes known about particular genes influential in virulence it will be possible nol only to delete or modify these genes but also to construct vaccines with any designated nucleotide sequence. Indeed, the day may come when licensing authorities demand that new vaccines be fully defined genetically, that is, that the complete nucleotide sequence of the vaccine virus be known and perhaps even stipulated.
Live Recombinant Viruses or Bacteria as Antiviral Vaccines
It is ironic that, no sooner had the World Health Organization (WHO) recommended the abandonment of vaccination following the eradication of smallpox, than an exciting new use was discovered for vaccinia virus. Recombinant DNA technology has opened a novel approach to vaccination which could prove to be of widespread applicability. The concept is to insert the gene for the protective surface protein of any chosen virus into the genome of an avirulent virus, which can then be administered as a live vaccine. Cells in which the vector virus replicates in vivo will produce this foreign protein and the body will mount both a humoral and a T-cell-mediated immune response to it.
In the original construct the hepatitis B surface antigen (HBsAg) gene, flanked by the nonessential vaccinia gene for thymidine kinase (TK) and its promoter, was inserted into a bacterial plasmid (Fig. 13-1). Mammalian cells infected with vaccinia virus were then transfected with this chimeric plasmid. Recombination occurred between the vaccinia DNA and the plasmid DNA. Selection for virus with a TK" phenotype enabled vaccinia virions containing the HBsAg gene to be recovered. This construct and analogous vaccinia recombinants incorporating genes from a range of other viruses have been
demonstrated to protect animals against the diseases they cause. For example, a vaccinia-rabies recombinant protects foxes and raccoons against rabies when incorporated in bait dropped from the air. Because the large vaccinia genome can accommodate at least a dozen foreign genes and still be packaged satisfactorily within the virion, it would theoretically be possible to construct a single recombinant virus as a vaccine capable of protecting against most of the common viral (and nonviral) infectious diseases of childhood. The potential benefits, particularly to the Third World, are self-evident.
DNA viruses of the families Poxviridae, Herpesviridae, and Adenoviridae can all be used as vectors, but vaccinia virus offers obvious advantages (Table 13-2). The major problem with vaccinia virus is that with unmodified smallpox vaccine there were always minor side effects, and in about 1 in 100,000 persons there were severe complications, with a substantially higher incidence in severely immunocompromised individuals. This problem has in principle been overcome by making deletion mutants of vaccinia virus that produce
Live Vaccinia Virus as Vector for Cloned Viral Genes Advantages
Large genome can accommodate several foreign inserts Production inexpensive Relatively stable, even in tropics Delivery (multiple puncture) by nonmedical personnel Cell-mediated immunity also elicited Disadvantages
Rare serious side effects with regular vaccinia virus vaccine" Revaccinatton with vaccinia vector within 5 years may be unreliable
" Attenuated vectors are now available, most have not yet been tested in large numbers of people, to determine the possible occurence of rare severe complications trivial lesions but still satisfactorily express incorporated foreign genes. It has also been found that birdpox viruses, which undergo restricted replication in mammalian cells, can express foreign genes effectively In both systems, the incorporation of genes encoding certain cytokines may greatly enhance the immune response. Recombinant poxviruses produced by these approaches are currently undergoing clinical trials.
Recombinant DNA technology also allows the expression of a viral epitope on the surface of a bacterium. The general approach is to insert the DNA encoding a protective viral epitope into a region of the genome of a bacterium that encodes a prominent surface domain on a protein normally situated on the exterior of the organism. Provided that the added loop protruding from the resultant hybrid protein does not seriously interfere with its transport, packaging, stability, or function, that organism will present the passenger epitope to the immune system of the host. Enteric bacteria which multiply naturally in the gut would seem to be ideal expression vectors for presenting protective epitopes of virulent enteric viruses to the gut-associated lymphoid tissue. The main candidates currently under development as potential vehicles are attenuated (vaccine) strains of Salmonella typhi, S typhimurium, Escherichia coli, and BCG, the world's most widely used live bacterial vaccine.
