Protection of an increasingly vulnerable population (60
) and the development of safer smallpox vaccines (22
) remain ongoing research concerns. In this study, we attempted to (i) establish the black-tailed prairie dog as a novel smallpox vaccine testing model and (ii) contribute to the body of knowledge regarding protection of first-, second-, and third-generation smallpox vaccines when used prophylactically.
To establish the prairie dog as a valid vaccine model, we first evaluated the safety of current smallpox vaccines in this model by comparing any disease manifestations and virus shedding to those of animals infected with a high-dose of VACV-WR. We demonstrated that VACV-WR (106 PFU) m.p. infection in prairie dogs caused weight loss, local and disseminated lesions, ocular infections, presence of viral DNA in blood, and viral DNA and infectious virus in oral swabs. Conversely, m.p. inoculation of prairie dogs with human doses of Dryvax or Acambis2000 (105 PFU; 5 times less virus than the VACV-WR infection) or s.c. inoculation of IMVAMUNE (108 TCID50) did not induce any demonstrable illness (), indicating that any disease we see in MPXV challenge studies is due to challenge virus and not vaccine.
To compare vaccine-induced immune responses between one- and two-dose IMVAMUNE regimens, we measured total anti-OPXV IgG antibodies for all three vaccines at the peak of antibody induction (with day 0 at 30 days postvaccination). At this time point, vaccine-induced total antibodies were greater in Dryvax-vaccinated (3.55 × 104
) and Acambis2000-vaccinated (2.74 × 103
) animals than in animals vaccinated with one dose of IMVAMUNE (1.04 × 102
). However, 30 days after a second dose of IMVAMUNE was administered, total anti-OPXV Abs in these animals (1.35 × 103
) were at levels similar to Dryvax-vaccinated (2.68 × 103
) or Acambis2000-vaccinated (1.18 × 103
) animals (). Our results confirmed the boosting effect of the second dose of IMVAMUNE (17
), as Abs induced with a single dose were 12.9-fold lower than the level induced by two doses.
Because vaccine-induced NAbs are considered a vital immune response for protection against monkeypox challenge in NHP (15
) and to better compare our experiments to previously reported animal studies, we measured NAb titers for animals vaccinated with Dryvax, Acambis2000, or two doses of IMVAMUNE. Our results showed that two-dose IMVAMUNE (6.26 × 102
) induced a higher peak level of NAbs (with day 0 at 30 days after the second vaccination) than Dryvax (2.38 × 102
) or Acambis2000 (8.94 × 101
). This is consistent with previous reports that “first-generation” vaccines induce similar higher levels of anti-VACV NAbs than “second-generation” vaccines (22
), and it recapitulates findings (17
) where, in human volunteers, two doses of MVA generated higher levels of NAbs than Dryvax alone. Similarly, in MPXV-infected NHP (13
) and RPXV-infected rabbit challenge models (18
), a two-dose MVA regimen elicited higher levels of NAbs than Dryvax. In summary, the observation that vaccinated animal serum showed induction of total Abs and NAbs consistent with previous studies, coupled with the evidence of a “take” seen in Dryvax-vaccinated and Acambis2000-vaccinated animals, led us to hypothesize that smallpox vaccination would be efficacious in protecting prairie dogs from MPXV challenge.
