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The basic goal of vaccination is to stimulate protective immunity while avoiding disease from the vaccine itself. The first generation of viral vaccines relied on empirical attenuation by repeated passage in cultured cells. Several LAVs meet both criteria for vaccines; they elicit a strong and protective immune response with a low risk of disease from the vaccine itself. Despite recent successes in the development of LAVs for rotavirus and several arboviruses, the classical attenuation process is somewhat unpredictable and has not always been applicable. In the present regulatory environment, the use of LAVs has also been limited by safety concerns, including reversion to wild-type virulence. Because LAVs are shed from vaccinees, they sometimes present a risk to unvaccinated individuals with impaired immunity.

These safety concerns have led to a shift toward the use of inactivated viruses or viral subunits as vaccines. Despite notable successes like the inactivated poliovirus vaccine1, inactivated viruses are generally less immunogenic than their LAV counterparts, and this strategy is limited to viruses for which there are good culture and production systems. Subunit vaccines, which use viral proteins as immunogens, have become a major focus of vaccine development and have led to several successfully licensed vaccines, including vaccines against hepatitis B virus, influenza viruses and papillomaviruses2. Production is more easily controlled and efficient than that of LAVs or inactivated viruses. Even so, this strategy has not achieved universal success, as many subunit vaccines have failed to elicit a protective immune response in the host. Although adjuvants have increased the immunogenicity of subunit vaccines, newer methods of subunit delivery mimic a natural immune response by incorporating more viral components. There are several approaches to this end, including liposome delivery of antigens3,4, viruslike particles5 and virosomes, which are reconstituted viral envelopes lacking any viral genetic material6. Another approach to increase the immunogenicity of subunit vaccines is to recombinantly encode a pathogenic antigen in a nonpathogenic, yet infectious poxvirus or adenovirus vector7,8. Although there have been some notable successes, the major concern with this strategy is that the vector vaccines will not induce adequate immunological responses in hosts who have preexisting antibodies against the vector.

As vaccines become more complex and 'virus-like,' unsurprisingly, live, attenuated vaccines have received a second look. Advances in molecular virology and the advent of recombinant-virus systems have led to the identification of many viral genes associated with virulence and immunogenicity. Researchers have used this information to better control the replication and pathogenesis of vaccine candidates, thereby avoiding the unpredictability of empirical attenuation.

Here we first review attempts to use mutation or deletion of replication genes to create attenuated virus. We then discuss the application of four new methods—altered replication fidelity, codon deoptimization, and control by microRNAs (miRNAs) or zinc finger nucleases (ZFNs)—to rationally design vaccines. These novel LAV designs each allow limited viral replication and antigen production, and because the host immune response is not required to limit viral spread, such LAVs may be safer than classic LAVs, even in immunocompromised patients.

Attenuation through deletion or mutation

The identification of genes essential for viral replication and assembly led to the first generation of rationally designed, live-virus vaccines (Table 1). Deletion or mutation of these genes results in a 'defective virus,' which cannot replicate in the host (for an excellent review of defective-virus vaccines, see Dudek and Knipe8). These defective viruses are propagated in 'helper' cells that express the missing gene(s). Although the virus is unable to replicate its genome, viral genes are still expressed, which can induce a strong immune response in the inoculated host. 'Single-cycle viruses,' which are defective in a viral protein required for assembly or spread, are a variation on this theme. Although these viruses can replicate their genome through a single cycle, they do not produce infectious virus9.

