Validation of animal models for use as surrogates for vaccination studies is of paramount interest when human experimentation is not feasible. Monkeypox virus infection of prairie dogs produces disease that is highly similar to that in humans after systemic OPV infection, and protection from disease occurs when animals are vaccinated prior to challenge with monkeypox virus (24
). Additionally, since vaccination in humans produces an antibody response correlated with protection, a highly similar response in prairie dogs would further support their use as a surrogate animal model of OPV vaccination. This in turn would allow testing of additional smallpox vaccines in this model for efficacy against systemic OPV infections. Here, we used a protein microarray spotted with select VACV WR proteins and compared the antibody response profiles from VACV-vaccinated humans and prairie dogs. The profiles were highly heterogeneous, with one or more individuals in each group reacting to 15 of the 61 proteins on the chip, yet also contained strong and consistent reactions to several immunodominant and known neutralizing proteins. We further characterized the immune response after MPXV challenge of naïve and vaccinated prairie dogs. Upon challenge, the response profiles became much more homogeneous, with an average of 27.4 antigens recognized by each prairie dog and 21 of those antigens recognized by more than 75% of all challenged animals.
One concern when using nonclonal and unpurified proteins was the potential for background reactivity due to aberrant protein production or the complexity of the crude mixture spotted. A high degree of correlation between our results and those of others using quantified or purified protein arrays would indicate that this is not an issue. In one of the more complete efforts to date, 193 of 273 vaccinia virus Copenhagen strain proteins were expressed and purified from a baculovirus protein system; it was found that <10% of the proteome was reactive to VIG or vaccinated individuals (42
). Using a rabbit reticulocyte system, Duke-Cohan et al. (41
) selected 25 surface-exposed or known antigenic proteins of the 218 ORFs of the WR proteome to produce proteins in a micro-ELISA format. Reactivity was found to 17 proteins, although only 10 were strongly reactive. The correlations of vaccinated individuals and VIG profiles were similar in both sets of experiments. In our experiments, 13 proteins were recognized by more than one vaccinee, and 26 proteins were recognized by VIG. Overall, among all sets of results, there was good concordance when looking at strongly reactive proteins, such as D13, H3, D8, I1, A33, A27, and B5. When comparing less-reactive proteins, although there was variability as to which individual proteins were recognized, our experimental method did not produce significantly different hits than either the Schmid or Duke-Cohan data sets. A comparative table of reactivity observed by each system can be found in Table S2 in the supplemental material.
Our results show significant reactivity to membrane proteins in vaccinated humans and prairie dogs, with WR148, A13, H3, D8, and A17 commonly seen. The last four MV proteins have all been shown to be effective targets for virus neutralization and thus critical targets of the immune system to inactivate and limit virus spread (50
). However, A17 was recognized less frequently in prairie dogs, with only 6 of 34 animals responding to it, and four of those were in the Acam2000 group. A17 is an MV protein that is associated with viral morphogenesis and is essential for replication (51
). The N-terminal region has been identified as the target of neutralizing antibodies, as well as the region that interacts with A14 and A27 (52
). As such, reduced reactivity may be indicative of subtle changes that limit the accessibility of the external N-terminal region, or the region may simply be less immunogenic in the prairie dog. A fifth MV neutralizing target, A27, was seen in nearly 50% of Dryvax-vaccinated individuals of both species but failed to react with any Acam2000 or Imvamune vaccinee. Protein L1 was excluded from the chip, even though it has historically been shown to produce neutralizing antibodies (53
). At the time of writing, L1 failed to produce a response in human sera and was only minimally reactive in concentrations higher than normal (data not shown). L1 is notable for significant disulfide bonding; thus, the limitation is likely structural, and some gains have since been realized (44
). With baculovirus expression, detection ranged from 0 to 30% recognition (42
), and in a rabbit reticulocyte lysate expression system, recognition was <25%, although a strong correlation with neutralization in 4 samples with anti-L1 antibodies was noted (41
There were fewer and more varied responses to EV proteins after vaccination regimens in both species with serorecognition of A33 and sometimes B5, as indicated by low-level but significant fluorescence intensity in humans and serorecognition of A56 and B5 in prairie dogs. Interestingly, upon MPXV challenge, prairie dogs showed nearly universal responses to A56, B5, and F13 and lower responses to A34 and A36. This finding is interesting, since EV virions are thought to aid in virus dissemination in natural infection (55
