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. 2018 Sep 12;92(19):e01012-18.
doi: 10.1128/JVI.01012-18. Print 2018 Oct 1.

Multiantigenic Modified Vaccinia Virus Ankara Vaccine Vectors To Elicit Potent Humoral and Cellular Immune Reponses Against Human Cytomegalovirus in Mice

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Multiantigenic Modified Vaccinia Virus Ankara Vaccine Vectors To Elicit Potent Humoral and Cellular Immune Reponses Against Human Cytomegalovirus in Mice

Flavia Chiuppesi et al. J Virol. .
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Abstract

As human cytomegalovirus (HCMV) is a common cause of disease in newborns and transplant recipients, developing an HCMV vaccine is considered a major public health priority. Yet an HCMV vaccine candidate remains elusive. Although the precise HCMV immune correlates of protection are unclear, both humoral and cellular immune responses have been implicated in protection against HCMV infection and disease. Here we describe a vaccine approach based on the well-characterized modified vaccinia virus Ankara (MVA) vector to stimulate robust HCMV humoral and cellular immune responses by an antigen combination composed of the envelope pentamer complex (PC), glycoprotein B (gB), and phosphoprotein 65 (pp65). We show that in mice, multiantigenic MVA vaccine vectors simultaneously expressing all five PC subunits, gB, and pp65 elicit potent complement-independent and complement-dependent HCMV neutralizing antibodies as well as mouse and human MHC-restricted, polyfunctional T cell responses by the individual antigens. In addition, we demonstrate that the PC/gB antigen combination of these multiantigenic MVA vectors can enhance the stimulation of humoral immune responses that mediate in vitro neutralization of different HCMV strains and antibody-dependent cellular cytotoxicity. These results support the use of MVA to develop a multiantigenic vaccine candidate for controlling HCMV infection and disease in different target populations, such as pregnant women and transplant recipients.IMPORTANCE The development of a human cytomegalovirus (HCMV) vaccine to prevent congenital disease and transplantation-related complications is an unmet medical need. While many HCMV vaccine candidates have been developed, partial success in preventing or controlling HCMV infection in women of childbearing age and transplant recipients has been observed with an approach based on envelope glycoprotein B (gB). We introduce a novel vaccine strategy based on the clinically deployable modified vaccinia virus Ankara (MVA) vaccine vector to elicit potent humoral and cellular immune responses by multiple immunodominant HCMV antigens, including gB, phosphoprotein 65, and all five subunits of the pentamer complex. These findings could contribute to development of a multiantigenic vaccine strategy that may afford more protection against HCMV infection and disease than a vaccine approach employing solely gB.

Keywords: ADCC; HLA; cytomegalovirus; glycoprotein B; neutralizing antibodies; pentamer; phosphoprotein 65; polyfunctional T cells; vaccines; vaccinia.

