The use of BSL-2 or ABSL-2 practices and facilities are required for the manipulation of viruses or animals infected with non-highly attenuated strains. In addition to performing procedures inside a certified biological safety cabinet and avoidance of sharps and aerosol generating procedures, the use of double gloves and eye protection is recommended to minimize risk of infection.
Principal Investigators are ultimately responsible for assessing risk and providing a safe laboratory environment at all times. UVA-WorkMed will notify Principal Investigators when medically counseled personnel do not receive vaccination for any reason. In such cases, Principal Investigators are advised to consult with appropriate University resources e. References MacNeil, A. Risks associated with vaccinia virus in the laboratory. Virology Traditionally, a dose of 2.
Historically, protection against smallpox in humans has been found to be due to the presence of neutralizing antibodies in the sera of infected individuals Downie and McCarthy, ; Mack et al. However, the induction of protection does not always correlate with levels of neutralizing antibodies induced upon vaccination. In humans, immunization with 2. Further support for the importance of an early and non-specific cell-mediated response is seen in immunization of B-cell-deficient mice.
Upon challenge, these animals display an initial weight loss but recover completely Belyakov et al. The use of live animals for the production of vaccine material has changed due to current unacceptability of this process and quality control issues regarding microbial contamination.
VACV vaccine prepared in embryonated chicken eggs was used in large scale during the eradication campaign in South America and is the standard commercial vaccine in Israel. However, experience with vaccine material produced in cell culture is limited.
Vaccine was prepared on chick embryo fibroblasts in Japan before eradication of the disease, but its effectiveness is not well documented Fenner, Therefore substantial work is being directed toward the development of safer, yet immunogenic vaccines. One commonly used technique to attenuate VACVs involves multiple passaging of the wt viruses in tissue culture cells from alternative hosts, which has been shown to alter properties such as viral host range, virulence, and genome composition.
Passaging of viruses through alternative hosts has been used for over 80 years to obtain attenuated viral strains Monath, Arguably the most effective human viral vaccine, the 17D yellow fever virus vaccine, was obtained by nearly passages of the yellow fever virus through primary mouse and chicken cells in culture. The Lister virus was initially passaged 36 times through PRK cells, and individual clones were then evaluated for growth on monkey kidney Vero cells, in order to gauge their ability to replicate in primate tissues.
LC16, which grew to the lowest titer in Vero cells, was passaged 6 more times under identical conditions. LC16m0, which formed medium-sized pocks on chick chorioallantoic membranes CAM , was isolated from this stock and passaged 3 more times on PRK cells. In vivo, LC16, LC16m0 and LC16m8 strains have a take rate similar to the Lister strain, but are temperature-restricted, have limited host range and greatly reduced adverse effects, including neurovirulence Kenner et al.
The in vitro phenotype of the LC16m8 clone was attributed to a frame-shifting single nucleotide deletion in the B5R gene, in contrast to the Lister and LC16m0 viruses Morikawa et al.
EVs are necessary for effective virus dissemination, both in cells in culture and within an infected host Herrera et al. Presumably the lack of EV formation in cells infected with LC16m8 leads to the small plaque phenotype in RK cells, and the failure to form plaques in Vero cells. Since this phenotype is caused by a single base pair deletion, the LC16m8 strain can easily revert to the wild type version, and the small plaque phenotype in particular is unstable on passage Kidokoro et al. A stabilized version of LC16m8, containing a deletion of the entire B5R gene, has been developed to overcome this problem Kidokoro et al.
The mutation s responsible for the temperature restriction and in vivo attenuation phenotypes of LC16, LC16m0 and LC16m8 may be likewise unstable. Further, they also reported that the deletion of B5R in conjunction with use of the thymidine kinase TK locus for expression of a heterologous protein resulted in a vector with reduced virulence Viner et al.