) and typical vaccine strains produce only a single localized lesion. However, it is unclear whether this difference in reactivity to EV proteins is due to limited spread from the vaccination site or simply due to less immunologic insult than the systemic spread commonly found with monkeypox virus infection. It is also possible that since EV proteins would likely be posttranslationally modified, this low reactivity is an artifact of the E. coli
system. However, a low frequency of reactivity to EV proteins after immunization was also noted in similar assays using different expression systems (41
). Reactivity to EV proteins using single-antigen ELISA showed increases in response after vaccination (56
), but the absence of individual pre- and postvaccination sera makes comparison difficult. It is also notable that while very few sera were reactive with multiple EV proteins, 8 of 15 human vaccinees and 20 of 34 prairie dogs were reactive to at least one EV protein, and over 40% of all vaccinees were reactive to two or more EV proteins. This variability at the whole-protein level correlates with recent studies of antibody response after smallpox vaccination, where redundant neutralizing antibodies targeted a multitude of viral epitopes as opposed to a single immunodominant epitope, and shows that the specificities of the response may differ substantially by individual (39
Antibody responses to core proteins after vaccination were highly similar between virus strains and species. As core proteins, the accessibility of each would be limited until lysis of infected cells and then would be expected to be somewhat correlated with their relative abundances and the availability of immunogenic epitopes. Reports of relative abundances in MV virions (57
) showed that A4 and A10 were predominant proteins, with I1 representing as little as 1/100 of their total mass. This relationship of antigen mass to antibody recognition was reversed here, as I1 was immunodominant.
The recognition of E3 by Dryvax-vaccinated prairie dogs, but not humans, was also interesting. The protein was recognized only weakly in one human vaccinee and was previously seen with equivocal recognition by humans and no recognition by primate vaccinees (40
). In comparison, strong recognition of E3 was seen in mice and rabbits given the pathogenic VACV WR strain (40
), and primates challenged with MPXV show reactivity to E3, and in particular the truncated MPXV homologue of E3 (59
). Similar to the absence of reactivity to A17, reactivity to E3 after vaccination may result from species-specific sensitivity to available epitopes, and only upon challenge (VACV WR or MPXV) is the response sufficient to ensure that it is measurable. It is also possible that stronger recognition is at least partially a function of the pathogenicity of the challenge virus or the degree of systemic spread of virus during disease.
There were three commonly recognized proteins after vaccination that did not fit into one of the above-mentioned groups. D13 is a scaffold protein that is lost prior to MV maturation (60
). However, its necessity as a structural protein in crescent formation and its ubiquity confirm it as a likely immunologic target. A11 is a nonstructural protein also associated with crescent formation (61
). Its near absence in Acam2000-vaccinated prairie dogs was somewhat surprising but, upon examination of signal intensities, may simply be a function of low levels of response making it indistinguishable from background. While A11 was recognized in all other vaccination groups, it typically had an average intensity of only 2 to 3 times the cutoff value and for all groups was in the bottom third of reactive proteins by ranked average intensity.
Consistent with previous reports of vaccine immunogenicity (62
), we saw no difference between Dryvax and Acam2000 array responses in humans, with an average of 10.0 and 9.3 array hits per sample, respectively. However, in prairie dogs, there was a trend of lower response after Acam2000 than after Dryvax, including many instances where Dryvax vaccinees responded to a given protein 100% of the time whereas Acam2000 vaccinees were less frequently recognized but still greater than the 50% designation shown in . A similar overall reduction was observed after Imvamune vaccination, as well, only part of which can be accounted for by the loss of reactivity to WR148. Relative to Dryvax, reduced antibody response has also been seen in Imvamune vaccination of humans (63
) and included absent or reduced reactivity to WR148 and A17 but also significantly lower responses to A56, A27, and A33. We observed no reactivity to each of these antigenic targets in Imvamune-vaccinated prairie dogs, as well. The lower overall response between viruses resulted in a drop in the number of recognized antigens from an average of 11.7 hits per sample after Dryvax vaccination to 7.8 and 7.9 hits per sample after Acam2000 or Imvamune vaccination. This difference accounts for an ~15% loss in positive hits over the entire proteome between the vaccines. ELISA geometric mean titers (GMTs) also showed this reduced response, with both Acam2000 and Imvamune having between 2- and 10-fold-reduced peak GMTs relative to Dryvax.
