Human Cytomegalovirus mRNA-1647 Vaccine Candidate Elicits Potent and Broad Neutralization and Higher Antibody-Dependent Cellular Cytotoxicity Responses Than the gB/MF59 Vaccine.

BACKGROUND: MF59-adjuvanted gB subunit (gB/MF59) vaccine demonstrated approximately 50% efficacy against human cytomegalovirus (HCMV) acquisition in multiple clinical trials, suggesting that efforts to improve this vaccine design might yield a vaccine suitable for licensure. METHODS: A messenger RNA (mRNA)-based vaccine candidate encoding HCMV gB and pentameric complex (PC), mRNA-1647, is currently in late-stage efficacy trials. However, its immunogenicity has not been compared to the partially effective gB/MF59 vaccine. We assessed neutralizing and Fc-mediated immunoglobulin G (IgG) effector antibody responses induced by mRNA-1647 in both HCMV-seropositive and -seronegative vaccinees from a first-in-human clinical trial through 1 year following third vaccination using a systems serology approach. Furthermore, we compared peak anti-gB antibody responses in seronegative mRNA-1647 vaccinees to that of seronegative gB/MF59 vaccine recipients. RESULTS: mRNA-1647 vaccination elicited and boosted HCMV-specific IgG responses in seronegative and seropositive vaccinees, respectively, including neutralizing and Fc-mediated effector antibody responses. gB-specific IgG responses were lower than PC-specific IgG responses. gB-specific IgG and antibody-dependent cellular phagocytosis responses were lower than those elicited by gB/MF59. However, mRNA-1647 elicited higher neutralization and antibody-dependent cellular cytotoxicity (ADCC) responses. CONCLUSIONS: Overall, mRNA-1647 vaccination induced polyfunctional and durable HCMV-specific antibody responses, with lower gB-specific IgG responses but higher neutralization and ADCC responses compared to the gB/MF59 vaccine. CLINICAL TRIALS REGISTRATION: NCT03382405 (mRNA-1647) and NCT00133497 (gB/MF59).

Vaccination with a replication-defective cytomegalovirus vaccine elicits a glycoprotein B-specific monoclonal antibody repertoire distinct from natural infection.

Human Cytomegalovirus (HCMV) is the leading infectious congenital infection globally and the most common viral infection in transplant recipients, therefore identifying a vaccine for HCMV is a top priority. Humoral immunity is a correlate of protection for HCMV infection. The most effective vaccine tested to date, which achieved 50% reduction in acquisition of HCMV, was comprised of the glycoprotein B protein given with an oil-in-water emulsion adjuvant MF59. We characterize gB-specific monoclonal antibodies isolated from individuals vaccinated with a disabled infectious single cycle (DISC) CMV vaccine, V160, and compare these to the gB-specific monoclonal antibody repertoire isolated from naturally-infected individuals. We find that vaccination with V160 resulted in gB-specific antibodies that bound homogenously to gB expressed on the surface of a cell in contrast to antibodies isolated from natural infection which variably bound to cell-associated gB. Vaccination resulted in a similar breadth of gB-specific antibodies, with binding profile to gB genotypes 1-5 comparable to that of natural infection. Few gB-specific neutralizing antibodies were isolated from V160 vaccinees and fewer antibodies had identifiable gB antigenic domain specificity compared to that of naturally-infected individuals. We also show that glycosylation of gB residue N73 may shield binding of gB-specific antibodies.

The pentameric complex is not required for vertical transmission of cytomegalovirus in seronegative pregnant rhesus macaques.