However, the lack of induction of immunity to B5R may make LC16m8 a less efficient vaccine for protection against orthopoxviruses. This highly attenuated strain is unable to replicate in human cells and in clinical trials presented no adverse reactions Sutter and Moss, MVA was safely used to vaccinate over , people in Germany Mayr, , yet its effectiveness against smallpox remains untested.
These regions of the genome contain non-essential genes that are often involved in evasion of the host innate immune response, and genes necessary for maintaining the broad host range of VACV. MVA has several additional large deletions in the terminal arms, and contains numerous point mutations relative to fully replication-competent VACV strains Antoine et al.
In all, MVA appears to have lost nearly 30 kb of genomic information. Replication is restricted in all human cells that have been tested Meyer et al. In most cells MVA makes early, intermediate, and late proteins, but only immature virions are formed Sutter and Moss, This is unique, in that most other host range restricted VACVs that have been identified lead to a block in viral protein synthesis Meng et al. These additional mutations all appear to affect non-overlapping reading frames in the left terminal region of the genome, and restoration of this region in its entirety leads to a near normal host range Wyatt et al.
It is considered effective because it provides a complete antigenic dose by completing the already initiated replication cycle, an important characteristic for the generation of antibodies against late viral envelope proteins that are required for neutralization of VACVs, yet safe because it does not go through further cycles in primary human cells Blanchard et al. MVA has been shown to induce a protective immune response against monkeypox virus in non-human primates Earl et al.
Lack of these immunomodulators affects the viral replication cycle by blocking host protein synthesis more effectively and by inducing apoptosis more rapidly which may augment antigen presentation and immunogenicity of MVA Chahroudi et al.
However, a comparison study of VACV-based smallpox vaccines suggests that higher doses or multiple doses of MVA are required to achieve the immune protection seen with a single dose of wt replicating VACV.
The DI strain was first noticed as forming tiny plaques on CAM, in contrast to the parental strain, and was isolated after several rounds of pock formation on CAM. DIs contains a Consequently, DIs has a highly restricted host range, which is not simply restored with replacement of K1L and C7L, suggesting the existence of additional and yet unknown host-range genes in this region Ishii et al.
Of note, this deletion in the host-range determining region of VACV is similar to, although more extensive than, the region deleted in the left arm of the genetically engineered third generation smallpox vaccine NYVAC Paoletti et al. Nonetheless, while there are some similarities among the three viruses, there are numerous differences as well. DIs and MVA were both obtained by passage in chicken cells, and both have large deletions in the host range determining region, which makes both viruses replication defective in most mammalian cells.
MVA was passaged more extensively than DIs, which may be responsible for the additional large deletions, and numerous small insertions and deletions present in MVA, in both the left and right terminal arms of the virus.
These additional mutations in innate immune evasion genes in MVA may be responsible for the unique ability of this virus, among non-genetically engineered orthopoxviruses, to induce proinflammatory signal transduction, proinflammatory gene expression, and to lead to an increased migration of immune cells to the site of immunization Guerra et al.
The ability of MVA to induce proinflammatory cytokines may have a profound influence on its ability to induce adaptive immunity. Induction of proinflammatory signaling and proinflammatory gene expression by DIs has not been reported. LC16m8 was obtained by passage through rabbit kidney cells, and rather than having large deletions it has one single base pair deletion in B5R, and likely has other small mutations that alter gene function to give the temperature restriction and low pathogenicity of this virus.
LC16m8 has an intact host range determining region, and appears to be replication competent in most mammalian cells. Thus, the properties of these viruses as vaccines may be very different. However, the failure to make EV may at least potentially affect the efficacy of LC16m8 as a replacement smallpox vaccine.
With the advances in biotechnology that allow insertion, deletion and interruption of genes in specific genomic sites, targeted attenuation of viruses became a practical goal. For VACV this has involved deletion of immune-modulating, host-range and accessory nucleotide metabolism genes, as well as deletion of essential genes, that can be complemented by cell lines expressing the targeted VACV gene. The goal of deleting genes from VACV is to attenuate the virus while maintaining or increasing immunogenicity.
