Attenuate Newcastle disease virus by codon modification of the glycoproteins and phosphoprotein genes.

A codon modification strategy was used to attenuate the avian pathogenicity of an oncolytic mesogenic Newcastle disease virus (NDV) by targeting the three major virulence factors: the fusion (F) protein, hemagglutinin neuraminidase (HN) and phosphoprotein (P). Recoding the F and HN genes with rare codons greatly reduced expression of both F and HN proteins and resulted in their low incorporation into virions. The F and HN recoded virus was partially attenuated in chickens even when the F protein cleavage site was modified. Full attenuation was achieved when the 5' portion of the P gene was recoded. The recoded P, F and HN triple gene mutant exhibited delayed cell death in human cancer cells with prolonged expression of a GFP transgene. While this engineered attenuated NDV strain has lower oncolytic potency, its capacity for prolonged transgene expression may allow its use as a vaccine or gene delivery vector.

HBeAg seroconversion is associated with a more effective PD-L1 blockade during chronic hepatitis B infection.

BACKGROUND & AIMS: Current therapies for chronic hepatitis B virus (HBV) infection control viral replication but do not eliminate the risk of progression to hepatocellular carcinoma. HBV-specific CD8 T cells are necessary for viral control, but they are rare and exhausted during chronic infection. Preclinical studies have shown that blockade of the PD-1:PD-L1 axis can restore HBV-specific T cell functionality. The aim of this study was to analyze how the clinical and treatment status of patients impacts the ability of HBV-specific T cells to respond to PD-L1 blockade. METHODS: Expression patterns of the PD-1:PD-L1/PD-L2 axis were analyzed in healthy donors and chronically infected patients in different clinical phases of disease. A functional assay was performed to quantify baseline HBV-specific T cell responses in chronically infected patients. Baseline responses were then compared to those attained in the presence of an anti-PD-L1 monoclonal antibody (MEDI2790). RESULTS: Chronically infected patients were characterized by the upregulation of PD-1 within the T cell compartment and a concomitant upregulation of PD-L1 on myeloid dendritic cells. The upregulation was maximal in HBV e antigen (HBeAg)-positive patients but persisted after HBeAg negativization and was not restored by long-term treatment. HBV reactivity, measured as frequency of HBV-specific T cells, was significantly higher in HBeAg-negative patients with lower HBV DNA levels, independently of HBV surface antigen or alanine aminotransferase levels. Anti-PD-L1 blockade with MEDI2790 increased both the number of IFN-γ-producing T cells and the amount of IFN-γ produced per cell in 97% of patients with detectable HBV reactivity, independently of patients' clinical or treatment status. CONCLUSION: Patients with lower levels of HBV DNA and the absence of HBeAg have more intact HBV-specific T cell immunity and may benefit the most from PD-L1 blockade as a monotherapy. LAY SUMMARY: Hepatitis B virus (HBV)-specific T cell responses during chronic infection are weak due to the upregulation of inhibitor molecules on the immune cells. In this study we show that the inhibitory PD-1:PD-L1 axis is upregulated during chronic HBV infection and successful antiretroviral therapy does not restore normal levels of PD-1 and PD-L1 expression. However, in HBV e antigen-negative patients, treatment with an anti-PD-L1 antibody can increase the functionality of HBV-specific T cell responses by an average of 2-fold and is a promising new therapy for patients with chronic HBV infection.

Prevalence and Significance of Substitutions in the Fusion Protein of Respiratory Syncytial Virus Resulting in Neutralization Escape From Antibody MEDI8897.

