Genotype-Specific Neutralization and Protection by Antibodies against Dengue Virus Type 3

Dengue viruses (DENV) comprise a family of related positive-strand RNA viruses that infect up to 100 million people annually. Currently, there is no approved vaccine or therapy to prevent infection or diminish disease severity. Protection against DENV is associated with the development of neutralizing antibodies that recognize the viral envelope (E) protein. Here, with the goal of identifying monoclonal antibodies (MAbs) that can function as postexposure therapy, we generated a panel of 82 new MAbs against DENV-3, including 24 highly neutralizing MAbs. Using yeast surface display, we localized the epitopes of the most strongly neutralizing MAbs to the lateral ridge of domain III (DIII) of the DENV type 3 (DENV-3) E protein. While several MAbs functioned prophylactically to prevent DENV-3-induced lethality in a stringent intracranial-challenge model of mice, only three MAbs exhibited therapeutic activity against a homologous strain when administered 2 days after infection. Remarkably, no MAb in our panel protected prophylactically against challenge by a strain from a heterologous DENV-3 genotype. Consistent with this, no single MAb neutralized efficiently the nine different DENV-3 strains used in this study, likely because of the sequence variation in DIII within and between genotypes. Our studies suggest that strain diversity may limit the efficacy of MAb therapy or tetravalent vaccines against DENV, as neutralization potency generally correlated with a narrowed genotype specificity.Dengue viruses (DENV) cause the most common arthropod-borne viral infection in humans worldwide, with ∼50 million to 100 million people infected annually and ∼2.5 billion people at risk (13, 61). Infection by four closely related but serologically distinct viruses of the Flavivirus genus (DENV serotypes 1, 2, 3, and 4 [DENV-1 to -4, respectively]) cause dengue fever (DF), an acute, self-limiting, yet severe, febrile illness, or dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), a potentially fatal syndrome characterized by vascular leakage and a bleeding diathesis. Specific treatment or prevention of dengue disease is supportive, as there is no approved antiviral therapy or vaccine available.DENV has an ∼11-kb, single-stranded, positive-sense RNA genome that is translated into a polyprotein and is cleaved posttranslationally into three structural (envelope [E], pre/membrane [prM], and capsid [C]) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The three structural proteins encapsidate a single infectious RNA of the DENV genome, whereas the nonstructural proteins have key enzymatic or regulatory functions that promote replication. Additionally, several DENV proteins are multifunctional and modulate cell-intrinsic and cell-extrinsic host immune responses (10).Most flavivirus-neutralizing antibodies recognize the structural E protein (reviewed in reference 40). Based on X-ray crystallographic analysis (32, 33), the DENV E protein is divided into three domains: domain I (DI), which is an 8-stranded β-barrel, domain II (DII), which consists of 12 β-strands, and domain III (DIII), which adopts an immunoglobulin-like fold. Mature DENV virions are covered by 90 antiparallel E protein homodimers, arranged flat along the surface of the virus with quasi-icosahedral symmetry (25). Studies with mouse monoclonal antibodies (MAbs) against DENV-1 and DENV-2 have shown that highly neutralizing anti-DENV antibodies are serotype specific and recognize primarily the lateral-ridge epitope on DIII (15, 49, 53). Additionally, subcomplex-specific MAbs, which recognize some but not all DENV serotypes, recognize a distinct, adjacent epitope on the A β-strand of DIII and also may be inhibitory (16, 28, 42, 53, 56). Complex-specific or flavivirus cross-reactive MAbs recognize epitopes in both DII and DIII and are generally less strongly neutralizing (8, 53).Beyond having genetic complexity (the E proteins of the four distinct serotypes are 72 to 80% identical at the amino acid level), viruses of each serotype can be further divided into closely related genotypes (43, 44, 57). DENV-3 is divided into 4 or 5 distinct genotypes (depending on the study), with up to 4% amino acid variation between genotypes and up to 2% amino acid variation within a genotype (26, 58, 62). The individual genotypes of DENV-3 are separated temporally and geographically (1), with genotype I (gI) strains located in Indonesia, gII strains in Thailand, and gIII strains in Sri Lanka and the Americas. Few examples of strains of gIV and gV exist from samples isolated after 1980 (26, 62). Infection with one DENV serotype is believed to confer long-term durable immunity against strains of the homologous but not heterologous DENV serotypes due to the specificity of neutralizing antibodies and protective CD8⁺ T cells (45). Indeed, epidemiological studies suggest that a preexisting cross-reactive antibody (7, 24) and/or T cells (34, 35, 64) can enhance the risk of DHF/DSS during challenge with a distinct DENV serotype. Nonetheless, few reports have examined how intergenotypic or even strain variation within a serotype affects the protective efficacy of neutralizing antibodies. This concept is important because the development of tetravalent DENV vaccines with attenuated prototype strains assumes that neutralizing antibody responses, which are lower during vaccination than during natural infection, will protect completely against all genotypes within a given serotype (60). However, a recent study showed markedly disparate neutralizing activities and levels of protection of individual anti-DENV-1 MAbs against different DENV-1 genotypes (49).Herein, we developed a panel of 82 new DENV-3 MAbs and examined their cross-reactivities, epitope specificities, neutralization potential at the genotype level in cell culture, and protective capacities in vivo. The majority of strongly neutralizing MAbs in this panel mapped to specific sites in DIII of the E protein. Remarkably, because of the scale of the sequence variation of DENV-3 strains, most of the protective antibodies showed significant strain specificity in their functional profiles.     

Passage of Dengue Virus Type 4 Vaccine Candidates in Fetal Rhesus Lung Cells Selects Heparin-Sensitive Variants That Result in Loss of Infectivity and Immunogenicity in Rhesus Macaques

