Phase 1 study of the pharmacokinetics and clinical proof-of-concept activity of a biofilm-disrupting human monoclonal antibody in patients with chronic prosthetic joint infection of the knee or hip.

We report the results of a first-in-human phase 1 clinical study to evaluate TRL1068, a native human monoclonal antibody that disrupts bacterial biofilms with broad-spectrum activity against both Gram-positive and Gram-negative species. The study population consisted of patients with chronic periprosthetic joint infections (PJIs) of the knee or hip, including both monomicrobial and polymicrobial infections, that are highly resistant to antibiotics due to biofilm formation. TRL1068 was administered via a single pre-surgical intravenous infusion in three sequentially ascending dose groups (6, 15, and 30 mg/kg). Concomitant perioperative antibiotics were pathogen-targeted as prescribed by the treating physician. In this double-blinded study, 4 patients were randomized to receive placebo and 11 patients to receive TRL1068 on day 1, as well as targeted antibiotics for 7 days prior to the scheduled removal of the infected implant and placement of an antibiotic-eluting spacer as the first stage of the standard of care two-stage exchange arthroplasty. No adverse events attributable to TRL1068 were reported. TRL1068 serum half-life was 15-18 days. At day 8, the concentration in synovial fluid was approximately 60% of the blood level and thus at least 15-fold above the threshold for biofilm-disrupting activity in vitro. Explanted prostheses were sonicated to release adherent bacteria for culture, with elimination of the implant bacteria observed in 3 of the 11 patients who received TRL1068, which compares favorably to prior PJI treatments. None of the patients who received TRL1068 had a relapse of the original infection by the end of the study (day 169). CLINICAL TRIALS: This study is registered with ClinicalTrials.gov as NCT04763759.

Structure-Based Design and Antigenic Validation of Respiratory Syncytial Virus G Immunogens.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of children, the elderly, and immunocompromised individuals. Currently, there are no FDA-approved RSV vaccines. The RSV G glycoprotein is used for viral attachment to host cells and impairment of host immunity by interacting with the human chemokine receptor CX3CR1. Antibodies that disrupt this interaction are protective against infection and disease. Nevertheless, development of an RSV G vaccine antigen has been hindered by its low immunogenicity and safety concerns. A previous study described three engineered RSV G proteins containing single-point mutations that induce higher levels of IgG antibodies and have improved safety profiles compared to wild-type RSV G (H. C. Bergeron, J. Murray, A. M. Nuñez Castrejon, et al., Viruses 13:352, 2021, https://doi.org/10.3390/v13020352). However, it is unclear if the mutations affect RSV G protein folding and display of its conformational epitopes. In this study, we show that the RSV G S177Q protein retains high-affinity binding to protective human and mouse monoclonal antibodies and has equal reactivity as wild-type RSV G protein to human reference immunoglobulin to RSV. Additionally, we determined the high-resolution crystal structure of RSV G S177Q protein in complex with the anti-RSV G antibody 3G12, further validating its antigenic structure. These studies show for the first time that an engineered RSV G protein with increased immunogenicity and safety retains conformational epitopes to high-affinity protective antibodies, supporting its further development as an RSV vaccine immunogen. IMPORTANCE Respiratory syncytial virus (RSV) causes severe lower respiratory diseases of children, the elderly, and immunocompromised populations. There currently are no FDA-approved RSV vaccines. Most vaccine development efforts have focused on the RSV F protein, and the field has generally overlooked the receptor-binding antigen RSV G due to its poor immunogenicity and safety concerns. However, single-point mutant RSV G proteins have been previously identified that have increased immunogenicity and safety. In this study, we investigate the antibody reactivities of three known RSV G mutant proteins. We show that one mutant RSV G protein retains high-affinity binding to protective monoclonal antibodies, is equally recognized by anti-RSV antibodies in human sera, and forms the same three-dimensional structure as the wild-type RSV G protein. Our study validates the structure-guided design of the RSV G protein as an RSV vaccine antigen.