Figures

FIG 1
FIG 1
Construction of MVA expressing multiple HCMV antigens. (A and B) Vector construction. Utilizing MVABAC-TK, P2A-linked polycistronic and single antigen expression constructs of the PC subunits (gH, gL, UL128, UL130, and UL131A), gB (without TM), and pp65 were inserted as indicated into different intergenic regions (44/45, 64/45, 69/70, and 148/149), the deletion 3 site (Del3), or a restructured Del3 site (Del3 res.) to generate MVAB-7Ag1, MVAB-7Ag2, MVAB-7Ag3, and MVAB-7Ag4. Different P2A codon sequences (P2A1 to P2A5) were used to link the antigens. mH5, modified H5 promoter; B, BAC vector. (C) P2A codon sequences. The lower 5 lines indicate the different P2A codon sequences with mutated nucleotides (marked in colors) that were used for the vector construction as shown in panels A and B. The upper line shows the amino acid sequence of the P2A peptide. (D and E) BAC removal. A genomic duplication (blue in panel D) was utilized to seamlessly remove the BAC sequences (cat, OriS, sopA, RFP, and GFP) of the different MVA vectors by homologous recombination, resulting in MVA-7Ag1, MVA-7Ag2, MVA-7Ag3, and MVA-7Ag4. TK, thymidine kinase gene; P11, vaccinia virus P11 promoter. Primers P1 and P2 flanking the TK gene and primers specific for cat and sopA were used to confirm the restoration of the TK gene and absence of residual BAC sequences via PCR in DNA isolated from BHK cells infected with rMVA. BHK cells infected with MVAB-7Ag1 containing the BAC vector or parental MVA as well as uninfected cells were analyzed for controls.
FIG 2
FIG 2
HCMV antigen expression by multiantigenic MVA vectors. (A and B) Immunoblot (A) and flow cytometry (B) analyses were used to compare the HCMV antigen expression by MVAB-7Ag1 and the control vectors (MVAB-PC/gB, MVAB-PC/pp65, MVAB-PC, MVAB-gB, and MVAB-pp65). Monoclonal (gH, UL130, gB, and pp65) and polyclonal (gL, UL128, and UL131A) antibody preparations were used to detect the HCMV antigens in whole-cell lysates of MVA-infected CEF via immunblotting (A). NAb specific for epitopes formed by UL128/130/131A (1B2 and 12E2), UL130/131A (54E11), UL128 (13B5), or gH (21E9 and 62-11) were used to detect cell surface expression of the PC subunits on live, nonpermeabilized BHK cells infected with rMVA (B). (C to E) Immunoblot analysis as described for panel A was used to evaluate the HCMV antigen expression by MVAB-7Ag1 following 10 (lanes 0 to 10) virus passages in CEF (C), by MVA-7Ag1, MVA-7Ag2, MVA-7Ag3, and MVA-7Ag4 following BAC vector removal (D), and by MVA-7Ag1 and MVA-7Ag3 following 10 virus passages in CEF (E). Vaccinia virus BR5 was detected in panels A and C to E for loading control. Arrows in panels A and C to E indicate the precursor protein (PP) and C-terminal cleavage product (CT) of gB. CEF infected with MVA expressing the fluorescence marker Venus (MVA-Venus) or uninfected cells (Ctrl.) were analyzed as additional controls for panels A and C to E. The x axis in B represents the log10 of fluorescence intensity, and the y axis represents cell count.
FIG 3
FIG 3
HCMV NAb induction by MVAB-7Ag1 and control vectors in C57BL/6 mice. C57BL/6 mice (n = 4 or 5) were immunized three times in 4-week intervals by the intraperitoneal (i.p.) route with MVAB-7Ag1 or the control vectors (MVAB-PC/gB, MVAB-PC/pp65, MVAB-PC, MVAB-gB, or MVAB-pp65) and HCMV immune responses were evaluated 1 week postimmunization. MVA-pp65/IE and MVA-Venus were used as additional controls. NAb titers (NT50; geometric mean titer) were measured in mouse immune sera on ARPE-19 or MRC-5 cells against HCMV strain TB40/E (A and C), TR (B and D), Towne (E), or AD169 (F) in the absence (−) or presence (+) of 5% guinea pig complement (abbreviated C). Dotted lines indicate the lowest serum dilution analyzed. Bars represent geometric means with 95% confidence intervals. Statistical significance of differences between NAb titers were calculated using two-way ANOVA, followed by Sidak's multiple-comparison test.
FIG 4
FIG 4
ADCC and T cell stimulation by MVAB-7Ag1 and control vectors in C57BL/6 mice. The induction of ADCC (A and B), antigen-specific binding antibodies (C and D), and mouse MHC-restricted T cells (E and F) was evaluated in mice immunized with MVAB-7Ag1 or the control vectors (MVAB-PC/gB, MVAB-PC/pp65, MVAB-PC, MVAB-gB, or MVAB-pp65) as described in the legend to Fig. 3. (A and B) Serial dilutions of the mouse immune sera were tested for activity to promote ADCC following antibody binding to ARPE-19 cells infected with TB40/E (A) or TR (B) using an ADCC surrogate reporter assay. Bars represent SDs of duplicates. (C and D) PC-specific (C) and gB-specific (D) binding IgG endpoint titers were evaluated via ELISA using purified gB and PC protein. Dotted lines indicate the minimum serum dilution analyzed. Bars represent geometric means with 95% confidence intervals. (E and F) Ex vivo T cell responses were determined by IFN-γ ELISpot assay utilizing previously described mouse H2-b-restricted immunoreactive peptides of pp65 (E) and the PC subunits (F). Horizontal lines represent median values with interquartile ranges. Statistical significance of differences comparing each group in panels C to F was calculated using one-way analysis of variance (ANOVA) followed by Tukey's multiple-comparison test (NS, P > 0.05). SFCs, spot-forming units. Note that in panel F the measured IFN-γ responses, with only a single exception, were higher than the threshold, and therefore, differences could not be evaluated.
FIG 5
FIG 5