Table 1 Current vaccine strategies
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The first example of a replication-defective virus used as a vaccine was a herpes simplex virus-1 (HSV-1) strain with a deletion of a gene essential for genome replication10. This virus stimulated an immune response similar to natural infection and protected against wild-type virus challenge in a mouse model of infection11. Replication-defective HSV-2 strains, which lack genes essential for viral DNA synthesis (UL5) and viral replication (UL29), have also been described12. These viruses were more effective than subunit vaccines in eliciting protective immunity in mice13 and did not establish latency14, an important consideration in herpesviruses. HSV-1 and HSV-2 strains have also been created that lack glycoprotein H and are unable to spread from cell to cell or produce infectious progeny. These single-cycle viruses protect against wild-type challenge in rodent models15,16, but the block in viral spread may be leaky. Similar strategies are now being applied to viruses other than HSV. The newer smallpox vaccines are replication-defective viruses8, and an influenza nuclear export protein (formerly referred to as the NS-2) knockout and hemagglutinin cleavage site mutants have been shown to provide protective immunity in mice17,18. Likewise, flaviviruses with a deletion in the core nucleocapsid protein C function are single-cycle viruses as they cannot spread between cells or encapsidate virus. These viruses can elicit a potent immune response and protect against wild-type challenge19.

Even with progress in the attenuation of viruses by deleterious gene mutation, this approach has not led to a safe and effective vaccine for human disease. On the one hand, this can be attributed to the relatively short time this field has been in existence; on the other, vaccines based on deleterious gene mutation also often evoke only a weak immune response because the antigen is only expressed at the site of inoculation. There are also safety concerns about the completeness of the block in viral spread in single-cycle viruses19. As with conventional LAVs, it has proven very difficult to balance immunogenicity with safety, even with the rational design of replication-defective viruses.

Riboviral replication fidelity—failure then success

Although LAV vaccines have been developed for many RNA viruses, the mutability of these pathogens presents unique challenges for vaccine design. The RNA-dependent RNA polymerases of RNA viruses exhibit characteristically low fidelity, with measured mutation rates of 10−3 to 10−5 mutations per nucleotide copied per replication cycle20. These mutation rates are orders of magnitude greater than those of nearly all DNA-based viruses and organisms. Because the genomes of RNA viruses typically comprise <10,000 nucleotides (nt), this mutation rate translates to roughly 0.1–10 mutations per genome replicated. Work in our laboratory (R.A. and colleagues21) estimates that each viral replication cycle generates every possible point mutation and many double mutations, which may be present within the population at any time. This impressive diversity has important biological implications. First, low-frequency variants within the population may contain, or quickly acquire, mutations in key epitopes, which mediate escape from vaccine-elicited neutralizing antibody or cytotoxic T cells22. Antigenic drift within the hemagglutinin and neuraminidase proteins of influenza virus is the best example of this process and the primary reason for annual reevaluation of vaccine strains23. Second, many RNA viruses, including HIV and the hepatitis C virus, exhibit such marked intra- and interindividual genetic diversity that it has been difficult to identify stable, conserved epitopes that provide universal protection against all strains22. Finally, the mutability of RNA viruses has triggered real concerns about the potential reversion of live, attenuated vaccines to pathogenic strains. Both mutation and recombination probably have a role in this process, and the sporadic emergence of vaccine-derived polioviruses is a cautionary tale24.

A large body of work in recent years suggests that because of their mutation rates, the evolution of RNA viruses may differ fundamentally from that of DNA-based organisms. Much of this work builds on the mathematical framework of quasi-species theory and seeks to understand the importance of genetic diversity at the population level25. According to quasi-species theory, the mutation rates of RNA viruses place them near a critical 'error threshold.' Below this threshold, the mutant spectrum within the population favors adaptability, and low-fitness variants are tolerated so long as the majority remains viable. Beyond the error threshold, too many mutations accumulate, genetic information is lost and the population becomes inviable26. Indeed, mutagenic nucleosides increase viral mutation rate and cause population collapse, thus providing an effective treatment for several RNA viruses. Even so, several groups have identified mutants in poliovirus and foot-and-mouth disease virus that were resistant to nucleoside analogs27,28,29. Further studies in our laboratory (R.A. and colleagues30) have revealed that these variants replicate with higher fidelity by virtue of mutations within the viral RNA-dependent RNA polymerase. As a result, drug-resistant mutants give rise to populations with appreciably less genetic diversity than the wild type30. Significantly, this decrease in diversity was responsible for attenuation in a transgenic mouse model of infection30,31.