To determine if these vaccines protected the animals from death from MPXV challenge, we administered a high- or low-dose MPXV challenge to various vaccinated animal groups and compared survival to that observed in unvaccinated controls. All previously vaccinated animals survived and manifested less morbidity than was seen in unvaccinated animals, indicating that the vaccines were protective. Although these were designed as 17× and 170× LD50
studies, we did not observe 100% lethality for unvaccinated challenged animals. Our sample sizes for each group were chosen to minimize animal usage while maintaining statistical significance and used mortality estimates based on an LD50
of 5.9 × 103
PFU/ml, calculated using the Reed-Muench equation (27
). Based on multiple publications (27
) and additional unpublished work in our lab (P. Hudson, C. L. Hutson, and S. K. Smith), we expected a lethal challenge with unvaccinated control animals requiring euthanasia around day 12 of the study. Although that did not occur, the one control animal in the high-dose challenge study that survived to the end of the study (day 28) was severely ill and had not fully recovered from illness at the study end point. Although all vaccinated survivor animals were gaining weight and their lesions had resolved by study day 28, the survivor control animal barely maintained 87% of its initial weight and its lesions were incompletely resolved at day 28; however, the surviving control animal did not fully meet euthanasia criteria. Despite this, there was a statistically significant difference between survival of any of the vaccinated group arms and unvaccinated control arm in the high-dose MPXV-challenged animals (), leading us to conclude that all three vaccines were protective against mortality in this animal model.
In order to identify broad differences in the abilities of each vaccine to protect the animals from MPXV-induced illness, we evaluated the effects of vaccination on weight loss, inflammation at the site of inoculation of MPXV challenge virus, and disseminated lesions. Unvaccinated animals had demonstrable weight loss () and nasal involvement and rash illness () at both doses. The Dryvax- and Acambis2000-vaccinated animals demonstrated no weight loss () and no disseminated rash illness () regardless of MPXV challenge dose. After high-dose MPXV challenge, the Dryvax- and Acambis2000-vaccinated animals had very mild nasal involvement throughout the illness course compared to the unvaccinated animals (), indicating the overall effectiveness of Dryvax and Acambis2000 against systemic OPXV disease. This recapitulates findings from an NHP intravenous (i.v.) challenge study with ~10 times the LD50
(3.8 × 107
PFU), which demonstrated that vaccination with Acambis2000 was protective against both death and rash illness in macaques. All protective markers in the Acambis2000-vaccinated animals were virtually indistinguishable from the Dryvax-vaccinated animals challenged with the same dose (47
), which is similar to what we observed in both our 17× LD50
and 170× LD50
The prairie dogs that were previously vaccinated with one or two doses of IMVAMUNE did not show a demonstrable weight loss () after MPXV challenge. Interestingly, both groups of IMVAMUNE-vaccinated/MPXV-challenged animals did have significantly more signs of illness than the Dryvax- or Acambis2000-vaccinated/MPXV-challenged animals (), although this illness was attenuated in IMVAMUNE-vaccinated compared to unvaccinated animals. This recapitulates findings from previously reported animal studies that demonstrated that two-dose IMVAMUNE vaccination, although protective against mortality, does not prevent all morbidity upon MPXV challenge. In one NHP ~10× LD50
(5 × 107
PFU) MPXV i.v. challenge study, 6 of 6 MVA-vaccinated animals developed lesions, compared to none of the Dryvax-vaccinated animals (13
), which is comparable the results of our high-dose (106
PFU) prairie dog study when we used a two-dose IMVAMUNE regimen. Using this same NHP model, a single dose of MVA 30 days prior to challenge resulted in 4 of 4 MVA-vaccinated animals developing lesions, whereas the Dryvax-vaccinated animals did not (14
). This was similar to what we observed in our low-dose (105
PFU) study when we administered a single dose of IMVAMUNE.
Because the i.v. route of infection used in the studies above is not typical of human systemic OPXV infection, and our model uses an i.n. route of infection, we also compared our study to an NHP study that used intratracheal (i.t.) administration of 107
PFU MPXV after vaccination with Elstree-RIMV or 2 doses of MVA-BN. In this study, no lesions were seen in animals vaccinated with Elstree-RIMV, but 1 (of 6) MVA-BN-vaccinated animals developed rash illness, similar to 3 (of 3) unvaccinated control animals (47
). In the RPXV-infected rabbit animal model, vaccination with Dryvax or two doses of IMVAMUNE protected against death from an ~500× LD50
aerosolized RPXV challenge, but a comparison of rash illness cannot be done, as the rabbits succumbed to disease prior to cutaneous rash formation (18
). We feel that our results from this study of first-, second-, and third-generation vaccines in the prairie dog model augment results found in comparable animal model studies, which have shown that while MVA vaccines protect from mortality, some “breakthrough” of disease, albeit attenuated, does occur.