Three dengue virus type 4 (DENV-4) vaccine candidates containing deletions in the 3′ noncoding region were prepared by passage in DBS-FRhL-2 (FRhL) cells. Unexpectedly, these vaccine candidates and parental DENV-4 similarly passaged in the same cells failed to elicit either viremia or a virus-neutralizing antibody response. Consensus sequence analysis revealed that each of the three viruses, as well as the parental DENV-4 when passaged in FRhL cells, rapidly acquired a single Glu₃₂₇-Gly substitution in domain III (DIII) of the envelope protein (E). These variants appear to have accumulated in response to growth adaptation to FRhL cells as shown by growth analysis, and the mutation was not detected in the virus following passage in C6/36 cells, primary African green monkey kidney cells, or Vero cells. The Glu₃₂₇-Gly substitution was predicted by molecular modeling to increase the net positive charge on the surface of E. The Glu₃₂₇-Gly variant of the full-length DENV-4 selected after three passages in FRhL cells showed increased affinity for heparan sulfate compared to the unpassaged DENV-4, as measured by heparin binding and infectivity inhibition assays. Evidence indicates that the Glu₃₂₇-Gly mutation in DIII of the DENV-4 E protein was responsible for reduced infectivity and immunogenicity in rhesus monkeys. Our results point out the importance of cell substrates for vaccine preparation since the virus may change during passages in certain cells through adaptive selection, and such mutations may affect cell tropism, virulence, and vaccine efficacy.Dengue virus (DENV) infects humans via the bite of infected mosquitoes, principally Aedes aegypti. DENV infections can be asymptomatic or cause a spectrum of illnesses that range from mild dengue fever to a severe, life-threatening disease characterized by dengue hemorrhagic fever/dengue shock syndrome (13, 38). The four DENV serotypes (DENV type 1 [DENV-1] to DENV-4) are the most important members of the genus Flavivirus in terms of morbidity, geographic distribution, and socioeconomic burden (1, 12). Several other members of the flaviviruses, including yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus, and tick-borne encephalitis virus, are also important human pathogens.The flavivirus virion is a spherical enveloped particle with icosahedral symmetry. It has a relatively simple structure, consisting of an inner nucleocapsid-virus RNA core and an outer lipid bilayer membrane into which a small ∼9-kDa membrane protein (M) and a larger ∼54-kDa envelope protein (E) are embedded. The E protein, which is approximately 500 amino acids in length, is the major antigen responsible for attachment to the cell surface, viral entry mediated by endocytosis, fusion with endosomal membranes, and the eliciting of host immune responses. There are 180 copies of E in the form of homodimers arranged in a tight array on the smooth virion surface without major spikes (21, 37, 48). Structural analysis indicates that each E monomer is folded into three structurally distinct domains, termed domains I, II, and III (DI, DII, and DIII, respectively). DIII has an immunoglobulin-like fold, a structural feature shared by many cell-adhesive molecules and receptor-binding proteins. DIII has been proposed to be responsible for binding interaction with cell surface receptors (16, 48). A number of mosquito-borne flavivirus E proteins contain a sequence motif in DIII that is recognized by integrin receptors. Mutations affecting cell attachment that cluster in this region are associated with attenuation of virulence and cell tropism (26, 27, 29, 43, 53).A specific cell surface receptor has not been clearly identified for DENV or any other flavivirus. Studies focusing on the mechanisms of viral binding and entry in mosquito C6/36 cells (42, 56) or mammalian cells (35, 41) have suggested a number of proteins of various sizes that are capable of binding the DENV virion. Recently, the C-type lectin DC-SIGN was found to be capable of facilitating DENV infection of dendritic cells (51, 52). It has been proposed that flaviviruses could also utilize other less specific molecules on the cell surface as coreceptors for initial adsorption and infection. Infection of DENV-2 was first found to depend on heparan sulfate (HS), a major constituent of the extracellular matrix and a surface component of most mammalian cells, for binding interaction and infectivity of cultured cells (6). In that study the authors identified sequences of two HS binding sites in E, one in DIII and the other in the junction between DI and DIII. Although HS is essential for coordination of various cellular functions (10), the role of HS in mediating viral entry for infection of susceptible mammalian hosts or insect vectors is less defined. Studies have shown that propagation of wild-type strains of DENV adaptively selects variants to replicate in certain mammalian cell cultures, including rodent-derived BHK-21 (kidney fibroblast) and human SW-13 (adrenal carcinoma) cell lines (28, 33). These variants acquire mutations in E, creating new HS binding sites and facilitating interactions to gain an entry into these cells. Such adaptive selection of variants involving binding to HS also appears to be a common mechanism for other single-stranded RNA viruses, including alphaviruses and foot-and-mouth disease virus. Analysis of the biological properties of these HS binding variants demonstrated attenuation of virulence and restriction of cell tropism (2, 4, 15, 18, 26, 49).Passage of wild-type YFV in animals and in cell cultures was successfully employed to produce the live-attenuated 17D vaccine several decades ago, and, more recently, the live-attenuated Japanese encephalitis vaccine SA14-14-2 was similarly produced (17, 39). In an effort to develop a DENV vaccine, investigators have sought to attenuate the virus by serial passage in primary dog kidney (PDK) cells or selection of small plaque-forming viruses in cultured cells (9, 14). Depending on the DENV serotype, various passage levels in PDK cells have been empirically derived in order to produce attenuated live vaccines. Final passage in fetal rhesus lung (FRhL) cells is frequently used for virus seed and vaccine lot production (8). The FRhL cell strain is a normal diploid cell suitable for the production of vaccines for human use (55). These cells have been shown to support the replication of all four DENV serotypes to high titers (9, 31).The availability of DENV cDNA clones has made it possible to modify the viral genome in order to derive growth-restricted and chimeric DENV mutants for the production of live vaccine candidates (7, 22, 23). Earlier, a series of DENV-4 mutants containing deletions in the 3′ noncoding region (NCR) was generated and shown to be attenuated for replication in cultured cells and in nonhuman primates (36). In an effort to develop DENV vaccine candidates with defined attenuating mutations, these viruses were propagated in FRhL cells for vaccine production and then tested in rhesus macaques. Unexpectedly, the animals failed to develop either antibody responses or viremia after inoculation. In the present report, we present evidence that passage of these DENV-4 constructs in FRhL cells rapidly selected for variants containing a single mutation in E that resulted in increased virus binding to heparin, a highly sulfated form of HS, and the loss of infectivity for primates.     

Identification of Five Interferon-Induced Cellular Proteins That Inhibit West Nile Virus and Dengue Virus Infections

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-α for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-α is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause invasive neurological diseases and lethal hemorrhagic fever in humans, respectively (6, 32). Since its first incursion into New York City in 1999, WNV has rapidly spread throughout the continental United States and has recently reached South America (29, 34). In most cases, WNV infection of people resolves as an asymptomatic or a mild febrile illness. However, approximately 1% of infections result in severe neurological disorders, such as encephalitis and meningitis (27). Unlike WNV, for which people are only accidental hosts, DENV has fully adapted to humans (32). It has apparently lost the need for an enzootic cycle and causes a range of diseases in people, from acute febrile illness to life-threatening dengue hemorrhagic fever/dengue shock syndrome (6). Four distinct serotypes of DENV have spread throughout the tropical and subtropical parts of the world, with an estimated 50 to 100 million human cases annually and about 2.5 billion people worldwide being at risk of infection (32). Effective antiviral therapies and vaccines to treat or prevent WNV and DENV infections in humans are not yet available.Type I interferons (IFNs), represented by IFN-α and IFN-β, have been demonstrated to play an essential role in defending against WNV and DENV infections. For example, mice with deficiencies in the induction of type I IFNs and the receptor or JAK-STAT signal transduction pathway of the cytokines are vulnerable to WNV and DENV infections (7, 38, 42, 49-51). In addition, a strain of WNV that fails to block the type I IFN signal transduction pathway is phenotypically attenuated in mice (23, 50). Clinically, during acute DENV infection, innate immune responses play a key role in determining disease outcome (35), and resolution of WNV infection requires effective IFN-mediated innate host responses (23, 43, 53). Therefore, understanding how the IFN-mediated innate immune response functions is one of the critical frontiers in the molecular biology of WNV and DENV pathogenesis (1, 44).IFNs inhibit virus infection by induction of IFN-stimulated genes (ISGs) that disrupt distinct steps of the viral replication cycle (47). However, although IFN treatment of cells induces the expression of hundreds of cellular genes (9), only approximately a dozen ISGs have been experimentally demonstrated to instigate an antiviral state against selected viruses (41). As mentioned above, although there is ample evidence suggesting that IFN-mediated innate immunity plays a critical role in defending against WNV and DENV infections, the underlying antiviral mechanism of the cytokines remains to be understood (6, 16, 31). With WNV, previous studies suggested that mice lacking double-stranded-RNA-activated protein kinase (PKR) and RNase L were more susceptible to the virus infection and had increased viral loads in multiple peripheral organs and neuronal tissues, in comparison with congenic wild-type mice (43). In addition, genetic studies showed that a nonsense mutation in the gene encoding the 2′,5′-oligoadenylate synthetase 1b (OAS1b) isoform was associated with WNV susceptibility in mice, and expression of wild-type OAS1b in mouse fibroblasts efficiently inhibited WNV infection (22, 33, 37, 45). For DENV, it was reported recently that viperin was among the highly induced ISGs in DENV-infected cells and overexpression of viperin in A549 cells significantly reduced DENV replication (13).In principle, to understand how IFNs inhibit DENV and WNV infections, it is essential to know the repertoire of ISGs that are directly implicated in antiviral action and understand how these antiviral ISGs work individually and coordinately to limit virus replication. To achieve this goal, we set out to systematically identify the ISGs that are able to inhibit infection with the two viruses and elucidate their antiviral mechanisms.     

