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.     

Structure and Function Analysis of Therapeutic Monoclonal Antibodies against Dengue Virus Type 2

Dengue virus (DENV) is the most prevalent insect-transmitted viral disease in humans globally, and currently no specific therapy or vaccine is available. Protection against DENV and other related flaviviruses is associated with the development of antibodies against the viral envelope (E) protein. Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV type 2 (DENV-2), none have compared simultaneously the inhibitory activity against a genetically diverse range of strains in vitro, the protective capacity in animals, and the localization of epitopes. Here, with the goal of identifying MAbs that can serve as postexposure therapy, we investigated in detail the functional activity of a large panel of new anti-DENV-2 mouse MAbs. Binding sites were mapped by yeast surface display and neutralization escape, cell culture inhibition assays were performed with homologous and heterologous strains, and prophylactic and therapeutic activity was evaluated with two mouse models. Protective MAbs localized to epitopes on the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of DII, and the lateral ridge, C-C′ loop, and A strand of DIII. Several MAbs inefficiently inhibited at least one DENV-2 strain of a distinct genotype, suggesting that recognition of neutralizing epitopes varies with strain diversity. Moreover, antibody potency generally correlated with a narrowed genotype and serotype specificity. Five MAbs functioned efficiently as postexposure therapy when administered as a single dose, even 3 days after intracranial infection of BALB/c mice. Overall, these studies define the structural and functional complexity of antibodies against DENV-2 with protective potential.Dengue virus (DENV), a member of the Flaviviridae family of RNA viruses, is related to several other human pathogens of global concern, including yellow fever and tick-borne, West Nile, and Japanese encephalitis viruses. DENV infection in humans occurs after Aedes aegypti or Aedes albopictus mosquito inoculation and results in clinical disease, ranging from a febrile illness (dengue fever [DF]) to a life-threatening hemorrhagic and capillary leak syndrome (dengue hemorrhagic fever [DHF]/dengue shock syndrome [DSS]). Globally, there is significant diversity among DENV strains, including four distinct serotypes (DENV type 1 [DENV-1], DENV-2, DENV-3, and DENV-4) that differ at the amino acid level by 25 to 40%. Additional complexity occurs within each serotype, as genotypes vary from one another by up to 3% at the amino acid level (21, 49). No approved antiviral treatment is currently available, and several candidate tetravalent vaccines remain in clinical development (reviewed in reference 11). Because of the increased geographic range of its mosquito vectors, urbanization, and international travel, DENV continues to spread worldwide and now causes an estimated 50 to 100 million infections and 250,000 to 500,000 cases of DHF/DSS per year, with 2.5 billion people at risk (68).DENV is an enveloped icosahedral virus with a single-stranded, positive-polarity RNA genome. The 10.7-kb genome is translated as a single polyprotein, which is cleaved into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by host and viral proteases. The mature DENV virion is ∼500 Å in diameter, with a highly organized outer protein shell, a 50-Å lipid membrane bilayer, and a nucleocapsid core (26). Mature DENV virions are covered by 90 anti-parallel E protein homodimers, arranged flat along the surface with quasi-icosahedral symmetry. The immature virion, which lacks cleavage of the prM protein, has a rough surface with 60 spikes each composed of three prM-E heterodimers (7, 73). Exposure to mildly acidic conditions in the trans-Golgi network promotes virus maturation through a structural rearrangement of the flavivirus E proteins and cleavage of prM to M by a furin-like protease (29, 66, 69, 70). The ectodomain of DENV E protein is comprised of three discrete domains (34-36, 39). Domain I (DI) is a central, eight-stranded β-barrel, which contains a single N-linked glycan in most DENV strains. DII is a long, finger-like protrusion from DI, with the highly conserved fusion peptide at its distal end and a second N-linked glycan that recognizes DC-SIGN (37, 38, 46, 59). DIII, which adopts an immunoglobulin-like fold, has been suggested to contain cell surface receptor recognition sites (5, 64, 71). Several groups have recently defined contact residues for type-specific, subcomplex-specific, and cross-reactive monoclonal antibodies (MAbs) that recognize DIII of DENV-2 (16, 17, 31, 47, 57, 61). Type-specific MAbs with neutralizing activity against DENV-2 localized to the BC, DE, and FG loops on the lateral ridge of DIII, whereas subcomplex-specific MAbs recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310.To date, no study has compared the in vitro inhibitory activity of MAbs in cells against a genetically diverse range of DENV-2 strains and their protective capacity in animals. Here, we had the goal of generating strongly neutralizing MAbs that would recognize virtually all DENV-2 strains and function as a possible postexposure therapy. Twenty-four new anti-DENV-2 mouse MAbs were generated with moderate or strong neutralizing activity against the homologous virus in cell culture assays. Binding sites were mapped for the majority of these by yeast surface display, identifying distinct epitopes in regions in DI (lateral ridge), DII (dimer interface, lateral ridge, and fusion loop), and DIII (lateral ridge, C-C′ loop, and A strand). Several MAbs failed to neutralize efficiently at least one DENV-2 strain of a distinct genotype, suggesting that antibody recognition of neutralizing epitopes varies among DENV-2 genotypes.To begin to assess the utility of this new panel of inhibitory MAbs as possible therapeutics against DENV-2, we evaluated their protective capacity in a stringent intracranial challenge model in BALB/c mice. Among the 16 neutralizing MAbs tested in mice, most were protective when given as prophylaxis. Seven of these had postexposure therapeutic activity when administered as a single dose by intraperitoneal route even 3 days after intracranial infection. For the MAbs with the greatest therapeutic potential, protection was confirmed with an antibody-enhanced vascular leakage mouse model (2, 72) of DENV-2 infection.     