On the basis of these observations, our group (R.A. and colleagues32) proposed that the attenuation of these high-fidelity variants could be exploited for vaccine design. We focused on glycine 64 of the poliovirus polymerase, which regulates fidelity through a complex hydrogen-bond network and mediates sensitivity to nucleoside analogs27. Of the 19 possible amino acid substitutions at this position, only 13 gave rise to viable virus and 8 of these were unstable. The other 5 mutants had lower mutation rates than wild type and were less adaptable in cell culture. In the transgenic mouse model, these high-fidelity variants were markedly attenuated and shed less efficiently than wild type. Three of the viruses stimulated high titers of neutralizing antibody in infected mice, an order of magnitude greater than the Sabin 1 vaccine strain. They also induced long-lasting immunity. Mice vaccinated with G64S, G64A or G64L survived a lethal challenge of wild-type virus at 1 or 6 months via the intraperitoneal or intramuscular route.

This work suggests that controlling replication fidelity is a promising approach for engineering live, attenuated vaccines. Even so, several important questions remain. Although it is clear that such a strategy could be successful for other picornaviruses, which have structurally conserved polymerases, it may be difficult to identify the relevant residues in other viral RNA-dependent RNA polymerases. In these cases, selection for nucleoside analog resistance may be an unbiased way of discovering promising mutants for further characterization. Reversion to wild type is another potential problem, because viruses containing the lower fidelity wild-type polymerase appear to have a selective advantage. Although high-fidelity variants would certainly revert at a lower frequency, their mutation rate is still considerably higher than that of DNA viruses20,30. We found no evidence for reversion of the G64S mutation after either 20 passages in HeLa cells or five mouse-to-mouse passages over 25 days32. Although these results are encouraging, further experiments along these lines will probably be required before regulatory approval. Finally, the mouse model for poliovirus pathogenesis is an imperfect one, and the level of attenuation observed here may not reflect the situation in human vaccinees. Nevertheless, the high-fidelity variants could still be useful in the ongoing polio eradication campaign, as safer seed strains will be needed for large-scale production of the inactivated polio vaccine in a postpolio world33.

Attenuation by substitution

It is well known that many organisms exhibit a codon bias, using some synonymous codons or codon pairs more frequently than others34. In bacteria and simple eukaryotes, codon preference is related to amounts of the corresponding transfer RNA and affects translational efficiency35,36. The reasons for the observed codon bias are less clear in mammals. Because viruses rely on the host-cell machinery for nearly all aspects of replication, it is not surprising that codon bias has been described in many viral genomes. In bacteriophage, codon usage closely mirrors that of the host37. The bias is more pronounced in the highly expressed structural genes, suggesting optimization for translational efficiency38,39. Most mammalian viruses also have a strong preference against CpG dinucleotides, although their overall GC content is highly variable38. Studies of HIV and influenza suggest that codons in highly variable surface proteins may be optimized for their volatility, the probability that a codon will mutate to a different amino acid class40,41. This would presumably facilitate immune escape and suggests that there has been selection for genetic plasticity in these highly mutable viruses.

Recent studies of poliovirus have addressed the importance of codon bias for viral replication and pathogenesis. Burns and colleagues performed large-scale mutagenesis of the Sabin 2 vaccine strain, replacing up to 50% of the capsid codons with synonymous codons that are less preferred in the human genome42. Although these codon-deoptimized viruses exhibit minimal defects in viral gene expression, they produce fewer infectious progeny and overall fitness is markedly reduced. A subsequent study found that synonymous changes that increase the frequency of CpG and UpA dinucleotides have similar effects on viral fitness43. Mueller and colleagues44 took a similar approach but used gene synthesis technology to design poliovirus genomes with completely deoptimized codons in the capsid region. They also found a marked reduction in replicative fitness and a reduction in infectious progeny. Even so, their data imply that codon-deoptimized viruses have reduced translational efficiency compared with wild type. They obtained similar results with viruses in which synonymous changes are determined by codon pair bias. In both cases they found that codon-deoptimized polioviruses are attenuated by 1,000-fold on a per-particle basis compared with wild type.