Native Human Antibody to Shr Promotes Mice Survival After Intraperitoneal Challenge With Invasive Group A Streptococcus.

BACKGROUND: A vaccine against group A Streptococcus (GAS) has been actively pursued for decades. The surface receptor Shr is vital in GAS heme uptake and provides an effective target for active and passive immunization. Here, we isolated human monoclonal antibodies (mAbs) against Shr and evaluated their efficacy and mechanism. METHODS: We used a single B-lymphocyte screen to discover the mAbs TRL186 and TRL96. Interactions of the mAbs with whole cells, proteins, and peptides were investigated. Growth assays and cultured phagocytes were used to study the mAbs' impact on heme uptake and bacterial killing. Efficacy was tested in prophylactic and therapeutic vaccination using intraperitoneal mAb administration and GAS challenge. RESULTS: Both TRL186 and TRL96 interact with whole GAS cells, recognizing the NTR and NEAT1 domains of Shr, respectively. Both mAbs promoted killing by phagocytes in vitro, but prophylactic administration of only TRL186 increased mice survival. TRL186 improved survival also in a therapeutic mode. TRL186 but not TRL96 also impeded Shr binding to hemoglobin and GAS growth on hemoglobin iron. CONCLUSIONS: Interference with iron acquisition is central for TRL186 efficacy against GAS. This study supports the concept of antibody-based immunotherapy targeting the heme uptake proteins to combat streptococcal infections.

Conformational Flexibility in Respiratory Syncytial Virus G Neutralizing Epitopes.

Respiratory syncytial virus (RSV) is a top cause of severe lower respiratory tract disease and mortality in infants and the elderly. Currently, no vaccine or effective treatment exists for RSV. The RSV G glycoprotein mediates viral attachment to cells and contributes to pathogenesis by modulating host immunity through interactions with the human chemokine receptor CX3CR1. Antibodies targeting the RSV G central conserved domain are protective in both prophylactic and postinfection animal models. Here, we describe the crystal structure of the broadly neutralizing human monoclonal antibody 3G12 bound to the RSV G central conserved domain. Antibody 3G12 binds to a conformational epitope composed of highly conserved residues, explaining its broad neutralization activity. Surprisingly, RSV G complexed with 3G12 adopts a distinct conformation not observed in previously described RSV G-antibody structures. Comparison to other structures reveals that the RSV G central conserved domain is flexible and can adopt multiple conformations in the regions flanking the cysteine noose. We also show that restriction of RSV G flexibility with a proline mutation abolishes binding to antibody 3G12 but not antibody 3D3, which recognizes a different conformation of RSV G. Our studies provide new insights for rational vaccine design, indicating the importance of preserving both the global structural integrity of antigens and local conformational flexibility at antigenic sites, which may elicit a more diverse antibody response and broader protection against infection and disease.IMPORTANCE Respiratory syncytial virus (RSV) causes severe respiratory infections in infants, young children, and the elderly, and currently, no licensed vaccine exists. In this study, we describe the crystal structure of the RSV surface glycoprotein G in complex with a broadly neutralizing human monoclonal antibody. The antibody binds to RSV G at a highly conserved region stabilized by two disulfide bonds, but it captures RSV G in a conformation not previously observed, revealing that this region is both structured and flexible. Importantly, our findings provide insight for the design of vaccines that elicit diverse antibodies, which may provide broad protection from infection and disease.

Advances in the Development of Therapeutics for Cytomegalovirus Infections.