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.     

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.     

Safety and Immunogenicity of Novel Recombinant BCG and Modified Vaccinia Virus Ankara Vaccines in Neonate Rhesus Macaques

Although major inroads into making antiretroviral therapy available in resource-poor countries have been made, there is an urgent need for an effective vaccine administered shortly after birth, which would protect infants from acquiring human immunodeficiency virus type 1 (HIV-1) through breast-feeding. Bacillus Calmette-Guérin (BCG) is given to most infants at birth, and its recombinant form could be used to prime HIV-1-specific responses for a later boost by heterologous vectors delivering the same HIV-1-derived immunogen. Here, two groups of neonate Indian rhesus macaques were immunized with either novel candidate vaccine BCG.HIVA⁴⁰¹ or its parental strain AERAS-401, followed by two doses of recombinant modified vaccinia virus Ankara MVA.HIVA. The HIVA immunogen is derived from African clade A HIV-1. All vaccines were safe, giving local reactions consistent with the expected response at the injection site. No systemic adverse events or gross abnormality was seen at necropsy. Both AERAS-401 and BCG.HIVA⁴⁰¹ induced high frequencies of BCG-specific IFN-γ-secreting lymphocytes that declined over 23 weeks, but the latter failed to induce detectable HIV-1-specific IFN-γ responses. MVA.HIVA elicited HIV-1-specific IFN-γ responses in all eight animals, but, except for one animal, these responses were weak. The HIV-1-specific responses induced in infants were lower compared to historic data generated by the two HIVA vaccines in adult animals but similar to other recombinant poxviruses tested in this model. This is the first time these vaccines were tested in newborn monkeys. These results inform further infant vaccine development and provide comparative data for two human infant vaccine trials of MVA.HIVA.Close to 2.3 million of children globally are infected with human immunodeficiency virus type 1 (HIV-1). The majority of neonatal infections occur in utero or intrapartum and, in the absence of preventative interventions, up to 29% of infants breast-fed by infected mothers acquire HIV-1 (6). Furthermore, HIV-1-infected children face a worse prognosis than adults in that, without antiretroviral treatment (ART), 25% of perinatally infected children progress to AIDS within 1 year (10), and the median time to AIDS for the remaining children is less than 7 years (2). It is now clearly established that maternal and extended infant ART can substantially reduce transmission of HIV-1 through breast-feeding (23). However, in a resource-poor setting, many logistical barriers to implementation of the ART-based prevention of mother-to-child-transmission (PMTCT) remain (23). Because nutrition and hygiene makes breast milk an important determinant of infant survival (22, 28), formula feeding as a protective measure against HIV-1 acquisition is recommended only if it is AFASS (acceptable, feasible, affordable, sustainable, and safe). Unfortunately, AFASS it is still not for majority of infected mothers in sub-Saharan Africa. Also, mixed bottle and breast feeding is associated with a 10-fold increase in HIV-1 transmission relative to exclusive breast-feeding (4). Thus, an effective infant vaccine against HIV-1 infection is the best and safest solution for PMTCT of HIV-1 with the added practical option of