HCMV glycoprotein B nucleoside-modified mRNA vaccine elicits antibody responses with greater durability and breadth than MF59-adjuvanted gB protein immunization

A vaccine to prevent maternal acquisition of human cytomegalovirus (HCMV) during pregnancy is a primary strategy to reduce the incidence of congenital disease. Similarly, vaccination of transplant recipients against HCMV has been proposed to prevent transplant-associated HCMV morbidity. The MF59-adjuvanted glycoprotein B protein subunit vaccine (gB/MF59) is the most efficacious tested to-date for both indications. We previously identified that gB/MF59 vaccination elicited poor neutralizing antibody responses and an immunodominant response against gB antigenic domain 3 (AD-3). Thus, we sought to test novel gB vaccines to improve functional antibody responses and reduce AD-3 immunodominance. Groups of juvenile New Zealand White rabbits were administered 3 sequential doses of full-length gB protein with an MF59-like squalene adjuvant (analogous to clinically-tested vaccine), gB ectodomain protein (lacking AD-3) with squalene adjuvant, or lipid nanoparticle (LNP)-packaged nucleoside-modified mRNA encoding full-length gB. The AD-3 immunodominant IgG response following human gB/MF59 vaccination was closely mimicked in rabbits, with 78% of binding antibodies directed against this region in the full-length gB protein group compared to 1% and 46% in the ectodomain and mRNA-LNP-vaccinated groups, respectively. All vaccines were highly immunogenic with similar kinetics and comparable peak gB-binding and functional antibody responses. Although gB ectodomain subunit vaccination reduced targeting of non-neutralizing epitope AD-3, it did not improve vaccine-elicited neutralizing or non-neutralizing antibody functions. gB nucleoside-modified mRNA-LNP-immunized rabbits exhibited enhanced durability of IgG binding to soluble and cell membrane-associated gB protein as well as HCMV-neutralizing function. Furthermore, the gB mRNA-LNP vaccine enhanced breadth of IgG binding responses against discrete gB peptide residues. Finally, low-magnitude gB-specific T cell activity was observed in the full-length gB protein and mRNA-LNP vaccine groups, though not in ectodomain-vaccinated rabbits. Altogether, these data suggest that the gB mRNA-LNP vaccine candidate, aiming to improve upon the partial efficacy of gB/MF59 vaccination, should be further evaluated in preclinical models. Author summary Human cytomegalovirus (HCMV) is the most common infectious cause of infant birth defects, resulting in permanent neurologic disability for one newborn child every hour in the United States. Furthermore, this virus causes significant morbidity and mortality in immune-suppressed transplant recipients. After more than a half century of research and development, we remain without a clinically-licensed vaccine or therapeutic to reduce the burden of HCMV-associated disease. In this study, we sought to improve upon the glycoprotein B protein vaccine (gB/MF59), the most efficacious HCMV vaccine evaluated in clinical trial, via targeted modifications to either the protein structure or vaccine formulation. An attempt to alter the protein structure to focus the immune response on vulnerable epitopes (‘gB ectodomain’) had little effect on the quality or function of the vaccine-elicited antibodies. However, a novel vaccine platform, nucleoside-modified mRNA formulated in lipid nanoparticles, increased the durability and breadth of vaccine-elicited immune responses. We propose that an mRNA-based gB vaccine may ultimately prove more efficacious than the gB/MF59 vaccine and should be further evaluated for its ability to elicit antiviral immune factors that can prevent both infant and transplant-associated disease caused by HCMV infection.

140 gB mRNA vaccinated rabbits had slightly reduced total peptide binding compared to gB FL 141 (Figure 3B; median peptide-binding MFI sum: FL = 96,629, mRNA = 48,051, p=ns, Kruskal-Wallis 142 + post hoc Mann-Whitney U test), though both gB FL and gB mRNA had greater total peptide 143 binding than the gB ecto group (both p<0.05, Kruskal-Wallis + post hoc Mann-Whitney U test).
144 Importantly, there was enhanced breadth of peptide-binding responses in gB mRNA vaccinated 145 rabbits compared to both gB FL and gB ecto groups (Figure 3D  166 similar peak neutralization at 10 weeks (median AD169r ID 50 in fibroblast at 10 weeks +C: FL = 167 158, ecto = 184, mRNA = 174). gB mRNA vaccinated rabbits had two-fold higher virus 168 neutralization at 20 weeks, indicating superior response durability, though this distinction was not 169 statistically significant (median AD169r ID 50 in fibroblast at 20 weeks +C: FL = 49, ecto = 62, 170 mRNA = 120, p=0.12, Kruskal-Wallis + post hoc Mann-Whitney U test). Notably, neutralizing 171 responses measured in both fibroblast and epithelial cells were similar in magnitude, as might be 172 expected for gB-specific antibodies. In addition to heterologous AD169r, we also measured 173 neutralization of autologous (vaccine strain) Towne virus in fibroblast cells (Figure S2A,B), but 174 identified little difference between vaccination groups.