The development of therapeutics for cytomegalovirus (CMV) infections, while progressing, has not matched the pace of new treatments of human immunodeficiency virus (HIV) infections; nevertheless, recent developments in the treatment of CMV infections have resulted in improved human health and perhaps will encourage the development of new therapeutic approaches. First, the deployment of ganciclovir and valganciclovir for both the prevention and treatment of CMV infections and disease in transplant recipients has been further improved with the licensure of the efficacious and less toxic letermovir. Regardless, late-onset CMV disease, specifically pneumonia, remains problematic. Second, the treatment of congenital CMV infections with valganciclovir has beneficially improved both hearing and neurologic outcomes, both fundamental advances for these children. In these pediatric studies, viral load was decreased but not eliminated. Thus, an important lesson learned from studies in both populations is the need for new antiviral agents and the necessity for combination therapies as has been shown to be beneficial in the treatment of HIV infections, among others. The development of monoclonal antibodies, sirtuins, and cyclopropovir may provide new treatment options.

Comparative neutralizing potencies of antibodies suggest conservation as well as mechanistic differences in human cytomegalovirus entry into epithelial and endothelial cells.

Antibody neutralization of cytomegalovirus (CMV) entry into diverse cell types is a key consideration for development of vaccines and immunotherapeutics. CMV entry into fibroblasts differs significantly from entry into epithelial or endothelial cells: fibroblast entry is mediated by gB and gH/gL/gO, whereas both epithelial and endothelial cell entry require an additional pentameric complex (PC) comprised of gH/gL/UL128/UL130/UL131A. Because PC-specific antibodies in CMV-seropositive human sera do not affect fibroblast entry but potently block entry into epithelial or endothelial cells, substantially higher neutralizing potencies for CMV-positive sera are observed when assayed using epithelial cells as targets than when using fibroblasts. That certain sera exhibit similar discordances between neutralizing potencies measured using epithelial vs. endothelial cells (Gerna G. et al.J Gen Virol, 89:853-865, 2008) suggested that additional mechanistic differences may also exist between epithelial and endothelial cell entry. To further explore this issue, neutralizing potencies using epithelial and endothelial cells were simultaneously determined for eight CMV-positive human sera, CMV-hyperimmune globulin, and a panel of monoclonal or anti-peptide antibodies targeting specific epitopes in gB, gH, gH/gL, or the PC. No significant differences were observed between epithelial and endothelial neutralizing potencies of epitope-specific antibodies, CMV-hyperimmune globulin, or seven of the eight human sera. However, one human serum exhibited a six-fold higher potency for neutralizing entry into epithelial cells vs. endothelial cells. These results suggest that epitopes exist that are important for epithelial entry but are less critical, or perhaps dispensable, for endothelial cell entry. Their existence should be considered when developing monoclonal antibody therapies or subunit vaccines representing limited epitopes.

Respiratory Syncytial Virus: Targeting the G Protein Provides a New Approach for an Old Problem.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection (LRTI) annually affecting >2 million children in the United States <5 years old. In the elderly (>65 years old), RSV results in ∼175,000 hospitalizations annually in the United States with a worldwide incidence of ∼34 million. There is no approved RSV vaccine, and treatments are limited. Recently, a phase 3 trial in the elderly using a recombinant RSV F protein vaccine failed to meet its efficacy objectives, namely, prevention of moderate-to-severe RSV-associated LRTI and reduced incidence of acute respiratory disease. Moreover, a recent phase 3 trial evaluating suptavumab (REGN2222), an antibody to RSV F protein, did not meet its primary endpoint of preventing medically attended RSV infections in preterm infants. Despite these setbacks, numerous efforts targeting the RSV F protein with vaccines, antibodies, and small molecules continue based on the commercial success of a monoclonal antibody (MAb) against the RSV F protein (palivizumab). As the understanding of RSV biology has improved, the other major coat protein, the RSV G protein, has reemerged as an alternative target reflecting progress in understanding its roles in infecting bronchial epithelial cells and in altering the host immune response. In mouse models, a high-affinity, strain-independent human MAb to the RSV G protein has shown potent direct antiviral activity combined with the alleviation of virus-induced immune system effects that contribute to disease pathology. This MAb, being prepared for clinical trials, provides a qualitatively new approach to managing RSV for populations not eligible for prophylaxis with palivizumab.

Native Human Monoclonal Antibodies with Potent Cross-Lineage Neutralization of Influenza B Viruses.