prolonging breast-feeding.Neonatal immunity is immature compared to the adult immune system (25). The differences include naivety of the immune cells, a tendency to develop Th2 responses (5) and antigen-presenting cells with inefficient cytokine production (35). For example, human cord blood T cells proliferated poorly and produced low levels of interleukin-2 (IL-2) and gamma interferon (IFN-γ) when endogenous antigen-presenting cells presented the antigen (35, 44). Also, infant myeloid dendritic cells are less efficient in priming Th1 responses because of their decreased responsiveness to Toll-like receptor stimulation, lower levels of surface costimulatory molecules, and lower production of IL-12 (8, 27). In several infections, qualitative and quantitative differences between human newborn and adult responses were detected (1, 9, 26, 37). In contrast, other studies of infants reported proliferation as well as IL-2 and IFN-γ production by T cells equal to that of adults following T-cell receptor-independent activation (21, 46). These latter observations indicate that neonate T cells are not intrinsically “locked” into an immature phenotype but, given the correct stimuli, they can develop mature immune responses (25). The requirement for specific stimuli will likely differ for different pathogens and vaccine vectors.Mycobacterium bovis bacillus Calmette-Guérin (BCG) is commonly delivered at birth as an antituberculosis vaccine as a part of the WHO Expanded Programme on Immunization (EPI). It has been reported by several studies to promote an adultlike Th1 response in newborns (16, 24, 34, 43), although it was also suggested that delaying the BCG delivery to 10 weeks of age benefits the quantity and quality of BCG-induced CD4 T-cell responses (20). BCG and related mycobacterial vectors have been explored as vaccines against other infectious agents, including human and simian immunodeficiency viruses (19), and in adult animals showed immunogenicity and protection (3, 36, 39, 47, 48). The only clinical study of recombinant BCG (rBCG) in adults failed to provide consistent efficacy (7). We have suggested the use of rBCG expressing an HIV-1-derived immunogen as the priming component of a heterologous vaccine platform for PMTCT of HIV-1 through infected breast milk (18), where it is critical to prime HIV-1-specific responses as soon as possible after birth. These responses could be boosted a few weeks later or shortly after the already busy EPI by heterologous vaccines delivering the same HIV-1-derived immunogen. To this extent, we constructed the novel candidate vaccine BCG.HIVA⁴⁰¹ (36) by inserting a gene coding for the HIV-1 clade A-derived immunogen HIVA (14) into recombinant BCG strain AREAS-401 (40). AERAS-401 is a newly developed strain that displayed enhanced safety (40) and immunogenicity (11, 15) in murine models relative to its parental BCG vaccine strain Danish SSI-1331. Increased safety represents an important feature should the BCG.HIVA⁴⁰¹ vaccine be deployed in babies born to HIV-1-infected mothers. We showed that BCG.HIVA⁴⁰¹ in a heterologous combination with recombinant modified vaccinia virus Ankara MVA.HIVA and recombinant ovine atadenovirus OAdV.HIVA induced robust polyfunctional HIV-1-specific T-cell responses in adult macaques (36). Here, we assess the safety and immunogenicity of the BCG.HIVA prime-MVA.HIVA boost regimen in newborn rhesus macaques.     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.     