While attenuation can certainly be attained by deleting genes from VACV, there has been limited evidence that immunogenicity can be increased by deleting genes from VACV. At best, immunogenicity appears to be maintained after deleting genes from VACV, although often only at higher doses compared to a more wt parent. This may be due to the need to delete gene families rather than single genes or to the limitations of historical analyses of immune responses.
A summary of modifcations through genetic engineering is shown in Table 3. Consequently, a one log higher dose than MVA is required for seroconversion, probably due to lack of late stage protein expression Ferrier-Rembert et al.
Yet the deletion of B8R did not prevent replication of the virus in human or mouse cells in vitro. Additionally, there were no significant differences in humoral and cellular immune responses of immunocompetent mice infected with a B8R deleted-VACV or the parental virus, and despite prior studies suggesting that the B8R gene does not affect virulence of VACV in mice Alcami and Smith, a ; Alcami and Smith, b , the mutant caused much less morbidity in nude mice than the wt virus, even at higher doses Denes et al.
Deletion of either of the serine protease inhibitor serpin genes, B13R and B22R has been shown by Legrand, et al. Both mutant viruses were replication-competent in murine cells, but only the B13R-deleted virus was able to replicate in human cells.
Most mutations of immune modulating genes have been analyzed in a WR background. Recently several mutations in the VTT strain have been analyzed. Dai et al. Deletion of the C12L gene in ectromelia virus has also been shown to be an attenuating mutation Born et al.
Mutations in the host range region of VTT have also been used to attenuate this virus. Zhu et al. With IC inoculation of mice, this mutant virus was attenuated by fold in comparison to wild type virus.
The virus was replication-incompetent in several cell lines, including HeLa human , RK rabbit , and Vero African green monkey. Deletion of the host range gene K1L reduces replication in RK cells, and deletion of this gene along with serine protease inhibitor K2L, ankyrin-like protein M1L, and the early M2L gene reduces in vitro replication further. Vaccination requires a prime-boost regimen for neutralizing antibody titers and cell-mediated response to approach that observed after wt VACV vaccination Zhu et al.
Several viruses containing mutations in accessory nucleotide metabolism genes and genes encoding membrane or secreted proteins have been analyzed for attenuation and immunogenicity. Following the work done to develop vaccinia vectors to express foreign antigens in the early s Panicali et al. This virus was attenuated by 4 logs, compared to a wt control. One of the first applications for genetic engineering of VACVs was a vaccine used to combat rabies in wildlife.
Even though the TK-deleted virus is attenuated, one case of human infection was reported Rupprecht et al. In immunocompetent mice, this virus replicates poorly in the nasal mucosa but still induces a protective immune response Vijaysri et al.
In order to overcome this obstacle vD4-ZG must be grown in a complementing cell line that expresses the missing early viral gene Holzer and Falkner, This non-replicating late defective mutant makes it attractive as a vaccine vector due to its ability to elicit potent immune responses through the expression of early genes. Furthermore, the virus was rapidly cleared from immune-suppressed mice even at the highest vaccination dose of 10 6 pfu Holzer et al.
However, the lack of expression of structural gene products may make this virus unsuitable as a vaccine for protection against orthopoxviruses. Flexner et al. Another example is expression of IL by VACVs resulting in a several-thousand-fold reduction in lethality in athymic nude mice Perera et al.
Mice vaccinated with the ILexpressing VACV survived a lethal IN challenge with the wt version 10 months after vaccination, whereas mice which had received a homologous vaccination succumbed Perera et al. Expression of immune-stimulating genes may on the other hand enhance immunogenicity of highly attenuated non-replicating VACVs.
For instance, MVA has been shown to infect antigen-presenting cells, including dendritic cells, B cells, and macrophages Chahroudi et al. Therefore, expression by MVA of cytokines known to enhance activation of antigen-presenting cells could augment the overall immune response to the vaccine.
