Neutralizing antibodies (NAb) interfering with glycoprotein complex-mediated virus entry into host cells are thought to contribute to the protection against herpesvirus infection. However, using herpesvirus glycoprotein complexes as vaccine antigens can be complicated by the necessity of expressing multiple subunits simultaneously to allow efficient complex assembly and formation of conformational NAb epitopes. By using a novel bacterial artificial chromosome (BAC) clone of the clinically deployable Modified Vaccinia Ankara (MVA) vector and exploiting ribosomal skipping mediated by 2A peptides, MVA vectors were generated that expressed self-processing subunits of the human cytomegalovirus (HCMV) pentamer complex (PC) composed of gH, gL, UL128, UL130, and UL131A. These MVA vectors expressed 2A-linked HCMV PC subunits that were efficiently cleaved and transported to the cell surface as protein complexes forming conformational neutralizing epitopes. In addition, vaccination of mice by only two immunizations with these MVA vectors resulted in potent HCMV NAb responses that remained stable over a period of at least six months. This method of eliciting NAb by 2A-linked, self-processing HCMV PC subunits could contribute to develop a HCMV vaccine candidate and may serve as a template to facilitate the development of subunit vaccine strategies against other herpesviruses.

As human cytomegalovirus (HCMV) is the most common infectious cause of fetal anomalies during pregnancy, development of a vaccine that prevents HCMV infection is considered a global health priority. Although HCMV immune correlates of protection are only poorly defined, neutralizing antibodies (NAb) targeting the envelope pentamer complex (PC) composed of the subunits gH, gL, UL128, UL130, and UL131A are thought to contribute to the prevention of HCMV infection. Here, we describe a continuous target sequence within UL128 that is recognized by a previously isolated potent PC-specific NAb termed 13B5. By using peptide-based scanning procedures, we identified a 13-amino-acid-long target sequence at the UL128 C terminus that binds the 13B5 antibody with an affinity similar to that of the purified PC. In addition, the 13B5 binding site is universally conserved in HCMV, contains a previously described UL128/gL interaction site, and interferes with the 13B5 neutralizing function, indicating that the 13B5 epitope sequence is located within the PC at a site of critical importance for HCMV neutralization. Vaccination of mice with peptides containing the 13B5 target sequence resulted in the robust stimulation of binding antibodies and, in a subset of immunized animals, in the induction of detectable NAb, supporting that the identified 13B5 target sequence constitutes a PC-specific neutralizing epitope. These findings provide evidence for the discovery of a continuous neutralizing epitope within the UL128 subunit of the PC that could be an important target of humoral immune responses that are involved in protection against congenital HCMV infection.IMPORTANCE Neutralizing antibodies (NAb) targeting the human cytomegalovirus (HCMV) envelope pentamer complex (PC) are thought to be important for preventing HCMV transmission from the mother to the fetus, thereby mitigating severe developmental disabilities in newborns. However, the epitope sequences within the PC that are recognized by these potentially protective antibody responses are only poorly defined. Here, we provide evidence for the existence of a highly conserved, continuous, PC-specific epitope sequence that appears to be located within the PC at a subunit interaction site of critical importance for HCMV neutralization. These discoveries provide insights into a continuous PC-specific neutralizing epitope, which could be an important target for a vaccine formulation to interfere with congenital HCMV infection.

Since neutralizing antibodies (NAb) targeting the human cytomegalovirus (HCMV) pentamer complex (PC) potently block HCMV host cell entry, anti-PC NAb induction is thought to be important for a vaccine formulation to prevent HCMV infection. By developing a vaccine strategy based on soluble PC protein and using a previously generated Modified Vaccinia Ankara vector co-expressing all five PC subunits (MVA-PC), we compared HCMV NAb induction by homologous immunization using prime-boost vaccine regimen employing only PC protein or MVA-PC and heterologous immunization using prime-boost combinations of PC protein and MVA-PC. Utilizing a recently isolated anti-PC NAb, we produced highly pure soluble PC protein that displayed conformational and linear neutralizing epitopes, interfered with HCMV entry, and was recognized by antibodies induced by HCMV during natural infection. Mice vaccinated by different immunization routes with the purified PC protein in combination with a clinically approved adjuvant formulation elicited high-titer and durable HCMV NAb. While MVA-PC and soluble PC protein either alone or in combination elicited robust HCMV NAb, significantly different potencies of these vaccine approaches were observed in dependence on immunization schedule. Using only two immunizations, vaccination with MVA-PC alone or prime-boost combinations of MVA-PC and PC protein was significantly more effective in stimulating HCMV NAb than immunization with PC protein alone. In contrast, with three immunizations, NAb induced by soluble PC protein either alone or combined with two boosts of MVA-PC increased to levels that exceeded NAb titer stimulated by MVA-PC alone. These results provide insights into the potency of soluble protein and MVA to elicit NAb by the HCMV PC via homologous and heterologous prime-boost immunization, which may contribute to develop clinically deployable vaccine strategies to prevent HCMV infection.

The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes. DOI:http://dx.doi.org/10.7554/eLife.00444.001.

Glycogen and lipids are major storage forms of energy that are tightly regulated by hormones and metabolic signals. We demonstrate that feeding mice a high-fat diet (HFD) increases hepatic glycogen due to increased expression of the glycogenic scaffolding protein PTG/R5. PTG promoter activity was increased and glycogen levels were augmented in mice and cells after activation of the mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target SREBP1. Deletion of the PTG gene in mice prevented HFD-induced hepatic glycogen accumulation. Of note, PTG deletion also blocked hepatic steatosis in HFD-fed mice and reduced the expression of numerous lipogenic genes. Additionally, PTG deletion reduced fasting glucose and insulin levels in obese mice while improving insulin sensitivity, a result of reduced hepatic glucose output. This metabolic crosstalk was due to decreased mTORC1 and SREBP activity in PTG knockout mice or knockdown cells, suggesting a positive feedback loop in which once accumulated, glycogen stimulates the mTORC1/SREBP1 pathway to shift energy storage to lipogenesis. Together, these data reveal a previously unappreciated broad role for glycogen in the control of energy homeostasis.