A Chimeric Alphavirus Replicon Particle Vaccine Expressing the Hemagglutinin and Fusion Proteins Protects Juvenile and Infant Rhesus Macaques from Measles

Measles remains a major cause of child mortality, in part due to an inability to vaccinate young infants with the current live attenuated virus vaccine (LAV). To explore new approaches to infant vaccination, chimeric Venezuelan equine encephalitis/Sindbis virus (VEE/SIN) replicon particles were used to express the hemagglutinin (H) and fusion (F) proteins of measles virus (MV). Juvenile rhesus macaques vaccinated intradermally with a single dose of VEE/SIN expressing H or H and F proteins (VEE/SIN-H or VEE/SIN-H+F, respectively) developed high titers of MV-specific neutralizing antibody and gamma-interferon (IFN-γ)-producing T cells. Infant macaques vaccinated with two doses of VEE/SIN-H+F also developed neutralizing antibody and IFN-γ-producing T cells. Control animals were vaccinated with LAV or with a formalin-inactivated measles vaccine (FIMV). Neutralizing antibody remained above the protective level for more than 1 year after vaccination with VEE/SIN-H, VEE/SIN-H+F, or LAV. When challenged with wild-type MV 12 to 17 months after vaccination, all vaccinated juvenile and infant monkeys vaccinated with VEE/SIN-H, VEE/SIN-H+F, and LAV were protected from rash and viremia, while FIMV-vaccinated monkeys were not. Antibody was boosted by challenge in all groups. T-cell responses to challenge were biphasic, with peaks at 7 to 25 days and at 90 to 110 days in all groups, except for the LAV group. Recrudescent T-cell activity coincided with the presence of MV RNA in peripheral blood mononuclear cells. We conclude that VEE/SIN expressing H or H and F induces durable immune responses that protect from measles and offers a promising new approach for measles vaccination. The viral and immunological factors associated with long-term control of MV replication require further investigation.Measles remains a major cause of child mortality despite the availability of a safe and effective live attenuated virus vaccine (LAV). Recent efforts to improve routine vaccination and implement national immunization days have moved measles control toward the World Health Organization''s goal of a 90% reduction in mortality by 2010 compared to 2000 (7). One persistent impediment to measles control in many countries remains the inability to successfully immunize young infants due to the immaturity of the immune system and interference of maternal antibodies with immune responses to LAV (1, 15, 65).Because the decrease in maternal antibody varies from one infant to another, many children in areas with high measles virus (MV) transmission rates are at risk of acquiring measles prior to vaccination (3, 5, 12). Immaturity also affects the quality and quantity of antibody produced in response to the current vaccine, with lower levels of neutralizing antibody and deficient avidity and isotype maturation in younger than in older infants (15, 16, 37, 59). As a result, the recommended age for vaccination is generally 9 months in developing countries to balance the risk of infection with the likelihood of response to the vaccine (24).A vaccine that could be given to children under the age of 6 months would improve measles control by allowing delivery with other infant vaccines and by closing the window of susceptibility prior to delivery of the current vaccine. Increasing the dose of LAV improved the antibody responses in young infants but resulted in an unexpected increase in mortality for girls, so this is not an acceptable approach to lowering the age of vaccination (18, 26, 29). Experience with a formalin-inactivated measles vaccine (FIMV) in the 1960s also led to unexpected complications. FIMV provided only short-term protection, and vaccinated individuals were at risk for more severe disease (atypical measles) upon infection with wild-type MV (14, 36, 54). Therefore, other strategies are necessary for development of a vaccine for young infants.One particularly promising approach for delivery of vaccine antigens is the use of alphavirus replicon particles (55). Alphaviruses are small positive-strand RNA viruses with the nonstructural replicase proteins encoded in the 5′ two-thirds of the genome and the structural proteins in the 3′ one-third. A subgenomic promoter is used to synthesize an abundant, smaller RNA from which the structural proteins are translated (61). Replicons contain the nonstructural protein genes, the 5′ and 3′ end cis-active replication sequences, and the subgenomic promoter that directs expression of a heterologous gene rather than the viral structural proteins. The replicon RNA can be packaged into virus-like particles by providing the structural proteins in trans using transient transfection (6, 33) or with stable packaging cell lines (51) and can be engineered for efficient delivery to antigen-presenting cells (17). Advantages include high-level expression of the vaccine antigen (68), stimulation of innate immunity (25, 31, 32, 64), and general lack of preexisting immunity in the human population.MV encodes six structural proteins of which two, hemagglutinin (H) and fusion (F), are surface glycoproteins involved in attachment and entry. Antibodies that inhibit MV infection in neutralization assays are directed primarily against the H protein, which also contains important CD8⁺ T-cell epitopes (39, 41). Nonhuman primates, particularly rhesus macaques, develop a disease similar to that of humans and offer the opportunity for assessing both protection from wild-type MV challenge and priming for enhanced disease after immunization with new experimental vaccines (2, 48, 50, 66). Because protection from measles correlates best with the quality and quantity of neutralizing antibodies at the time of exposure (9, 50), most experimental vaccines have used H alone or H and F for induction of MV protective immunity (44, 50, 65, 70).Alphaviruses that have been used for construction of replicon particle vaccines include Sindbis virus (SINV) (6, 68), Semliki Forest virus (33), and Venezuelan equine encephalitis virus (VEEV) (53). Each of the alphavirus vectors studied has its own advantages and disadvantages. For instance, VEEV replicon particles have high levels of gene expression (47), but vaccine production is disadvantaged by the requirement for biosafety level 3 manufacturing. SINV replicon particles avoid the safety concerns of VEEV, but expression levels are lower. Previous studies of a SINV-based replicon particle vaccine expressing MV H (SIN-H) in macaques showed good induction of neutralizing antibody and T-cell responses and protection from rash (44). However, vaccinated monkeys developed viremias after challenge, indicating that they were not protected from infection. In this study, we sought to improve the alphavirus replicon particle approach to vaccination for measles by using a chimeric VEE/SIN vaccine (47) expressing both the MV H and F proteins.     

Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.Hantaviruses cause 2 human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (50). HPS and HFRS are multifactorial in nature and cause thrombocytopenia, immune and endothelial cell responses, and hypoxia, which contribute to disease (7, 11, 31, 42, 62). Although these syndromes sound quite different, they share common components which involve the ability of hantaviruses to infect endothelial cells and induce capillary permeability. Edema, which results from capillary leakage of fluid into tissues and organs, is a common finding in both HPS and HFRS patients (4, 7, 11, 31, 42, 62). In fact, both diseases can present with renal or pulmonary sequelae, and the renal or pulmonary focus of hantavirus diseases is likely to result from hantavirus infection of endothelial cells within vast glomerular and pulmonary capillary beds (4, 7, 11, 31, 42, 62). All hantaviruses predominantly infect endothelial cells which line capillaries (31, 42, 44, 61, 62), and endothelial cells have a primary role in maintaining fluid barrier functions of the vasculature (1, 12, 55). Although hantaviruses do not lyse endothelial cells (44, 61), this primary cellular target underlies hantavirus-induced changes in capillary integrity. As a result, understanding altered endothelial cell responses following hantavirus infection is fundamental to defining the mechanism of permeability induced by pathogenic hantaviruses (1, 12, 55).Pathogenic, but not nonpathogenic, hantaviruses use β₃ integrins on the surface of endothelial cells and platelets for attachment (19, 21, 23, 39, 46), and β₃ integrins play prominent roles in regulating vascular integrity (3, 6, 8, 24, 48). Pathogenic hantaviruses bind to basal, inactive conformations of β₃ integrins (35, 46, 53) and days after infection inhibit β₃ integrin-directed endothelial cell migration (20, 46). This may be the result of cell-associated virus (19, 20, 22) which keeps β₃ in an inactive state but could also occur through additional regulatory processes that have yet to be defined. Interestingly, the nonpathogenic hantaviruses Prospect Hill virus (PHV) and Tula virus (TULV) fail to alter β₃ integrin functions, and their entry is consistent with the use of discrete α₅β₁ integrins (21, 23, 36).On endothelial cells, α_vβ₃ integrins normally regulate permeabilizing effects of vascular endothelial growth factor receptor-2 (VEGFR2) (3, 24, 48, 51). VEGF was initially identified as an edema-causing vascular permeability factor (VPF) that is 50,000 times more potent than histamine in directing fluid across capillaries (12, 14). VEGF is responsible for disassembling adherens junctions between endothelial cells to permit cellular movement, wound repair, and angiogenesis (8, 10, 12, 13, 17, 26, 57). Extracellular domains of β₃ integrins and VEGFR2 reportedly form a coprecipitable complex (3), and knocking out β₃ causes capillary permeability that is augmented by VEGF addition (24, 47, 48). Pathogenic hantaviruses inhibit β₃ integrin functions days after infection and similarly enhance the permeability of endothelial cells in response to VEGF (22).Adherens junctions form the primary fluid barrier of endothelial cells, and VEGFR2 responses control adherens junction disassembly (10, 17, 34, 57, 63). Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific adherens junction protein and the primary determinant of paracellular permeability within the vascular endothelium (30, 33, 34). Activation of VEGFR2, another endothelial cell-specific protein, triggers signaling responses resulting in VE-cadherin disassembly and endocytosis, which increases the permeability of endothelial cell junctions (10, 12, 17, 34). VEGF is induced by hypoxic conditions and released by endothelial cells, platelets, and immune cells (2, 15, 38, 52). VEGF acts locally on endothelial cells through the autocrine or paracrine activation of VEGFR2, and the disassembly of endothelial cell adherens junctions increases the availability of nutrients to tissues and facilitates leukocyte trafficking and diapedesis (10, 12, 17, 55). The importance of endothelial cell barrier integrity is often in conflict with requirements for endothelial cells to move in order to permit angiogenesis and repair or cell and fluid egress, and as a result, VEGF-induced VE-cadherin responses are tightly controlled (10, 17, 18, 32, 33, 59). This limits capillary permeability while dynamically responding to a variety of endothelial cell-specific factors and conditions. However, if unregulated, this process can result in localized capillary permeability and edema (2, 9, 10, 12, 14, 17, 29, 60).Interestingly, tissue edema and hypoxia are common findings in both HPS and HFRS patients (11, 31, 62), and the ability of pathogenic hantaviruses to infect human endothelial cells provides a means for hantaviruses to directly alter normal VEGF-VE-cadherin regulation. In fact, the permeability of endothelial cells infected by pathogenic Andes virus (ANDV) or Hantaan virus (HTNV) is dramatically enhanced in response to VEGF addition (22). This response is absent from endothelial cells comparably infected with the nonpathogenic TULV and suggests that enhanced VEGF-induced endothelial cell permeability is a common underlying response of both HPS- and HFRS-causing hantaviruses (22). In these studies, we comparatively investigate responses of human endothelial cells infected with pathogenic ANDV and HTNV, as well as nonpathogenic TULV.     