Ultimately, a combination of strategies may be needed to attenuate wt-VACV while enhancing specific immune pathways. Following September 11, , the potential use of the smallpox virus VARV as a bioterrorism agent was brought into special attention, as it constitutes an ideal terrorist weapon of mass destruction LeDuc et al. Moreover, there also remains a low but very real risk of VARV release, whether intentional or accidental Thorne et al. All these reasons make the requirement of a post-exposure prophylaxis of smallpox one of the ultimate goals not only nationwide but also worldwide.
Post-exposure vaccination with VACV has been suggested as an effective means to minimize casualties from smallpox exposure, as long as the vaccine is administered within four days of initial exposure Mortimer, While there is anecdotal evidence for efficacy of post-exposure vaccination, this has not been definitively studied in humans, and while no animal model of post-exposure vaccination has thus far been consistently described, several animal studies were done and are worthy of mention.
However, these immunization protocols failed to completely prevent clinical signs even when given within three hours of challenge and were completely ineffective when given one to four days post-exposure. Thus, post-exposure vaccination with these strains, in this particular animal model, seems to be limited. This study suggested that a multi-dose antiviral treatment within 24 hours post-infection could reduce mortality and cutaneous lesions in comparison to standard post-exposure vaccination Stittelaar et al.
Recent studies have shown that MVA given via a subcutaneous injection at the same time as an IN lethal dose of ectromelia virus ECTV was able to protect mice from death and that it is able to protect TLR-9 deficient mice when administered up to two days post-challenge Samuelsson et al. Moreover, protection was affected by the vaccine dose, which improved the survival rate vaccination at days two and three post-infection Paran et al.
This extended period observed for the onset of the disease may be essential to allow time for the T and B cells to mount a solid immune response as recombination activating gene 1-deficient mice fail to confer immunity after short-term vaccination Paran et al.
Another approach for post-exposure treatment involves the use of VACV as an expression vector for foreign genes. Conversely, expression of IL has been shown to clear a VACV infection when co-expressed with IL, indicating a synergistic action of both cytokines Gherardi et al. In conclusion, various approaches for post-exposure prophylaxis have shown some promise in being effective on preventing disease in some animal models.
Although the ECTV model for post-exposure prophylaxis seems promising, a model using the more neuropathogenic WR strain could indeed be more useful when finding post-exposure treatment to a harsher onset of the disease.
Future studies will determine if either replication competent or highly attenuated non-replicating VACV strains can be safely and effectively use as post-exposure prophylaxis approaches in humans. The eradication of smallpox combined with the advent of genetically engineered recombinant poxvirus technology demonstrated the feasibility of using VACVs to protect against specific pathogens. With the relative ease of generating recombinant VACVs to express heterologous genes, widespread attention has been given to the idea of using these vectors as a vehicle for an antigen delivery system against different diseases Moss, ; Paoletti, To date, recombinant poxvirus vaccine vectors have been generated to target many human and veterinary infectious diseases and cancer Perkus et al.
This campaign has successfully eliminated rabies in parts of Europe and significantly reduced the incidence of rabies in the United States. Consequently, this vaccine is the only oral rabies vaccine licensed for use in animals in the United States and Europe this vaccine is not licensed for use in humans and is still currently in use.
In an effort to circumvent the adverse effects of VACV, technology has turned to the development of highly attenuated strains of VACV and exploited the use of divergent host-restricted poxviruses in an attempt to generate a safe vaccine platform.
Currently, various parameters such as mode of inoculation, routes of vaccine administration and immunization schedules are also being investigated to optimize immunization protocols and achieve efficacy and safety Gomez et al. Addressing the safety issues surrounding vaccine development, extensive studies have been done using replication-deficient poxviruses as vaccine vectors. Both MVA and NYVAC are attenuated VACVs containing multiple gene deletions that prevent a productive replication cycle in mammalian cells, whereas avipoxviruses have a natural host range restriction and cannot replicate in non-avian cells.