Background: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children. To date, no vaccine is approved for the broad population of healthy infants. MEDI8897, a potent anti-RSV fusion antibody with extended serum half-life, is currently under clinical investigation as a potential passive RSV vaccine for all infants. As a ribonucleic acid virus, RSV is prone to mutation, and the possibility of viral escape from MEDI8897 neutralization is a potential concern. Methods: We generated RSV monoclonal antibody (mAb)-resistant mutants (MARMs) in vitro and studied the effect of the amino acid substitutions identified on binding and viral neutralization susceptibility to MEDI8897. The impact of resistance-associated mutations on in vitro growth kinetics and the prevalence of these mutations in currently circulating strains of RSV in the United States was assessed. Results: Critical residues identified in MARMs for MEDI8897 neutralization were located in the MEDI8897 binding site defined by crystallographic analysis. Substitutions in these residues affected the binding of mAb to virus, without significant impact on viral replication in vitro. The frequency of natural resistance-associated polymorphisms was low. Conclusions: Results from this study provide insights into the mechanism of MEDI8897 escape and the complexity of monitoring for emergence of resistance.

Oncolytic VSV Primes Differential Responses to Immuno-oncology Therapy.

Vesicular stomatitis virus encoding the IFNß transgene (VSV-IFNß) is a mediator of potent oncolytic activity and is undergoing clinical evaluation for the treatment of solid tumors. Emerging preclinical and clinical data suggest treatment of tumors with oncolytic viruses may sensitize tumors to checkpoint inhibitors and increase the anti-tumor immune response. New generations of immuno-oncology molecules including T cell agonists are entering clinical development and could be hypothesized to enhance the activity of oncolytic viruses, including VSV-IFNß. Here, we show that VSV-IFNß exhibits multiple mechanisms of action, including direct cell killing, stimulation of an innate immune response, recruitment of CD8 T cells, and depletion of T regulatory cells. Moreover, VSV-IFNß promotes the establishment of a CD8 T cell response to endogenous tumor antigens. Our data demonstrate a significant enhancement of anti-tumor function for VSV-IFNß when combined with checkpoint inhibitors, but not OX40 agonists. While the addition of checkpoint inhibitors to VSV-IFNß generated robust tumor growth inhibition, it resulted in no increase in viral replication, transgene expression, or immunophenotypic changes beyond treatment with VSV-IFNß alone. We hypothesize that tumor-specific T cells generated by VSV-IFNß retain activity due to a lack of immune exhaustion when checkpoint inhibitors were used.

S. aureus blocks efferocytosis of neutrophils by macrophages through the activity of its virulence factor alpha toxin.

Bacterial pneumonia, such as those caused by Staphylococcus aureus, is associated with an influx of inflammatory neutrophils into the lung tissue and airways. Regulation and clearance of recruited neutrophils is essential for preventing tissue damage by "friendly fire", a responsibility of macrophages in a process called efferocytosis. We hypothesized that S. aureus impairs efferocytosis by alveolar macrophages (AMs) through the activity of the secreted virulence factor alpha toxin (AT), which has been implicated in altering the antimicrobial function of AMs. Infection of mice lacking AMs resulted in significantly increased numbers of neutrophils in the lung, while clearance of neutrophils delivered intranasally into uninfected mice was reduced in AM depleted animals. In vitro, sublytic levels of AT impaired uptake of apoptotic neutrophils by purified AMs. In vivo, the presence of AT reduced uptake of neutrophils by AMs. Differential uptake of neutrophils was not due to changes in either the CD47/CD172 axis or CD36 levels. AT significantly reduced lung expression of CCN1 and altered AM surface localization of DD1α, two proteins known to influence efferocytosis. We conclude that AT may contribute to tissue damage during S. aureus pneumonia by inhibiting the ability of AM to clear neutrophils at the site of infection.

Genetic Modification of Oncolytic Newcastle Disease Virus for Cancer Therapy.