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neonatal neurological impairment but essential virological determinants of transplacental CMV transmission remain unclear. The pentameric complex (PC), composed of five subunits, glycoproteins H (gH), gL, UL128, UL130, and UL131A, is essential for efficient entry into non-fibroblast cells in vitro . Based on this role in cell tropism, the PC is considered a possible target for CMV vaccines and immunotherapies to prevent cCMV. To determine the role of the PC in transplacental CMV transmission in a non-human primate model of cCMV, we constructed a PC-deficient rhesus CMV (RhCMV) by deleting the homologues of the HCMV PC subunits UL128 and UL130 and compared congenital transmission to PC-intact RhCMV in CD4+ T cell-depleted or immunocompetent RhCMV-seronegative, pregnant rhesus macaques (RM). Surprisingly, we found that the transplacental transmission rate was similar for PC-intact and PC-deleted RhCMV based on viral genomic DNA detection in amniotic fluid. Moreover, PC-deleted and PC-intact RhCMV acute infection led to similar peak maternal plasma viremia. However, there was less viral shedding in maternal urine and saliva and less viral dissemination in fetal tissues in the PC-deleted group. As expected, dams inoculated with PC-deleted RhCMV demonstrated lower plasma IgG binding to PC-intact RhCMV virions and soluble PC, as well as reduced neutralization of PC-dependent entry of the PC-intact RhCMV isolate UCD52 into epithelial cells. In contrast, binding to gH expressed on the cell surface and neutralization of entry into fibroblasts by the PC-intact RhCMV was higher for dams infected with PC-deleted RhCMV compared to those infected with PC-intact RhCMV. Our data demonstrates that the PC is dispensable for transplacental CMV infection in our non-human primate model. One Sentence Summary: Congenital CMV transmission frequency in seronegative rhesus macaques is not affected by the deletion of the viral pentameric complex.

A single, improbable B cell receptor mutation confers potent neutralization against cytomegalovirus.

Cytomegalovirus (CMV) is a leading cause of infant hearing loss and neurodevelopmental delay, but there are no clinically licensed vaccines to prevent infection, in part due to challenges eliciting neutralizing antibodies. One of the most well-studied targets for CMV vaccines is the viral fusogen glycoprotein B (gB), which is required for viral entry into host cells. Within gB, antigenic domain 2 site 1 (AD-2S1) is a target of potently neutralizing antibodies, but gB-based candidate vaccines have yet to elicit robust responses against this region. We mapped the genealogy of B cells encoding potently neutralizing anti-gB AD-2S1 antibodies from their inferred unmutated common ancestor (UCA) and characterized the binding and function of early lineage ancestors. Surprisingly, we found that a single amino acid heavy chain mutation A33N, which was an improbable mutation rarely generated by somatic hypermutation machinery, conferred broad CMV neutralization to the non-neutralizing UCA antibody. Structural studies revealed that this mutation mediated key contacts with the gB AD-2S1 epitope. Collectively, these results provide insight into potently neutralizing gB-directed antibody evolution in a single donor and lay a foundation for using this B cell-lineage directed approach for the design of next-generation CMV vaccines.

Characterization of Plasma Immunoglobulin G Responses in Elite Neutralizers of Human Cytomegalovirus.

BACKGROUND: Human cytomegalovirus (HCMV) is the most common infectious complication of organ transplantation and cause of birth defects worldwide. There are limited therapeutic options and no licensed vaccine to prevent HCMV infection or disease. To inform development of HCMV antibody-based interventions, a previous study identified individuals with potent and broad plasma HCMV-neutralizing activity, termed elite neutralizers (ENs), from a cohort of HCMV-seropositive (SP) blood donors. However, the specificities and functions of plasma antibodies associated with EN status remained undefined. METHODS: We sought to determine the plasma antibody specificities, breadth, and Fc-mediated antibody effector functions associated with the most potent HCMV-neutralizing responses in plasma from ENs (n = 25) relative to that from SP donors (n = 19). We measured antibody binding against various HCMV strains and glycoprotein targets and evaluated Fc-mediated effector functions, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). RESULTS: We demonstrate that ENs have elevated immunoglobulin G binding responses against multiple viral glycoproteins, relative to SP donors. Our study also revealed potent HCMV-specific antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis activity of plasma from ENs. CONCLUSIONS: We conclude that antibody responses against multiple glycoprotein specificities may be needed to achieve potent plasma neutralization and that potently HCMV elite-neutralizing plasma antibodies can also mediate polyfunctional responses.

Common Polymorphisms in the Glycoproteins of Human Cytomegalovirus and Associated Strain-Specific Immunity.

Human cytomegalovirus (HCMV), one of the most prevalent viruses across the globe, is a common cause of morbidity and mortality for immunocompromised individuals. Recent clinical observations have demonstrated that mixed strain infections are common and may lead to more severe disease progression. This clinical observation illustrates the complexity of the HCMV genome and emphasizes the importance of taking a population-level view of genotypic evolution. Here we review frequently sampled polymorphisms in the glycoproteins of HCMV, comparing the variable regions, and summarizing their corresponding geographic distributions observed to date. The related strain-specific immunity, including neutralization activity and antigen-specific cellular immunity, is also discussed. Given that these glycoproteins are common targets for vaccine design and anti-viral therapies, this observed genetic variation represents an important resource for future efforts to combat HCMV infections.