Preclinical studies have shown these vectors induce some level of protection against many heterologous agents when used as a vaccine in both human and veterinary medicine Bisht et al. MVA and NYVAC have been studied extensively and a comprehensive review has been published comparing these vectors in detail and their various applications as vaccine vectors Gomez et al.
Prime-boost regimens including different combinations of these non-replicating strains have become commonplace to enhance immunity and to avoid complications with preexisting immunity to VACV vectors Webster et al. Despite studies that show these highly attenuated vectors are capable of inducing protective immunity against a variety of pathogens, their limited replication still generates concern as to their ability to induce an immune response as effective as their replication-competent counterparts Abaitua et al.
Studies comparing the two vectors in vaccine studies clearly demonstrate that in an effort for safety, often long term protection and efficacy are lost. For instance, in a quest for a safer rabies vaccine, a recent study compared the efficacies of various rabies vaccine candidates in different VACV strains.
Recombinant MVA proved to be inferior in mounting an immune response in mice when given by oral immunization compared to Cop and WR strain. Furthermore, oral administration of this vector failed to elicit memory responses in previously vaccinated dogs and raccoons and was therefore deemed not suitable for a replacement of the current oral vaccine, V-RG Weyer et al.
In addition, a recent smallpox vaccine study has further emphasized the superiority of replication competent vectors to non replicating vectors in long term protection and immunogenicity against lethal cowpox challenge Ferrier-Rembert et al. Replication-competent recombinant VACV-based vaccines have received increasing attention as potential vaccine vectors for many infectious diseases, since they are presumably able to elicit potent humoral and cell mediated immune responses, and confer lasting protection.
Different approaches have been taken to lower the virulence and enhance the immune response in an effort to generate a safe vaccine platform. Many have turned to deleting genes encoding virulence factors whose function is to evade the host immune response.
Immunogenicity of vectors attenuated through deletion of these genes was not greatly affected. In two independent studies, the deletion of the B8R gene Denes et al. Immunologic responses between the deletion mutant and parental virus were analyzed and no substantial differences were observed.
Another example of attenuation by reduction of virulence factor functionality is the work performed by our group with E3L, as previously discussed Jentarra et al. Collectively, these live vaccine vectors represent a promising potential as safe effective vaccine platforms. Many of the genetically modified poxviruses are replication-defective, a characteristic which has long been considered critical in developing safe vaccines in an era of immune deficiency resulting from chemotherapy, organ transplant, HIV infection, or other diseases impacting the immune system.
However, the necessity for replication- competence in vaccine vectors has become an increasingly theorized potential solution to the lack of efficacy in some of the vaccines that have been tested in human trials recently. Replication competence does not exclude attenuation, as was evidenced in a report by Parker et al.
Parker et al. The virus can be grown to higher titers than a replication-defective HSV vector and expresses the heterologous antigens at higher levels.
VACV encodes a number of proteins involved in inhibiting the host anti-viral, immunological and inflammatory responses. Mutagenesis or deletion of these genes often leads to viral constructs which are replication-competent but with reduced pathogenesis.
As discussed in this review, mutations of this gene in VACVs resulted in several replication-competent viruses with greatly reduced virulence in animals, which retained the ability to protect against an orthopoxvirus challenge Vijaysri et al.
Therefore, we are confident that continued efforts to genetically modify poxvirus vectors will not only improve our understanding of these viruses but also allow preservation of safety while taking advantage of the immunogenic benefits of replication competence. After the challenge, the mice were observed daily for illness and weight loss.
PBS-inoculated mice developed weight loss beginning on day 4 Fig. In sharp contrast, all immunized mice were protected from significant weight loss. Lungs were harvested on days 4, 8, and 12 after the challenge, and vaccinia virus titers were determined Fig. The viral titers reflected the illness of the PBS-inoculated mice: the vaccinia virus titers per gram of lung were 7.