UNLABELLED: Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has a modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198-nucleotide sequence into the HN-L intergenic region, resulting in reduced viral gene expression and replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene while still retaining the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus. IMPORTANCE: Avian paramyxovirus type 1, NDV, has been an attractive oncolytic agent for cancer virotherapy. However, this virus can cause epidemic disease in poultry, and concerns about the potential environmental and economic impact of an NDV outbreak have precluded its clinical development. Here we describe generation and characterization of a highly potent oncolytic NDV variant that is unlikely to cause Newcastle disease in its avian host, representing an essential step toward moving NDV forward as an oncolytic agent. Several attenuation mechanisms have been genetically engineered into the recombinant NDV that reduce chicken pathogenicity to a level that is acceptable worldwide without impacting viral production in cell culture. The selective tumor replication of this recombinant NDV, both in vitro and in vivo, along with efficient tumor cell killing makes it an attractive oncolytic virus candidate that may provide clinical benefit to patients.

A multifunctional bispecific antibody protects against Pseudomonas aeruginosa.

Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa-induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4αPa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens.

Targeting the programmed cell death 1: programmed cell death ligand 1 pathway reverses T cell exhaustion in patients with sepsis.

INTRODUCTION: A major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections. Although many immunosuppressive mechanisms exist, increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-L1) are thought to play key roles. The newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-L1 antibodies to prevent apoptosis and improve lymphocyte function in septic patients. METHODS: Blood was obtained from 43 septic and 15 non-septic critically-ill patients. Effects of anti-PD-1, anti-PD-L1, or isotype-control antibody on lymphocyte apoptosis and interferon gamma (IFN-γ) and interleukin-2 (IL-2) production were quantitated by flow cytometry. RESULTS: Lymphocytes from septic patients produced decreased IFN-γ and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-L1 expression compared to non-septic patients (P<0.05). Monocytes from septic patients had increased PD-L1 and decreased HLA-DR expression compared to non-septic patients (P<0.01). CD8 T cell expression of PD-1 increased over time in ICU as PD-L1, IFN-γ, and IL2 decreased. In addition, donors with the highest CD8 PD-1 expression together with the lowest CD8 PD-L1 expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype. Treatment of cells from septic patients with anti-PD-1 or anti-PD-L1 antibody decreased apoptosis and increased IFN-γ and IL-2 production in septic patients; (P<0.01). The percentage of CD4 T cells that were PD-1 positive correlated with the degree of cellular apoptosis (P<0.01). CONCLUSIONS: In vitro blockade of the PD-1:PD-L1 pathway decreases apoptosis and improves immune cell function in septic patients. The current results together with multiple positive studies of anti-PD-1 and anti-PD-L1 in animal models of bacterial and fungal infections and the relative safety profile of anti-PD-1/anti-PD-L1 in human oncology trials to date strongly support the initiation of clinical trials testing these antibodies in sepsis, a disorder with a high mortality.

Infection and propagation of human rhinovirus C in human airway epithelial cells.

Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors.

Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening.

Pseudomonas aeruginosa is a leading cause of hospital-associated infections in the seriously ill, and the primary agent of chronic lung infections in cystic fibrosis patients. A major obstacle to effective control of P. aeruginosa infections is its intrinsic resistance to most antibiotic classes, which results from chromosomally encoded drug-efflux systems and multiple acquired resistance mechanisms selected by years of aggressive antibiotic therapy. These factors demand new strategies and drugs to prevent and treat P. aeruginosa infections. Herein, we describe a monoclonal antibody (mAb) selection strategy on whole P. aeruginosa cells using single-chain variable fragment phage libraries derived from healthy individuals and patients convalescing from P. aeruginosa infections. This approach enabled identification of mAbs that bind three distinct epitopes on the product of the Psl. This exopolysaccharide is important for P. aeruginosa attachment to mammalian cells, and for the formation and maintenance of biofilms produced by nonmucoid and mucoid P. aeruginosa isolates. Functional screens revealed that mAbs to one epitope exhibit superior activity in opsonophagocytic killing and cell attachment assays, and confer significant protection in multiple animal models. Our results indicate that Psl is an accessible serotype-independent surface feature and promising novel protective antigen for preventing P. aeruginosa infections. Furthermore, our mAb discovery strategy holds promise for application to other bacterial pathogens.