Inactivated Virus and Virus Subunit Vaccines
Inactivated ("killed") vaccines are made from virulent virus by chemically destroying its infectivity while retaining its immunogenicity. The traditional inactivating agent was formaldehyde, but this is being supplanted by P-propiolactone and ethylenimines; one of the advantages of p-propiolactone is that it is completely hydrolyzed within hours to nontoxic products Being noninfectious, such vaccines are generally safe but need to be injected in large amounts to elicit an antibody response commensurate with that attainable by a much smaller dose of live-virus vaccine Normally, even the primary course comprises two or three injections, and further ("booster") doses may be required at intervals over the succeeding years to revive waning immunity.
Large doses of inactivated virions often produce a febrile response as well as local reaction at the injection site, especially in young children. It seems logical to remove all nonessential components of the virion and inoculate only the relevant immunogen, namely, the particular surface (envelope or outer capsid) protein against which neutralizing antibodies are directed (Table 13-3). For example, the HA and NA glycoproteins can be extracted from influenza virions with detergent and used as a subunit vaccine.
To produce viral proteins more inexpensively on an industrial scale, recombinant DNA technology is now being widely exploited to clone the appropriate genes in prokaryotic or eukaryotic cells. Particular advantages of this approach are that safe, noninfectious vaccines can be made against viruses which cannot be cultured satisfactorily in vitro, for example, HIV or hepatitis B, or for which it has proved difficult to develop safe, effective live-virus vaccines.
Once the critical structural protein conferring protection has been identified, its gene (or, in the case of an RNA virus, a cDNA copy of the gene) may be cloned in bacteria, yeasts, insect cells, or mammalian cells. The yield of functional viral protein from bacteria is often poor, for a variety of reasons. Yeast cells have some capacity to glycosylate and secrete proteins, and the industrial technology for the large-scale cultivation of yeasts is well developed. The current hepatitis B vaccine, produced in Saccharomyces cerevisiae,
Defined Antigen Vaccines
Protein "cloned" by recombinant DNA technology (in yeasts, insect, or mammalian cells} Synthetic peptide Advantages Production and quality control simple Nontoxic, nonallergenic Feasible even if virus cannot be cultured
Safer if virus is highly virulent (e g , HiV), persistent (p g , heipesvirus), or carcinogenic (e.g , hepatitis B virus) Disadvantages Less immunogenic than whole virus Requires adjuvant Requires booster injections May (ail to elicit cell-mcdiated immunity was the first genetically engineered vaccine to be licensed for use in humans. Mammalian cells present advantages over cells from lower eukaryotes in that they possess the machinery for posttranslational processing of mammalian viral proteins including glycosylation and secretion. A wide variety of mammalian DNA virus vectors have been constructed to facilitate or prevent viral replication as desired, or to regulate gene expression. A fascinating alternative is the use of insect viruses of the baculovirus family growing in cultured moth cells (or caterpillars!). The promoter for the gene encoding the nonessential viral polyhedrin protein is so strong that the product of a foreign gene inserted within the polyhedrin gene may comprise up to half of all the protein the infected cells make.
Techniques have been developed for locating and defining epitopes on viral proteins, and it is possible to synthesize peptides corresponding to these antigenic domains (Table 13-4). Such synthetic peptides have been shown to elicit neutralizing antibodies against HIV and a few other viruses, but in general have been disappointing. The main reason for this is that, in the native antigen, most of the epitopes recognized by antibodies are not continuous but assembled, that is, not a linear array of contiguous amino acids but an assemblage of amino acids which, although separated in the primary sequence, are brought into close apposition by the folding of the polypeptide chain(s). In contrast, the epitopes recognized by T lymphocytes are short, linear peptides (bound to MHC protein). Further, some of these "T-cell epitopes" are conserved between strains of viruses and therefore elicit a cross-reactive T-cell response. Thus, attention is moving toward the construction of artificial heteropolymers of T-cell epitopes and 15-ceII epitopes, perhaps coupled to a peptide facilitating fusion with cell membranes to enhance uptake. Such constructs might prime T cells to respond more vigorously when boosted with an inactivated whole-virus vaccine, or on natural challenge.