By day 8, there was no detectable vaccinia virus in any immunized mouse. Thus, clinical protection was achieved after a single-dose immunization with MVA and correlated directly with a reduction in the overall viral burden and the rapid clearance of vaccinia virus.
Mice immunized with MVA demonstrate absence of weight loss and reduced vaccinia virus titers in the lungs after an intranasal challenge with vSC8.
A PBS-inoculated mice demonstrated weight loss that peaked on days 6 and 7. MVA- and vSC8-immunized mice were protected from weight loss and clinical illness. B Lungs were harvested on days 4 and 8, and vaccinia virus titers were determined and expressed as log 10 PFU per gram of lung tissue.
The limit of detection for this assay was 2. On day 4, there was a significant decline in vaccinia virus titer in MVA-immunized mice compared to PBS-inoculated mice. By day 8, there was a complete clearance of vaccinia virus from the lungs of mice that were immunized with MVA. Vaccinia virus neutralization was performed by use of a plaque reduction assay with samples collected 2 and 4 weeks after immunization.
A dose-dependent humoral response was elicited by MVA immunization Fig. The peak response was seen on day 14 and persisted to at least day MVA elicits dose-dependent humoral and cellular immune responses.
Data represent means from two independent experiments, resulting in data for 10 mice per group. The data are for five mice in each group from one representative experiment of four total experiments. Similarly, the magnitude of the cellular response was dependent on the number of MVA immunizations. In order to determine the efficacy of MVA immunization against a vaccinia virus strain with enhanced virulence, we challenged mice intranasally with a recombinant vaccinia virus encoding murine IL-4, vSC8-mIL4.
As with the vSC8 parent strain, mice immunized with one or two doses of MVA were protected from the development of clinical illness, as measured by weight loss Fig. Nonimmunized mice lost weight rapidly, and all mice died or were euthanized because of extreme illness by day 8 after the challenge.
Nonimmunized mice demonstrated significantly higher vaccinia virus titers in the lungs than did MVA-immunized mice Fig. Lung histopathologies were evaluated to compare the inflammatory processes in immunized versus nonimmunized mice. MVA-immunized mice showed only a minimal infiltrate on day 8, while there was a profound alteration of the lung architecture in nonimmunized mice, with severe mononuclear cell peribronchiolar and interstitial infiltrates, alveolar edema and exudate, and epithelial necrosis Fig.
A PBS-inoculated mice demonstrated a rapid decline in weight loss that resulted in death. MVA-immunized mice were protected from weight loss and clinical illness. B Lungs were harvested on days 4 and 8, and vaccinia virus titers in the lungs were determined and expressed as log 10 PFU per gram of lung tissue. Similar to the case for the vSC8 challenge, there was a significant decline in viral titer in MVA-immunized mice compared to PBS-inoculated mice on day 4, with a complete clearance of vaccinia virus by day 8.
Lung histopathology in mice challenged with vSC8-mIL4. Both Dryvax and MVA immunization provided significant protection from the extensive peribronchiolar and interstitial inflammation, alveolar exudates and edema, and epithelial necrosis seen in the lungs of unimmunized mice.
PBS-inoculated mice developed significant illness and mortality by day 8 after the challenge Fig. On day 8, vaccinia virus was still detectable in the lungs of CD4- and CD8-depleted mice, while all other MVA-immunized mice had cleared the vaccinia virus. The most severe illness was associated with higher residual vaccinia virus titers on both days 4 and 8 in mice that were depleted of both T-cell subsets.
T-cell-depleted mice that are immunized with MVA are partially protected from a vaccinia virus intranasal challenge. A Changes in baseline weight were determined daily.