Improvement of RG1-VLP vaccine performance in BALB/c mice by substitution of alhydrogel with the next generation polyphosphazene adjuvant PCEP.

Current human papillomavirus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain for which vaccine coverage is lacking. In addition, all current HPV vaccines rely on aluminum salt-based adjuvant formulations that function through unclear mechanisms with few substitutes available. In an effort to expand the toolbox of available adjuvants suitable for HPV vaccines, we compared the immunogenicity of the RG1-VLP (virus-like particle) vaccine in BALB/c mice when formulated with either the aluminum hydroxide adjuvant Alhydrogel or the novel polyphosphazene macromolecular adjuvant poly[di (carboxylatoethylphenoxy) phosphazene] (PCEP). PCEP-formulated RG1-VLPs routinely outperformed VLP/Alhydrogel in several measurements of VLP-specific humoral immunity, including consistent improvements in the magnitude of antibody (Ab) responses to both HPV16-L1 and the L2 RG1 epitope as well as neutralizing titers to HPV16 and cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39. Dose-sparing studies indicated that RG1-VLPs could be reduced in dose by 75% and the presence of PCEP ensured activity comparable to a full VLP dose adjuvanted by Alhydrogel. In addition, levels of HPV16-L1 and -L2-specific Abs were achieved after two vaccinations with PCEP as adjuvant that were equivalent to or greater than levels achieved with three vaccinations with Alhydrogel alone, indicating that the presence of PCEP resulted in accelerated immune responses that could allow for a decreased dose schedule. Given the extensive clinical track record of polyphosphazenes, these data suggest that substitution of alum-based adjuvants with PCEP for the RG1-VLP vaccine could lead to rapid seropositivity requiring fewer boosts, the dose-sparing of commercial VLP-based vaccines, and the establishment of longer-lasting humoral responses to HPV.

Next generation polyphosphazene immunoadjuvant: Synthesis, self-assembly and in vivo potency with human papillomavirus VLPs-based vaccine.

Poly[di(carboxylatomethylphenoxy)phosphazene] (PCMP), a new member of polyphosphazene immunoadjuvant family, is synthesized. In vitro assessment of a new macromolecule revealed hydrolytic degradation profile and immunostimulatory activity comparable to its clinical stage homologue PCPP; however, PCMP was characterized by a beneficial reduced sensitivity to the ionic environment. In vivo evaluation of PCMP potency was conducted with human papillomavirus (HPV) virus-like particles (VLPs) based RG1-VLPs vaccine. In contrast with previously reported self-assembly of polyphosphazene adjuvants with proteins, which typically results in the formation of complexes with multimeric display of antigens, PCMP surface modified VLPs in a composition dependent pattern, which at a high polymer-to VLPs ratio led to stabilization of antigenic particles. Immunization experiments in mice demonstrated that PCMP adjuvanted RG1-VLPs vaccine induced potent humoral immune responses, in particular, on the level of highly desirable protective cross-neutralizing antibodies, and outperformed PCPP and Alhydrogel adjuvanted formulations.

Optimization of RG1-VLP vaccine performance in mice with novel TLR4 agonists.