Synthetic Peptides as Potential Vaccines Advantages
Short defined amino acid sequence representing piotective epitope Safe, nontoxic, stable
T-cell epitopes are naturally presented in the form of peptides
Artificial constructs may be engineered to contain B-cell and T-cell epitopes, or epitopes of one or more proteins Disadvantages
Poorly immunogenic; adjuvant, liposome, iscom, or polymer needed
Most B-cell epitopes are assembled (discontinuous)
May be too specific, not protecting against natural varianls
Single-epitope vaccine will readily select mutants
No response in those lacking appropriate class II MHC antigen
An even more revolutionary technique for vaccination may be in the wings. To the surprise of immunologists, it has been found that intramuscular injection of cloned viral DNA, m a plasmid with suitable promoters, can produce long-lasting antibody and cell-mediated immune responses to the protein(s) encoded by that DNA. Likewise, the immunomodulatory effects of various cytokines can be produced by the intramuscular injection of the relevant DNA. The potential of this technique as a method of vaccination is being vigorously explored by pharmaceutical companies, although all concerned realize that much research and development lie ahead if it is ever to become a practical proposition.
It will be necessary, for example, to obtain detailed information on such critical aspects as the location (especially elsewhere than in muscle cells) and fate of the injected plasmids, their persistence, the mechanism of expression, including the processing of peptides through cytosolic and endosomal pathways and presentation via class I and II MHC antigens to Tc and Th lymphocytes, the con tinuity of priming of various classes of memory T cells, the mecha-nism of protection m vivo in the shot t and long term, the mass of DNA required in humans as opposed to mice, and the optimum methods of presentation (packaging) and protection of the injected DNA. Above all, licensing authorities will need to be convinced that there is no realistic possibility of integration of the foreign nucleic acid into the host genome which might lead to cancer.
The antigen-binding site of the antibody produced by each B-cell clone contains a unique amino acid sequence known as its idiotypic determinant or id-iotype (id). Antibodies can be raised against this idiotype; they are known as anti-idiotypic antibodies (anti-Id). Because the original antigen binds to the same variable region of the antibody molecule as does anti-Id, they might be expected to have similar conformations. If so, anti-Id could be employed as a surrogate antigen, that is, as a vaccine to elicit an antiviral immune response.
There are now a number ot examples of anti-idiotypic antibodies that elicit specific antiviral B-cell and/or T-cell responses. It is still uncertain whether this points the way to a novel vaccine strategy.
The immunogenicity of inactivated vaccines, and especially of purified protein vaccines and synthetic peptides, usually needs to be enhanced in some way. This may be achieved by mixing the antigen with an adjuvant or incorporating it into liposomes or into an iinimimsHimilating complex (tscom)
Adjuvants are substances that, when mixed with vaccines, potentiate the immune response, humoral and/or cellular, so that a lesser quantity of anti gen and/or fewer doses will suffice. Adjuvants differ greatly m their chemistry and in their modes of action, which may include the following' (1) prolongation of release of antigen; (2) activation of macrophages, leading to secretion of cytokines and attraction of lymphocytes, (3) mitogenicity for lymphocytes. Alum is the only adjuvant currently licensed lor use in humans, and has been widely used for years, but it is not particularly effective. Mineral oil adjuvants are used in animals but are too reactogenic to be acceptable in humans. There is a clear requirement for better adjuvants, preferably chemically defined and of known mode of action. For instance, there are now a number of experimental formulations based on muramyl dipeptide, which can also be chemically coupled to synthetic antigens or incorporated into liposomes.
Liposomes consist of artificial lipid membrane spheres into which proteins can be incorporated. When purified viral envelope proteins are used, the resulting "virosomes" (or "immunosomes") somewhat resemble the original envelope of the virion. This enables one not only to reconstitute virus envelope-like structures lacking nucleic acid and other viral components, but also to select nonpyrogenic lipids and to incorporate substances with adjuvant activity. Although liposomes have not fully lived up to expectations as immunogens, other types of membrane-bound micelles (i.e., aggregates of protein molecules) can fully restore the immunogenicity lost when viral glycoprotein is removed from its original milieu.