Because all changes are synonymous, the proteins expressed from codon-deoptimized viruses are identical to wild type and similarly immunogenic. Mueller and colleagues45, therefore, proposed that their marked attenuation would make them ideal live vaccines. In their second study, they show that deoptimized viruses provoke a robust neutralizing antibody response following three weekly intraperitoneal inoculations. All immunized mice survive subsequent lethal challenge with wild-type poliovirus, demonstrating the vaccine efficacy of the engineered viruses.

As a general strategy for vaccine development, codon deoptimization offers several advantages. First, attenuation does not affect antigenicity, and the immune response should closely mimic a natural infection. Second, because attenuation is systematic and not empirical, it may be easily applied to other viruses. Finally, codon-deoptimized viruses encode hundreds of point mutations, each with a fairly small individual effect on fitness. Consequently, there is little risk of reversion to virulence with even a handful of point mutations. Both Mueller et al.42 and Burns et al.44 found that codon-deoptimized viruses are genetically stable and remain attenuated after repeated passage. The marked sequence divergence of such engineered viruses from circulating strains may also reduce the frequency of recombination and the risk of pathogenic, vaccine-derived variants.

Much work remains to be done before codon-deoptimized viruses are employed as live, attenuated vaccines. Although the results among the studies are consistent, the mechanism of attenuation is still debated. This would certainly be an issue for regulatory bodies, and the lack of clarity makes it difficult to determine whether codon-based attenuation is a unique aspect of picornaviruses, or a more generalizable approach to vaccine design. As in the case of the high-fidelity variants, the mouse model may not be the best system for assessing vaccine efficacy and safety. Nevertheless, codon deoptimization is a promising approach that has already generated significant interest in the virology community.

miRNA-controlled LAVs

The discovery of RNA interference (RNAi) just a decade ago has resulted in an explosion of research into this novel form of gene regulation. The two main effectors of RNAi are small interfering RNA (siRNA) and miRNA46. Although there has been intense interest in using siRNAs to combat mammalian RNA viruses47, miRNAs are now also being used to limit viral pathogenesis. miRNAs are genomically encoded and have a major role in endogenous gene regulation48. They are transcribed as long precursor pri-miRNAs, which are processed by the nuclear RNase Drosha to ∼60-nt hairpin intermediates, which are then transported to the cytoplasm where they are trimmed by Dicer to roughly ∼22 nt (Fig. 1). Similar to siRNAs, mature miRNAs are loaded into the RNA-induced silencing complex, where they mediate either degradation or translational repression of target messages. The human genome encodes over 400 miRNAs, many of which have tissue-specific or developmental expression patterns. Several DNA viruses also express miRNAs49. These virally derived miRNAs modulate pathogenesis and host immunity through regulation of viral and cellular transcripts, respectively.

Figure 1: The microRNA (miRNA)-virus vaccine strategy.
figure 1

Viral replication can be regulated in a tissue-specific manner by incorporating miRNA target sites into the viral genome. In cells that express the miRNA (e.g., brain, top cell), the miRNAs are processed and transported to the cytoplasm, where they mediate cleavage of viral RNA. Viral replication is restricted to cells in which the miRNA is not expressed (e.g., intestine, bottom cell). The engineered virus can therefore trigger a natural immune response in target tissues without the associated risk of dissemination and disease.