Current human papilloma virus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain that lack vaccine coverage. The novel RG1-VLP (virus-like particle) vaccine candidate utilizes the HPV16-L1 subunit as a backbone to display an inserted HPV16-L2 17-36 a.a. "RG1" epitope; the L2 RG1 epitope is conserved across many HPV types and the generation of cross-neutralizing antibodies (Abs) against which has been demonstrated. In an effort to heighten the immunogenicity of the RG1-VLP vaccine, we compared in BALB/c mice adjuvant formulations consisting of novel bacterial enzymatic combinatorial chemistry (BECC)-derived toll-like receptor 4 (TLR4) agonists and the aluminum hydroxide adjuvant Alhydrogel. In the presence of BECC molecules, consistent improvements in the magnitude of Ab responses to both HPV16-L1 and the L2 RG1 epitope were observed compared to Alhydrogel alone. Furthermore, neutralizing titers to HPV16 as well as cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39 were augmented in the presence of BECC agonists as well. Levels of L1 and L2-specific Abs were achieved after two vaccinations with BECC/Alhydrogel adjuvant that were equivalent to or greater than levels achieved with 3 vaccinations with Alhydrogel alone, indicating that the presence of BECC molecules resulted in accelerated immune responses that could allow for a decreased dose schedule for VLP-based HPV vaccines. In addition, dose-sparing studies indicated that adjuvantation with BECC/Alhydrogel allowed for a 75% reduction in antigen dose while still retaining equivalent magnitudes of responses to the full VLP dose with Alhydrogel. These data suggest that adjuvant optimization of HPV VLP-based vaccines can lead to rapid immunity requiring fewer boosts, dose-sparing of VLPs expensive to produce, and the establishment of a longer-lasting humoral immunity.

Vaccines for Perinatal and Congenital Infections-How Close Are We?

Congenital and perinatal infections are transmitted from mother to infant during pregnancy across the placenta or during delivery. These infections not only cause pregnancy complications and still birth, but also result in an array of pediatric morbidities caused by physical deformities, neurodevelopmental delays, and impaired vision, mobility and hearing. Due to the burden of these conditions, congenital and perinatal infections may result in lifelong disability and profoundly impact an individual's ability to live to their fullest capacity. While there are vaccines to prevent congenital and perinatal rubella, varicella, and hepatitis B infections, many more are currently in development at various stages of progress. The spectrum of our efforts to understand and address these infections includes observational studies of natural history of disease, epidemiological evaluation of risk factors, immunogen design, preclinical research of protective immunity in animal models, and evaluation of promising candidates in vaccine trials. In this review we summarize this progress in vaccine development research for Cytomegalovirus, Group B Streptococcus, Herpes simplex virus, Human Immunodeficiency Virus, Toxoplasma, Syphilis, and Zika virus congenital and perinatal infections. We then synthesize this evidence to examine how close we are to developing a vaccine for these infections, and highlight areas where research is still needed.

The BDLF3 gene product of Epstein-Barr virus, gp150, mediates non-productive binding to heparan sulfate on epithelial cells and only the binding domain of CD21 is required for infection.

The cell surface molecules used by Epstein-Barr virus (EBV) to attach to epithelial cells are not well-defined, although when CD21, the B cell receptor for EBV is expressed epithelial cell infection increases disproportionately to the increase in virus bound. Many herpesviruses use low affinity charge interactions with molecules such as heparan sulfate to attach to cells. We report here that the EBV glycoprotein gp150 binds to heparan sulfate proteoglycans, but that attachment via this glycoprotein is not productive of infection. We also report that only the aminoterminal two short consensus repeats of CD21 are required for efficient infection, This supports the hypothesis that, when expressed on an epithelial cell CD21 serves primarily to cluster the major attachment protein gp350 in the virus membrane and enhance access of other important glycoproteins to the epithelial cell surface.

Important but differential roles for actin in trafficking of Epstein-Barr virus in B cells and epithelial cells.

Epstein-Barr virus (EBV) uses different virus and cell proteins to enter its two major targets, B lymphocytes and epithelial cells. The routes that the virus takes into the two cell types are also different. To determine if these differences extend to movement from the cell surface to the nucleus, we examined the fate of incoming virus. Essentially all virus that entered a B cell remained stable for at least 8 h. In contrast, up to 80% of virus entering an epithelial cell was degraded in a compartment sensitive to inhibitors of components involved in autophagy. Inhibitors of actin remodeling blocked entry into a B cell but had no effect or enhanced entry into an epithelial cell. Inhibitors of the microtubule network reduced intracellular transport in both cell types, but movement to the nucleus in an epithelial cell also required involvement of the actin cytoskeleton. Deletion of the cytoplasmic tail of CR2, which in an epithelial cell interacts with the actin nucleator FHOS/FHOD when cross-linked by EBV, had no effect on infection. However, inhibitors of downstream signaling by integrins reduced intracellular transport. Cooperation of the microtubule and actin cytoskeletons, possibly activated by interaction with integrin binding proteins in the envelope of EBV, is needed for successful infection of an epithelial cell.