After MPXV challenge, the response to a much greater number of antigens (n
= 27.4) than after vaccination (n
= 9.1) may reflect the more systemic presentation of the virus. In Dryvax revaccination cases, only a modest increase in the number of recognized targets has been seen (40
). We also recently showed that disease severity, as judged by weight loss, lesion count, and nasal involvement, was significantly limited upon challenge after vaccination, particularly after Dryvax or Acam2000 vaccination (25
). Although the limited number of immunodominant antigens after vaccination may have been expected to continue dominating the profile, along with the addition of a few novel targets that may have been recognized during vaccination but failed to generate a detectable antibody response, the large increase here suggests that differences in virus pathogenicities or life cycles affect immune recognition. It was observed that pathogenic WR virus infection of naïve rabbits induced a large and robust response to a broad range of proteins compared to vaccination alone (44
), and a recent report showed that the antibody response to monkeypox virus infection of naïve macaques also targeted a larger number of antigens than human vaccination (59
). Thus, dramatic increases in the frequency and intensity of responses to multiple targets upon challenge, but not repeat vaccination, provides evidence that increased pathogenicity drives the stronger immune response. Added support for pathogenicity affecting immune response is shown in the shift from intermediate- and late-promoter genes (64
), which are typical of structural virion core and membrane proteins and are present on the surfaces of infected cells (67
), to early-promoter genes. In the present study, all seven antigens reactive in greater than 50% of vaccinated prairie dogs were intermediate- or late-promoter driven. Of 27 commonly reactive antigens after challenge, 13 were classified as intermediate or late promoter, but 6 early/immediate-early and 8 early/late promoters were seen. One final piece of evidence supporting virus pathogenesis driving the response to novel antigens includes mounting of a secondary IgM response noted in vaccinated individuals during the 2003 monkeypox outbreak in the United States (48
). The IgM assay utilized whole VACV virions (Dryvax) to assess post-monkeypox IgM induction, and as such, a response would indicate new immune priming by previously unrecognized proteins and epitopes after vaccination. An increase in pathogenicity between highly similar yet different viruses, such as VACV and then MPXV, would allow for the large number of additional antigenic targets to highly homologous proteins recognized here and the observed secondary IgM response. One additional correlative observation was the relationship between antigens recognized by proteome analysis and disease severity upon challenge of the vaccinated animals. Prairie dogs given Imvamune responded to fewer antigens by our analysis and showed some signs of morbidity upon challenge (25
). No morbidity was observed when challenging Dryvax-vaccinated animals. Although there were fewer targets overall, there was no consistently absent antibody target that correlated with protective or known neutralizing targets, nor were there statistically significant differences in the average numbers of neutralizing targets recognized. However, we also saw fewer responses during Acam2000 vaccination yet no increase in morbidity after challenge, suggesting that multiple factors are involved in immunogenicity and protection. It would also be of interest to gain greater insight into the full spectrum of antibody responses before and after challenge using an MPXV proteomic approach. Keasey et al. observed greater increases in antibody responses to several MPXV proteins than to VACV proteins by aerosol MPXV-challenged macaques (59
), indicating that although broad cross-reactivity exists, a stronger response may be directed toward MPXV-specific proteins. Differences in neutralization titers have also been observed, depending on the strain of OPV utilized in the PRNT assays (68
). While a direct comparison between titration methods is not possible using the vaccinia virus-based HCS-GFP assay, future experiments that shed light on the virus specificity of this broader response and the virus-specific neutralization capacity after vaccination and challenge would be of interest.
In conclusion, we have shown that the antibody response in prairie dog vaccination closely resembles that in human vaccination when examined at the whole-proteome level. While some differences were observed, including variability in the recognition of proteins A17, A9, A56, E3, and WR169 after vaccination, a clear set of proteins recognized by all vaccinees in both species remained. Of the proteins known to be targeted by neutralizing antibodies, only the absence of A17 recognition in prairie dogs was observed. However, there was no significant difference in neutralizing capacity, as demonstrated by similar 50% RPR titers in our HSC-GFP assay, suggesting this loss of reactivity alone is not critical in protection, nor is it a salient difference in the animal model system. We observed fewer responses after vaccination with the attenuated Imvamune strain, which correlated with increases in morbidity when the prairie dogs were challenged with MPXV. We have also shown that challenge with pathogenic MPXV induced a larger-magnitude response to a greater number of targets than vaccination, regardless of prechallenge status (i.e., vaccinated or naïve). This dramatic increase in the frequency of antigen recognition after challenge may be relevant as safer, and likely more attenuated, vaccines are developed. The increase in the breadth of response after challenge also supports observations of lifelong immunity following smallpox but allows for wide variations in waning immunity years after vaccination (70