Human Monoclonal Antibodies against West Nile Virus Induced by Natural Infection Neutralize at a Postattachment Step

West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies against the DIII-lr epitope comprise, at best, a minor component of the human anti-WNV antibody response. Herein, we characterize in detail two WNV-specific human MAbs, CR4348 and CR4354, that were isolated from B-cell populations of convalescent patients. These MAbs strongly neutralize WNV infection of cultured cells, protect mice against lethal infection in vivo, and yet poorly recognize recombinant forms of the E protein. Instead, CR4348 and CR4354 bind determinants on intact WNV virions and subviral particles in a pH-sensitive manner, and neutralization is altered by mutations at the dimer interface in domain II and the hinge between domains I and II, respectively. CR4348 and CR4354 human MAbs neutralize infection at a postattachment step in the viral life cycle, likely by inhibiting acid-induced fusion within the endosome.West Nile encephalitis virus (WNV) is a positive-polarity, single-stranded RNA virus of the genus Flavivirus within the family Flaviviridae. Other members of this genus that cause significant human disease include dengue virus (DENV), St. Louis encephalitis virus, Japanese encephalitis virus (JEV), yellow fever virus, and tick-borne encephalitis virus (TBEV). Flaviviruses are translated as a single polypeptide, which is then cleaved by host and viral proteases into three structural (capsid [C], premembrane [prM], and envelope [E]) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (reviewed in references 42 and 43).WNV cycles in nature between several species of birds and Culex mosquitoes, with humans and other mammals as dead-end hosts (25, 62). Infection causes syndromes ranging from a mild febrile illness to severe encephalitis and death (13, 72). WNV has spread globally and causes outbreaks with thousands of severe human cases annually in the United States. An age of greater than 55 years, a compromised immune status, and a CC5Δ32 genotype have been associated with more-severe disease (15, 20). There is currently no approved vaccine or therapy for WNV infection.The mature WNV virion has a ∼500-Å diameter and consists of a single RNA genome surrounded by the capsid protein, a lipid bilayer, and a shell of the prM/M and E proteins (31, 55). X-ray crystallography studies have elucidated the three-domain structure of the flavivirus E protein (30, 48, 50, 58, 67). Domain I (DI) is a central, eight-stranded β-barrel, which contains the only N-linked glycosylation site in WNV E. Domain II (DII) is a long, finger-like protrusion from DI and contains the highly conserved fusion peptide at its distal end. Domain III (DIII) adopts an immunoglobulin-like fold at the opposite end of DI and is believed to contain a site for receptor attachment (6, 8, 40).Within an infected cell, progeny WNV are assembled initially as immature particles. In immature virions, three pairs of E and prM interact as trimers and form 60 spiked projections with icosahedral symmetry (85, 86). Exposure to mildly acidic conditions in the trans-Golgi secretory pathway promotes virus maturation through a structural rearrangement of the E proteins and cleavage of prM to M by a furin-like protease (41, 83). Mature WNV virions are covered by 90 antiparallel E protein homodimers, which are arranged flat along the surface in a herringbone pattern with quasi-icosahedral symmetry (55).Upon binding to poorly characterized cell surface receptors, internalization of WNV is believed to occur through receptor-mediated, clathrin-dependent endocytosis (1, 79, 80). After trafficking to Rab5- and/or Rab7-positive endosomes (38, 79), the mildly acidic pH within the lumen of the endosome induces structural alterations in the flavivirus E protein (7, 49), which includes changes in its oligomeric state (7, 49, 77). During this process, also known as type II fusion, the hydrophobic peptide on the fusion loop of DII of the E protein inserts into the endosomal membrane, thus physically joining the host and viral membranes, which allows the infectious RNA genome to enter the cytoplasm (32, 33).Humoral immunity is an essential component of the protective host response against flaviviruses including WNV (reviewed in references 64 and 68). Studies by several groups have shown that the neutralization of WNV can occur after antibodies bind to a series of discrete epitopes on all three domains of the E protein (3, 12, 22, 59, 61, 71). To date, the most potently neutralizing monoclonal antibodies (MAbs) localize to an epitope on the lateral ridge of DIII (DIII-lr). One well-characterized strongly neutralizing mouse MAb, E16, blocks infection primarily at a postattachment step (57) and requires the engagement of only a fraction of its epitopes on the surface of the virion (66). Studies of the human antibody response to WNV infection reveal that, in contrast to mice, antibodies that bind the DIII-lr epitope comprise a minor component of the neutralizing humoral response in most individuals (60).In this study, we characterized two strongly neutralizing novel human MAbs (CR4348 and CR4354) that were selected from an antibody phage display library constructed from B cells of subjects that survived WNV infection (78). We demonstrate that both MAbs are WNV specific, bind weakly to recombinant or yeast surface-displayed E proteins, exhibit pH-sensitive binding to viral particles, and protect against lethal infection in mice. Our experiments suggest that these human MAbs map to distinct epitopes and neutralize infection at a postattachment stage, likely by inhibiting the acid-catalyzed viral fusion step.     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

An immunogenic and protective alphavirus replicon particle-based dengue vaccine overcomes maternal antibody interference in weanling mice