B Vaccinia virus titers in the lungs were determined on days 4 and 8 after the challenge. Clinical illness corresponded to vaccinia virus titers in MVA-immunized mice. MVA-immunized double-depleted mice had significantly higher vaccinia virus titers on days 4 and 8 than did MVA-immunized mice treated with the isotype control or an antibody to either CD4 or CD8. B The vaccinia virus titers in the lungs were determined on days 4 and 8 after challenge and demonstrated the same patterns as those seen in the weight loss curves and were also consistent with the patterns seen after the lower-dose vSC8-mIL4 challenge.
In order to assess the contribution of the humoral immune response to vaccine-induced immunity, we immunized mice lacking mature B cells, B6. Neutralization assays were performed on day 28 postimmunization with B6. The vaccinia virus lung titers measured on day 4 postinfection revealed a significant decline in titer in the B6. Both B6. These data support a significant role for the humoral immune response in protection against vaccinia virus, particularly in controlling the peak of vaccinia virus replication, but mice deficient in neutralizing antibodies were eventually able to clear the virus without significant clinical illness.
C Vaccinia virus titers in the lungs were determined and expressed as log 10 PFU per gram of lung tissue. The viral burden in MVA-immunized B6. Following the anthrax attacks in the fall of , discussions about the potential use of other virulent pathogens as biological weapons have increased. The threat of a smallpox outbreak elicits great concerns because of its acute and significant morbidity and mortality rates and the lack of significant immunity against poxviruses among civilians born after , despite a large vaccination campaign effort among the military and medical first responders Discussions about the widespread use of the present FDA-approved vaccine Dryvax have resulted in much debate in the medical community.
While the vaccine is efficacious, the complications associated with Dryvax make the development of a safer second-generation smallpox vaccine a medical priority. For the present study, we evaluated the efficacy and immune response of a highly attenuated vaccinia virus, MVA, by use of a mouse model.
Investigations of this vaccinia virus strain began in the late s after attenuation of the parent strain CVA in chicken embryo fibroblasts. Since that time, MVA has been evaluated in several animal models as well as in humans 22 , 30 , During the s, more than , people were immunized with MVA Reports reflect a good safety profile for MVA, including safe administration to immunocompromised animals 38 , However, the specific determinants of MVA-induced immunity compared with those induced by replication-competent vaccinia virus have only recently been studied 5 , We demonstrated the ability of a single dose of MVA to protect against both intradermal and intranasal challenges with vaccinia virus.
In particular, we demonstrated that MVA immunization can protect against a lethal respiratory challenge with a molecularly modified recombinant vaccinia virus expressing murine IL While previous studies have shown a dose-related effect following single-dose escalation 4 , 11 , we showed that a multidose schedule of MVA may enhance the overall immune response and provide added protection against vaccinia virus replication.
While clinical illness was reduced in all MVA-immunized mice after an intranasal challenge, the vaccinia virus titers in the lungs were higher for mice that received a single immunization. In mice receiving multiple immunizations, the immune responses were enhanced, suggesting that strategies to optimize the protection of humans against poxviruses such as variola virus and monkeypox virus may necessitate a multidose immunization regimen.
Although it was not evaluated in these studies, the route of immunization is also important. For example, MVA was administered intramuscularly in our studies, and the responses compared favorably to those induced by replication-competent vaccinia virus given by the intramuscular or intradermal route.
Therefore, additional exploration of the route of poxvirus immunization may be warranted. With the threat of bioterrorism, there is concern not only about the potential release of virulent pathogens, but also about the purposeful manipulation of pathogens in an effort to augment their virulence. Importantly, we have demonstrated that MVA immunization can protect against a lethal pulmonary challenge with a molecularly modified recombinant vaccinia virus expressing murine IL Prior studies have demonstrated the importance of cytokine balance in the pathogenesis of viral, bacterial, and parasitic diseases.
Disruption of the Th1-Th2 balance has been shown to adversely affect viral clearance and protection from dissemination. As previously described 24 , 36 , we demonstrated that the virulence of vaccinia virus is enhanced in the setting of excess IL-4 production, with a prolonged elevation of lung viral titers and death in mice infected with vSC8-mIL4 Fig.