HCMV immune stimulation by multiantigenic MVA vectors in BALB/c mice. BALB/c mice were immunized three times i.p. in 4-week intervals (triangles) with the multiantigenic MVA vectors, and HCMV NAb and T cell responses were evaluated. HCMV NAb titers were measured at the indicated time points against HCMV TB40/E on ARPE-19 cells in the absence of complement, and on MRC-5 cells in the absence or presence of 5% guinea pig complement. T cell responses were determined ex vivo by IFN-γ ELISpot assay utilizing pp65 and gB peptide libraries. Panels A to C show NAb and T cell responses of BALB/c mice (n = 4 or 5) immunized with MVA-7Ag1 in either standard dose (5 × 107 PFU) or low dose (LD; 1 × 107 PFU) or control vectors MVA-PC and MVA-Venus in standard dose. NAb titers shown in panels A and B were measured over a period of 30 weeks. Ex vivo T cell responses shown in panel C were measured at week 31 after an additional booster immunization at week 30. Panels D to F show NAb and T cell responses of BALB/c mice (n = 8) immunized with MVA-7Ag1 that was derived before (P0) and after (P10) 10 virus passages on CEF. Panels G to I show NAb and T cell responses of BALB/c mice (n = 6 or 7) immunized with MVA-7Ag2, MVA-7Ag3, or MVA-7Ag4. Bars in panels A, B, D, E, G, and H represent geometric means with 95% confidence intervals. Bars in panels C, F, and I represent median values with interquartile ranges. Statistical significance of differences comparing each group in panels A, B, D, E, G, and H was calculated using ANOVA followed by Sidak's multiple-comparison test. Differences between vaccine groups in panels C, F, and I were calculated by multiple t test (NS, P > 0.05).
FIG 6
FIG 6
Human MHC-restricted T cell responses elicited by MVA-7Ag1. Transgenic C57BL/6 mice (n = 5) expressing HLA-A*0201 (A2) (A) or HLA-B*0702 (B7) (B) class I molecules were immunized two times in a 4-week interval with MVA-7Ag1 or control vector MVA-Venus. One week following the booster immunization, antigen-specific T cell responses were determined by IFN-γ ELISpot assay using pp65- and gB-specific peptide libraries, HLA-A*0201- or HLA-B*0702-restricted immunodominant peptide epitopes of pp65, or pools of mouse H-2b-restricted peptides of gB or the PC. Bars represent median values with interquartile ranges. Significance of the difference between the groups was calculated using two-way ANOVA followed by Sidak's multiple-comparison test.
FIG 7
FIG 7
HLA-B*0702-restricted polyfunctional CD8+ T cells elicited by MVA-7Ag1. HLA-B*0702 (B7) transgenic mice were immunized two times in 4-week intervals with MVA-7Ag1, MVAB-pp65 (n = 5), or MVA-Venus (n = 1). One week postimmunization, antigen-specific T cell responses were evaluated by multicytokine ICS following stimulation with pp65- and PC-specific peptide libraries or an HLA-B*0702-restricted immunodominant pp65 peptide epitope. (A to C) Shown are the percentages of CD8+ T cells secreting IFN-γ (A), TNF-α (B), or IL-2 (C) following stimulation of splenocytes from B7 immunized mice with different stimuli. (D to F) Frequency of antigen-specific CD8+ T cells producing all combinations of IFN-γ, TNF-α, and IL-2 cytokines following in vitro stimulation with the HLA-B*0702-restricted pp65 peptide epitope (D), pp65-specific peptide library (E), or PC-specific peptide library (F) in MVA-7Ag1, MVAB-pp65, or MVA-Venus immunized B7 mice. Horizontal bars represent the median values. (G) Pie charts show the relative contribution of each polyfunctional subset among the total polyfunctional response to the pp65 epitope, pp65 library, or PC library in MVA-7Ag1- or MVAB-pp65-immunized B7 mice. Each pie chart represents the mean response across the immunized mice to the three different antigen stimulations. The average total percentage of CD8+ T cells responding to the peptide stimulation is shown under each pie chart. Polyfunctional subsets and functions (Fn°) are indicated in the color key. Significance of the difference between the groups was calculated using multiple t test.
FIG 8
FIG 8
HLA-A*0201-restricted polyfunctional CD8+ T cells elicited by MVA-7Ag1. HLA-A*0201 (A2) transgenic mice were immunized two times in 4-week intervals with MVA-7Ag1, MVAB-pp65 (n = 5), or MVA-Venus (n = 1). One week postimmunization, antigen-specific T cell responses were evaluated by multicytokine ICS following stimulation with pp65- and PC-specific peptide libraries or an HLA-A*0201-restricted immunodominant pp65 peptide epitope. (A to C) Shown are the percentages of CD8+ T cells secreting IFN-γ (A), TNF-α (B), or IL-2 (C) following stimulation of splenocytes from A2 immunized mice with different stimuli. (D to F) Frequency of antigen-specific CD8+ T cells producing all combinations of IFN-γ, TNF-α, and IL-2 cytokines following in vitro stimulation with the HLA-A*0201-restricted peptide epitope (D), pp65-specific peptide library (E), or PC-specific peptide library (F) in MVA-7Ag1-, MVAB-pp65-, or MVA-Venus-immunized B7 mice. Horizontal bars represent median values. (G) Pie charts show the relative contribution of each polyfunctional subset among the total polyfunctional response to the pp65 epitope, pp65 library, or PC library in MVA-7Ag1- or MVAB-pp65-immunized A2 mice. Each pie chart represents the mean response across the immunized mice to the three different antigen stimulations. The average total percentage of CD8+ T cells responding to the peptide stimulation is shown under each pie chart. Polyfunctional subsets and functions (Fn°) are indicated in the color key. Significance of the difference between the groups was calculated using multiple t test.

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