A candidate pediatric dengue virus (DENV) vaccine based on nonpropagating Venezuelan equine encephalitis virus replicon particles (VRP) was tested for immunogenicity and protective efficacy in weanling mice in the presence and absence of potentially interfering maternal antibodies. A gene cassette encoding envelope proteins prM and E from mouse-adapted DENV type 2 (DENV2) strain NGC was cloned into a VEE replicon vector and packaged into VRP, which programmed proper in vitro expression and processing of DENV2 envelope proteins upon infection of Vero cells. Primary immunization of 3-week-old weanling BALB/c mice in the footpad with DENV2 VRP resulted in high levels of DENV-specific serum immunoglobulin G antibodies and significant titers of neutralizing antibodies in all vaccinates. A booster immunization 12 weeks after the prime immunization resulted in increased neutralizing antibodies that were sustained for at least 30 weeks. Immunization at a range of doses of DENV2 VRP protected mice from an otherwise-lethal intracranial DENV2 challenge. To model vaccination in the presence of maternal antibodies, weanling pups born to DENV2-immune or DENV2-naïve dams were immunized with either DENV2 VRP or live DENV2 given peripherally. The DENV2 VRP vaccine induced neutralizing-antibody responses in young mice regardless of the maternal immune status. In contrast, live-DENV2 vaccination performed poorly in the presence of preexisting anti-DENV2 antibodies. This study demonstrates the feasibility of a VRP vaccine approach as an early-life DENV vaccine in populations with high levels of circulating DENV antibodies and suggests the utility of VRP-based vaccines in other instances where maternal antibodies make early vaccination problematic.Dengue viruses (DENV) are members of the family Flaviviridae and one of the most important groups of emerging viruses of global significance today (36, 66). There are four distinct antigenic serotypes (DENV1, DENV2, DENV3, and DENV4), all of which are capable of causing a spectrum of diseases in humans ranging from asymptomatic infections to debilitating classical dengue fever and severe and often fatal dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (36, 68). DENV is transmitted to humans primarily by the mosquito Aedes aegypti. The lack of effective mosquito control, as well as demographic and economic changes, has contributed to the dramatic expansion and worldwide distribution of DENV epidemic activity in tropical and subtropical areas (36). It is estimated that up to 100 million infections and several hundred thousand cases of DHF/DSS occur each year, with more than 2.5 billion people living in areas at risk of infection in 2004 (21, 68). DHF is a leading cause of hospitalization and death among children in many countries in Southeast and South Asia, and the WHO has reported a rising trend in disease over the past decade (68). At the peak of epidemic times, as many as 70 children with severe DHF may present to a single hospital in a day, 20 of them with potentially fatal DSS (58). Although DHF/DSS in infants has not been comprehensively studied, it is estimated that more than 5% of all DHF/DSS cases occur in infants (26, 33, 41, 43, 56, 67, 70).In the absence of vector control effective on a global scale, there is a clear need for a DENV vaccine. However, the development of a DENV vaccine has faced significant challenges that have resulted in the lack of a licensed vaccine after 70 years of research (17). In many areas where there is cocirculation of two or more serotypes, there is a high probability that individuals will be infected more than once in their lifetimes. Preexisting homotypic immunity protects from a secondary infection with the same serotype, and this protection seems to last for life (24, 25). However, preexisting heterotypic nonneutralizing immunity to a secondary infection with a different DENV serotype is a risk factor for the development of severe DHF/DSS (23, 27, 61). These considerations suggest that a safe and efficacious DENV vaccine must be tetravalent and induce a long-term and balanced immune response to all four serotypes simultaneously in order to avoid sensitizing the vaccine recipient to a more severe outcome during a subsequent DENV infection. Additionally, primary infections during the first year of life that result in DHF/DSS have been associated with the presence of subneutralizing levels of maternal anti-DENV antibodies, which may increase the risk of enhanced infection and disease by antibody-mediated enhancement (26, 33, 41, 56). To protect infants and children in dengue-endemic countries from severe dengue, the ideal DENV vaccine should be given during the first 6 months of life. In addition, an infant DENV vaccine has to be effective in the face of circulating anti-DENV maternal antibodies, which in dengue-endemic countries are present in more than 95% of newborns and have disappeared by 12 months of age (63).There are a number of DENV vaccine candidates in preclinical and clinical trials (reviewed in references 10 and 66), including live attenuated virus, DNA plasmids (49), subunit vaccines (11, 16), and adenovirus vectors (29, 31). Live attenuated virus vaccines are the more advanced candidates in phase I and II clinical trials. They have been attenuated either empirically (4), by engineering attenuating mutations into a DENV cDNA infectious clone (5, 15), or by chimerization with other flaviviruses (22, 39, 46). Further clinical development of these candidates has been delayed due to several problems. (i) Balanced immune responses to the four serotypes have proven difficult to achieve with tetravalent cocktails of live vaccine candidates, in which each component differs in its level of attenuation or in which interference among the live components of the vaccine may occur. (ii) Determination of virulence in primate models may not accurately predict attenuation for humans. In fact, an attenuated DENV3 candidate vaccine that was deemed safe in mice and primates produced dengue fever in human volunteers (51). (iii) In many DENV-endemic regions of Asia, the dengue seroprevalence is very high, and over 95% of children born have maternal dengue antibody. Human safety as assessed in a phase I trial in seronegative populations may not accurately reflect safety in persons seropositive for one of the DENV serotypes or infants with maternal antibodies. (iv) The presence of such antibodies also might interfere with live attenuated dengue vaccines. If vaccine is administered during the first year of life, passively transferred anti-DENV maternal antibodies would likely interfere with the replication and immunogenicity of one or more components of the tetravalent cocktail. If the vaccine is administered later in childhood or in adulthood, antibodies to an earlier natural infection may be boosted and yet interfere with the immunogenicity of a heterologous component of the multivalent live vaccine.We propose that nonpropagating Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) are well suited to address the difficulties faced in DENV vaccine development. Three properties of the VEE vectors may contribute to their ability to overcome maternal-antibody interference to a significant degree. (i) The DENV antigens are not exposed on the VRP surface; therefore, preexisting DENV-neutralizing antibodies should not affect delivery of the DENV genes to the target cells. (ii) Unlike live attenuated vaccines that depend on multiple rounds of replication and are thus more susceptible to interference by preexisting anti-DENV antibodies, nonpropagating VRP vectors express high levels of the heterologous gene in a single round of infection. (iii) Due to the tropism mediated by the VEE glycoproteins that targets the VRP to the lymph node (35), and due to the adjuvant activity of the VRP (57), antigen presentation is facilitated and enhanced.The safety of nonpropagating VEE replicon vectors has been tested in many different animals, including over 2,000 rodents, 100 macaques, and more than 20 horses. No clinical signs of disease have been observed with any of these animals, including neonatal mice inoculated intracranially (i.c.) with 5 × 10⁷ infectious units (IU) and RAG^−/− mice inoculated with 10⁷ IU of a VRP vaccine (48; A. West and N. Davis, personal communication). Safety has also been demonstrated in young adult volunteers in the United States, South Africa, and Botswana undergoing phase I clinical trials with a VRP expressing the Gag protein of clade C human immunodeficiency virus type 1. No serious adverse events were reported with doses as high as 10⁸ IU (12). VRP vectors confer long-lived humoral and cellular immune responses to a wide variety of viral and bacterial antigens tested in animal models, resulting in strong and complete protective immune responses to influenza virus in rodents and chickens (48, 52), Lassa fever and ebola viruses in rodents (69), equine arteritis virus in rodents and horses (2), and Marburg virus in primates (28).Here, we demonstrate the ability of VRP vaccine vectors to deliver the immunogenic membrane prM and E protein genes of DENV2 into young mice and to induce a protective humoral immune response, even in the presence of maternal antibodies that otherwise interfere with immunization with a model live DENV2 vaccine. This study shows the feasibility of a VRP vaccine approach as an early-life DENV vaccine to protect infants during that window of time when maternal antibodies are no longer protective but still may interfere with active immunization induced by a live attenuated vaccine.     

Identification of B-Cell Epitopes on Virus-Like Particles of Cutaneous Alpha-Human Papillomaviruses

Human papillomavirus (PV) (HPV) types 2, 27, and 57 are closely related and, hence, represent a promising model system to study the correlation of phylogenetic relationship and immunological distinctiveness of PVs. These HPV types cause a large fraction of cutaneous warts occurring in immunocompromised patients. Therefore, they constitute a target for the development of virus-like particle (VLP)-based vaccines. However, the immunogenic structure of HPV type 2, 27, and 57 capsids has not been studied yet. Here we provide, for the first time, a characterization of the B-cell epitopes on VLPs of cutaneous alpha-HPVs using a panel of 94 monoclonal antibodies (MAbs) generated upon immunization with capsids from HPV types 2, 27, and 57. The MAbs generated were characterized regarding their reactivities with glutathione S-transferase-L1 fusion proteins from 18 different PV types, the nature of their recognized epitopes, their isotypes, and their ability to neutralize HPV type 2, 27, 57, or 16. In total, 33 of the 94 MAbs (35%) showed type-specific reactivity. All type-specific MAbs recognize linear epitopes, most of which map to the hypervariable surface loop regions of the L1 amino acid sequence. Four of the generated MAbs neutralized pseudovirions of the inoculated HPV type efficiently. All four MAbs recognized epitopes within the BC loop, which is required and sufficient for their neutralizing activity. Our data highlight the immunological distinctiveness of individual HPV types, even in comparison to their closest relatives, and they provide a basis for the development of VLP-based vaccines against cutaneous alpha-HPVs.Recently licensed prophylactic vaccines confer efficient protection against infections by human papillomavirus (PV) (HPV) types 16 and 18, thereby aiming to prevent approximately 70% of all cervical cancer cases (17, 39). These vaccines are composed of virus-like particles (VLPs), which spontaneously assemble from the major capsid protein L1 via 72 pentamers (capsomeres) as subunits (2, 23, 26).In the process of vaccine development, monoclonal antibodies (MAbs) proved to be valuable tools for the immunological analysis of recombinantly produced capsids and capsomeres (51) as well as for serological studies (25, 49, 56). Moreover, the identification and characterization of many neutralizing epitopes of HPV types 11 and 16 have been facilitated by the employment of MAbs (6, 11, 30-32, 41, 42, 55). Such epitopes to neutralizing antibodies are mostly conformation dependent, but a few neutralizing MAbs that recognize linear epitopes have also been generated (16, 18). Most neutralizing MAbs are HPV type specific due to the hypervariable nature of their respective epitopes, which typically reside in the surface-exposed loop regions of the L1 protein (10). In contrast, cross-reactive MAbs targeting rather conserved L1 epitopes are generally nonneutralizing.HPV types 2, 27, and 57 are the three members of Alphapapillomavirus species 4 (20). They are very closely related, and HPV types 2 and 27 hardly fulfill the requirement of more than 10% nucleotide variation in the L1 open reading frame to be classified as distinct types (8). Therefore, they represent a promising model system to study the immunological distinctiveness of closely related HPV types. Pathologically, HPV types 2, 27, and 57 infect primarily the cutaneous epithelia, thereby causing common skin warts, which often occur ubiquitously and confluently in immunocompromised patients (1, 24, 28). It is our long-term goal to develop a prophylactic L1 VLP-based vaccine to alleviate the burden provoked by HPV-induced skin lesions in these patients. However, to date, neither the structure nor the immunogenicity of HPV type 2, 27, and 57 capsids has been elucidated.The purpose of the present study was twofold. First, we sought to generate MAbs specific for HPV types 2, 27, and 57 as tools for type-specific diagnostic assays. Second, we aimed to exploit the generated MAbs for an investigation of the B-cell epitopes on capsids of HPV types 2, 27, and 57.     