Natural polymorphisms and resistance-associated mutations in the fusion protein of respiratory syncytial virus (RSV): effects on RSV susceptibility to palivizumab.

Specific mutations in respiratory syncytial virus (RSV) fusion protein can cause palivizumab resistance. We assessed the incidence of sequence polymorphisms and palivizumab resistance in clinical RSV isolates collected from immunoprophylaxis-naive subjects. Polymorphisms were identified at low frequency, and only polymorphic mutations in antigenic site A (<1% of all polymorphisms) conferred palivizumab resistance.

Respiratory syncytial virus infection is associated with an altered innate immunity and a heightened pro-inflammatory response in the lungs of preterm lambs.

INTRODUCTION: Factors explaining the greater susceptibility of preterm infants to severe lower respiratory infections with respiratory syncytial virus (RSV) remain poorly understood. Fetal/newborn lambs are increasingly appreciated as a model to study key elements of RSV infection in newborn infants due to similarities in lung alveolar development, immune response, and susceptibility to RSV. Previously, our laboratory demonstrated that preterm lambs had elevated viral antigen and developed more severe lesions compared to full-term lambs at seven days post-infection. Here, we compared the pathogenesis and immunological response to RSV infection in lungs of preterm and full-term lambs. METHODS: Lambs were delivered preterm by Caesarian section or full-term by natural birth, then inoculated with bovine RSV (bRSV) via the intratracheal route. Seven days post-infection, lungs were collected for evaluation of cytokine production, histopathology and cellular infiltration. RESULTS: Compared to full-term lambs, lungs of preterm lambs had a heightened pro-inflammatory response after infection, with significantly increased MCP-1, MIP-1α, IFN-γ, TNF-α and PD-L1 mRNA. RSV infection in the preterm lung was characterized by increased epithelial thickening and periodic acid-Schiff staining, indicative of glycogen retention. Nitric oxide levels were decreased in lungs of infected preterm lambs compared to full-term lambs, indicating alternative macrophage activation. Although infection induced significant neutrophil recruitment into the lungs of preterm lambs, neutrophils produced less myeloperoxidase than those of full-term lambs, suggesting decreased functional activation. CONCLUSIONS: Taken together, our data suggest that increased RSV load and inadequate immune response may contribute to the enhanced disease severity observed in the lungs of preterm lambs.

Respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children worldwide. The understanding of neonatal RSV pathogenesis depends on using an animal model that reproduces neonatal RSV disease. Previous studies from us and others demonstrated that the neonatal lamb model resembles human neonatal RSV infection. Here, we provide an extensive and detailed characterization of the histopathology, viral load, cellular infiltration, and cytokine production in lungs and tracheobronchial lymph nodes of lambs inoculated with human RSV strain A2 over the course of infection. In the lung, RSV titers were low at day 3 postinfection, increased significantly by day 6, and decreased to baseline levels at day 14. Infection in the lung was associated with an accumulation of macrophages, CD4(+) and CD8(+) T cells, and a transcriptional response of genes involved in inflammation, chemotaxis, and interferon response, characterized by increased IFNγ, IL-8, MCP-1, and PD-L1, and decreased IFNß, IL-10, and TGF-ß. Laser capture microdissection studies determined that lung macrophage-enriched populations were the source of MCP-1 but not IL-8. Immunoreactivity to caspase 3 occurred within bronchioles and alveoli of day 6-infected lambs. In lung-draining lymph nodes, RSV induced lymphoid hyperplasia, suggesting an ability of RSV to enhance lymphocytic proliferation and differentiation pathways. This study suggests that, in lambs with moderate clinical disease, RSV enhances the activation of caspase cell death and Th1-skewed inflammatory pathways, and complements previous observations that emphasize the role of inflammation in the pathogenesis of RSV disease.