When viral envelope glycoproteins or synthetic peptides are mixed with cholesterol plus a glycoside known as Quil A, a spherical cagelike structure 40 nm in diameter is formed. These iscoms (irnmunostimulating complexes) have been shown experimentally to display significantly enhanced immunogenicity but have not yet been developed commercially as vaccines.
The relative advantages and disadvantages of live-virus vaccines compared with inactivated or subunit vaccines are summarized in Table 13-5 and discussed below
Naturally acquired immunity to reinfection is virtually lifelong in the case of most of the viruses that reach their target organ(s) via systemic (viremic) spread. This solid immunity is attributable to antibody of the IgG class, which successfully neutralizes the incoming challenge virus. Immunity to those respiratory and enteric viruses whose pathogenic effects are manifested mainly at the site of entry is attributable mainly to antibodies of the IgA class and tends to be of shorter duration. Thus the principal objective of artificial immunization by vaccine is to elicit a high titer of neutralizing antibodies of the appropriate class, directed against the relevant epitopes on the surface of the
Advantages and Limitations of Live and Inactivated Vaccines
Route of administration Dose of virus, cost Number of doses Need for adjuvant Duration of immunity Antibody response Cell-mediated response Heat lability Interference Side effects Reversion to virulence
Natural or injection Low
Single, generally No
IgG, IgA (mucosal route)
Oral poliovaccine only
Rarely; oral poliovaccine only
Generally less than live vaccines
Occasional, local No
No No virion, in the hope of preventing initiation of infection by neutralization of the challenge virus.
This ideal is not always realizable, but it may not matter if the progress of the infection can be curtailed sufficiently to allow time for the emergence of the quite different set of immunologic mechanisms that contribute to recovery from viral infection. Provided that enough memory T and B cells are still present at the time of challenge, anamnestic T-celi- and B-cell-mediated responses will be mounted without undue delay. In particular, recovery will be accelerated by cytotoxic T-cell-mediated cytolysis of infected cells. Such "memory-dependent immunity" may be particularly important in diseases with a relatively long incubation period.
It has proved difficult to produce effective vaccines against three classes of viral diseases: respiratory infections, sexually transmitted diseases, and persistent infections. Mucosal immunity, mediated by IgA, is important in both respiratory and sexually transmitted diseases; there is evidence that vaccination by the convenient oral route may generate satisfactory mucosal immunity in the respiratory and genital tracts, as well as in the intestinal tract, as a result of lymphocyte trafficking between different compartments of the "common mucosal pathway." Antigenic drift and shift pose yet another problem, circumvention of which requires constant revision of the antigenic composition of vaccines such as those used to control influenza. Special difficulties also attend vaccination against viruses known to establish persistent infections, such as herpesviruses and retroviruses; a vaccine must be outstandingly effective if it is to prevent not only the primary disease but also the establishment of lifelong latency.
Subclinical infection is, by and large, extremely effective, inducing lifelong immunity following systemic infection. Live avirulent vaccines, preferably but not necessarily delivered via the natural route, are obviously the nearest approach to this ideal. The track record of live vaccines against major human diseases such as smallpox, yellow fever, poliomyelitis, and measles is clearly superior to that of most of the inactivated vaccines devised so far. Why is this so?
First, one must consider the quantitative advantage of a live immunogen which replicates many millionfold following delivery. Second, whether delivered via the natural route or not, live virus will be presented to the various arms of the immune system in a natural way. Not only will virions be processed and presented via the endosomal pathway to MHC class II restricted helper T cells, but peptides derived from newly synthesized viral proteins in infected cells will be presented via the cytosolic pathway lo class I restricted cytotoxic T cells. Thus live vaccines are generally found to be much more effective in eliciting cell-mediated immunity. Furthermore, live vaccines may elicit a broader immunologic response, because many cytotoxic and helper T cells are directed to conserved epitopes on internal or nonstructural proteins that are shared between different strains of virus. There is also the possibility that live vaccine virus persists in some form for years or that larger amounts of relevant antigens are sequestered, on follicular dendritic cells or elsewhere, for longer than with other types of vaccines. It does appear that memory T and B lymphocytes are more effectively recruited by live than by inactivated vaccines, though very little is known about the optimal conditions of antigen presentation for eliciting B- or T-cell memory. ' '
The efficacy of an immunogen depends not only on the dose of protein delivered but also on the form in which that protein is presented to the immune system. For example, immunogen in certain forms may elicit a stronger response in some classes of T cells than others, and it may be crucial to avoid, say, a suppressor T-cell response at the expense of a helper or cytotoxic response. An excessive DTH response or IgE response might also be detrimental under some circumstances. There have been some unforeseen immu-nopathologic consequences of immunization with certain inactivated respiratory vaccines, for example.