Virus-Specific Cytolytic Antibodies to Nonstructural Protein 1 of Japanese Encephalitis Virus Effect Reduction of Virus Output from Infected Cells

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.The genus Flavivirus, many of whose more than 70 members are arthropod-borne human pathogens, such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), tick-borne encephalitis virus, and Japanese encephalitis virus (JEV), has assumed increasing public health importance in recent years. The single-strand, positive-sense RNA genomes of flaviviruses encode a single polyprotein, which is cotranslationally cleaved to produce three structural proteins (capsid [C], membrane [M], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). NS1, a glycoprotein, is perhaps the most versatile among these, being involved both in vital processes such as viral RNA synthesis and in multiple interactions with the host, in ways that appear to benefit both pathogen and host. Following translocation into the lumen of the endoplasmic reticulum through a hydrophobic signal sequence that is encoded by the carboxyl terminus of E (17), NS1 undergoes glycosylation followed by rapid dimerization (44, 52). In DENV infection of cultured mammalian cells, extracellular NS1 was additionally detected as hexamers (19, 51). Despite the apparent absence of a canonical hydrophobic membrane anchor domain, the NS1s from JEV, Kunjin virus, DENV, and YFV have all been shown to be present on the surface of virus-infected cells (8, 23, 50). The mechanistic details of this membrane anchor still remain uncertain.The ability of DENV NS1 to bind host complement (9, 49) pointed to a role for this protein in DENV pathogenesis. Serum NS1 levels in both DENV and WNV patients correlate directly with disease severity (1, 36). Promotion of immune complex formation (54), ability to elicit autoantibodies with reactivity to platelets and extracellular matrix (10), and damage inflicted on endothelial cells (34) are some of the mechanisms proposed to explain pathogenesis mediated by DENV NS1. Recent studies with WNV NS1 demonstrated its ability to bind human complement factor H, suggesting a role in reducing the host''s ability to bring about complement-mediated control of early virus replication (11).Critical differences between the functions of NS1s encoded by different pathogenic flaviviruses and their contributions to pathology are evident from the published reports, with DENV NS1 believed to be involved in complement activation and the consequent capillary leak syndrome of dengue hemorrhagic fever (6), while WNV NS1 appears relatively more benign and has more to do with modulation of the host innate immune response (11). We have not encountered reports of adverse impacts of JEV NS1 in infected individuals.Paradoxically, several studies have pointed to a role for flavivirus NS1-specific immune responses in protection against flaviviruses. Passive immunization studies using monoclonal antibodies (MAbs) (24, 28, 29, 55) as well as immunization of mice using naked DNA constructs expressing NS1 (35, 40) revealed that antibodies directed to prM or E of DENV and NS1 of DENV and JEV are protective. Studies by different groups have shown that active immunization with purified NS1 or passive immunization with MAbs against YFV and DENV NS1 provides protection from lethal viral challenge in the absence of neutralizing antibodies (24, 45, 48). A panel of anti-WNV NS1 MAbs revealed multiple antibody-mediated mechanisms for protection, some mediated through complement and others via the Fc receptor (12). Those authors went on to show that anti-NS1 MAbs that facilitate phagocytosis and clearance of WNV-infected cells through Fc-γ receptors I and/or IV belonged to the IgG2a subclass and bound to cell surface-associated NS1 (13).Earlier studies also pointed to the cytolytic potential of NS1 antibodies, a property that might contribute significantly to their protective ability. Passive immunization experiments using a panel of anti YFV NS1-specific MAbs showed a significant correlation between protection and in vitro complement-mediated cytolysis of YFV-infected mouse neuroblastoma cells (47). Additionally, immunization of mice with a DNA vaccine construct carrying JEV NS1 induced a strong antibody response exhibiting complement-mediated cytolysis of JEV-infected cells (35), but no neutralizing activity, and resulted in protection against subsequent challenge with virus. Cell-mediated immune responses directed to NS1 of JEV have also been reported to play a role in cytotoxic T-lymphocyte-mediated killing of JEV-infected murine target cells (41). Thus, NS1 appears to contribute to protection in the murine model by inducing both humoral and cell-mediated arms of the immune response.It was therefore of interest to query whether NS1-specific antibodies in humans exposed to JEV exhibit cytolytic activity and to determine if these antibodies are capable of reducing virus production by infected cells. In this study we report for the first time the existence of detectable levels of anti-NS1 antibodies in a significant proportion of sera from humans infected with JEV and demonstrate their ability to induce antibody-dependent complement-mediated cytolysis of cells expressing JEV NS1 on the surface. These sera failed to cause lysis of cells infected with WNV or DENV, both of which cocirculate with JEV in the Indian subcontinent and have been reported in the region where we enrolled our volunteers, revealing stringent specificity and absence of flaviviral cross-reactivity for these cytolytic antibodies. Furthermore, we demonstrate the ability of NS1-specific antibodies elicited in mice to limit virus production in infected human SW-13 cell monolayers, which may explain, at least in part, the widely reported protective ability of flavivirus NS1. Significantly, we found no evidence for the ability of NS1 from JEV to bind human complement factor H, in contrast to the case for WNV NS1 (11). Taken together, these findings suggest that JEV NS1 may positively and significantly affect virus-specific protective immune responses.     

A Short N-Terminal Peptide Motif on Flavivirus Nonstructural Protein NS1 Modulates Cellular Targeting and Immune Recognition

