Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.Human cytomegalovirus (HCMV) is a member of the β-herpesvirus subfamily with >60% seropositivity in adults worldwide (1). HCMV infection is typically asymptomatic, but can cause severe disease or death in immunocompromised solid organ and hematopoietic stem cell transplant recipients. In addition, HCMV can infect the placenta and cross this barrier to infect developing fetuses, causing severe birth defects (2). Given the severity and importance of this disease, obtaining an effective vaccine is considered a public health priority (3).The ability of HCMV to cause disease in a wide range of organs and tissue types is reflected at the cellular level by the virus infecting epithelial cells, endothelial cells, fibroblasts, dendritic cells, hepatocytes, neurons, macrophages, and leukocytes (4). Similar to other herpesviruses, the envelope glycoproteins gB and gH/gL form the conserved fusion machinery required for viral entry (5, 6). Recent structural and mutagenesis analysis suggested that gB is responsible for mediating virus and host membrane fusion during viral entry (7, 8). The role of gH/gL in fusion is less clear because crystal structures of herpes simplex virus 2 (HSV-2), pseudo-rabies virus (PrV), and Epstein–Barr virus (EBV) gH/gL did not reveal any similarity to known viral fusion proteins (9–11). It has been proposed that gH/gL is involved in the entry process through activation of gB (12). In addition to gB and gH/gL, most herpesviruses encode additional glycoproteins that are able to interact with gH/gL and are capable of either mediating binding to specific cellular receptors or regulating the activity of the gH/gL–gB complex (5, 6).HCMV entry into both epithelial and endothelial cells requires a pentameric glycoprotein complex (Pentamer) formed between gH/gL and the UL128, UL130, and UL131A proteins (13, 14). Mutations in the UL131A–UL128 gene locus are sufficient to eliminate epithelial/endothelial tropism and occur spontaneously within only a few passages of wild-type (WT) HCMV in fibroblasts (15, 16). In addition, Pentamer cell surface overexpression interferes with HCMV entry into epithelial cells, but not into fibroblasts, suggesting the presence of a cell-type-specific Pentamer receptor (17).HCMV entry into fibroblasts is mediated by the gH/gL/gO complex at the cell surface at neutral pH (18–21). gO is a highly glycosylated protein and has been shown to covalently interact with gH/gL (22, 23). It has been proposed that gO might function as a molecular chaperone to promote gH/gL incorporation, but not gH/gL/gO, into the virion (21). However, it has been recently demonstrated that gH/gL/gO and Pentamer are much more abundant on the HCMV envelope than gH/gL alone (24).Highly potent HCMV-neutralizing monoclonal antibodies were isolated from the memory B-cell repertoire of HCMV-immune donors and shown to bind the Pentamer. These antibodies were capable of neutralizing HCMV infection of epithelial/endothelial cells, but not fibroblasts (25, 26). In addition, several studies have demonstrated that the Pentamer is the main target of the neutralizing humoral response to HCMV infection in epithelial/endothelial cells (27–29). Consistent with these observations, immunization with the Pentamer has been shown to elicit a strong neutralizing antibody response in mouse, rabbit, and rhesus macaque models (30–32). Together these data indicate that the Pentamer represents a key antigenic target for vaccine development against HCMV infection.Here we report the purification and biochemical characterization of HCMV gH/gL, gH/gL/gO, and Pentamer. In addition, we describe the architecture of these complexes by electron microscopy (EM) and characterize their interaction with MSL-109, a previously described HCMV-neutralizing antibody isolated from the spleen of a HCMV-seropositive individual (33, 34). Our data provide new insights into the structure and function of the HCMV gH/gL/gO and Pentamer complexes.     

A viral regulator of glycoprotein complexes contributes to human cytomegalovirus cell tropism

Viral glycoproteins mediate entry of enveloped viruses into cells and thus play crucial roles in infection. In herpesviruses, a complex of two viral glycoproteins, gH and gL (gH/gL), regulates membrane fusion events and influences virion cell tropism. Human cytomegalovirus (HCMV) gH/gL can be incorporated into two different protein complexes: a glycoprotein O (gO)-containing complex known as gH/gL/gO, and a complex containing UL128, UL130, and UL131 known as gH/gL/UL128-131. Variability in the relative abundance of the complexes in the virion envelope correlates with differences in cell tropism exhibited between strains of HCMV. Nonetheless, the mechanisms underlying such variability have remained unclear. We have identified a viral protein encoded by the UL148 ORF (UL148) that influences the ratio of gH/gL/gO to gH/gL/UL128-131 and the cell tropism of HCMV virions. A mutant disrupted for UL148 showed defects in gH/gL/gO maturation and enhanced infectivity for epithelial cells. Accordingly, reintroduction of UL148 into an HCMV strain that lacked the gene resulted in decreased levels of gH/gL/UL128-131 on virions and, correspondingly, decreased infectivity for epithelial cells. UL148 localized to the endoplasmic reticulum, but not to the cytoplasmic sites of virion envelopment. Coimmunoprecipitation results indicated that gH, gL, UL130, and UL131 associate with UL148, but that gO and UL128 do not. Taken together, the findings suggest that UL148 modulates HCMV tropism by regulating the composition of alternative gH/gL complexes.The lipid bilayer membranes of living cells pose an existential challenge to viruses. In enveloped viruses, viral glycoproteins execute a highly regulated fusion event between virion and cellular membranes, thereby delivering the viral genome and other contents of the virion into the host cell. Antibody responses that block entry are considered neutralizing and represent an important host defense against viral pathogens.In many enveloped viruses, one or two viral glycoproteins suffice to carry out binding and membrane fusion events that mediate entry. In herpesviruses, however, at least four envelope glycoproteins are typically involved. The core machinery for herpesvirus entry comprises three highly conserved viral glycoproteins, glycoprotein B (gB), glycoprotein H (gH), and glycoprotein L (gL), along with one or more accessory glycoproteins necessary for binding to cell surface receptors (reviewed in refs. 1, 2). gB is thought to be the proximal mediator of membrane fusion, whereas gH and gL form a complex, termed gH/gL, which has been found to regulate the fusogenic activity of gB (3–6). In a number of beta and gamma herpesviruses, including the human pathogens human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6), and Epstein–Barr virus (EBV), two different gH/gL complexes are found on the virion envelope and are necessary for the viruses to enter the full range of cell types that they infect in vivo.Of the two gH/gL complexes expressed in HCMV virions, the gH/gL complex with glycoprotein O (gO), gH/gL/gO, suffices for entry into fibroblasts, a cell type in which fusion events at the plasma membrane initiate infection (7). Infection of several other types of cells, including monocytes, dendritic cells, endothelial cells, and epithelial cells, requires the pentameric complex of gH/gL and three small glycoproteins—UL128, UL130, and UL131 (UL128-131)—and appears to involve fusion at endosomal membranes (8–16). Strains of HCMV, such as AD169 and Towne, that have undergone extensive serial passage in cultured fibroblasts fail to express the pentameric gH/gL/UL128-131 complex on virions and thus are unable to infect epithelial and endothelial cells (12, 13, 15); however, repair of a frameshift mutation in the UL131 gene of strain AD169 restores expression of gH/gL/UL128-131 (11, 12) and expands its cell tropism.Less extensively passaged HCMV strains that retain expression of gH/gL/UL128-131 can efficiently infect epithelial and endothelial cells (13, 17, 18). Nonetheless, several such strains replicate to ∼1,000-fold lower titers on epithelial cells compared with strain AD169 repaired for UL131 (11). AD169 lacks a ∼15-kb region at the end of the unique long genome region, termed the ULb′ (19). We were intrigued by the rather striking differences in cell tropism between laboratory strain AD169 repaired for expression of the pentameric gH/gL/UL128-131 complex, and strains, such as TB40/E, that have largely intact ULb′ regions and maintain expression of gH/gL/UL128-131. We hypothesized the ULb′ region encodes an additional factor involved in HCMV cell tropism. Our studies addressing this hypothesis led us to identify a new function for UL148, a gene within the ULb′. We found that UL148 encodes an endoplasmic reticulum (ER) resident glycoprotein that influences virion cell tropism by regulating the composition of alternative gH/gL complexes.     