Although antibodies that effectively neutralize a broad set of influenza viruses exist in the human antibody repertoire, they are rare. We used a single-cell screening technology to identify rare monoclonal antibodies (MAbs) that recognized a broad set of influenza B viruses (IBV). The screen yielded 23 MAbs with diverse germ line origins that recognized hemagglutinins (HAs) derived from influenza strains of both the Yamagata and Victoria lineages of IBV. Of the 23 MAbs, 3 exhibited low expression in a transient-transfection system, 4 were neutralizers that bound to the HA head region, 11 were stalk-binding nonneutralizers, and 5 were stalk-binding neutralizers, with 4 of these 5 having unique antibody sequences. Of these four unique stalk-binding neutralizing MAbs, all were broadly reactive and neutralizing against a panel of multiple strains spanning both IBV lineages as well as highly effective in treating lethal IBV infections in mice at both 24 and 72 h postinfection. The MAbs in this group were thermostable and bound different epitopes in the highly conserved HA stalk region. These characteristics suggest that these MAbs are suitable for consideration as candidates for clinical studies to address their effectiveness in the treatment of IBV-infected patients.

Impact of Antibodies and Strain Polymorphisms on Cytomegalovirus Entry and Spread in Fibroblasts and Epithelial Cells.

Cytomegalovirus (CMV) entry into fibroblasts differs from entry into epithelial cells. CMV also spreads cell to cell and can induce syncytia. To gain insights into these processes, 27 antibodies targeting epitopes in CMV virion glycoprotein complexes, including glycoprotein B (gB), gH/gL, and the pentamer, were evaluated for their effects on viral entry and spread. No antibodies inhibited CMV spread in fibroblasts, including those with potent neutralizing activity against fibroblast entry, while all antibodies that neutralized epithelial cell entry also inhibited spread in epithelial cells and a correlation existed between the potencies of these two activities. This suggests that exposure of virions to the cell culture medium is obligatory during spread in epithelial cells but not in fibroblasts. In fibroblasts, the formation of syncytiumlike structures was impaired not only by antibodies to gB or gH/gL but also by antibodies to the pentamer, suggesting a potential role for the pentamer in promoting fibroblast fusion. Four antibodies reacted with linear epitopes near the N terminus of gH, exhibited strain specificity, and neutralized both epithelial cell and fibroblast entry. Five other antibodies recognized conformational epitopes in gH/gL and neutralized both fibroblast and epithelial cell entry. That these antibodies were strain specific for neutralizing fibroblast but not epithelial cell entry suggests that polymorphisms external to certain gH/gL epitopes may influence antibody neutralization during fibroblast but not epithelial cell entry. These findings may have implications for elucidating the mechanisms of CMV entry, spread, and antibody evasion and may assist in determining which antibodies may be most efficacious following active immunization or passive administration.IMPORTANCE Cytomegalovirus (CMV) is a significant cause of birth defects among newborns infected in utero and morbidity and mortality in transplant and AIDS patients. Monoclonal antibodies and vaccines targeting humoral responses are under development for prophylactic or therapeutic use. The findings reported here (i) confirm that cell-to-cell spread of CMV is sensitive to antibody inhibition in epithelial cells but not fibroblasts, (ii) demonstrate that antibodies can restrict the formation in vitro of syncytiumlike structures that resemble syncytial cytomegalic cells that are associated with CMV disease in vivo, and (iii) reveal that neutralization of CMV by antibodies to certain epitopes in gH or gH/gL is both strain and cell type dependent and can be governed by polymorphisms in sequences external to the epitopes. These findings serve to elucidate the mechanisms of CMV entry, spread, and antibody evasion and may have important implications for the development of CMV vaccines and immunotherapeutics.

A Native Human Monoclonal Antibody Targeting HCMV gB (AD-2 Site I).