Flavivirus NS1 is a versatile nonstructural glycoprotein, with intracellular NS1 functioning as an essential cofactor for viral replication and cell surface and secreted NS1 antagonizing complement activation. Even though NS1 has multiple functions that contribute to virulence, the genetic determinants that regulate the spatial distribution of NS1 in cells among different flaviviruses remain uncharacterized. Here, by creating a panel of West Nile virus-dengue virus (WNV-DENV) NS1 chimeras and site-specific mutants, we identified a novel, short peptide motif immediately C-terminal to the signal sequence cleavage position that regulates its transit time through the endoplasmic reticulum and differentially directs NS1 for secretion or plasma membrane expression. Exchange of two amino acids within this motif reciprocally changed the cellular targeting pattern of DENV or WNV NS1. For WNV, this substitution also modulated infectivity and antibody-induced phagocytosis of infected cells. Analysis of a mutant lacking all three conserved N-linked glycosylation sites revealed an independent requirement of N-linked glycans for secretion but not for plasma membrane expression of WNV NS1. Collectively, our experiments define the requirements for cellular targeting of NS1, with implications for the protective host responses, immune antagonism, and association with the host cell sorting machinery. These studies also suggest a link between the effects of NS1 on viral replication and the levels of secreted or cell surface NS1.West Nile virus (WNV) is a single-stranded, positive-sense enveloped RNA Flavivirus that cycles in nature between birds and Culex mosquitoes. It is endemic in parts of Africa, Europe, the Middle East, and Asia, and outbreaks occur annually in North America. More than 29,000 human cases of severe WNV infection have been diagnosed in the United States since its entry in 1999, and millions have been infected and remain undiagnosed (9). Humans can develop a febrile illness that progresses to a flaccid paralysis, meningitis, or encephalitis syndrome (59). Dengue virus (DENV) is a genetically related flavivirus that is transmitted by Aedes aegypti and Aedes albopictus mosquitoes and causes clinical syndromes in humans, ranging from an acute self-limited febrile illness (dengue fever [DF]) to a severe and life-threatening vascular leakage and bleeding diathesis (dengue hemorrhagic fever/dengue shock syndrome [DHF/DSS]). Globally, DENV causes an estimated 50 million infections annually, resulting in 500,000 hospitalizations and ∼22,000 deaths (45).The ∼10.7-kb Flavivirus RNA genome is translated as a single polyprotein, which is then cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by virus- and host-encoded proteases (39). The multifunctional NS proteins include an RNA-dependent RNA polymerase and methyltransferase (NS5), a helicase and protease (NS3), accessory proteins that form part of the viral replication complex, and immune evasion molecules (33, 34). Flavivirus NS1 is a 48-kDa nonstructural glycoprotein with two or three N-linked glycans, depending on the flavivirus, and is absent from the virion. The Japanese encephalitis virus (JEV) serogroup (West Nile, Japanese, Murray Valley, and St. Louis encephalitis viruses) generate NS1 and NS1′ proteins, the latter of which is a product of a ribosomal frameshift event that occurs at a heptanucleotide motif located at the beginning of the NS2A gene (25, 47).NS1 is an essential gene as it is required for efficient viral RNA replication (34, 41, 44). In infected mammalian cells, NS1 is synthesized as a soluble monomer, dimerizes after posttranslational modification in the lumen of the endoplasmic reticulum (ER), and accumulates extracellularly as higher-order oligomers, including hexamers (16, 26, 64, 65). Soluble NS1 binds back to the plasma membrane of uninfected cells through interactions with sulfated glycosaminoglycans (5). In infected cells, NS1 is also directly transported to and expressed on the plasma membrane although it lacks a transmembrane domain or canonical targeting motif. The mechanism of cell surface expression of flavivirus NS1 in infected cells remains uncertain although some fraction may be linked through an atypical glycosyl-phosphatidylinositol anchor (30, 50) or lipid rafts (49).NS1 has been implicated in having pathogenic consequences in flavivirus infection. The high levels of NS1 in the serum of DENV-infected patients correlate with severe disease (4, 37). NS1 has been proposed to facilitate immune complex formation (4), elicit auto-antibodies that react with host matrix proteins (21), damage endothelial cells via antibody-dependent complement-mediated cytolysis (38), or directly enhance infection (1). Flavivirus NS1 also has direct immune evasion functions and antagonizes complement activation on cell surfaces and in solution. WNV NS1 attenuates the alternative pathway of complement activation by binding the complement-regulatory protein factor H (11, 36), and DENV, WNV, and YFV NS1 proteins bind C1s and C4 in a complex to promote efficient degradation of C4 to C4b (3).Although NS1 is absent from the virion, antibodies against it can protect against infection in vivo. Immunization with purified NS1 or passive administration of some anti-WNV, anti-yellow fever virus (YFV), and anti-DENV NS1 monoclonal antibodies (MAbs) protect mice against lethal virus challenge (12, 13, 17, 22, 27, 29, 31, 32, 56-58). Initial studies with isotype switch variants and F(ab′)₂ fragments of anti-YFV NS1 MAbs suggested that the Fc region of anti-NS1 MAbs was required for protection (58). Subsequent mechanistic studies with WNV NS1 indicated that only MAbs recognizing cell surface-associated NS1 trigger Fc-γ receptor I- and/or IV-mediated phagocytosis and clearance of infected cells (13).In this study, we identify a reciprocal relationship between the secretion and cell surface expression patterns of WNV and DENV NS1s. Using WNV-DENV NS1 chimeras and point mutants, we identified a novel short peptide motif immediately C-terminal to the signal sequence cleavage position that directs NS1 for secretion or to the plasma membrane. These studies begin to explain how NS1 regulates its localization to several cellular compartments (ER, cell surface, and extracellular space) and have implications for viral infectivity, association with the host cell sorting machinery, and protective immune responses.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC₉₀] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5, 33, 45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12, 23, 64, 65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21, 37, 44, 54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17, 43, 44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1, 18, 21, 36, 62, 63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15, 43, 55, 61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22, 28, 48, 63).In immunocompetent individuals T-cell and antibody responses efficiently control HCMV infection and reduce pathological consequences of maternal-fetal transmission (13, 67), although this is usually not sufficient to eradicate the virus. Albeit with controversial results, HCMV immunoglobulins (Igs) have been administered to transplant patients in association with immunosuppressive treatments for prophylaxis of HCMV disease (56, 57), and a recent report suggests that they may be effective in controlling congenital infection and preventing disease in newborns (32). These products are plasma derivatives with relatively low potency in vitro (46) and have to be administered by intravenous infusion at very high doses in order to deliver sufficient amounts of neutralizing antibodies (4, 9, 32, 56, 57, 66).The whole spectrum of antigens targeted by HCMV-neutralizing antibodies remains poorly characterized. Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6, 14, 26, 29, 34, 41, 52, 60). Remarkably, we have recently shown that human sera exhibit a more-than-100-fold-higher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20). Similarly, CMV hyperimmunoglobulins have on average 48-fold-higher neutralizing activities against epithelial cell entry than against fibroblast entry (10). However, epitopes that are targeted by the antibodies that comprise epithelial or endothelial cell-specific neutralizing activity of human immune sera remain unknown.In this study we report the isolation of a large panel of human monoclonal antibodies with extraordinarily high potency in neutralizing HCMV infection of endothelial and epithelial cells and myeloid cells. With the exception of a single antibody that recognized a conserved epitope of UL128, all other antibodies recognized conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Inhibition of Dengue Virus Polymerase by Blocking of the RNA Tunnel

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)₂₀]. Chemical modification of the initial “hit” improved the compound potency to an IC₅₀ (that is, a concentration that inhibits 50% RdRp activity) of 0.7 μM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC₅₀ of 5 μM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC₅₀s of >100 μM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.The family Flaviviridae consists of three genera: Flavivirus, Pestivirus, and Hepacivirus. The genus Flavivirus contains about 73 viruses, many of which are arthropod-borne and pose major public health threats worldwide (15). The four serotypes of dengue virus infect 50 to 100 million people each year, with approximately 500,000 cases developing into life-threatening dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS), leading to about 20,000 deaths. In addition to DENV, West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV) also cause significant human diseases. No antiviral therapy is currently available for treatment of flavivirus infections. Human vaccines are only available for YFV, JEV, and TBEV (15). Development of antiviral therapy and new vaccines is urgently needed for flaviviruses.The flavivirus genome is a single-stranded RNA of plus-sense polarity. The genomic RNA contains a 5′ untranslated region (UTR), a single open reading frame, and a 3′ UTR. The single open reading frame encodes a long polyprotein that is processed by viral and host proteases into 10 mature viral proteins. Three structural proteins (Capsid [C], premembrane [prM], and envelope [E]) are components of virus particles. Seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are responsible for viral replication (40), virion assembly (19, 21, 24, 33), and innate immunity antagonism (4, 16, 23, 29, 30). Two viral proteins encode enzymatic activities that have been targeted for antiviral development. NS3 functions as a protease (with NS2B as a cofactor), helicase, 5′-RNA triphosphatase, and nucleoside triphosphatase (7, 14, 42). The N-terminal part of NS5 is a methyltransferase that methylates the N7 and 2′-O positions of the viral RNA cap structure (13, 18, 37); the C-terminal part of NS5 has an RNA-dependent RNA polymerase (RdRp) activity (1, 39). The RdRp activity is unique to RNA viruses and therefore represents an attractive antiviral target.Two types of inhibitors could be developed to suppress viral polymerases. Type 1 inhibitors are nucleoside/nucleotide analogs that function as RNA or DNA chain terminators; about half of the current antiviral drugs are nucleotide analogs (10). For flaviviruses, a nucleoside analog (7-deaza-2′-C-methyl-adenosine), originally developed for hepatitis C virus (HCV) RdRp, showed anti-DENV activity (32, 38). We recently reported a similar adenosine analog (7-deaza-2′-C-acetylene-adenosine) that potently inhibited DENV both in cell culture and in mice; unfortunately, this compound showed side effects during a 2-week in vivo toxicity study (44). Nevertheless, these studies have proved the concept that nucleoside analogs could potentially be developed for flavivirus therapy. Type 2 inhibitors are non-nucleoside inhibitors (NNI) which bind to allosteric pockets of protein to block enzymatic activities; the mechanism of action of NNI includes structural alteration of polymerase to an inactive conformation, blocking the conformational switch from polymerase initiation to elongation, or impeding the processivity of polymerase elongation (11). A broad range of chemical classes have been identified as NNI, including inhibitors of HIV (9, 35) and HCV (3, 5, 11, 25).In the present study, we performed high-throughput screening (HTS) to search for NNI of DENV RdRp. The HTS and chemistry synthesis led to the identification of N-sulfonylanthranilic acid derivatives as inhibitors of DENV RdRp. The compounds specifically inhibit DENV RdRp. UV cross-linking experiments mapped the compound binding site to the RdRp domain of DENV NS5. Amino acid Met343, located at the entrance of RNA template tunnel of the DENV RdRp, was cross-linked to the compound. These results, together with biochemistry experiments, suggest that the compound blocks the RdRp activity through binding to the RNA template tunnel of the polymerase.     

