In this paper we demonstrate that strains of VACV containing mutations in the E3L virulence gene are highly attenuated in several mouse models, yet still induce potent protective immune responses. In the C57BL/6 intra-nasal model, these viruses are from 3 logs to greater than 5 logs less neurovirulent than wtVACV (WR strain). All of the viruses tested, with the exception of VACVΔE3L, replicated to high titers in the nasal mucosa, but failed to spread to the lungs or brain. Somewhat surprisingly two of the viruses that are highly attenuated in C57BL/6 mice (VACVE3LΔ83N and VACVΔE3L
Orf E3L) were quite pathogenic in SCID mice. Since the defect in virulence for VACVΔE3L
Orf E3L maps to its C-terminal dsRNA-binding domain [26
], loss of pathogenicity for these two viruses and restoration of pathogenicity in SCID mice, appears to be mechanistically unlinked. Nonetheless, the data suggest that an intact immune response is responsible for attenuation of these viruses, but not for viruses that are highly attenuated in both models (VACVE3LΔ7C, Δ54N and ΔE3L). While VACVE3LΔ7C and Δ54N were highly attenuated in the SCID model, they did cause disease when used at high doses (>106
pfu IN), after long latent periods (30 days for VACVE3LΔ7C and 97 days for VACVE3LΔ54N). Both viruses established at least somewhat persistent infections in SCID mice, which in the case of VACVE3LΔ54N may have led to the selection of a more virulent virus. VACVE3LΔ54N encodes an unstable E3L protein, such that increased virulence could be attained by selection for a variant that encodes a stable E3L protein. VACVΔE3L was apathogenic for over 150 days even after infection of SCID mice with 106
pfu. Deletion of E3L in a Copenhagen or NYCBH vaccine strain background further attenuated these already attenuated human vaccine strains. Since VACV deleted for E3L either in a Copenhagen or a NYCBH background replicate to high titers in CEF cells, which are appropriate substrates for vaccine production, these viruses have the potential for development as a vaccine for use in humans.
All of the viruses tested here induced a potent protective immune response after vaccination intra-nasally with 100–20,000 pfu. Some of the viruses induced partial protection with intra-nasal immunization with as little as 10 pfu. These doses are over 3 logs lower than the IN LD50
for these viruses. For the viruses that replicate to high titers in the nasal mucosa this was not surprising, but we were surprised that VACVΔE3L, which does not replicate efficiently in the nasal mucosa, could induce a protective immune response. Immunohistochemical analysis of the nasal mucosa of mice infected with VACVΔE3L indicates that this virus can replicate efficiently in localized patches of the mucosa, unlike wtVACV, which essentially infects the entire mucosa (data not shown). Even this very limited replication appears to be necessary for vaccination, since UV-inactivated VACV does not induce a protective immune response. Induction of a protective immune response despite limited replication (a 3 log lower viral load than a fully replication-competent virus) suggests that VACVΔE3L has inherent adjuvancy activity that increases immunogenicity of this virus. We have shown that VACVΔE3L leads to activation of IRF3, ATF-2 and NFκB in infected cells [34
]. This is likely at least partially due to the presence of unbound dsRNA in cells infected with VACVΔE3L [31
]. Thus, the presence of this danger signal may act as a stimulator of the innate immune response, which may lead to the increased immunogenicity of this virus. VACVΔE3L was a potent inducer of protective immunity even in a Copenhagen and a NYCBH vaccine strain background when administered intra-nasally.
VACVs have proven to be one of the most promising vaccination strategies in infectious disease ranging from malaria [2
] to HIV [22
] in humans. Attenuated VACV are also being investigated for use as therapeutic vaccines against various tumors [35
]. However due to safety issues associated with replicating VACV, most of these vaccination regimens use non-replicating recombinant VACV. When using non-replicating VACV vectors such as MVA, high doses ranging from 106
pfu are commonly employed for induction of immune response to the transgene [18
]. Often augmentation of immunization protocols using attenuated strains of VACV with DNA or peptide vaccines in ‘prime-boost strategies’ yield better results than immunization with attenuated VACV alone. In fact this approach is being widely studied in potential vaccines against HIV and malaria. But the need for multiple inoculations [2
] often defeats the purpose of using live vaccines, which are expected to provide protection after a single inoculation, an advantage that may offset the additional risks often associated with these vaccines. In this paper we demonstrate that highly attenuated vaccines can induce a protective immune response after vaccination intra-nasally with a single dose of as little as 100 pfu.
In contrast to non-replicating strains of VACV, a single dose of replicating VACV vector appears to be sufficient to induce a robust immune response against the transgene [36
]. This is evident from the studies conducted by Ohishi et al. [48
] using VACV encoding the haemagglutinin protein of rinderpest virus. They demonstrated that a single dose of this recombinant VACV expressing the H gene of rinderpest virus was sufficient to induce a protective immune response against rinderpest in cattle for over 3 years. Use of replicating VACV expressing rabies glycoprotein as an oral vaccine has led to elimination of sylvatic rabies from large areas of land in Europe and preliminary data from field trials in the United States indicate a significant reduction of rabies in vaccinated areas [37
]. Replicating VACV was used globally for eradication of the deadly disease, smallpox.
Replicating VACV is also in clinical trials as vectors for tumor antigens against prostatic cancer [38
], cervical carcinoma [39
] and breast cancer [40
]. Co-expression of cytokines like IL-4 and GM-CSF in vivo
along with tumor-associated antigens using the VACV system enhances the anticancer activity of these vaccines [41
]. However, safety has been a major concern involved in the use of these vectors.
Replication on mucosal surfaces, such as the intra-nasal epithelium, confers an added advantage to these vectors as it gives these viruses an opportunity to stimulate a mucosal immune response [43
]. Since the vast majority of infections take place by a mucosal route the goal of developing a mucosal immune response is better achieved by administration of the vaccine by the intra-nasal route rather than parenterally [44
]. Work done by Belyakov et al. [49
] suggests that mucosal vaccination may overcome the barrier to recombinant VACV immunization caused by pre-existing poxvirus immunity. Apart from the efficacious immune response elicited by administration of immunogen by the intra-nasal route, ease of administration is also an added benefit.
For any live virus vaccine reversion of attenuation is a concern. All of the viruses tested here contain deletions for all or part of the E3L gene, which should make it difficult for reversion to occur. We have not succeeded in selecting for a phenotypic revertant of VACVΔE3L, despite repeated attempts (unpublished observations). Reversion by recombination with another poxvirus, which could donate a functional E3L gene, is a potential concern, as it would be with any strain containing a single attenuating mutation. However, not all poxvirus E3L genes can complement for deletion of VACV E3L [26
], and several poxviruses contain N-terminal deletions in E3L [45
] suggesting that reversion by recombination with another poxvirus may be limited.
In conclusion, we have characterized several highly attenuated strains of vaccinia virus containing mutations in the E3L interferon-resistance gene, that induce protective immune responses when delivered mucosally, either prior to or 1 day post-challenge. These viruses have the potential for being improved vaccines for protection against smallpox and improved vaccine vectors.