Hyperimmune globulin (HIG) has shown efficacy against human cytomegalovirus (HCMV) for both transplant and congenital transmission indications. Replicating that activity with a monoclonal antibody (mAb) offers the potential for improved consistency in manufacturing, lower infusion volume, and improved pharmacokinetics, as well as reduced risk of off-target reactivity leading to toxicity. HCMV pathology is linked to its broad cell tropism. The glycoprotein B (gB) envelope protein is important for infections in all cell types. Within gB, the antigenic determinant (AD)-2 Site I is qualitatively more highly-conserved than any other region of the virus. TRL345, a high affinity (Kd = 50 pM) native human mAb to this site, has shown efficacy in neutralizing the infection of fibroblasts, endothelial and epithelial cells, as well as specialized placental cells including trophoblast progenitor cells. It has also been shown to block the infection of placental fragments grown ex vivo, and to reduce syncytial spread in fibroblasts in vitro. Manufacturing and toxicology preparation for filing an IND (investigational new drug) application with the US Food and Drug Administration (FDA) are expected to be completed in mid-2019.

A Human Biofilm-Disrupting Monoclonal Antibody Potentiates Antibiotic Efficacy in Rodent Models of both Staphylococcus aureus and Acinetobacter baumannii Infections.

Many serious bacterial infections are antibiotic refractory due to biofilm formation. A key structural component of biofilm is extracellular DNA, which is stabilized by bacterial proteins, including those from the DNABII family. TRL1068 is a high-affinity human monoclonal antibody against a DNABII epitope conserved across both Gram-positive and Gram-negative bacterial species. In the present study, the efficacy of TRL1068 for the disruption of biofilm was demonstrated in vitro in the absence of antibiotics by scanning electron microscopy. The in vivo efficacy of this antibody was investigated in a well-characterized catheter-induced aortic valve infective endocarditis model in rats infected with a methicillin-resistant Staphylococcus aureus (MRSA) strain with the ability to form thick biofilms, obtained from the blood of a patient with persistent clinical infection. Animals were treated with vancomycin alone or in combination with TRL1068. MRSA burdens in cardiac vegetations and within intracardiac catheters, kidneys, spleen, and liver showed significant reductions in the combination arm versus vancomycin alone (P < 0.001). A trend toward mortality reduction was also observed (P = 0.09). In parallel, the in vivo efficacy of TRL1068 against a multidrug-resistant clinical Acinetobacter baumannii isolate was explored by using an established mouse model of skin and soft tissue catheter-related biofilm infection. Catheter segments infected with A. baumannii were implanted subcutaneously into mice; animals were treated with imipenem alone or in combination with TRL1068. The combination showed a significant reduction of catheter-adherent bacteria versus the antibiotic alone (P < 0.001). TRL1068 shows excellent promise as an adjunct to standard-of-care antibiotics for a broad range of difficult-to-treat bacterial infections.

Maternal Antibody Responses and Nonprimary Congenital Cytomegalovirus Infection of HIV-1-Exposed Infants.

Risk of congenital cytomegalovirus (cCMV) transmission is highly dependent on the presence of preexisting maternal immunity, with the lowest rates observed in CMV-seroimmune populations. Among infants of CMV-seroimmune women, those who are exposed to human immunodeficiency virus (HIV) have an increased risk of acquiring cCMV infection as compared to HIV-unexposed infants. To better understand the risk factors of nonprimary cCMV transmission in HIV-infected women, we performed a case-control study in which CMV-specific plasma antibody responses from 19 CMV-transmitting and 57 CMV-nontransmitting women with chronic CMV/HIV coinfection were evaluated for the ability to predict the risk of cCMV infection. Primary multivariable conditional logistic regression analysis revealed an association between epithelial-tropic CMV neutralizing titers and a reduced risk of cCMV transmission (odds ratio [OR], 0.18; 95% confidence interval [CI], .03-.93; P = .04), although this effect was not significant following correction for multiple comparisons (false-discovery rate, 0.12). Exploratory analysis of the CMV specificity of plasma antibodies revealed that immunoglobulin G (IgG) responses against the glycoprotein B (gB) neutralizing epitope AD-2 had a borderline association with low risk of transmission (OR, 0.72; 95% CI, .51-1.00; P = .05), although this was not confirmed in a post hoc plasma anti-AD-2 IgG blocking assay. Our data suggest that maternal neutralizing antibody responses may play a role in protection against cCMV in HIV/CMV-coinfected populations.