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176 Vaccine-elicited engagement of F c γ receptors and non-neutralizing effector functions 177 We next investigated the ability of vaccine-elicited antibodies to engage F c γ receptors 178 (specifically F c γRI, F c γRIIa, F c γRIIb, and F c γRIIIa) which is a prerequisite for F c -mediated effector 179 functions (Figure 5A-D). Intriguingly, a single dose of gB mRNA vaccine (but not gB FL or gB 180 ecto) resulted in the rapid development of antigen-specific IgG that could engage with F c receptors 181 (Figure S3), which might be due to robust induction of T follicular helper cells that facilitate B cell 182 maturation and class switching [21]. This distinction was most notable for F c γRIIa and F c γRIIb (2 183 weeks median F c γRIIa MFI: FL = 0 vs. mRNA = 128, p=0.02, Kruskal-Wallis + post hoc Mann-184 Whitney U test). At peak immunogenicity (10 weeks), gB-specific antibody engagement of F c γ 185 receptors was similar between vaccine groups. However, at the time of necropsy we generally 186 noted enhanced median binding to F c γR's among gB mRNA-vaccinated rabbits vs. gB FL, though   254 though the precise mechanism remains unknown. Lastly, HCMV-specific CD4 +   276 Importantly, while HCMV gB mRNA vaccines have been tested previously in a preclinical model 277 [48], this is the first such investigation to test a gB mRNA-based vaccine alongside the partially-278 effective gB/MF59 protein subunit vaccination and to directly compare the epitope specificity, 279 durability, and neutralizing/non-neutralizing function of antibodies elicited by these two vaccine 280 platforms.

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In this investigation, we specifically focused on the implications of the gB/MF59-elicited 282 immune-dominant response directed against gB AD-3 [25]. We hypothesized the absence of 283 neutralizing antibodies in gB/MF59 vaccinees may be attributable to the dominant responses 284 against the AD-3 'decoy epitope', which diverted antibody targeting away from 'more functional' 285 epitopes [24]. We directly tested this hypothesis with our gB ecto group by excluding AD-3 from 286 the immunogen. Intriguingly, we failed to see any consistent increase in the magnitude of 287 functional neutralizing/non-neutralizing antibodies in gB ecto vaccinated rabbits, suggesting that 288 inclusion of the AD-3 epitope in the vaccine immunogen does not hinder the development of more 289 functional antibodies. Is it therefore possible that AD-3 directed antibodies have any functional or 290 protective role that might account for the 50% vaccine efficacy observed in gB/MF59 vaccinees?
291 In this study we noted that AD-3-specific antibodies can mediate non-neutralizing antibody 292 effector functions including whole virion phagocytosis (Figure 6). Consequently, the high-293 magnitude AD-3 response in gB FL-vaccinated rabbits likely accounts for our observation of 294 robust and durable phagocytosis activity in these animals ( Figure 5E).

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A limitation to this study is dissimilarity in vaccine dose and route of delivery between 300 Depending on the antigen and vaccine formulation, intradermal vaccination may be as much as 301 10 times more potent than intramuscular dosing [50]. Therefore, we cannot rule out the possibility 302 that our results of enhanced breadth and durability in gB mRNA-immunized rabbits are 303 dose/method dependent -that a higher dose or different delivery method of gB FL protein might 304 not have achieved similar results to gB mRNA-LNP vaccinated rabbits. Furthermore, while rabbits 305 provide an excellent model to study vaccine immunogenicity, this investigation was restricted by 306 the rabbit immunologic toolbox. We were able to measure F c γ receptor engagement in this study, 307 though lacked the ability to identify the mechanism behind variable F c γ receptor engagement (e.g. 308 IgG subclass or F c glycosylation). Furthermore, while we were able to identify gB-specific T cells 309 in spleen, we were unable to: 1) identify antigen-specific T cells in peripheral blood, and 2) parse 310 out T cell subsets (CD4 + , CD8 + , etc). Nevertheless, the results described are sufficient justification