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.     

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.     

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.     

Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.Arenaviruses are bisegmented, single-stranded RNA viruses that use an ambisense coding strategy to express four proteins: NP (nucleoprotein), Z (matrix protein), L (polymerase), and GP (glycoprotein). The viral GP is sufficient to direct entry into host cells, and retroviral vectors pseudotyped with GP recapitulate the entry pathway of these viruses (5, 13, 24, 31). GP is a class I fusion protein comprising two subunits, GP1 and GP2, cleaved from the precursor protein GPC (4, 14, 16, 18, 21). GP1 contains the receptor binding domain (19, 28), while GP2 contains structural elements characteristic of viral membrane fusion proteins (8, 18, 20, 38). The N-terminal stable signal peptide (SSP) remains associated with the mature glycoprotein after cleavage (2, 39) and plays a role in transport, maturation, and pH-dependent fusion (17, 35, 36, 37).The New World arenaviruses are divided into clades A, B, and C based on phylogenetic relatedness (7, 9, 11). Clade B contains the human pathogenic viruses Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia, and Chapare, which cause severe hemorrhagic fevers in South America (1, 10, 15, 26, 34). Clade B also contains the nonpathogenic viruses Amapari (AMAV), Cupixi, and Tacaribe (TCRV), although mild disease has been reported for a laboratory worker infected with TCRV (29).Studies with both viruses and GP-pseudotyped retroviral vectors have shown that the pathogenic clade B arenaviruses use the human transferrin receptor type 1 (hTfR1) to gain entry into human cells (19, 30). In contrast, GPs from nonpathogenic viruses, although capable of using TfR1 orthologs from other species (1), cannot use hTfR1 (1, 19) and instead enter human cells through as-yet-uncharacterized hTfR1-independent pathways (19). In addition, human T-cell lines serve as useful tools to distinguish these GPs, since JUNV, GTOV, and MACV pseudotyped vectors readily transduce CEM cells, while TCRV and AMAV GP vectors do not (27; also unpublished data). These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3, 22, 25).     

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.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).