Antibody-driven design of a human cytomegalovirus gHgLpUL128L subunit vaccine that selectively elicits potent neutralizing antibodies

The use of neutralizing antibodies to identify the most effective antigen has been proposed as a strategy to design vaccines capable of eliciting protective B-cell immunity. In this study, we analyzed the human antibody response to cytomegalovirus (human cytomegalovirus, HCMV) infection and found that antibodies to glycoprotein (g)B, a surface glycoprotein that has been developed as a HCMV vaccine, were primarily nonneutralizing. In contrast, most of the antibodies to the complex formed by gH, gL, protein (p)UL128, pUL130, and pUL131 (the gHgLpUL128L pentamer) neutralized HCMV infection with high potency. Based on this analysis, we developed a single polycistronic vector encoding the five pentamer genes separated by “self-cleaving” 2A peptides to generate a stably transfected CHO cell line constitutively secreting high levels of recombinant pentamer that displayed the functional antigenic sites targeted by human neutralizing antibodies. Immunization of mice with the pentamer formulated with different adjuvants elicited HCMV neutralizing antibody titers that persisted to high levels over time and that were a hundred- to thousand-fold higher than those found in individuals that recovered from primary HCMV infection. Sera from mice immunized with the pentamer vaccine neutralized infection of both epithelial cells and fibroblasts and prevented cell-to-cell spread and viral dissemination from endothelial cells to leukocytes. Neutralizing monoclonal antibodies from immunized mice showed the same potency as human antibodies and targeted the same as well as additional sites on the pentamer. These results illustrate with a relevant example a general and practical approach of analytic vaccinology for the development of subunit vaccines against complex pathogens.Human cytomegalovirus (HCMV) is a ubiquitously distributed member of the Herpesviridae family that establishes a lifelong infection and represents a major threat for human health. Primary infection during pregnancy is the most frequent cause of congenital birth defects, with an overall 0.6% incidence, whereas severe infections develop in immunocompromised patients (1, 2). In addition, HCMV has been proposed as an agent associated with immune senescence (3) and atherosclerosis (4).HCMV has a broad cell tropism and exploits multiple glycoprotein complexes present on the virion envelope for binding and fusion with host cells. Some glycoproteins (g), such as gM/gN and gB, are used to infect several cell types, whereas glycoprotein complexes containing gH and gL mediate cell type-specific virus entry (5, 6). A pentameric complex comprising gH, gL, protein (p)UL128, pUL130, and pUL131 [gHgLpUL128locus (L)] was shown to be required by clinical HCMV isolates to infect endothelial, epithelial, and myeloid cells (7–10). In vitro cultured HCMV viruses with mutations in the UL128–131 locus lose tropism for endothelial and epithelial cells but retain the expression of the gHgL-containing complex, which is sufficient to infect fibroblasts (11).Because of the high incidence rate of HCMV infections and its impact on public health, considerable efforts have been made in the last decade to develop treatments or vaccines capable of preventing HCMV infection (12). The major target populations for a HCMV vaccine are seronegative women of childbearing age, whereas infants represent another potential population contributing to viral dissemination (13). In addition, patients on a list for organ transplantation (especially those with HCMV-seronegative who are at risk for life-threatening HCMV disease) would benefit from a HCMV vaccine. The administration of the HCMV-attenuated Towne vaccine prevented the development of disease in kidney transplant recipients, although it did not prevent infection (14).The abundant virion protein gB was shown to elicit vigorous T-cell and antibody responses and represents the basis of most vaccines developed so far (15). However, in recent phase II trials, a MF59-adjuvanted gB vaccine showed modest efficacy in preventing infection (16) and reducing duration of viremia in transplant recipients (17). These findings may be explained by the finding that most antibodies induced by the vaccines lack virus-neutralizing activity (18), whereas those that neutralized did not block efficiently infection of epithelial cells (19). Therefore, a HCMV vaccine capable of eliciting neutralizing antibodies that prevent the infection of multiple cellular targets and block viral dissemination is considered a high priority (20).Passively administered polyclonal antibodies isolated from seropositive donors were suggested to be effective in preventing infection of the fetus (21). These findings were not confirmed in a recent randomized study where the same antibody preparation showed a modest, not significant, effect on the rate of congenital HCMV infection, possibly due to the low level of neutralizing antibodies contained in Ig preparation (22).We previously isolated from HCMV immune donors antibodies that bound to conformational epitopes on the gHgLpUL128L pentameric complex and were extraordinarily potent in neutralizing HCMV infection of epithelial, endothelial, and myeloid cells (23). The pentamer-specific antibodies neutralized viral infection at picomolar concentrations and were a thousand-fold more potent than antibodies to gB, gH, or gMgN complex (23). More recently, we showed that an early antibody response to the pentamer was associated with lack of viral transmission to the fetus from HCMV-infected pregnant mothers, suggesting that pentamer-specific antibodies are responsible for the inhibition of viral spread in vivo (24).In this study, we report a systematic analysis of the human antibody response to HCMV infection, which indicates that the gHgLpUL128L pentamer is the target of the most effective neutralizing antibodies. Based on this information, we developed a novel process to produce in a secreted form a recombinant pentamer vaccine from a mammalian CHO cell line stably transfected by a single polycistronic vector encoding the five different HCMV pentamer genes separated by autonomous “self-cleaving” 2A peptides. We found that this vaccine can elicit in mice titers of neutralizing antibodies 100–1,000-fold higher than those induced by natural infection. These antibodies neutralized infection of both epithelial cells and fibroblasts and prevented viral dissemination from endothelial cells to leukocytes.     

Mechanism for neutralizing activity by the anti-CMV gH/gL monoclonal antibody MSL-109