Prophylactic and therapeutic benefits of a monoclonal antibody against the fusion protein of human metapneumovirus in a mouse model.

Human metapneumovirus (HMPV) is a paramyxovirus causing acute respiratory tract infections in humans. The effects of a monoclonal antibody (MAb 338, MedImmune, Inc.) directed against the HMPV fusion protein were assessed in vivo. Different groups of BALB/c mice received an intraperitoneal injection of 25 or 50mg/kg of MAb 338 either 24h before or 48h after viral infection. Lung samples were collected on days 5 and 42 after infection for determination of viral titers and histopathological changes. Pulmonary functions were also evaluated by plethysmography. On day 5 post-infection, lung viral titers were significantly decreased in mice treated with 25 or 50mg/kg before or after viral infection compared to HMPV-infected control mice. Similarly, HMPV copy numbers on day 42 were decreased for all prophylactic and therapeutic interventions. Histopathological changes were also less severe in all treated groups of mice on days 5 and 42 post-infection, correlating with decreased airways obstruction. Finally, on day 42, all treated groups had a significant decrease in airways hyperresponsiveness following treatment with MAb 338. Both prophylactic and, to a lesser extent, therapeutic administration of MAb 338 improved acute and late consequences of HMPV infection in a relevant mouse model.

Combination of rituximab with blinatumomab (MT103/MEDI-538), a T cell-engaging CD19-/CD3-bispecific antibody, for highly efficient lysis of human B lymphoma cells.

We have compared the cytotoxic activity of rituximab with that of blinatumomab (MT103/MEDI-538), a single-chain CD19-/CD3-bispecific antibody engaging human T cells. Blinatumomab consistently led to a higher degree of lysis of human lymphoma lines than rituximab, and was active at much lower concentration. The cytotoxicity mediated by blinatumomab and rituximab both caused a potent activation of pro-caspases 3 and 7 in target cells, a key event in induction of granzyme-mediated apoptotic cell death. Combination of rituximab with blinatumomab was found to greatly enhance the activity of rituximab, in particular at low effector-to-target cell ratios and at low antibody concentration.

Macrophage impairment underlies airway occlusion in primary respiratory syncytial virus bronchiolitis.

Although respiratory syncytial virus (RSV) infection is the most important cause of bronchiolitis in infants, the pathogenesis of RSV disease is poorly described. We studied histopathologic changes in a panel of lung tissue specimens obtained from infants with fatal cases of primary RSV infection. In these tissues, airway occlusion with accumulations of infected, apoptotic cellular debris and serum protein was consistently observed. Similar observations were found after RSV infection in New Zealand black (NZB) mice, which have constitutive deficiencies in macrophage function, but not in BALB/c mice. A deficiency in the number of alveolar macrophages in NZB mice appears to be central to enhanced disease, because depletion of alveolar macrophages in BALB/c mice before RSV exposure resulted in airway occlusion. In mice with insufficient numbers of macrophages, RSV infection yielded an increased viral load and enhanced expression of type I interferon-associated genes at the height of disease. Together, our data suggest that innate, rather than adaptive, immune responses are critical determinants of the severity of RSV bronchiolitis.

Identification of antibody neutralization epitopes on the fusion protein of human metapneumovirus.

Human metapneumovirus (hMPV) is genetically related to respiratory syncytial virus (RSV); both cause respiratory tract illnesses ranging from a mild cough to bronchiolitis and pneumonia. The F protein-directed monoclonal antibody (mAb) palivizumab has been shown to prevent severe lower respiratory tract RSV infection in animals and humans. We have previously reported on a panel of mAbs against the hMPV F protein that neutralize hMPV in vitro and, in two cases, in vivo. Here we describe the generation of hMPV mAb-resistant mutants (MARMs) to these neutralizing antibodies. Sequencing the F proteins of the hMPV MARMs identified several neutralizing epitopes. Interestingly, some of the epitopes mapped on the hMPV F protein coincide with homologous regions mapped previously on the RSV F protein, including the site against which the broadly protective mAb palivizumab is directed. This suggests that these homologous regions play important, conserved functions in both viruses.