A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model.

Many serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observedin vitroat an antibody concentration of 1.2 µg/ml over 12 h. The effect of TRL1068in vivowas evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistantStaphylococcus aureus(MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections.

Human cytomegalovirus infection interferes with the maintenance and differentiation of trophoblast progenitor cells of the human placenta.

UNLABELLED: Human cytomegalovirus (HCMV) is a major cause of birth defects that include severe neurological deficits, hearing and vision loss, and intrauterine growth restriction. Viral infection of the placenta leads to development of avascular villi, edema, and hypoxia associated with symptomatic congenital infection. Studies of primary cytotrophoblasts (CTBs) revealed that HCMV infection impedes terminal stages of differentiation and invasion by various molecular mechanisms. We recently discovered that HCMV arrests earlier stages involving development of human trophoblast progenitor cells (TBPCs), which give rise to the mature cell types of chorionic villi-syncytiotrophoblasts on the surfaces of floating villi and invasive CTBs that remodel the uterine vasculature. Here, we show that viral proteins are present in TBPCs of the chorion in cases of symptomatic congenital infection. In vitro studies revealed that HCMV replicates in continuously self-renewing TBPC lines derived from the chorion and alters expression and subcellular localization of proteins required for cell cycle progression, pluripotency, and early differentiation. In addition, treatment with a human monoclonal antibody to HCMV glycoprotein B rescues differentiation capacity, and thus, TBPCs have potential utility for evaluation of the efficacies of novel antiviral antibodies in protecting and restoring placental development. Our results suggest that HCMV replicates in TBPCs in the chorion in vivo, interfering with the earliest steps in the growth of new villi, contributing to virus transmission and impairing compensatory development. In cases of congenital infection, reduced responsiveness of the placenta to hypoxia limits the transport of substances from maternal blood and contributes to fetal growth restriction. IMPORTANCE: Human cytomegalovirus (HCMV) is a leading cause of birth defects in the United States. Congenital infection can result in permanent neurological defects, mental retardation, hearing loss, visual impairment, and pregnancy complications, including intrauterine growth restriction, preterm delivery, and stillbirth. Currently, there is neither a vaccine nor any approved treatment for congenital HCMV infection during gestation. The molecular mechanisms underlying structural deficiencies in the placenta that undermine fetal development are poorly understood. Here we report that HCMV replicates in trophoblast progenitor cells (TBPCs)-precursors of the mature placental cells, syncytiotrophoblasts and cytotrophoblasts, in chorionic villi-in clinical cases of congenital infection. Virus replication in TBPCs in vitro dysregulates key proteins required for self-renewal and differentiation and inhibits normal division and development into mature placental cells. Our findings provide insights into the underlying molecular mechanisms by which HCMV replication interferes with placental maturation and transport functions.

A high-affinity native human antibody neutralizes human cytomegalovirus infection of diverse cell types.

Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation.

Intrauterine growth restriction caused by underlying congenital cytomegalovirus infection.