Cytomegalovirus (CMV) is a widespread opportunistic pathogen that causes birth defects when transmitted transplacentally and severe systemic illness in immunocompromised individuals. MSL-109, a human monoclonal IgG isolated from a CMV seropositive individual, binds to the essential CMV entry glycoprotein H (gH) and prevents infection of cells. Here, we suggest a mechanism for neutralization activity by MSL-109. We define a genetic basis for resistance to MSL-109 and have generated a structural model of gH that reveals the epitope of this neutralizing antibody. Using surface-based, time-resolved FRET, we demonstrate that gH/gL interacts with glycoprotein B (gB). Additionally, we detect homodimers of soluble gH/gL heterodimers and confirm this novel oligomeric assembly on full-length gH/gL expressed on the cell surface. We show that MSL-109 perturbs the dimerization of gH/gL:gH/gL, suggesting that dimerization of gH/gL may be required for infectivity. gH/gL homodimerization may be conserved between alpha- and betaherpesviruses, because both CMV and HSV gH/gL demonstrate self-association in the FRET system. This study provides evidence for a novel mechanism of action for MSL-109 and reveals a previously undescribed aspect of viral entry that may be susceptible to therapeutic intervention.Human CMV is a β-group herpesvirus that causes severe complications in immunocompromised individuals. CMV infects between 60% and 80% of the adult population worldwide (1). As with other herpesviruses, CMV establishes a lifelong latency in the host but is largely asymptomatic among infected immunocompetent individuals (2). However, during severe immunosuppression (e.g., in the setting of hematopoietic stem cell transplantation and solid organ transplantation, or advanced HIV/AIDS), CMV reactivation or primary infection can result in life-threatening disease. In addition, the acquisition of primary CMV infection during pregnancy, although of little consequence to the mother, can have severe clinical consequences in the developing fetus (3, 4). The current therapy for CMV disease is treatment with either ganciclovir or valganciclovir, which are associated with significant toxicity and not approved for use in pregnant women or for congenitally damaged infants (5). CMV hyperimmunoglobulin (CMV-HIG; pooled human IgG from CMV-positive individuals) has demonstrated efficacy in certain solid organ transplant recipients and more recently found to show limited success in protecting infants from congenital CMV disease (1, 6, 7). These findings suggest that more potent or differently targeted antibody therapy may prove to be an effective and safe alternative to the current forms of CMV therapy.Like other herpesviruses, CMV uses multiprotein entry complexes to initiate infection of host cells. Three glycoproteins, gB, gH, and gL, known as the “core fusion machinery,” are conserved in all herpesviruses and are required for entry (8, 9). gB, the most conserved of these glycoproteins, exists as a homotrimer and catalyzes membrane fusion during viral entry (9, 10). gH and gL form a heterodimer, and aid in conferring cell-type specificity to different herpesviruses, although their precise role has not been demonstrated. Recent work in herpes simplex virus-1 (HSV-1) indicates that when glycoprotein gD binds to its cellular receptor, it associates with gH/gL, and in turn gH/gL binds gB and subsequently triggers fusion as a result of a direct interaction (11, 12). These data suggest that, at least in HSV-1, gH/gL facilitates the activation and not the repression of gB. Complexes of gB:gH/gL have been detected from both HSV and CMV (13, 14). CMV lacks gD and requires the minimal complex of gB:gH/gL for entry into fibroblasts. For entry into monocytes, macrophages, epithelial cells, and endothelial cells, CMV requires the pentameric complex gH/gL/UL128/UL130/UL131 in addition to gB (10, 15–17). Although the mechanisms for formation and regulation of these multiprotein complexes have been elucidated in HSV, they are less well understood in CMV. Specifically, there remains uncertainty about the nature of these complexes, their role in infection, and how these complexes are targets of neutralization by antibodies.Recently, the crystal structure of gH was determined from HSV-2, Epstein–Barr virus (EBV), and pseudorabies virus (PRV), and although the overall protein sequence conservation is low, the core structure of gH is conserved (12, 18, 19). gH has three distinct domains: the N-terminal domain that binds gL (domain H1), the central helical domain (domain H2), and the C-terminal β-sandwich domain (domain H3) (9, 12, 20). Domain H1 is the most divergent, whereas domains H2 and H3 are more conserved and present the same fold in gH from HSV-2, EBV, and PRV (9, 12, 20). The HSV-neutralizing antibody LP11 binds H1 and prohibits the interaction of gB with gH/gL, as shown by bimolecular complementation, revealing a face of gH involved in gB binding (12, 21, 22). Interestingly, the same face of gH/gL is implicated for gB binding in EBV, albeit more N-terminal on the heterodimer, within gL (23, 24). Another anti-HSV neutralizing antibody, 52S, inhibits cell–cell fusion and binds to gH at H2/H3 border (21). Compared with the position of LP11 epitope, 52S binds to the opposite face of gH/gL, consistent with the finding that 52S does not block the association of gH/gL with gB (21). It is unclear how this antibody blocks viral entry. Domain H3 of gH is the most highly conserved and several nonfunctional mutations map to H3 in both HSV-1 and EBV (22, 25). Moreover, a potent neutralizing mAb (CL59) for EBV is directed at this domain (25). Because this is the most conserved domain of gH, one can speculate that domain H3 serves a similar function in other herpesviruses, such as CMV. Of the prevalent herpesviruses, the least is known about CMV gH/gL, which lacks a solved structure and mechanistic studies involving neutralizing antibodies.MSL-109 is a human monoclonal IgG originally isolated from spleen cells of a CMV seropositive individual. MSL-109 recognizes CMV gH complexes, and in vitro, blocks the infection of fibroblasts by laboratory and clinical strains of CMV (26). Both the mechanism of neutralization and how MSL-109 engages gH is unknown. MSL-109 has been evaluated in the clinic for the prevention of CMV infection following allogeneic hematopoietic stem cell transplantation and as adjuvant therapy for CMV retinitis in HIV-infected individuals (27, 28). Although MSL-109 did not demonstrate benefit in all-comers, analysis of a subset of the transplantation patients who were at high risk for primary CMV infection (donor-positive/recipient-negative patients) demonstrated that MSL-109 could confer protection (27). In addition, MSL-109 failed to demonstrate efficacy in HIV patients with CMV retinitis, possibly because of the immune-privileged nature of the eye (29). Ultimately, MSL-109 was not developed further. A recent study demonstrated a nongenetic viral resistance to MSL-109 in vitro and suggested that the clinical failure of MSL-109 was because of this novel mechanism of resistance (30). However, this study did not reveal the binding site or mechanism of action of MSL-109, because the mode of resistance was nongenetic.In this study, MSL-109–resistant CMV was generated primarily through passage on epithelial cells and was found to have a genetic basis. Resistance mutations were mapped onto a structural model of gH, revealing a putative epitope for MSL-109 in the H2 domain of gH. Using a surface-based, time-resolved (TR) FRET method, we demonstrate interactions between viral glycoproteins in real-time, namely that gH/gL binds to gB and gH/gL binds to gH/gL, in the form of gH/gL:gH/gL homodimers. MSL-109 binding perturbs the gH/gL:gH/gL interaction but not the gB:gH/gL interaction, suggesting that MSL-109 engages gH at the gH/gL:gH/gL interface. Moreover, introduction of an epitope tag into the MSL-109 interface allows anti-tag antibody to prevent viral infection. This study suggests a mechanism of neutralization for MSL-109 in addition to revealing a previously undescribed homophilic interaction in CMV envelope glycoproteins required for viral entry.     

Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

Herpes simplex virus (HSV) is an important human pathogen. It enters cells through an orchestrated process that requires four essential glycoproteins, gD, gH/gL, and gB, activated in cascade fashion by receptor-binding and signaling. gH/gL heterodimer is conserved across the Herpesviridae family. HSV entry is enabled by gH/gL interaction with αvβ6- or αvβ8-integrin receptors. We report that the interaction of virion gH/gL with integrins resulted in gL dissociation and its release in the medium. gL dissociation occurred if all components of the entry apparatus—receptor-bound gD and gB—were present and was prevented if entry was blocked by a neutralizing monoclonal antibody to gH or by a mutation in gH. We propose that (i) gL dissociation from gH/gL is part of the activation of HSV glycoproteins, critical for HSV entry; and (ii) gL is a functional inhibitor of gH and maintains gH in an inhibited form until receptor-bound gD and integrins signal to gH/gL.Entry of herpesviruses into the cell is an orchestrated process that necessitates a multipartite glycoprotein system, rather than one-two glycoproteins, as is the case for the vast majority of viruses (1–4). For herpes simplex virus (HSV), the entry-fusion apparatus consists of four essential glycoproteins —gD, the heterodimer gH/gL, and gB—plus cognate cellular receptors. gD is the major determinant of HSV tropism. Structurally, it exhibits an Ig-folded core with N- and C-terminal extensions (5, 6). The structure of the gH/gL heterodimer does not resemble that of any known protein (7–9). The binding site of gL in gH maps to the N-terminal domain I. Whether gH/gL has a profusion activity in itself or is only an intermediate between gD and gB in the chain of activation of the glycoproteins is an open question (10–13). gB is considered the fusogenic glycoprotein, based on structural features, including a trimeric fold and a bipartite fusion loop (14, 15). gH, gL, and gB constitute the conserved-entry glycoproteins across the Herpesviridae family. The ability of gH to heterodimerize with gL is also conserved across the family, highlighting that the heterodimeric structure is critical to the function of two glycoproteins.The prevailing model of HSV entry envisions that following a first virion attachment to cells mediated by heparan sulfate glycosaminoglycans, the interaction of gD with one of its receptors, nectin1 and HVEM (herpesvirus entry mediator), results in conformational changes to gD, in particular to the ectodomain C terminus, which harbors the profusion domain (5, 6, 16, 17). The activated gD recruits gH/gL, which, in turn, recruits gB. gB executes the virus–cell fusion (2–4, 18, 19). We observed that the glycoproteins are already in complex in resting virions (17, 18). In contrast with the view that glycoproteins are stepwise-recruited to a complex, we favor the view that the process of activation of the viral glycoproteins results from the interaction of preassembled glycoproteins’ complexes with cellular receptors and from a signaling cascade, which is likely triggered by receptor-induced conformational changes (18).The more speculative part of the HSV-entry model concerns the roles of gH and of gL and why gH has evolved to be a heterodimer with gL. Recently, we discovered that αvβ6- and αvβ8-integrins serve as interchangeable receptors for HSV gH/gL. They play two distinct roles in infection (20). They enable virus entry, as inferred by inhibition of infection following integrin depletion by siRNAs or exposure of cells to anti-integrin antibodies. Second, they promote HSV endocytosis into acidic endosomes (20); the latter function is nonessential because the virus may enter some cells also by fusion with plasma membranes or with neutral endosomes (21–23). Remarkably, the use of integrins as receptors is a common feature among herpesviruses. Integrins serve as receptors also for gH/gL of EBV (Epstein Barr virus), of human cytomegalovirus and equine herpesvirus, and for gB of Kaposi’s sarcoma-associated herpesvirus (24–28). Most likely, they play a common role.Here, we asked whether αvβ6- or αvβ8-integrin induce conformational changes to HSV gH/gL, as part of the process of glycoprotein activation in virus entry. We report that αvβ6- and αvβ8-integrin promote the dissociation of gL from gH/gL. Conditions for the dissociation were the presence of gD, its receptor nectin1, and gB, i.e., conditions that lead to activation of the entry machinery, including the virion glycoproteins.     

A common solution to group 2 influenza virus neutralization

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.Influenza viruses are a significant and persistent threat to human health worldwide. Annual epidemics cause 3–5 million cases of severe illness and up to 0.5 million deaths (1), and periodic influenza pandemics have the potential to kill millions (2). Inhibitors against the viral surface glycoprotein neuraminidase are widely used for the treatment of influenza infections, but their efficacy is being compromised by the emergence of drug-resistant viral strains (3). Vaccination remains the most effective strategy to prevent influenza virus infection. However, protective efficacy is suboptimal in the highest risk groups: infants, the elderly, and the immunocompromised (1). Furthermore, because immunity after vaccination is typically strain-specific and influenza viruses evolve rapidly, vaccines must be updated almost annually. The antigenic composition of the vaccine is based on a prediction of strains likely to circulate in the coming year, therefore, mismatches between vaccine strains and circulating strains occur that can render the vaccine less effective (4). Consequently, there is an urgent need for new prophylactic and therapeutic interventions that provide broad protection against influenza.Immunity against influenza viruses is largely mediated by neutralizing antibodies that target the major surface glycoprotein hemagglutinin (HA) (5, 6). Identification of antigenic sites on HA indicates that influenza antibodies are primarily directed against the immunodominant HA head region (7), which mediates endosomal uptake of the virus into host cells by binding to sialic acid receptors (8). Because of high mutation rates in the HA head region and its tolerance for antigenic changes, antibodies that target the HA head are typically only effective against strains closely related to the strain(s) by which they were elicited, although several receptor binding site-targeting antibodies with greater breadth have been structurally characterized (9–15). In contrast, antibodies that bind to the membrane-proximal HA stem region tend to exhibit much broader neutralizing activity and can target strains within entire subtypes and groups (16–25) as well as across influenza types (24). These stem-directed antibodies inhibit major structural rearrangements in HA that are required for the fusion of viral and host endosomal membranes and thus, prevent the release of viral contents into the cell (8). The stem region is less permissive for mutations than the head and relatively well-conserved across divergent influenza subtypes.Anti-stem antibodies are elicited in some, but not all, individuals during influenza infection or vaccination (20, 26) and thus, hold great promise as potential broad spectrum prophylactic or therapeutic agents and for the development of a universal influenza vaccine (27–29). The majority of the known heterosubtypic stem binding antibodies neutralize influenza A virus subtypes belonging to group 1 (17–20, 23, 25). Furthermore, two antibodies that target a similar epitope in the HA stem, like most heterosubtypic group 1 antibodies, are able to more broadly recognize both group 1 and 2 influenza A viruses (22) or influenza A and B viruses (24). Strikingly, group 2-specific broadly neutralizing Abs (bnAbs) seem to be rare, because only one has been reported to date (21). CR8020 uniquely targets a distinct epitope in the stem in close proximity to the viral membrane at the HA base and binds lower down the stem than any other influenza HA antibody (21).In the discovery process that led to the isolation of bnAb CR8020, we recovered additional group 2-specific bnAbs. Here, we describe one such bnAb, CR8043, which recognizes a similar but nonidentical footprint on the HA as CR8020 and approaches the HA from a different angle. Furthermore, these two bnAbs are derived from different germ-line genes and, consequently, use distinct sets of interactions for HA recognition. Thus, the human immune system is able to recognize this highly conserved epitope in different ways using different germ-line genes. Hence, this valuable information can be used for the design of therapeutics and vaccines targeting this site of vulnerability in group 2 influenza A viruses that include the pandemic H3N2 subtype.     