The effect of dexamethasone on polyclonal T cell activation and redirected target cell lysis as induced by a CD19/CD3-bispecific single-chain antibody construct.

BiTE molecules comprise a new class of bispecific single-chain antibodies redirecting previously unstimulated CD8+ and CD4+ T cells for the elimination of target cells. One example is MT103 (MEDI-538; bscCD19xCD3), a CD19-specific BiTE that can induce lysis of normal and malignant B cells at low picomolar concentrations, which is accompanied by T cell activation. Here, we explored in cell culture the impact of the glucocorticoid derivative dexamethasone on various activation parameters of human T cells in response to MT103. In case cytokine-related side effects should occur with BiTE molecules and other T cell-based approaches during cancer therapy it is important to understand whether glucocorticoids do interfere with the cytotoxic potential of T cells. We found that MT103 induced in the presence of target cells secretion by peripheral T cells of interleukin (IL)-2, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, IL-10 and IL-4 into the cell culture medium. Production of all studied cytokines was effectively reduced by dexamethasone at a concentration between 1 and 3x10(-7) M. In contrast, upregulation of activation markers CD69, CD25, CD2 and LFA-1 on both CD4+ and CD8+ T cells, and T cell proliferation were barely affected by the steroid hormone analogue. Most importantly, dexamethasone did not detectably inhibit the cytotoxic activity of MT103-activated T cells against a human B lymphoma line as investigated with lymphocytes from 12 human donors. Glucocorticoids thus qualify as a potential co-medication for therapeutic BiTE molecules and other cytotoxic T cell therapies for treatment of cancer.

Isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo.

Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV), another member of the same subfamily. hMPV causes respiratory tract illnesses that, similar to human RSV, occur predominantly during the winter months and have symptoms that range from mild to severe cough, bronchiolitis, and pneumonia. Like RSV, the hMPV virus can be subdivided into two genetic subgroups, A and B. With RSV, a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV.

Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only.

An effective virus-like particle (VLP) based prophylactic vaccine designed to protect against persistent infection with human papillomavirus (HPV) types 16 and 18 and subsequent lesion development will need to induce a strong humoral and cellular immune response capable of providing long-term protection. Our objective was to evaluate the ability of an HPV16/18 L1 VLP vaccine formulated with the AS04 adjuvant system (3-O-desacyl-4'-monophosphoryl lipid A (MPL) and aluminium salt) to induce an immune response of higher magnitude and persistence compared to a vaccine formulated with aluminium salt only. We demonstrated that MPL adsorbed onto aluminium salt retains its capacity to activate an innate immune response as assessed by the production of TNFalpha by human monocytes (U937). In addition, vaccination of mice, monkeys or human subjects with AS04 formulations induced higher total anti-L1 VLP16 and L1 VLP18 antibody responses (1.6-8.5-fold) than the aluminium salt only formulations. The enhanced antibody response induced by the AS04 vaccine formulation (1.6-4.1-fold) in monkeys and humans was shown to be targeted to functional neutralising L1 VLP16 and L1 VLP18 epitopes as assessed by V5/J4 specific ELISAs or HPV16 and HPV18 pseudo-neutralization assays. The enhanced immune profile observed with the AS04 formulation in terms of both total, V5/J4 specific and neutralizing antibodies was shown to persist for at least 3.5-year post-vaccination in human subjects. Finally, using the newly developed B cell ELISPOT assay we also demonstrated that the AS04 formulation elicited an increased frequency (2.2-5.2-fold) of HPV L1 VLP specific memory B cells when compared with the aluminium salt only formulations. These data strongly support the role of the AS04 adjuvant, which includes the immunostimulant MPL, in triggering a persistent vaccine-induced immune response of high quality.