Special problems attend the choice of submolecular immunogens such as synthetic peptides. Because each individual in an outbred population carries a unique set of MHC molecules, any particular T-cell epitope is unlikely to be recognized by all humans. Therefore, if synthetic peptides or other sub-molecular constructs are to be used as vaccines, it will be necessary to ensure, that they include at least one T-cell epitope recognized by a class I molecule and one recognized by a class II molecule present in most people, as well as at least one B-cell epitope that is immunodominant in a large majority of the population and that elicits a protective antibody response.
To be acceptable, a vaccine must be both safe and efficacious. Licensing authorities have become extremely vigilant and have insisted on rigorous safety tests since residual live virulent virus in certain batches of inactivated vaccines caused a number of tragedies in pioneering days. Live attenuated vaccines present a different set of challenges that must be met before any particular product is released onto the market; these are discussed below.
Because vaccine viruses are grown in cells derived from humans or animals, there is always a possibility that a vaccine will be contaminated with another virus, derived from those cells or from the medium (especially the serum) in which the cells are cultured. For example, primary monkey kidney cell cultures, once widely use for the manufacture of poliovaccines, have, at one time or another, yielded over 75 simian viruses, some of which are pathogenic for humans. This danger has led to a swing away from primary cell cultures toward well-characterized continuous cell lines which can be subjected to comprehensive screening for endogenous agents before being certified as safe, then stored frozen for many years as seed lots.
Some excellent human viral vaccines in routine use, such as rubella and measles vaccines, produce some symptoms—in effect, a very mild case of the disease—in a minority of recipients. Attempts to attenuate virulence further by additional passages in cultured cells have been accompanied by a decline in the capacity of the virus to replicate in humans, with a corresponding loss of immunogenicity. Such trivial side effects as do occur with current human viral vaccines are of no real consequence and do not prove to be a significant disincentive to immunization, provided that parents of vaccinated children are adequately informed in advance.
A different problem occurs in the case of vaccine strains with an inherent tendency to revert toward virulence during replication in the recipient. The only example in a human vaccine in general usage is oral poliovaccine (OPV). Exceedingly rarely (less than once in every million vaccinees), "vaccine-associated" poliomyelitis is seen, either in a congenitally immunodeficient baby or, even more rarely, in an unvaccinated parent to whom the virulent revertant has spread. For this reason, inactivated poliovaccine is preferred in known immunocompromised individuals.
Live vaccines are vulnerable to inactivation by high ambient temperatures, which presents a particular problem in the tropics. Because most tropical countries also have underdeveloped health services, difficulties are encountered in maintaining the "cold chain" from manufacturer to the point of delivery, namely, the child in some remote rural village. To some extent the problem has been alleviated by the addition of stabilizing agents to the vaccines, and by packaging them in freeze-dried form for reconstitution immediately before administration. In other cases simple portable refrigerators have been developed and placed in the field.
Live vaccines delivered by mouth or nose depend for their efficacy on replication in the enteric and respiratory tract, respectively. Interference can occur between different live viruses contained in vaccines delivered via the natural route (e.g., between the three serotypes of poliovirus in OPV if their concentrations are not appropriately balanced), or between the vaccine virus and itinerant enteric or respiratory viruses that happen to be growing in the vaccinee at the time. The latter is the reason why OPV is routinely adminis-
Vir.il Vaccines Recommended for Human Use" ''
Vir.il Vaccines Recommended for Human Use" ''
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