Structural insights into the histone H1-nucleosome complex

Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1–nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo.Eukaryotic genomic DNA is packaged into chromatin through association with positively charged histones to form the nucleosome, the structural unit of chromatin (1–3). The nucleosome core consists of an octamer of histones with two copies of H2A, H2B, H3, and H4, around which ∼146 bp of DNA winds in ∼1.65 left-handed superhelical turns (4). At this level of the DNA packaging, chromatin resembles a beads-on-a-string structure, with the nucleosome core as the beads and the linker DNA between them as the strings (5). At the next level of DNA packaging, H1 histones bind to the linker DNA and the nucleosome to further condense the chromatin structure (6, 7). H1-mediated chromatin condensation plays important roles in cellular functions such as mitotic chromosome architecture and segregation (8), muscle differentiation (9), and regulation of gene expression (10, 11).Linker H1 histones typically are ∼200 amino acid residues in length, with a short N-terminal region, followed by a ∼70–80-amino acid structured globular domain (gH1) and a ∼100-amino acid unstructured C-terminal domain that is highly enriched in Lys residues. H1 stabilizes the nucleosome and facilitates folding of nucleosome arrays into higher-order structures (12–15). gH1 alone confers the same protection from micrococcal nuclease digestion to the nucleosome as the full-length H1 does (16). The N-terminal region of H1 is not important for nucleosome binding (16, 17), whereas the C terminus is required for H1 binding to chromatin in vivo (18, 19) and for the formation of a stem structure of linker DNA in vitro (17, 20, 21).The globular domain structures of avian H5 (22) and budding yeast Hho1 (23), which are both H1 homologs, have been determined at atomic resolution and show similar structures. In addition, numerous studies have indicated that gH1/gH5 binds around the dyad region of the nucleosome (14, 24), leading to many conflicting structural models for how the globular domain of H1/H5 binds to the nucleosome (SI Appendix, Fig. S1) (24–26). These models are divided into two major classes, symmetric and asymmetric, on the basis of the location of gH1/gH5 in the nucleosome. In the symmetric class, gH1/gH5 binds to the nucleosomal DNA at the dyad and interacts with both linker DNAs (16, 17, 27, 28). In the asymmetric class, gH1/gH5 binds to the nucleosomal DNA in the vicinity of the dyad axis and to 10 bp (27, 29–32) or 20 bp (19, 29, 33, 34) of one linker DNA, or is located inside the DNA gyres, where it interacts with histone H2A (35). In addition, Zhou and colleagues also characterized the orientation of gH5 in the gH5-nucleosome complex (29). The use of nonuniquely positioned nucleosomes and indirect methods may have contributed to the differences in these models (SI Appendix, Fig. S1).Multidimensional nuclear magnetic resonance (NMR), and in particular methyl-based NMR, provides a direct approach to the structural characterization of macromolecular complexes (36, 37). We have previously assigned chemical shifts of the methyl groups of the side chains of residues Ile, Leu, and Val in the core histones (38) and the backbone amides in the disordered histone tails (39), which provide the fingerprints for investigating the interactions between H1 and the nucleosome. Here, we used NMR, along with several other methods, to determine the location and orientation of the globular domain of a stable mutant of Drosophila H1 on a well-positioned nucleosome.     

Nonmuscle myosin heavy chain IIA mediates Epstein–Barr virus infection of nasopharyngeal epithelial cells

EBV causes B lymphomas and undifferentiated nasopharyngeal carcinoma (NPC). Although the mechanisms by which EBV infects B lymphocytes have been extensively studied, investigation of the mechanisms by which EBV infects nasopharyngeal epithelial cells (NPECs) has only recently been enabled by the successful growth of B lymphoma Mo-MLV insertion region 1 homolog (BMI1)-immortalized NPECs in vitro and the discovery that neuropilin 1 expression positively affects EBV glycoprotein B (gB)-mediated infection and tyrosine kinase activations in enhancing EBV infection of BMI1-immortalized NPECs. We have now found that even though EBV infected NPECs grown as a monolayer at extremely low efficiency (<3%), close to 30% of NPECs grown as sphere-like cells (SLCs) were infected by EBV. We also identified nonmuscle myosin heavy chain IIA (NMHC-IIA) as another NPEC protein important for efficient EBV infection. EBV gH/gL specifically interacted with NMHC-IIA both in vitro and in vivo. NMHC-IIA densely aggregated on the surface of NPEC SLCs and colocalized with EBV. EBV infection of NPEC SLCs was significantly reduced by NMHC-IIA siRNA knock-down. NMHC-IIA antisera also efficiently blocked EBV infection. These data indicate that NMHC-IIA is an important factor for EBV NPEC infection.EBV is a nearly ubiquitous human γ-herpesvirus that causes B-cell lymphomas and nasopharyngeal carcinoma (NPC), indicative of tropism for both cell types (1–3). Until recently, the molecular mechanisms of EBV infection of B lymphocytes were better understood than the mechanisms of epithelial cell infection (4). EBV attachment to the B-cell membrane is mediated by interactions between EBV glycoprotein 350 (gp350) and complement receptor type 2 (CR2 or CD21) (5) or CD35 (6). EBV gp42 binding to HLA class II triggers EBV fusion with B cells in the presence of EBV glycoprotein B (gB) and gH/gL (7, 8). For epithelial cells, gH/gL and gB are important for EBV infection (4, 9, 10). Epithelial cells lack HLA class II expression; thus, gp42 cannot trigger EBV and cell fusion. Instead, gp42 inversely suppresses the infection (11), and an antibody against gp350 can enhance infections of CD21/CD35-negative epithelial cells (12). The gH/gL heterodimer is required for virus entry (4) and may be involved in binding (13), as well as fusion of EBV (14–17). However, the crystal structure of EBV gH/gL does not show any known fusion domain (18). It is now thought that gH/gL regulates the fusion function of gB (19). Binding of gH/gL to a subset of αv integrins (e.g., αvβ₅, αvβ₆, or αvβ₈) provides the initial trigger for gB-mediated fusion (16, 20, 21). However, E1D1(gH/gL) antibody or CL59(gH) antibody, with a different epitope, can impair epithelial cell infection (20, 22). Thus, multiple gH/gL domains are critical to EBV infection, and gH/gL may interact with proteins in addition to integrins. Direct interaction of EBV gB amino acids 23–431 with neuropilin 1 (NRP1) and its associated tyrosine kinases is critical for EBV infection of nasopharyngeal epithelial cells (NPECs). NRP1 knock-down or EBV pretreatment with soluble NRP1 suppresses EBV NPEC infection, whereas NRP1 overexpression enhances EBV infection (10). Confocal microscopy and experiments with inhibitors of macropinocytosis indicate that EBV enters NPECs through macropinocytosis and not through clathrin-mediated endocytosis (10).The principal obstacle to identifying factors that may enable more efficient EBV infection of NPECs and better understanding of the role of EBV in NPC is that EBV is remarkably inefficient in infection of primary or B lymphoma Mo-MLV insertion region 1 homolog (BMI1)-immortalized NPECs. As a polycomb complex protein, BMI1 is a proto-oncogene. BMI1 has an important role in regulating proliferation, senescence, and self-renewal of stem cells (23, 24). BMI1 overexpression immortalizes human epithelial cells and mouse embryonic fibroblasts (25, 26). By optimizing the growth of BMI1-immortalized NPEC cultures, we found that BMI1-immortalized NPECs seeded at a 10-fold higher density than used in previous protocols and grew initially as a monolayer. “Spherical cells” then grew above the monolayer. Surprisingly, the spherical cells consistently supported an EBV infection efficiency of ∼20–30%, using an estimated EBV multiplicity of infection (MOI) of 300. Using this more efficient in vitro EBV infection protocol, we identified an interaction between nonmuscle myosin heavy chain IIA (NMHC-IIA) and gH/gL on the cell surface, which was critical for more efficient EBV NPEC infection.     

Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies

Broadly neutralizing HIV antibodies (bNAbs) can recognize carbohydrate-dependent epitopes on gp120. In contrast to previously characterized glycan-dependent bNAbs that recognize high-mannose N-glycans, PGT121 binds complex-type N-glycans in glycan microarrays. We isolated the B-cell clone encoding PGT121, which segregates into PGT121-like and 10-1074–like groups distinguished by sequence, binding affinity, carbohydrate recognition, and neutralizing activity. Group 10-1074 exhibits remarkable potency and breadth but no detectable binding to protein-free glycans. Crystal structures of unliganded PGT121, 10-1074, and their likely germ-line precursor reveal that differential carbohydrate recognition maps to a cleft between complementarity determining region (CDR)H2 and CDRH3. This cleft was occupied by a complex-type N-glycan in a “liganded” PGT121 structure. Swapping glycan contact residues between PGT121 and 10-1074 confirmed their importance for neutralization. Although PGT121 binds complex-type N-glycans, PGT121 recognized high-mannose-only HIV envelopes in isolation and on virions. As HIV envelopes exhibit varying proportions of high-mannose- and complex-type N-glycans, these results suggest promiscuous carbohydrate interactions, an advantageous adaptation ensuring neutralization of all viruses within a given strain.Antibodies are essential for the success of most vaccines (1), and antibodies against HIV appear to be the only correlate of protection in the recent RV144 anti-HIV vaccine trial (2). Some HIV-1–infected patients develop broadly neutralizing serologic activity against the gp160 viral spike 2–4 y after infection (3–10), but these antibodies do not generally protect infected humans because autologous viruses escape through mutation (11–13). Nevertheless, broadly neutralizing activity puts selective pressure on the virus (13) and passive transfer of broadly neutralizing antibodies (bNAbs) to macaques protects against simian/human immunodeficiency virus (SHIV) infection (14–24). It has therefore been proposed that vaccines that elicit such antibodies may be protective against HIV infection in humans (10, 25–28).The development of single-cell antibody cloning techniques revealed that bNAbs target several different epitopes on the HIV-1 gp160 spike (29–35). The most potent HIV-1 bNAbs recognize the CD4 binding site (CD4bs) (31, 34, 36) and carbohydrate-dependent epitopes associated with the variable loops (32, 33, 37, 38), including the V1/V2 (antibodies PG9/PG16) (33) and V3 loops (PGTs) (32). Less is known about carbohydrate-dependent epitopes because the antibodies studied to date are either unique examples or members of small clonal families.To better understand the neutralizing antibody response to HIV-1 and the epitope targeted by PGT antibodies, we isolated members of a large clonal family dominating the gp160-specific IgG memory response from the clade A-infected patient who produced PGT121. We report that PGT121 antibodies segregate into two groups, a PGT121-like and a 10-1074–like group, according to sequence, binding affinity, neutralizing activity, and recognition of carbohydrates and the V3 loop. The 10-1074 antibody and related family members exhibit unusual potent neutralization, including broad reactivity against newly transmitted viruses. Unlike previously characterized carbohydrate-dependent bNAbs, PGT121 binds to complex-type, rather than high-mannose, N-glycans in glycan microarray experiments. Crystal structures of PGT121 and 10-1074 compared with structures of their germ-line precursor and a structure of PGT121 bound to a complex-type N-glycan rationalize their distinct properties.     

A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL

Varicella-zoster virus (VZV), of the family Alphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.Varicella-zoster virus (VZV) is an enveloped, double-stranded DNA virus of the family Alphaherpesvirinae. Primary VZV infection initiates at the respiratory mucosa. T cells are infected in regional lymph nodes and disseminate virions to the skin, causing varicella (chickenpox) (1). VZV reaches sensory ganglia by the hematogenous route or by anterograde axonal transfer from skin lesions and establishes latency in neurons (2). VZV reactivation can lead to herpes zoster (shingles), which is particularly prevalent in the elderly and in immunocompromised individuals (1, 3). An estimated one third of people in the United States will develop herpes zoster and are at risk for the chronic pain syndrome referred to as postherpetic neuralgia.Live attenuated vaccines derived from the Oka strain are safe and effective against varicella and herpes zoster in healthy individuals but are contraindicated in most immunocompromised patients (4, 5). Subunit vaccines have the potential to be safe in these populations (6). High-titer VZV immunoglobulin given shortly after exposure decreases the incidence of varicella (7), indicating that antibodies against the VZV envelope glycoproteins can interfere with the initiation of infection and cell-to-cell spread. Thus, subunit vaccines made from one or more of these proteins could be an alternative to live attenuated VZV vaccines. Indeed in a phase 1/2 clinical trial, AS01_B-adjuvanted envelope glycoprotein gE has been reported to be more immunogenic than a live attenuated VZV vaccine in healthy individuals in the age groups of 18–30 or 50–70 y. Advances are occurring in the development of this gE-based vaccine candidate against herpes zoster (6).Similar to other herpesviruses, VZV entry into host cells is presumed to be initiated by virion attachment to the cell surface, followed by fusion between the virus envelope and the plasma membrane of the target cell. The envelope glycoproteins gB and the gHgL heterodimer (8) are highly conserved components of the herpesvirus entry machinery. VZV gB and gHgL are necessary and sufficient to induce cell fusion, which is considered a surrogate for virion envelope fusion (9, 10).The crystal structures of the gHgL heterodimer ectodomain for herpes simplex virus 2 (HSV-2) and Epstein–Barr virus (EBV) showed that gH and gL cofold to form a tightly packed complex with no similarity to any known viral fusion proteins (11, 12). In contrast, the structures of HSV-1 and EBV gB are highly similar to the vesicular stomatitis virus membrane fusion glycoprotein G, indicating that gB is the herpesvirus fusion protein (13–15). Fluorescence bimolecular complementation studies with HSV-1 envelope glycoproteins imply that gHgL interacts with gB during membrane fusion (16, 17). This interaction was inhibited by the anti-gHgL neutralizing monoclonal antibody (mAb) LP11 (11). How gHgL facilitates the conformational changes in gB required for membrane fusion and viral entry remains elusive.A homology model of VZV gH based on the HSV-2 gHgL structure was used to identify gH residues involved in membrane fusion (9). Incorporating gH mutations into recombinant VZV showed the importance of the gH ectodomain and cytoplasmic domain for viral replication and regulation of cell fusion in vitro and for infection of human skin xenografts in the SCID mouse model of VZV pathogenesis, as are gB mutations (9, 18, 19).The contribution of VZV gHgL to viral entry and spread was also demonstrated by the capacity of an anti-gH mouse mAb, mAb206, to neutralize VZV in vitro and to severely impair infection of human skin xenografts (20–22). Human neutralizing mAbs generated from a library of pooled B cells obtained from naturally immune individuals also target gHgL (23). Two of these, designated IgG-24 and IgG-94, and their respective fragment of antigen-binding (Fab), had the most VZV-neutralizing activity. Recently, a VZV-neutralizing human mAb (rec-RC IgG), which recognized the gHgL heterodimer in VZV infected cells, was isolated from plasmablasts recovered from an individual who was immunized with a zoster vaccine (24).The present study provides the first report, to our knowledge, on the structure of a herpesvirus gHgL heterodimer in complex with human mAbs and delineates the location of clinically relevant neutralizing epitopes on VZV gHgL. Structural and binding studies demonstrated that Fab-RC and Fab-94 target the same epitope formed by gH and gL residues, whereas Fab-24 binds to a distal site on gH. Immunization studies proved that the gHgL Fab-RC/-94 epitope is a critical target for neutralizing Abs against VZV. Compared with postfusion gB, gHgL elicited Abs in mice that more potently inhibited cell fusion and neutralized VZV. Vulnerability to Ab-mediated inhibition suggests the gHgL complex could be a component of an effective subunit vaccine.     

Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections

The 2009 H1N1 pandemic and recent human cases of H5N1, H7N9, and H6N1 in Asia highlight the need for a universal influenza vaccine that can provide cross-strain or even cross-subtype protection. Here, we show that recombinant monoglycosylated hemagglutinin (HA_mg) with an intact protein structure from either seasonal or pandemic H1N1 can be used as a vaccine for cross-strain protection against various H1N1 viruses in circulation from 1933 to 2009 in mice and ferrets. In the HA_mg vaccine, highly conserved sequences that were originally covered by glycans in the fully glycosylated HA (HA_fg) are exposed and thus, are better engulfed by dendritic cells (DCs), stimulated better DC maturation, and induced more CD8+ memory T cells and IgG-secreting plasma cells. Single B-cell RT-PCR followed by sequence analysis revealed that the HA_mg vaccine activated more diverse B-cell repertoires than the HA_fg vaccine and produced antibodies with cross-strain binding ability. In summary, the HA_mg vaccine elicits cross-strain immune responses that may mitigate the current need for yearly reformulation of strain-specific inactivated vaccines. This strategy may also map a new direction for universal vaccine design.HA glycoprotein on the surface of influenza virus is a major target for infectivity-neutralizing antibodies. However, the antigenic drift and shift of this protein mean that influenza vaccines must be reformulated annually to include HA proteins of the viral strains predicted for the upcoming flu season (1). This time-consuming annual reconfiguration process has led to efforts to develop new strategies and identify conserved epitopes recognized by broadly neutralizing antibodies as the basis for designing universal vaccines to elicit antibodies with a broad protection against various strains of influenza infection (2–6). Previous studies have shown that the stem region of HA is more conserved and able to induce cross-reactive and broadly neutralizing antibodies (7–9) to prevent the critical fusion of viral and endosomal membranes in the influenza lifecycle (10–14). Other broadly neutralizing antibodies have been found to bind regions near the receptor binding site of the globular domain, although these antibodies are fewer in number (15, 16).Posttranslational glycosylation of HA plays an important role in the lifecycle of the influenza virus and also contributes to the structural integrity of HA and the poor immune response of the infected hosts. Previously, we trimmed down the size of glycans on avian influenza H5N1 HA with enzymes and showed that H5N1 HA with a single N-linked GlcNAc at each glycosylation site [monoglycosylated HA (HA_mg)] produces a superior vaccine with more enhanced antibody response and neutralization activity against the homologous influenza virus than the fully glycosylated HA (HA_fg) (17). Here, to test whether the removal of glycans from HA contributes to better immune responses and possibly protects against heterologous strains of influenza viruses, we compared and evaluated the efficacy of HA glycoproteins with various lengths of glycans as potential vaccine candidates.     

Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice

Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.Monoclonal antibodies (mAbs) are a rapidly growing class of therapeutics that combine high binding affinities and specificities with long in vivo half-lives. A large number of mAbs have been approved for therapeutic use or are in development (1–6). Early therapeutic mAbs were derived from mouse sources, retained mouse sequences, and were thus immunogenic when used in human patients, limiting the ability to dose repeatedly. The use of humanized and/or fully human antibodies avoided immunogenicity problems and allowed long-term treatment of chronic diseases. Thus, a variety of systems were developed to create humanized or fully human therapeutic antibodies (7–18). One popular approach involves the in vitro isolation of antigen-specific antibodies using display strategies involving human antibody fragments expressed on the surface of phage, bacteria, or yeast (12). Although these synthetic approaches can be quite powerful and can rapidly generate leads, they potentially result in increased immunogenicity in vivo, and the initial display-derived antibody fragments can subsequently require extensive post hoc protein reengineering efforts when reformatted into conventional antibody formats to overcome issues such as insolubility, aggregation, and proteolysis (1, 12, 19). Natural selection of antibodies in vivo within mammalian systems tends to optimize desirable biochemical and pharmacokinetic properties, avoiding the need for extensive post hoc reengineering. Thus, as first proposed based on the finding that human Ig genes efficiently rearrange when introduced into mouse pre-B cells (20), another popular approach for generating human therapeutic mAbs was developed using transgenic mice genetically engineered to produce fully human antibodies (15–18). These so-called HumAb mice were engineered using a “KO-plus-transgenic” strategy in which the endogenous murine Ig genes were disrupted to eliminate the endogenous mouse immune response, whereas transgenic introduction of human Ig loci at different random genetic loci drove production of fully human antibodies.Although HumAb mice generated using this KO-plus-transgenic approach represented a transforming advance in the field, they suffered, however, from partial immune deficiencies compared with WT mice, limiting their ability to produce robust Ab responses to some antigens (21–23). The immune deficiencies of these first-generation HumAb mice may be due to the manner in which they were genetically engineered. First, the genomic context of the randomly inserted human transgenes may contribute to their inefficient functionality, as they may lack extended locus control regions such as the 3′ enhancers (24) and regulatory region (25) of the Ig heavy (IgH) locus, which have been shown to play critical roles in Ig expression and class switching or even alter the 3D location of the Ig genes within B-cell nuclei (26, 27) and thus perturb function in unanticipated ways. In addition, the immune deficiencies of the first-generation HumAb mice may be partly explained by their use of human constant regions. Immunoglobulins interact with other components of the B-cell receptor (BCR) signaling complex via their constant regions, and such interactions are required for appropriate signaling required for antigen-independent B-cell development in bone marrow, as well as antigen-dependent natural selection processes in the periphery (28–36). Others previously noted that interactions between constant regions and BCR coreceptors do not operate efficiently across species (29). Thus, we reasoned that the immune deficiencies in first-generation HumAb mice may in part be due to inefficient interspecies protein-protein interactions between human constant regions and the mouse coreceptors. Finally, the secreted human immunoglobulins produced in the HumAb mice may also interact inefficiently with various mouse Fc receptors, further adversely affecting the humoral immune response (37, 38).As described in the companion article (39), we attempted to exploit the advantages and overcome the limitations of the first-generation HumAb approaches by precisely replacing the entire mouse germ-line variable region gene repertoire with the equivalent human germ-line variable sequences in situ, while maintaining all mouse constant regions and all known gene expression control elements within the natural mouse genomic location. We reasoned that the introduced human Ig variable gene segments would rearrange normally, be linked to mouse constant regions, and furthermore be expressed from the endogenous mouse Ig loci at physiologically appropriate levels. Because these “reverse chimeric” antibody molecules would bear human antigen-binding variable domains fused to mouse constant domains, we presumed they would interact in a species appropriate way with mouse BCR coreceptors and mouse Fc receptors, resulting in a fully functional immune system. We refer to these humanized Ig variable domain mice as VelocImmune mice because they were generated using VelociGene technology (40).In this paper, we show that VelocImmune mice have a humoral immune system indistinguishable from that of WT mice, with normal cell populations at all stages of B-cell development and normal lymphoid organ structures. Sequences of antibodies derived from the humanized Ig loci exhibit normal variable segment rearrangement, somatic hypermutation, and class switching. Immunizations of VelocImmune mice generate robust humoral responses from which a large diversity of monoclonal antibodies can be isolated. Thus, VelocImmune mice are a unique platform for the efficient production of fully human antibodies, several of which have already entered clinical development.     