To attempt to better understand this attenuated disease and the effects of the vaccines on the level and duration of viral load/viral shedding, we measured DNAemia by RT-PCR in blood and infectious virus by tissue culture from oropharyngeal secretions and compared these results among vaccinated and unvaccinated animal groups (). A previous report for an NHP study using i.t. administration of 107
PFU MPXV after vaccination with Elstree-RIMV or two doses of MVA-BN indicated that the MVA-BN-vaccinated animals had viral loads (in plasma and throat swabs) that were higher than in the Elstree-RIMV-vaccinated animals but less than the unvaccinated controls (47
). In a similar study, using two-doses of MVA and an i.v. MPXV (107
PFU) challenge, MVA-vaccinated animals had viral genome levels (GE/ml) in blood that were significantly higher than the Dryvax-vaccinated animals but significantly lower than unvaccinated control animals (13
). We observed a similar result in our high-dose (106
PFU), two-dose IMVAMUNE study, which showed that IMVAMUNE- vaccinated animals had MPXV DNAemia levels that were statistically indistinguishable from those observed in unvaccinated animals and were slightly but significantly higher than those observed in Dryvax- and Acambis2000-vaccinated animals from day 7 through day 14. In addition, the infectious viral load in oral swabs was statistically indistinguishable between IMVAMUNE-vaccinated animals (106
PFU) and unvaccinated control animals (>106
PFU) and higher than Dryvax-vaccinated (105
PFU) or Acambis2000-vaccinated (105
PFU) animals on day 7. On day 14, only the unvaccinated (>106
PFU) and IMVAMUNE-vaccinated animals (>103
PFU) had any infectious virus in oral swabs, although notably, the IMVAMUNE-vaccinated animals excreted ~3 logs less virus. Our conclusions are that after challenge with high-dose MPXV, IMVAMUNE-vaccinated animals have similar DNAemia in blood but presumably shed less virus than unvaccinated animals, but they have more DNAemia in blood and presumably shed more virus and for a longer duration than Dryvax- or Acambis2000-vaccinated animals. Further studies measuring infectious virus in blood and transmission studies using MPXV-challenged animals after various vaccination regimens would help to clarify the significance of these observations.
Our study and the NHP studies referenced above indicate that even though MVA induces NAbs to levels similar to Dryvax by 30 days after the last vaccination, animals vaccinated with MVA, but not Dryvax, still exhibit attenuated rash illness upon challenge. While this may be a function of the high levels of challenge dose used in the study, it is more likely an indication of differences in the MVA-induced immune response, which fail to constrain viral replication before the secondary viremia, and seeding of the skin, with virus. In OPXV infections, examination of the effects and interactions of the humoral and cellular responses induced by smallpox vaccines is an active area of research (51
). NAbs appear to be more necessary for protection from challenge than cellular responses (9
), but there is compelling evidence that intramuscular administration of MVA protects B cell-deficient mice (79
), indicating that MVA can protect irrespective of Abs. The level of protection attributed to each immune response component appears to be highly variable across animal models (51
), as MVA-vaccinated macaques have been shown to have lower cellular immune responses than Dryvax-vaccinated macaques (54
), but human MVA vaccinees have higher VACV-specific CD8 T cell responses (56
). In addition, no correlation between survival and cellular cytokine production was seen in VACV-WR-challenged mice (50
), indicating a need for more studies of cellular immunity in animal models of smallpox vaccination (73
). In our study, all vaccinated animals, regardless of challenge dose (17× or 170× the LD50
) or vaccine regimen (one or two doses of IMVAMUNE), had higher levels of total Abs and NAbs than unvaccinated animals on day 0, and these responses increased by day 7 due to a robust anamnestic response ( and ). This may indicate that the attenuated disease seen in IMVAMUNE-vaccinated animals is due to differences in the vaccine-induced cellular response. We are currently performing studies to generate the immunological reagents necessary to study the cellular immune response in this model in an attempt to understand what role the cellular immune response plays in vaccine-induced protection.
