Highly stable and immunogenic CMV T cell vaccine candidate developed using a synthetic MVA platform

Abstract

Human cytomegalovirus (CMV) is the most common infectious cause of complications post-transplantation, while a CMV vaccine for transplant recipients has yet to be licensed. Triplex, a multiantigen Modified Vaccinia Ankara (MVA)-vectored CMV vaccine candidate based on the immunodominant antigens phosphoprotein 65 (pp65) and immediate-early 1 and 2 (IE1/2), is in an advanced stage of clinical development. However, its limited genetic and expression stability restricts its potential for large-scale production. Using a recently developed fully synthetic MVA (sMVA) platform, we developed a new generation Triplex vaccine candidate, T10-F10, with different sequence modifications for enhanced vaccine stability. T10-F10 demonstrated genetic and expression stability during extensive virus passaging. In addition, we show that T10-F10 confers comparable immunogenicity to the original Triplex vaccine to elicit antigen-specific T cell responses in HLA-transgenic mice. These results demonstrate improvements in translational vaccine properties of an sMVA-based CMV vaccine candidate designed as a therapeutic treatment for transplant recipients.

Fig. 1. Triplex protein expression stability.

WB analysis of the CMV proteins and the MVA control protein expression of Triplex passaged up to passage 7 in CEF. IEfusion was probed using an anti-IE1 mouse mAb (p63-27). Pp65 was probed using an anti-pp65 mouse mAb (28-103). Vaccinia virus BR5 was probed using an anti-pp65 rat mAb (19C2). “CEF” lane corresponds to uninfected cells. “MVA” lane corresponds to cells infected with the empty MVA vector. “(+)” corresponds to cells infected with virus stock used to generate clinical lots of Triplex. kDa: kilodalton.

Fig. 2. sMVA-T10 construct design.

Schematic representation of the T10 vaccine construct reconstituted using three sMVA fragments. The insertion of the IE2-4nt, IE1-4nt, and pp65 CMV antigens sequences in each corresponding insertion site (44/45, IGR3, Del3) as well as the approximate length of the sMVA fragments, the overlapping homologous sequences for recombination between fragments 1 and 2 and fragments 2 and 3 (indicated as grey boxes with a red cross), and the inverted terminal repeats (ITR) is indicated. kbp kilobase pairs.

Fig. 3. Antigen stability of non-mutated and IE2 M361I-mutated T10-derived virus isolates.

T10-derived virus isolates containing non-mutated (B4 and F2) or M361I-mutated (F1 and F10) IE2 antigen sequences were blind passaged ten times in CEF and evaluated for antigen stability by PCR and WB. a virus titer. Given are the virus titer (pfu/mL) of the T10 plaque-purified isolates (T10-B4 in red, T10-F2 in purple, T10-F1 in green and T10-F10 in blue) during the ten virus passages in CEF. b–e WB and PCR analysis of T10-B4 (b), T10-F2 (c), T10-F1 (d), T10-F10 (e) passaged up to passage 10 in CEF. Upper panels show WB for the CMV proteins and BR5. IE1 ( ~ 55 kDa) was probed using an anti-IE1 mouse monoclonal antibody (mAb) (p63-27). IE2 ( ~ 63 kDa) was probed using an anti-IE2 mouse mAb (2.9.5). Pp65 (~65 kDa) was probed using an anti-pp65 mouse mAb (28-103). Vaccinia virus BR5 ( ~ 43 kDa) was probed using an anti-pp65 rat mAb (19C2). Lower panels show PCR analysis of the CMV gene sequences in the corresponding insertion sites. Expected PCR products are: 2,936 bp for IE1-4nt in IGR3, 2,219 bp for IE2-4nt/IE2-4nt-M361I in 44/45 and 2,882 bp for pp65 in Del3. “s52” and “T10” lanes correspond to cells infected with the empty sMVA vector and the parental T10 respectively. “CEF” lanes correspond to uninfected CEF cells. kbp: kilobase pairs; kDa: kilodalton.

Fig. 4. IE2 gene sequencing of the T10 and T10-F10 plaque-purified isolates.

Schematic representation of the plaque-purification processes of T10 and T10-F10 where the IE2 gene sequencing results are indicated for the original stock (passage 0) of all isolates, passage 10 of the isolates used for the stability comparison assay and passage 12 of T10-F10. Sequencing results in blue boxes indicate original IE2-4nt sequences, green boxes indicate mutated IE2-4nt-M361I sequences and yellow boxes indicate aberrant sequences. Grey boxes with an “X” indicate sequencing not performed.

Fig. 5. T10-F10 antigen stability in CEF and AGE1.CR.PIX cells.

T10-F10 was blind passaged twelve times in CEF (a) and passaged twelve times in AGE1.CR.PIX (b) cells and evaluated for antigen stability by PCR and WB. Upper panels correspond to WB analysis of the CMV proteins and the MVA vector control expression. IE1 ( ~ 55 kDa) was probed using an anti-IE1 mAb (p63-27). IE2 ( ~ 63 kDa) was probed using an anti-IE2 mouse mAb (2.9.5). Pp65 (~65 kDa) was probed using an anti-pp65 mouse mAb (28-103). Vaccinia virus BR5 ( ~ 43 kDa) was probed using an anti-pp65 rat mAb (19C2). Lower panels correspond to PCR analysis of the CMV genes sequences in the corresponding insertion sites. Expected PCR products are: 2,936 bp for IE1-4nt in IGR3, 2,219 bp for IE2-4nt-M361I in 44/45 and 2,882 bp for pp65 in Del3. “s52” and “T10” lanes correspond to cells infected with the empty sMVA vector and the parental T10 respectively. “CEF” and “AGE1” lanes correspond to uninfected CEF and AGE1.CR.PIX cells. kbp: kilobase pairs; kDa: kilodalton.

Fig. 6. Potent stimulation of CMV-specific IFNγ-expressing T cells by T10-F10 in HLA-transgenic mice.

a HLA-A2 transgenic mice ELISpot analysis of IFNγ-secreting cells following stimulation with CMV pp65, IE1 and IE2 peptide libraries and pp65 and IE1 peptides containing the HLA-A*0201-restricted pp65- and IE1-specific immunodominant epitopes (pp65 495–503 and IE1 316–324). b HLA-B7 transgenic mice ELISpot analysis of IFNγ-secreting cells following stimulation with CMV pp65, IE1 and IE2 peptide libraries and pp65 peptide containing the HLA-B*0702-restricted pp65-specific immunodominant epitope (pp65 265–275). Two-way ANOVA with Tukey’s multiple comparison test was used to calculate significance of the difference between the groups. Data is presented as mean values ± SD; **0.01 <p < 0.001, ***0.001 <p < 0.0001, ****p < 0.0001; ns not significant.