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The diversity and complexity of cellular miRNAs means that many cell types will have a unique miRNA profile50. Several investigators have taken advantage of this property to better target viral gene therapy vectors51. Silencing of specific transcripts or the entire genome can be accomplished by inclusion of miRNA binding sites in the vector sequence. In many cases the miRNA system is used to provide a second level of control beyond receptor expression or tissue-specific promoter activity. For example, Brown and colleagues52 eliminated off-target expression from a hepatocyte-specific promoter in antigen-presenting cells by incorporating miR-142-3p binding sites in their lentiviral construct52. In a related study, muscle-specific miRNA binding sites were used to limit secondary replication of a coxsackievirus in a murine tumor model53. Improved targeting of adenoviral vectors has also been achieved by the addition of miRNA binding sites to the 3′ untranslated region of the E1A transcript54,55.

In LAV design, empirical attenuation of viruses is often accomplished by changing the tissue tropism of a virus through repeated passage in a new cell type. We (R.A. and colleagues)56 proposed that the same result could be achieved through miRNA restriction of poliovirus replication. Although poliovirus replicates in many tissues, disease onset is linked to lytic infection of the central nervous system (CNS). By incorporating binding sites for either let7a (a ubiquitous miRNA) or miR124 (a CNS-restricted miRNA) into the RNA genome of wild-type poliovirus (Fig. 1), we showed that viral replication is restricted in a cell type–dependent manner and that the effect is dependent on the cellular RNAi machinery. The miRNA-targeted viruses are largely restricted from the CNS in a murine model of infection and markedly attenuated as a result56. Experiments with viruses containing mutant target sequences confirm that the altered tropism is due to miRNA. The degree of attenuation exceeds 5 orders of magnitude, and neither let7a- nor miR124-targeted viruses are pathogenic in immunocompromised mice lacking the α/β-interferon receptor56. Both viruses are able to replicate in non-neuronal tissues and stimulate a strong neutralizing antibody response after a single intraperitoneal inoculation. The level of protection is impressive, as even the interferon receptor–knockout mice are protected from subsequent challenge with 10,000 times the lethal dose of wild-type virus56.

Although miRNA targeting is a promising approach to rational design of LAVs, the study has several caveats worth mentioning. The let7a virus replicates poorly in most tissues, whereas the mir124 virus is restricted only in the CNS56. As a result, the former stimulates a weaker immune response and is a less effective vaccine. On the other hand, widespread replication of the mir124 virus in non-neuronal tissues could allow the virus to accumulate mutations within the miRNA target sequence and thereby escape degradation. Indeed, several mice in the study had low titers of mir124 virus in the spinal cord, and sequence analysis showed mutations within the miRNA target sequences56. Work from our laboratory suggests that a single let7a site can accumulate escape mutations in as little as 24–48 h32. The risk of miRNA escape could be minimized by the inclusion of several target sequences for the same miRNA or different miRNAs with the same tissue distribution.

Another way of minimizing escape is highlighted in another article on species-specific restriction of influenza virus for vaccine production57. In this study Perez and colleagues57 incorporated nonavian miRNA target sequences into a region of the viral nucleoprotein open reading frame. Because the miRNA target sequence also serves as codons for conserved amino acids, escape mutations would alter protein structure and probably have a deleterious effect on viral replication.

We expect that as the RNAi field matures, investigators will find other ways of controlling the replication and mutability of miRNA-targeted vaccines, although the potential for reversion to wild type will have to be mitigated to the satisfaction of regulatory bodies.

