Evaluation of recombinant modified vaccinia Ankara virus-based rhesus cytomegalovirus vaccines in rhesus macaques
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- Yue, Y., Wang, Z., Abel, K. et al. Med Microbiol Immunol (2008) 197: 117. doi:10.1007/s00430-008-0074-5
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A vaccine consisting of rhesus cytomegalovirus (RhCMV) pp65-2, gB and IE1 expressed via modified vaccinia Ankara (MVA) was evaluated in rhesus macaques with or without prior priming with expression plasmids for the same antigens. Following two MVA treatments, comparable levels of anti-gB, pp65-2 and neutralizing antibody responses, and pp65-2- and IE1-specific cellular immune responses were detected in both vaccinated groups. Similar reductions in plasma peak viral loads were observed in these groups compared to untreated controls. This study demonstrates the immunogenicity and protective efficacy of rMVA-based RhCMV subunit vaccines in a primate host and warrants further investigation to improve the efficacy of subunit vaccines against CMV.
KeywordsCytomegalovirus vaccine Rhesus macaques Modified vaccinia Ankara virus
Development of an effective vaccine for human cytomegalovirus (HCMV) has been designated a “level 1” priority by the Institute of Medicine because of the devastating impact of congenital infection by HCMV which can have lifelong consequences . The strict species-specificity of the virus requires assessment of vaccine strategies in a cognate animal model. Rhesus CMV (RhCMV) infection of rhesus macaques (Macaca mulatta—Mmu) strongly recapitulates HCMV infection in humans in terms of the similarity of viral genome, persistence, pathogenesis and host immune responses [2, 3, 4, 26, 27], therefore, it represents a clinically relevant model to evaluate HCMV vaccine strategies.
Measurement of natural immunity to HCMV supports targeting the predominant neutralizing antibody response antigen gB and the strongly recognized cellular immunogens pp65 and IE1 as the optimal antigens in a vaccine formulated to prevent infection and control disease . Previously, a study in Mmu demonstrated that immunization with DNA vaccines expressing RhCMV pp65-2 and truncated gB with deletion of transmembrane and intracellular domains (gBΔ) stimulated antigen-specific CD8+ T cell and antibody responses and provided limited protection against challenge . A notable observation of this study was that a combination of DNA priming administered five times in 50 weeks followed by a heterologous boost (in this case, by the virus challenge) stimulated a rapid and profound increase in neutralizing antibody titers. In this report, we compared an approach in which recombinant modified vaccinia Ankara (rMVA) expressing RhCMV pp65-2, gBΔ and IE1 antigens was administered twice to Mmu versus an identical schedule of rMVA administration preceded by a single DNA prime. Our primary goal was to establish whether these sub-unit vaccines are effective for controlling an experimentally applied CMV infection and to test whether DNA priming enhances the effectiveness of rMVA to stimulate protective immunity in this model.
RhCMV-rMVA construction and expression
rMVA expressing either both RhCMV gBΔ and pp65-2 (Rh gB-pp65-MVA) or IE1 alone (Rh IE1-MVA) was generated on BHK-21 cells via homologous recombination. The protein expression levels for RhCMV gB, pp65-2 and IE1 in infected BHK-21 cells were confirmed by Western blot using polyclonal antibodies to RhCMV gB, pp65-2, and IE1 by chemiluminescence detection (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK) (Fig. 1b).
Although the genetic stability of the rMVA constructs was not directly evaluated, previous work using the same recombination plasmid pZWIIA to express HCMV soluble gB and/or pp65 and IE1 exon-4 demonstrated that the resultant rMVA constructs were stable through four passages [20, 21]. The genetic instability of rMVA that has been observed usually happens when attempting to express certain integral membrane proteins at high levels, which may be due to the toxicity of over-expressed proteins . The genetic stability of rMVA expressing membrane proteins can be enhanced after truncating the cytoplasmic domain [22, 23]. In this report, we used the same strategies and methods to construct the rMVAs expressing RhCMV truncated gB, pp65-2 and IE1. RhCMV gB, pp65-2 and IE1 are homologues of HCMV gB, pp65 and IE1 and the results in Fig. 1b confirm expression of the foreign inserts. Aliquots of the same rMVA stocks used to demonstrate expression in infected tissue culture cells (Fig. 1) were used to infect rhesus macaques.
Vaccination-induced immune responses
Plasma viral loads post-challenge
In addition to plasma viral loads, skin biopsies of two sites of RhCMV inoculation (SC) were obtained on days 7 and 10 post-challenge to evaluate RhCMV replication at local inoculation sites. IE1-expressing cells in skin tissue slides were analyzed by immunohistochemstry. No significant differences were observed between either the vaccinated and control groups (data not shown).