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners.With a steady increase of antibodies and antibody derivatives such as antibody drug conjugates and bispecific antibodies entering the clinic, monoclonal human antibodies are now an established source of new therapeutic agents (1, 2). The development of bispecific antibodies has generated particular interest, because it allows expansion of basic antibody functions (3, 4). Through binding two (or more) different targets, a bispecific antibody can simultaneously engage two epitopes of a disease agent, block/activate multiple ligands/receptors at once, or recruit immune effector cells (i.e., T cells or B cells) to a specific (tumor) site (5). There is a growing interest in bispecific antibodies with anticancer properties, which has led to an increase in bispecifics that have entered preclinical testing (5, 6).Bispecific antibodies with defined functions are generated by means of genetic or biochemical engineering. Many different methods exist to engineer immunoglobulins, with more than 45 bispecific antibody formats at last count (reviewed in ref. 5). These bispecific antibody formats fall into three broad subclasses (5): (i) single-chain double variable domain formats (50–100 kDa) (7–9): Generally these bispecifics consist of multiple variable domains that are connected via peptide linkers. (ii) IgG with multiple variable domains: In this type of bispecific antibody, a second variable domain is genetically linked to any desirable position in the IgG molecule (i.e., the C or N terminus of either the IgG heavy or light chain) (10–12). (iii) Asymmetric IgG molecules: In an asymmetric IgG antibody, two different variable domains are incorporated into a single, asymmetric, antibody molecule via heterodimerization of the constant domains. Heterodimerization may be achieved through engineering the C_H3 domain (13–16) or the hinge region of the antibody (17, 18). Depending on the engineering method, asymmetric IgGs can be made with a common light chain or with two different light chains (19).Each of these formats has its specific advantages and drawbacks. Most of the limitations arise from the fact that their formats deviate significantly from the natural, highly stable, IgG structure, which compromises stability and ease of manufacture.Here, we present a bispecific antibody format, in which two antibodies are fused at their C termini, using a combination of sortase transpeptidation and click chemistry (20), to create an IgG heterodimer. This C-C fusion does not require mutations within the antibody constant domains that might interfere with Fc-receptor binding or that would compromise antibody stability. Thus, the native antibody structure is fully retained in our format.C-to-C fusion is a two-step process (Fig. 1), using a combination of sortase transpeptidation and click chemistry (20). Sortase is a bacterial enzyme that functions to attach cell surface proteins bearing an “LPXTG” motif to the cell wall of Gram-positive bacteria via transacylation (21, 22). Sortase-catalyzed transpeptidation allows for efficient site-specific modifications under physiological conditions, with excellent specificity and near-quantitative yields (23–25). To facilitate site-specific linking of the C termini of two antibodies, the fusion partners are labeled with either an azide or a cyclooctyne (DIBAC) functional group. The modified proteins are then conjugated via a strain-promoted cycloaddition between the azide and the cyclooctyne. This reaction is highly specific and readily proceeds at room temperature in aqueous environments at neutral pH (26), allowing for efficient fusion under mild conditions.Open in a separate windowFig. 1.Approach for synthesis of C-to-C–fused antibodies. (A) Antibodies are labeled at the C terminus either with an azide (N₃) or DIBAC with a click peptide by using sortase. (B) Click-labeled antibodies are fused via the click reaction.To test the robustness of this process and determine the features of this bispecific antibody format, we fused two potent anti-influenza antibodies, each active against a different subgroup of the influenza A virus. Based on the hemagglutinin (HA) protein sequence, there are 18 different subtypes of influenza A, divided into two subgroups (27, 28). The HA protein is the common target of almost all neutralizing antibodies, and several antibodies with broadly neutralizing activity between influenza A subtypes in the same group exist (29–33). Combining two such potent subgroup-specific antibodies may result in an IgG heterodimer with even broader anti-influenza A activity. This type of molecule would have therapeutic relevance in a passive immunization setting, because influenza viruses continue to cause significant morbidity and mortality, despite efforts to contain them with seasonal vaccines (34). Because these vaccines are typically only effective against the specific seasonal viral strain, there is an urgent unmet medical need for new treatments active against multiple subtypes of the influenza virus (35).     

Maternal CD4+ T cells protect against severe congenital cytomegalovirus disease in a novel nonhuman primate model of placental cytomegalovirus transmission

Elucidation of maternal immune correlates of protection against congenital cytomegalovirus (CMV) is necessary to inform future vaccine design. Here, we present a novel rhesus macaque model of placental rhesus CMV (rhCMV) transmission and use it to dissect determinants of protection against congenital transmission following primary maternal rhCMV infection. In this model, asymptomatic intrauterine infection was observed following i.v. rhCMV inoculation during the early second trimester in two of three rhCMV-seronegative pregnant females. In contrast, fetal loss or infant CMV-associated sequelae occurred in four rhCMV-seronegative pregnant macaques that were CD4⁺ T-cell depleted at the time of inoculation. Animals that received the CD4⁺ T-cell–depleting antibody also exhibited higher plasma and amniotic fluid viral loads, dampened virus-specific CD8⁺ T-cell responses, and delayed production of autologous neutralizing antibodies compared with immunocompetent monkeys. Thus, maternal CD4⁺ T-cell immunity during primary rhCMV infection is important for controlling maternal viremia and inducing protective immune responses that prevent severe CMV-associated fetal disease.Human congenital cytomegalovirus (CMV) infection occurs in 0.7% of all pregnancies (1) and is a major cause of permanent sensorineural and neurocognitive disabilities in infants worldwide. The rate of congenital CMV transmission is as high as 50% among women who acquire primary CMV infection during pregnancy, compared with less than 2% in women with chronic infection (2). Furthermore, congenital CMV transmission following primary maternal infection causes the most severe fetal outcomes including microcephaly, intracranial cyst formation, seizures, and intrauterine growth restriction (3).Although evident, the protective immune correlates of preconceptual immunity have not been determined. Currently, the guinea pig animal model is used to study protective immune responses against congenital CMV infection, as it is the only other species known to be susceptible to placental transmission of its species-specific CMV (4). Despite having limited sequence homology to human CMV (HCMV) (5), guinea pig CMV (GPCMV) crosses the placental barrier at a similar rate following acute maternal infection and establishes fetal infection with comparable CMV-associated sequelae (6). Several vaccine strategies including live-attenuated virus (7, 8), passive immunization of antibodies specific for glycoprotein B (gB) and the gH/gL complex (9, 10), and recombinant gB subunit immunization (11) have proven to be effective at reducing the rate of congenital GPCMV infection and preventing fetal demise. In human clinical trials, gB immunization was only 50% effective in reducing postpartum maternal virus acquisition (12) and passive immunization with CMV hyperimmune globulin of women with primary CMV infection within 6 wk of presumed acquisition did not achieve a significant reduction in the rate of fetal infection compared with placebo controls (13). These findings address the need for a relevant large-animal model with a more closely related CMV genome and wider availability of tools to assess the maternal immune system.Nonhuman primate models are widely used in the preclinical evaluation of vaccine candidates, as they are anatomically, physiologically, and immunologically similar to humans. Furthermore, their widespread use has led to the development of an extensive set of immunological tools that allow rigorous probing and characterization of vaccine-elicited immune responses. Rhesus macaques, a common nonhuman primate animal model, have previously been used to study CMV pathogenesis in the setting of primary and secondary infection (14–16). Rhesus CMV (rhCMV), which has greater sequence and structural homology to HCMV than GPCMV (17–19), results in asymptomatic infection and establishment of a persistent and lifelong infection, similar to that in humans.. Importantly, previous studies have shown that rhCMV inoculated intraperitoneally or intracranially into the developing fetus can induce neurological defects similar to those observed in congenitally infected human infants (20, 21). However, because of the high rate of rhCMV seroprevalence among animals of reproductive age (22) and repeated exposure to rhCMV within breeding colonies where rhCMV is endemic (23), there have been no previous reports of fetal or neonatal macaques exhibiting sequelae consistent with congenital rhCMV infection.Here, to our knowledge, we report the first nonhuman primate model of congenital CMV transmission using rhCMV-seronegative rhesus monkeys. In this model, CD4⁺ T-cell–depleted or immunocompetent rhCMV-seronegative monkeys were inoculated with a defined mixture of rhCMV strains in the early second trimester of pregnancy. Following detection of placental rhCMV transmission, fetuses were observed for signs of rhCMV-associated sequelae, and the maternal immune responses between mothers with severely affected fetuses and asymptomatic or noninfected infants were evaluated to identify potential immune correlates of protection against congenital CMV disease in nonhuman primates.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.