Differences seen in protection from illness by these vaccines could also be due to differences in the neutralizing targets of vaccine-induced Abs. Protein microarrays comparing MVA or Dryvax vaccination in humans and macaques have indicated that the Ab profiles induced by each vaccine are broadly similar (10
), and macaques vaccinated with MVA or Dryvax mount similar Ab responses to the L1, A33, and B5 proteins (13
). In another study, MVA vaccination induced similar levels of NAbs as Dryvax, with MVA inducing an increased NAb response to extracellular enveloped virion proteins (56
). In our study, we observed that the IMVAMUNE-vaccinated animals had similar numbers of NAbs but lower total Abs on day 7 postchallenge compared to the Dryvax- or Acambis2000-vaccinated animals. IMVAMUNE-vaccinated animals also had the highest level of NAbs on day 0, but Dryvax- and Acambis2000-vaccinated animals had levels of NAbs that equaled or surpassed those of the IMVAMUNE-vaccinated animals by day 7. Interpretations of these observations are complicated by the use of different VACV strains in each assay (Wyeth for ELISA, WR for HCS-GFP), and we are currently investigating the proteins targeted by Abs induced by IMVAMUNE, Dryvax, and Acambis2000 in order to better understand this observation and to determine if differences in NAb targets correlate to the differences in protection from rash illness seen in this model.
Although MPXV-infected NHP are a frequently used animal model for the testing of smallpox vaccines and recent research continues to improve this model (19
), the expense of these animals and their relatively complicated husbandry make an inexpensive, easily obtainable, easily husbandried small animal model—like the prairie dog—attractive as an alternate model for use for initial testing of second- and third-generation vaccines. Comparisons with other small animal models, (1
) show that the pathogenesis of MPXV in prairie dogs is more similar to systemic OPXV infection in humans than other small animal models in both the route of transmission of challenge virus and length of viral incubation period. This 7- to 9-day incubation period is especially important, as it potentially makes the MPXV-infected prairie dog an ideal model for testing postexposure therapies, as have recently been tested in ECTV-infected mice (55
). In addition, unlike the recently described MPXV-infected STAT1−/−
) and inbred mouse models (2
), the prairie dog is an immunocompetent and natural host for MPXV. Although these animals have not been successfully bred in captivity, they are easily available; although there are a limited number of commercially available reagents for this model, the fact that the prairie dog is an outbred animal model should offer a more realistic view of the variability in protection that will occur in any human vaccination program. These characteristics make the prairie dog a useful small animal model for evaluation of pre- and postexposure medical countermeasures, such as vaccination.
In summary, our results demonstrate that the MPXV-challenged prairie dog animal model offers a novel alternative small animal model to further explore humoral and cellular immunity and the mechanisms of protection of prophylactic smallpox vaccination, and possibly to assess postexposure vaccine efficacy. In this study, Dryvax or Acambis2000 completely, and single-dose IMVAMUNE partially, protected animals from rash illness from a low-dose systemic OPXV challenge administered 30 days prior to challenge. Similarly, a two-dose IMVAMUNE vaccination regimen, Dryvax, and Acambis2000 regimens all protected animals from death from a high-dose systemic OPXV challenge when vaccination regimens were completed 30 days prior to challenge. In the high-dose challenge study, the amount of rash illness in IMVAMUNE-vaccinated animals was subjectively 10-fold lower than that seen in the unvaccinated animals but was significantly higher than that seen in Dryvax-vaccinated, Acambis2000-vaccinated, or uninfected animals.