Immune responses post-challenge
All the vaccinees exhibited vigorous memory antibody responses post-challenge. These included anti-IE1 antibodies in the rMVA animals, which had no detectable antibodies prior to challenge (Fig. 3). The relative increases in NT50 of DNA/rMVA and rMVA animals at 1 and 2 weeks post challenge, compared to the time of challenge, were 24 to 44-fold and 101 to 453-fold, respectively (Fig. 3). It was also noted that the NT50 of DNA/rMVA animals at 2 weeks post-challenge were approximately two- to fivefold above those of the rMVA animals. The normative range of NT50 in long-term RhCMV seropositive macaques is 231–3,348 (N = 24, mean 973, median 833) (unpublished data). DNA/rMVA and rMVA/rMVA animals produced comparable NT50 at 1-week post-challenge and higher NT50 at 2-week post-challenge as compared to long-term naturally infected Mmu, whereas the control animals had comparable NT50 after 4 weeks post-challenge. RhCMV challenge stimulated a rapid increase in the frequencies of IE1- and pp65-2-specific CD8+ T cells in one of the DNA/rMVA treated MMu (animal #2) and the frequency of pp65-2-specific CD8+ T cells in one of the rMVA treated animals (animal #4) at 1-week post-challenge (Fig. 4). The frequencies were comparable to or higher than those observed in two unvaccinated naturally RhCMV-seropositive animals, suggesting a memory T cell response in these animals. One unvaccinated control produced a very high frequency of IE1-specific CD8+ T cells at 7 weeks of challenge.
In this report, we demonstrate that rMVA, either given as the sole vaccine or part of a heterologous prime/boost, can result in a dramatic decrease in viral load after RhCMV challenge in the Mmu model. Noteworthy was the need for only a single application of DNA to effectively prime the immune system for a heterologous boosting to produce higher levels of immunogenicity than observed with three DNA applications without viral boosting .
Modified vaccinia Ankara has been established as a safe and potent antigen delivery system. Promising data obtained from both nonhuman primates and clinical trials have shown that a combination of DNA prime and rMVA boost is effective in stimulating effective cellular immune responses and providing high protective efficacy against HIV/SIV and malaria infection . In this study, following the first rMVA boosting, the monkeys primed with only a single application of DNA plasmids elicited earlier and stronger antibody and cellular immune responses than those without priming. However, similar magnitudes of anti-gB, pp65-2 and neutralizing antibodies and IE1- and pp65-2-specific CD4+ and CD8+ T cells were detected in both vaccinated groups after the second rMVA boost, with the exception of anti-IE1 antibodies, which were not detectable in rMVA animals.
Based on the level of IE1 expression in infected tissue culture cells (Fig. 1b), it was somewhat surprising that rMVA immunization in the absence of DNA priming did not stimulate detectable antibody responses. However, the results were not unprecedented. Other studies have shown that a recombinant canarypox expressing HCMV gB did not stimulate immune responses in vaccinated humans, despite expression both in vitro and in small animals (mice and guinea pigs) [1, 10]. Comparable to our study, the canarypox construct did prime the immune system as demonstrated by the memory response generated after infection with the live attenuated Towne vaccine. In our study, prior immunization with DNA or RhCMV infection post rMVA vaccination similarly demonstrated that RhCMV IE1 was recognized in vivo in the context of rMVA.
Both approaches provided comparable protection against challenge. It was also noted that the frequencies of pp65-2- and IE1-specific CD4+ and CD8+ T cells in both vaccine groups generally were low, and strong memory T cell responses were only found in one animal in each vaccination group post-challenge. The reasons for the relatively low level of IE1- and pp65-2-specific T cell responses in this study are unknown. Other studies have demonstrated readily detectable levels of T cell responses to these RhCMV proteins in naturally infected or vaccinated Mmu [25, 26]. Naturally infected Mmu with IE1 and pp65-2 responses were included as positive controls for all of the cellular immune assays. Future studies will address whether augmentation of vaccine-induced responses to IE1 and pp65-2 correlate with increased protection against RhCMV challenge.
An observation of interest in this study was the apparent influence of neutralizing antibody on the containment of viral challenge in which the levels of peak plasma viral load reduction were related to the levels of neutralizing antibodies prior to challenge. Although a larger numbers of Mmu are needed to confirm this observation, it further indicates the protective role of neutralizing antibodies against CMV [14, 17, 18, 24] and a gB-based vaccine strategy [15, 17, 25]. Natural immunity studies have suggested that a vaccine that stimulates immunity equivalent to levels associated with natural infection could reduce the morbidity due to congenital CMV infection by ∼80% [8, 9]. While the employment of either DNA/rMVA or rMVA to deliver RhCMV gB did not induce a relevant natural level, long-lived neutralizing antibody response, other approaches including co-administration of GM-CSF, an adjuvant for the induction of NT , either expressed via DNA or MVA are being explored.
In summary, this study for the first time demonstrates the capacity of rMVA-based CMV vaccines to induce CMV-specific antibody and cellular immune responses in Mmu, and the protective efficacy in reducing plasma viral loads following challenge. The data highlight the potential of rMVA-based CMV vaccines for humans and warrant continuing investigation of the DNA/rMVA approach in a larger number of Mmu to further test the possibility that subunit vaccines can interrupt horizontal transmission.
The authors wish to thank Linda Wyatt and Bernard Moss for MVA vectors and Dan Barouch for advice on prime-boost strategies. This work was supported by NIH grants to PAB (AI063356), DJD (CA30206-Prj3, CA077544 and AI062496), the California National Primate Research Center (RR000169), and City of Hope Cancer Center (CA33572).