A highly stable lyophilized mRNA vaccine for Herpes Zoster provides potent cellular and humoral responses

Abstract

Herpes zoster (HZ) is a painful vesicular rash that occurs upon varicella-zoster virus (VZV) reactivation in older adults and immunocompromised individuals. Although there is currently an approved vaccine for the prevention of shingles, its administration is commonly associated with high reactogenicity. This highlights the need to develop new vaccine alternatives with long lasting immunity and improved tolerability upon administration. In the present study, 10 different vaccine candidate designs using two different codon optimizations targeting the VZV glycoprotein E (gE) were generated. A subset of mRNA constructs were formulated into lipid nanoparticles and assessed for their ability to induce specific cellular and humoral immune responses following vaccination in mice. Notably, the selected mRNA vaccine candidates induced high levels of antibodies and robust CD4⁺ but also CD8⁺ immune responses. Moreover, we showed that our alternate lyophilized vaccine provides comparable immunogenicity to current liquid frozen formulations and is stable under long-term storage conditions.

Fig. 1. Design of mRNA constructs expressing different versions of varicella zoster virus (VZV) glycoprotein E (gE).

Schematic representation of the different gE designs that were screened and evaluated in vitro. The different gE constructs are characterized for containing an N-terminal signal peptide (SP) followed by an ectodomain and a transmembrane domain (TM) that is present in all membrane anchored constructs (gE WT, ms4, ms5, ms8, ms9, ms10, ms11 and ms12) but is partially present or completely absent in the gE secreted versions (ms3 and ms6). Additionally, some of the designs contain a cytoplasmic tail (CT) with the Y569A mutation which is responsible for targeting the gE to the TGN (ms9, ms10, ms11 and ms12) and/or a deletion of a phosphorylated acidic amino acid cluster that is associated with Golgi localization (ms8 and ms9). Other CT truncations of different lengths were included in gE_ms5, ms10 and ms12 as illustrated in the diagrams.

Fig. 2. mRNA gE constructs in vitro screening and candidate selection.

mRNA vaccine candidates generated were tested in HeLa cells. A total of 50 ng of RNA were transfected and analyzed 24 h later through indirect immunofluorescence assay (IFA). Cells were fixed, permeabilized and percentage positive cells and mean fluorescence intensity (MFI) were measured using a specific antibody against the VZV gE protein. a Percentage of gE positive cells and (b) mean fluorescence intensity (MFI) of membrane anchored mRNA VZV gE constructs were measured for two different codon optimized sequences based on different GC content (Co1 and Co2). c Percentage of gE positive cells and (d) MFI of soluble mRNA VZV gE constructs was measured for two different codon optimized sequences based on different GC content (Co1 and Co2).

Fig. 3. mRNA gE constructs expressing secreted versions of gE are highly expressed in cell supernatants.

Western blot of cell supernatants (a) and cell extracts (b) after transfecting HeLa cells with 2.5 µg of RNA with the indicated gE-expressing mRNA constructs (gE WT, gE_ms3 and gE_ms6) that were synthetized with two different codon optimizations (Co1 and Co2). Samples were collected and analyzed 24 h after transfection. Both gE soluble versions (gE_ms3 and gE_ms6) were barely detected in total cell lysates but were instead detected at high levels in cell supernatants as opposed to gE WT version which was mostly detected in whole cell extracts.

Fig. 4. Subcellular localization and characterization of selected mRNA VZV gE vaccine candidates in Vero cells.

VZV gE protein presence in the Golgi was assessed 24 h after transfection in Vero cells using spinning disk confocal microscopy. a DS from the three lead candidates: gE WT_Co2, gE_ms5 Co1 and gE_ms6 Co2 were transfected into Vero cells. 24 h later cells were washed, permeabilized and fixed. gE protein was detected using a human mAb against VZV gE and A488 secondary antibody. Golgi apparatus was stained using a specific antibody against N-acetylgalactosaminyltransferase 7 (GALNT7) and using A555 as a secondary antibody for detection. Nuclei were stained using DAPI (blue). Small rectangles represent the areas that were selected for analysis of colocalization. b A total of ten images were acquired per condition and colocalization levels between VZV gE protein and Golgi were measured for each mRNA construct using Pearson’s correlation coefficient based on regions of interest (ROI). One-way Analysis of Variance (ANOVA) with Tukey’s multiple comparison test was performed to determine statistical significance between the sample groups. ****p < 0.0001; ns: not significant.

Fig. 5. Total and cell surface in vitro expression assay (IVE) of selected mRNA VZV gE vaccine candidates in HEK 293T cells.

In vitro expression of mRNA VZV LNP formulated material for the three proposed vaccines candidates gE WT_Co2, ms5_Co1 and ms6_Co2 was measured by flow cytometry in HEK 293T cells and whole gE expression levels (a) or gE levels in the cell surface (b) were detected using a specific antibody against VZV gE. EC50 values (ng/well) were calculated from each curve and are shown in a table together with the (%) positive cells at 31 ng dose for both conditions tested.

Fig. 6. Immunogenicity and CMI responses elicited by mRNA VZV gE vaccine candidates in vivo.

Female C57BL/6 mice were immunized with a full human dose of live attenuated VZV vaccine (Varivax®) to create preexisting immunity against VZV. Animals were subsequently immunized intramuscularly (IM) at day 35 and day 63 with 1 µg of our mRNA vaccine lead candidate LNP formulated material: gE WT_Co2 (frozen and lyophilized (Lyo) version), as well as frozen ms5_Co1 and ms6_Co2. Shingrix was also included and used as a positive control in the study. a Schematics of immunization and vaccination regimen schedule. Created in BioRender. Munoz-Moreno, R. (2024) https://BioRender.com/x92w358. b Blood was collected via submandibular route and gE-specific IgG antibodies (μg/ml) were measured by Luminex on day 76. c (%) of antigen induced specific CD4⁺ T cell responses from mice splenocytes were measured by intracellular cytokine staining (ICS) assay on day 76. Data is shown as the mean with standard deviation (SD). d (%) of antigen specific CD8⁺ T cell responses from mice splenocytes were measured by intracellular cytokine staining (ICS) assay on day 76. Data shows the median from 5 animals/group. Data is shown as the mean with SD.

Fig. 7. Stability and immunogenic profile of lyophilized mRNA material over time.

In vitro expression (IVE) of frozen and lyophilized material for gE WT Co1 candidate was measured by flow cytometry in HEK 293 T cells at initial timepoints (a) as well as after 12 months (b) and 24 months (c). Whole gE expression levels were detected using a specific antibody against VZV gE. EC50 values (ng/well) were calculated from each curve and are shown in a table together with the (%) positive cells at 31 ng dose for all three conditions tested. Blood from mice previously immunized with 1 µg dose of Shingrix® or VZV gE WT Co1 (frozen or lyophilized) mRNA material preserved for 0 months (d), 12 months (e) or 24 months (f) was collected via submandibular route and gE-specific IgG antibodies (µg/ml) were measured by Luminex on day 76.