Despite this enhanced virulence, MVA immunization protected mice from a lethal pulmonary challenge with vSC8-mIL4 and eliminated clinical illness and weight loss. The ability to protect against a lethal molecularly modified vaccinia virus lends further support to MVA as a candidate vaccine against smallpox. While they are effective against vaccinia virus challenges, the determinants of immunity elicited by MVA and other vaccinia viruses have not been clearly defined.
The immunization of human subjects with Dryvax has been shown to elicit both humoral and cellular immunity Historical reports suggest that both arms of the immune system are relevant to protection from smallpox.
Investigations of villages during outbreaks of smallpox correlated the vaccine take and antibody response with immunity More recent reports point to the importance of the cellular immune response for containing disseminated vaccinia virus 7.
It becomes important in the evaluation of novel vaccines to dissect the role of each component of the immune response in order to demonstrate similar patterns of response to both replication-competent and attenuated vaccines.
While this demonstrates the importance of cellular immunity for vaccinia virus clearance, it suggests that T cells are not the sole factor in the defense against poxvirus infection and that the immune system has evolved redundant mechanisms to control this important class of pathogens.
Contributions from the humoral immune response following immunization are also likely to be important for protection against vaccinia virus infection. We demonstrated a dose-dependent increase in the antibody response to MVA immunization, which correlated with protection. However, in the absence of neutralizing antibodies there was minimal illness in this murine model from an intranasal vaccinia virus challenge Fig.
While MVA-immunized B-cell-deficient mice demonstrated significant viral replication in the lungs on day 4 after the intranasal challenge, they lacked signs of clinical illness and cleared the vaccinia virus by day 8. As for many other virus infections, we believe that the data support an important role for vaccine-induced antibodies in protection from infection, but effective T-cell responses also appear to be important for protection against severe poxvirus-induced disease.
These data are consistent with recent studies that evaluated the immune determinants of protection against the WR strain of vaccinia virus 5 and that indicated that vaccine-induced protection from poxviral challenge should not be restricted to a single arm of the immune response, but should include a combination of both cellular and humoral immune responses.
We demonstrated a robust immune response following MVA immunization in a murine model. MVA not only protected mice from a standard vaccinia virus challenge, but it was also able to prevent illness and limit viral replication after the administration of a lethal molecularly modified strain of vaccinia virus. These data support the further development of MVA as a stand-alone vaccine against smallpox and infections caused by other orthopoxviruses.
National Center for Biotechnology Information , U. Journal List J Virol v. J Virol. Lewis H. McCurdy , John A. Rutigliano , Teresa R. Johnson , Man Chen , and Barney S. John A. Teresa R. Barney S. Author information Article notes Copyright and License information Disclaimer.
Phone: Fax: E-mail: vog. Received Feb 19; Accepted Jul 7. Copyright notice. This article has been cited by other articles in PMC. Abstract Recent events have raised concern over the use of pathogens, including variola virus, as biological weapons.
Mouse immunization and challenge. In vivo replication of vaccinia virus. Vaccinia virus neutralization assay. Intracellular cytokine assay.
Antibody depletion. Statistical analysis. TABLE 1. MVA immunization protects against intradermal vSC8 challengea. Vaccination regimen Frequency of lesion formation no.
Open in a separate window. Mice were observed daily for pox lesion formation. Data are a combination of two experiments and demonstrate a significant decrease in lesion formation in all immunized mice. There was partial protection in mice that received a single injection of MVA, and complete protection was observed for mice immunized with two or three doses of MVA. MVA immunization protects against illness and reduces viral burden in mice challenged intranasally with vaccinia virus.
MVA induces both humoral and cellular immune responses. T-cell depletion reduces efficacy of MVA immunization. Mice lacking mature B cells. Alcami, A. Vaccinia, cowpox and camelpox viruses encode soluble interferon receptors with novel broad species specificity. Antoine, G. Scheiflinger, F.
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