Role of Complex Carbohydrates in Human Immunodeficiency Virus Type 1 Infection and Resistance to Antibody Neutralization

Complex N-glycans flank the receptor binding sites of the outer domain of HIV-1 gp120, ostensibly forming a protective “fence” against antibodies. Here, we investigated the effects of rebuilding this fence with smaller glycoforms by expressing HIV-1 pseudovirions from a primary isolate in a human cell line lacking N-acetylglucosamine transferase I (GnTI), the enzyme that initiates the conversion of oligomannose N-glycans into complex N-glycans. Thus, complex glycans, including those that surround the receptor binding sites, are replaced by fully trimmed oligomannose stumps. Conversely, the untrimmed oligomannoses of the silent domain of gp120 are likely to remain unchanged. For comparison, we produced a mutant virus lacking a complex N-glycan of the V3 loop (N301Q). Both variants exhibited increased sensitivities to V3 loop-specific monoclonal antibodies (MAbs) and soluble CD4. The N301Q virus was also sensitive to “nonneutralizing” MAbs targeting the primary and secondary receptor binding sites. Endoglycosidase H treatment resulted in the removal of outer domain glycans from the GnTI- but not the parent Env trimers, and this was associated with a rapid and complete loss in infectivity. Nevertheless, the glycan-depleted trimers could still bind to soluble receptor and coreceptor analogs, suggesting a block in post-receptor binding conformational changes necessary for fusion. Collectively, our data show that the antennae of complex N-glycans serve to protect the V3 loop and CD4 binding site, while N-glycan stems regulate native trimer conformation, such that their removal can lead to global changes in neutralization sensitivity and, in extreme cases, an inability to complete the conformational rearrangements necessary for infection.The intriguing results of a recent clinical trial suggest that an effective HIV-1 vaccine may be possible (97). Optimal efficacy may require a component that induces broadly neutralizing antibodies (BNAbs) that can block virus infection by their exclusive ability to recognize the trimeric envelope glycoprotein (Env) spikes on particle surfaces (43, 50, 87, 90). Env is therefore at the center of vaccine design programs aiming to elicit effective humoral immune responses.The amino acid sequence variability of Env presents a significant challenge for researchers seeking to elicit broadly effective NAbs. Early sequence comparisons revealed, however, that the surface gp120 subunit can be divided into discrete variable and conserved domains (Fig. ​(Fig.1A)1A) (110), the latter providing some hope for broadly effective NAb-based vaccines. Indeed, the constraints on variability in the conserved domains of gp120 responsible for binding the host cell receptor CD4, and coreceptor, generally CCR5, provide potential sites of vulnerability. However, viral defense strategies, such as the conformational masking of conserved epitopes (57), have made the task of eliciting bNAbs extremely difficult.Open in a separate windowFIG. 1.Glycan biosynthesis and distribution on gp120 and gp41. (A) Putative carbohydrate modifications are shown on gp120 and gp41 secondary structures, based on various published works (26, 42, 63, 74, 119, 128). The gp120 outer domain is indicated, as are residues that form the SOS gp120-gp41 disulfide bridge. The outer domain is divided into neutralizing and silent faces. Symbols distinguish complex, oligomannose, and unknown glycans. Generally, the complex glycans of the outer domain line the receptor binding sites of the neutralizing face, while the oligomannose glycans of the outer domain protect the silent domain (105). Asterisks denote sequons that are unlikely to be utilized, including position 139 (42), position 189 (26, 42), position 406 (42, 74), and position 637 (42). Glycans shown in gray indicate when sequon clustering may lead to some remaining unused, e.g., positions 156 and 160 (42, 119), positions 386, 392, and 397 (42), and positions 611 and 616 (42). There is also uncertainty regarding some glycan identities: glycans at positions 188, 355, 397, and 448 are not classified as predominantly complex or oligomannose (26, 42, 63, 128). The number of mannose moieties on oligomannose glycans can vary, as can the number of antennae and sialic acids on complex glycans (77). The glycan at position 301 appears to be predominantly a tetra-antennary complex glycan, as is the glycan at position 88, while most other complex glycans are biantennary (26, 128). (B) Schematic of essential steps of glycan biosynthesis from the Man₉GlcNAc₂ precursor to a mature multiantennary complex glycan. Mannosidase I progressively removes mannose moieties from the precursor, in a process that can be inhibited by the drug kifunensine. GnTI then transfers a GlcNAc moiety to the D1 arm of the resulting Man₅GlcNAc₂ intermediate, creating a hybrid glycan. Mannose trimming of the D2 and D3 arms then allows additional GlcNAc moieties to be added by a series of GnT family enzymes to form multiantennary complexes. This process can be inhibited by swainsonine. The antennae are ultimately capped and decorated by galactose and sialic acid. Hybrid and complex glycans are usually fucosylated at the basal GlcNAc, rendering them resistant to endo H digestion. However, NgF is able to remove all types of glycan.Carbohydrates provide a layer of protection against NAb attack (Fig. ​(Fig.1A).1A). As glycans are considered self, antibody responses against them are thought to be regulated by tolerance mechanisms. Thus, a glycan network forms a nonimmunogenic “cloak,” protecting the underlying protein from antibodies (3, 13, 20, 29, 39, 54, 65, 67, 74, 85, 96, 98, 117, 119, 120). The extent of this protection can be illustrated by considering the ways in which glycans differ from typical amino acid side chains. First, N-linked glycans are much larger, with an average mass more than 20 times that of a typical amino acid R-group. They are also usually more flexible and may therefore affect a greater volume of surrounding space. In the more densely populated parts of gp120, the carbohydrate field may even be stabilized by sugar-sugar hydrogen bonds, providing even greater coverage (18, 75, 125).The process of N-linked glycosylation can result in diverse structures that may be divided into three categories: oligomannose, hybrid, and complex (56). Each category shares a common Man₃GlcNAc₂ pentasaccharide stem (where Man is mannose and GlcNAc is N-acetylglucosamine), to which up to six mannose residues are attached in oligomannose N-glycans, while complex N-glycans are usually larger and may bear various sizes and numbers of antennae (Fig. ​(Fig.1B).1B). Glycan synthesis begins in the endoplasmic reticulum, where N-linked oligomannose precursors (Glc₃Man₉GlcNAc₂; Glc is glucose) are transferred cotranslationally to the free amide of the asparagine in a sequon Asn-X-Thr/Ser, where X is not Pro (40). Terminal glucose and mannose moieties are then trimmed to yield Man₅GlcNAc₂ (Fig. ​(Fig.1B).1B). Conversion to a hybrid glycan is then initiated by N-acetylglucosamine transferase I (GnTI), which transfers a GlcNAc moiety to the D1 arm of the Man₅GlcNAc₂ substrate (19) (Fig. ​(Fig.1B).1B). This hybrid glycoform is then a substrate for modification into complex glycans, in which the D2 and D3 arm mannose residues are replaced by complex antennae (19, 40, 56). Further enzymatic action catalyzes the addition of α-1-6-linked fucose moiety to the lower GlcNAc of complex glycan stems, but usually not to oligomannose glycan stems (Fig. ​(Fig.1B)1B) (21, 113).Most glycoproteins exhibit only fully mature complex glycans. However, the steric limitations imposed by the high density of glycans on some parts of gp120 lead to incomplete trimming, leaving “immature” oligomannose glycans (22, 26, 128). Spatial competition between neighboring sequons can sometimes lead to one or the other remaining