Zinc-finger nuclease-controlled LAVs

Zinc-finger (ZF) domains mediate nucleotide-specific binding of proteins to DNA, a property that defines a large family of DNA-binding proteins58. Each finger makes contact with a separate DNA triplet, and natural or recombinant ZFs have been created that can recognize almost any triplet59. The modular nature of the ZFs allows them to be joined in useful combinations. Typically, three ZFs are combined to bind to a specific 9–base pair (bp) DNA sequence, and these ZFs have been coupled to various functional domains to create artificial transcription factors that can activate or repress gene transcription with remarkable promoter specificity60. ZFs have also been fused to the nuclease domain of the restriction enzyme FokI to cleave double-stranded DNA at specific sequences61. The nuclease domain must dimerize to cleave DNA, and because the dimer interface is weak, two nuclease domains are typically brought into close proximity by pairs of ZFs binding to neighboring 9-bp sites, spaced 6 bp apart (Fig. 2)62,63. In this configuration the engineered ZFN recognizes a specific 18-bp sequence, which is long enough, by a few orders of magnitude, to be unique in the human genome. Because of this specificity, this same technology could be used to distinguish between human and virus DNA.

Figure 2: Zinc-finger nuclease (ZFN)-virus vaccine strategy.
figure 2

The ZFN is composed of two arrays of three ZF domains fused to a DNA nuclease domain (blue lightning bolt). The nuclease must dimerize to be activated, so each ZFN array is designed to bind the adjacent 9-bp sequences in the virus genome, spaced 5–6 bp apart, allowing the nuclease domains to dimerize and cleave the viral double-stranded DNA. ZFNs can be designed to target multiple, essential viral sequences. By encoding the ZFNs in the viral genome itself and temporally controlling the expression of the ZFNs using viral promoters, the virus can express immunogenic proteins before ZFN cleavage of circular episomal DNA to linear DNA, which is incapable of replication and establishment of latency.

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Several groups have used recombinant ZF proteins to control aspects of the viral life cycle. ZF proteins fused to the ZF protein 10 gene (ZNF10/KOX1) repression domain have been created that target the HSV-1–infected cell polypeptide 4 (ICP4) promoter64. These proteins bind the promoter with nanomolar affinity, with one able to markedly repress viral protein 16 (VP16)–activated transcription in vitro. This ZF-ZNF10 fusion, when delivered in trans into HSV-1–infected cells, was able to limit HSV-1 replication and reduce viral titer by 90%. In a similar strategy, recombinant ZF proteins have been designed to recognize the human papillomavirus 18 (HPV-18) replication origin65. When expressed in vitro, these ZFs were able to compete with the replication protein E2 for binding to viral DNA. This competitive antagonism led to reduced HPV replication in transient replication assays in mammalian cells. By fusing the origin-targeted ZF protein to a nuclease domain, this ZFN was able to cleave viral DNA and reduce viral replication in cultured cells66. These experiments demonstrate that ZFNs can effectively target and eliminate viral DNA in mammalian cells.

It may be feasible to deliver a therapeutic virus-specific ZFN in trans to eradicate latent viral DNA. Delivery of the ZFNs to all latently infected cells is, however, technically challenging. Alternatively, virus-specific ZFNs could be delivered using the viral genome itself and serve as a vaccine. In the ZFN-vaccine strategy, ZFNs targeting sequences for viral replication and other essential viral processes would be introduced into the viral genome (Fig. 2). Following inoculation, immunogenic viral genes and virus-specific ZFNs would be expressed. While the viral proteins would stimulate a natural immune response, the ZFNs would cleave viral DNA, and limit replication. ZFN-LAVs have potential both as prophylactic vaccines, protecting against wild-type challenge, as well as therapeutic vaccines, delivering ZFNs to cells already harboring latent viral DNA.

The immunogenicity of ZFN vaccines can be controlled by temporal and spatial regulation of ZF expression to balance viral protein expression with the ability of the ZFNs to eliminate all replication-competent viral DNA. This could best be accomplished using promoters that are temporally controlled by the virus itself. For instance, herpesvirus gene transcription occurs in at least three distinct stages: immediate-early (before most of viral protein synthesis), early (before viral replication) and late (after viral replication begins)67. Other DNA viruses for which ZFNs would be useful are similarly regulated. There is also the potential to encode ZFNs behind inducible promoters, so that ZFN expression would commence upon the administration of a small molecule68. Nuclease activity can also be controlled directly by addition of small molecule–sensitive residues to the ZFN69. These strategies would provide an ideal way to optimize the balance between ZFN-virus replication and nuclease activity.