unutilized, further distancing the final Env product from what might be expected based on its primary sequence (42, 48, 74, 119). An attempt to assign JR-FL gp120 and gp41 sequon use and types, based on various studies, is shown in Fig. ​Fig.1A1A (6, 26, 34, 35, 42, 63, 71, 74, 119, 128). At some positions, the glycan type is conserved. For example, the glycan at residue N301 has consistently been found to be complex (26, 63, 128). At other positions, considerable heterogeneity exists in the glycan populations, in some cases to the point where it is difficult to unequivocally assign them as predominantly complex or oligomannose. The reasons for these uncertainties might include incomplete trimming (42), interstrain sequence variability, the form of Env (e.g., gp120 or gp140), and the producer cell. The glycans of native Env trimers and monomeric gp120 may differ due to the constraints imposed by oligomerization (32, 41, 77). Thus, although all the potential sequons of HXB2 gp120 were found to be occupied in one study (63), some are unutilized or variably utilized on functional trimers, presumably due to steric limitations (42, 48, 75, 96, 119).The distribution of complex and oligomannose glycans on gp120 largely conforms with an antigenic map derived from structural models (59, 60, 102, 120), in which the outer domain is divided into a neutralizing face and an immunologically silent face. Oligomannose glycans cluster tightly on the silent face of gp120 (18, 128), while complex glycans flank the gp120 receptor binding sites of the neutralizing face, ostensibly forming a protective “fence” against NAbs (105). The relatively sparse clustering of complex glycans that form this fence may reflect a trade-off between protecting the underlying functional domains from NAbs by virtue of large antennae while at the same time permitting sufficient flexibility for the refolding events associated with receptor binding and fusion (29, 39, 67, 75, 98, 117). Conversely, the dense clustering of oligomannose glycans on the silent domain may be important for ensuring immune protection and/or in creating binding sites for lectins such as DC-SIGN (9, 44).The few available broadly neutralizing monoclonal antibodies (MAbs) define sites of vulnerability on Env trimers (reviewed in reference 52). They appear to fall into two general categories: those that access conserved sites by overcoming Env''s various evasion strategies and, intriguingly, those that exploit these very defensive mechanisms. Regarding the first category, MAb b12 recognizes an epitope that overlaps the CD4 binding site of gp120 (14), and MAbs 2F5 and 4E10 (84, 129) recognize adjacent epitopes of the membrane-proximal external region (MPER) at the C-terminal ectodomain of gp41. The variable neutralizing potencies of these MAbs against primary isolates that contain their core epitopes illustrate how conformational masking can dramatically regulate their exposure (11, 118). Conformational masking also limits the activities of MAbs directed to the V3 loop and MAbs whose epitopes overlap the coreceptor binding site (11, 62, 121).A second category of MAbs includes MAb 2G12, which recognizes a tight cluster of glycans in the silent domain of gp120 (16, 101, 103, 112). This epitope has recently sparked considerable interest in exploiting glycan clusters as possible carbohydrate-based vaccines (2, 15, 31, 70, 102, 116). Two recently described MAbs, PG9 and PG16 (L. M. Walker and D. R. Burton, unpublished data), also target epitopes regulated by the presence of glycans that involve conserved elements of the second and third variable loops and depend largely on the quaternary trimer structure and its in situ presentation on membranes. Their impressive breadth and potency may come from the fact that they target the very mechanisms (variable loops and glycans) that are generally thought to protect the virus from neutralization. Like 2G12, these epitopes are likely to be constitutively exposed and thus may not be subject to conformational masking (11, 118).The above findings reveal the importance of N-glycans both as a means of protection against neutralization as well as in directly contributing to unique neutralizing epitopes. Clearly, further studies on the nature and function of glycans in native Env trimers are warranted. Possible approaches may be divided into four categories, namely, (i) targeted mutation, (ii) enzymatic removal, (iii) expression in the presence of glycosylation inhibitors, and (iv) expression in mutant cell lines with engineered blocks in the glycosylation pathway. Much of the available information on the functional roles of glycans in HIV-1 and simian immunodeficiency virus (SIV) infection has come from the study of mutants that eliminate glycans either singly or in combination (20, 54, 66, 71, 74, 91, 95, 96). Most mutants of this type remain at least partially functional (74, 95, 96). In some cases these mutants have little effect on neutralization sensitivity, while in others they can lead to increased sensitivity to MAbs specific for the V3 loop and CD4 binding site (CD4bs) (54, 71, 72, 74, 106). In exceptional cases, increased sensitivity to MAbs targeting the coreceptor binding site and/or the gp41 MPER has been observed (54, 66, 72, 74).Of the remaining approaches for studying the roles of glycans, enzymatic removal is constrained by the extreme resistance of native Env trimers to many common glycosidases, contrasting with the relative sensitivity of soluble gp120 (67, 76, 101). Alternatively, drugs can be used to inhibit various stages of mammalian glycan biosynthesis. Notable examples are imino sugars, such as N-butyldeoxynojirimycin (NB-DNJ), that inhibit the early trimming of the glucose moieties from Glc₃Man₉GlcNAc₂ precursors in the endoplasmic reticulum (28, 38, 51). Viruses produced in the presence of these drugs may fail to undergo proper gp160 processing or fusion (37, 51). Other classes of inhibitor include kifunensine and swainsonine, which, respectively, inhibit the trimming of the Man₉GlcNAc₂ precursor into Man₅GlcNAc₂ or inhibit the removal of remaining D2 and D3 arm mannoses from the hybrid glycans, thus preventing the construction of complex glycan antennae (Fig. ​(Fig.1B)1B) (17, 33, 76, 104, 119). Unlike NB-DNJ, viruses produced in the presence of these drugs remain infectious (36, 76, 79, 100).Yet another approach is to express virus in insect cells that can only modify proteins with paucimannose N-glycans (58). However, the inefficient gp120/gp41 processing by furin-like proteases in these cells prevents their utility in functional studies (123). Another option is provided by ricin-selected GnTI-deficient cell lines that cannot transfer GlcNAc onto the mannosidase-trimmed Man₅GlcNAc₂ substrate, preventing the formation of hybrid and complex carbohydrates (Fig. ​(Fig.1B)1B) (17, 32, 36, 94). This arrests glycan processing at a well-defined point, leading to the substitution of complex glycans with Man₅GlcNAc₂ rather than with the larger Man₉GlcNAc₂ precursors typically obtained with kifunensine treatment (17, 32, 33, 104). With this in mind, here we produced HIV-1 pseudoviruses in GnTI-deficient cells to investigate the role of complex glycan antennae in viral resistance neutralization. By replacing complex glycans with smaller Man₅GlcNAc₂ we can determine the effect of “lowering the glycan fence” that surrounds the receptor binding sites, compared to the above-mentioned studies of individual glycan deletion mutants, whose effects are analogous to removing a fence post. Furthermore, since oligomannose glycans are sensitive to certain enzymes, such as endoglycosidase H (endo H), we investigated the effect of dismantling the glycan fence on Env function and stability. Our results suggest that the antennae of complex glycans protect against certain specificities but that glycan stems regulate trimer conformation with often more dramatic consequences for neutralization sensitivity and in extreme cases, infectious function.