The ability to create a ZFN vaccine that can prevent and eliminate persistent viral infections is a long way from being realized. As with any LAV, safety issues are always a concern. The ZFN vaccine approach would probably be limited to nonintegrating, DNA viruses, as random breaks in host chromosomal DNA caused by ZFN cleavage of integrated viral DNA could be catastrophic. There are many nonintegrating human viruses, includes the herpesviruses, polyomaviruses, adenoviruses and papillomaviruses, that establish a persistent infection and provide particularly difficult challenges for the treatment of their respective diseases. ZFN-based vaccines may offer a way to prevent or eliminate these hard-to-treat latent infections. Reversion to wild type is another concern, but the risk can be reduced by including ZFNs against multiple, essential viral sequences to ensure that the intrinsic mutation rate of the virus will not allow the mutation of every ZFN target site. It is also possible that DNA cleaved by ZFNs could be repaired via homologous recombination using uncleaved viral genomes. However, if the sequence is repaired accurately it would be subject to repeated cleavage; if it is repaired inaccurately the virus should be nonviable because of mutation of an essential sequence. Ideally, we will arrive at a live virus strain that will have limited replication, not establish latency and elicit a protective immune response. In essence, we would turn an otherwise detrimental latent infection into an asymptomatic, acute infection.

Conclusions

LAV vaccines have provided ideal protection from several major diseases but have not lived up to their potential as a result of limited applicability and safety concerns. Advances in molecular biology have opened the door to novel approaches to viral attenuation and may lead to a new generation of safer LAVs (Table 2). Although replication-defective LAVs have encountered some problems, this approach to attenuation is on the cusp of providing safe, effective vaccines for several diseases. Several other approaches to attenuation are poised to overcome other problems specifically associated with vaccine design for RNA and DNA viruses. For many RNA viruses wherein high mutation rates limit the efficacy of vaccines, altering the replication fidelity can attenuate the entire virus population, leading to population collapse without mutation of key immunogenic epitopes. Codon deoptimization provides a systematic means by which to attenuate any virus. Substituting synonymous codons throughout a viral genome avoids loss of immunogenicity and confers little risk of reversion to wild type. ZFNs and miRNAs can be used to control the replication of DNA and RNA viruses, respectively. Controlling viral replication temporally or spatially can cause a strong, natural immune response to be elicited before the virus is eliminated. These may be particularly useful approaches for designing vaccines against persistent or latent viruses, as ZFNs and miRNAs lead to the elimination of all viral DNA or RNA, thus preventing chronic infection.

Table 2 Approaches to viral attenuation for vaccine design
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Each of these approaches is aimed to address long-standing problems with LAV vaccine design. Although they could potentially change the way we think about attenuation, significant hurdles lie ahead. Live vaccines present an inherent trade-off between safety and efficacy, and regulatory bodies are right to be concerned about viral escape or reversion to wild type. The studies described here have largely been carried out in murine models with relatively short-term measures of immunogenicity and limited characterization of viral genetic stability. Much more work is needed in relevant animal models before an initial dosing and safety trial in humans can be contemplated. We expect that each strategy will have to be modified to optimize its safety and efficacy profile. Nevertheless, the efficacy demonstrated by available LAVs, particularly the recent success in developing safe and effective live, attenuated rotavirus, influenza and varicella zoster vaccines, is a strong incentive to redouble efforts to improve the safety characteristics of this type of vaccine. Rational attenuation may also facilitate the development of inactivated vaccines for high-risk agents by providing safer seed stocks for large-scale production. The next several years will clearly be an exciting time in vaccine research as advances in molecular biology are further translated into preventive strategies for viral disease.