BACKGROUND: Human cytomegalovirus (HCMV) is the major viral etiology of congenital infection and birth defects. Fetal transmission is high (30%-40%) in primary maternal infection, and symptomatic babies have permanent neurological, hearing, and vision defects. Recurrent infection is infrequently transmitted (2%) and largely asymptomatic. Congenital infection is also associated with intrauterine growth restriction (IUGR). METHODS: To investigate possible underlying HCMV infection in cases of idiopathic IUGR, we studied maternal and cord sera and placentas from 19 pregnancies. Anti-HCMV antibodies, hypoxia-related factors, and cmvIL-10 were measured in sera. Placental biopsy specimens were examined for viral DNA, expression of infected cell proteins, and pathology. RESULTS: Among 7 IUGR cases, we identified 2 primary and 3 recurrent HCMV infections. Virus replicated in glandular epithelium and lymphatic endothelium in the decidua, cytotrophoblasts, and smooth muscle cells in blood vessels of floating villi and the chorion. Large fibrinoids with avascular villi, edema, and inflammation were significantly increased. Detection of viral proteins in the amniotic epithelium indicated transmission in 2 cases of IUGR with primary infection and 3 asymptomatic recurrent infections. CONCLUSIONS: Congenital HCMV infection impairs placental development and functions and should be considered as an underlying cause of IUGR, regardless of virus transmission to the fetus.

Inhibition of human cytomegalovirus entry into mucosal epithelial cells.

Human cytomegalovirus (CMV) causes serious developmental disabilities in newborns infected in utero following oral acquisition by the mother. Thus, neutralizing antibodies in maternal saliva have potential to prevent maternal infection and, consequently, fetal transmission and disease. Based on standard cell culture models, CMV entry mediators (and hence neutralizing targets) are cell type-dependent: entry into fibroblasts requires glycoprotein B (gB) and a trimeric complex (TC) of glycoproteins H, L, and O, whereas endothelial and epithelial cell entry additionally requires a pentameric complex (PC) of glycoproteins H and L with UL128, UL130, and UL131A. However, as the mediators of mucosal cell entry and the potential impact of cellular differentiation remained unclear, the present studies utilized mutant viruses, neutralizing antibodies, and soluble TC-receptor to determine the entry mediators required for infection of mucocutaneus cell lines and primary tonsil epithelial cells. Entry into undifferentiated cells was largely PC-dependent, but PC-independent entry could be induced by differentiation. TC-independent entry was also observed and varied by cell line and differentiation. Infection of primary tonsil cells from some donors was entirely TC-independent. In contrast, an antibody to gB or disruption of virion attachment using heparin blocked entry into all cells. These findings indicate that CMV entry into the spectrum of cell types encountered in vivo is likely to be more complex than has been suggested by standard cell culture models and may be influenced by the relative abundance of virion envelope glycoprotein complexes as well as by cell type, tissue of origin, and state of differentiation.

Anti-G protein antibodies targeting the RSV G protein CX3C chemokine region improve the interferon response.

Background: Respiratory syncytial virus (RSV) is a poor inducer of antiviral interferon (IFN) responses which result in incomplete immunity and RSV disease. Several RSV proteins alter antiviral responses, including the non-structural proteins (NS1, NS2) and the major viral surface proteins, that is, fusion (F) and attachment (G) proteins. The G protein modifies the host immune response to infection linked in part through a CX3 C chemokine motif. Anti-G protein monoclonal antibodies (mAbs), that is, clones 3D3 and 2D10 that target the G protein CX3C chemokine motif can neutralize RSV and inhibit G protein-CX3CR1 mediated chemotaxis. Objectives: Determine how monoclonal antibodies against the RSV F and G proteins modify the type I and III IFN responses to RSV infection. Design: As the G protein CX3 C motif is implicated in IFN antagonism, we evaluated two mAbs that block G protein CX3C-CX3CR1 interaction and compared responses to isotype mAb control using a functional cellular assay and mouse model. Methods: Mouse lung epithelial cells (MLE-15 cells) and BALB/c mice were infected with RSV Line19 F following prophylactic mAb treatment. Cell supernatant or bronchoalveolar lavage fluid (BALF) were assayed for types I and III IFNs. Cells were interrogated for changes in IFN-related gene expression. Results: Treatment with an anti-G protein mAb (3D3) resulted in improved IFN responses compared with isotype control following infection with RSV, partially independently of neutralization, and this was linked to upregulated SOCS1 expression. Conclusions: These findings show that anti-G protein antibodies improve the protective early antiviral response, which has important implications for vaccine and therapeutic design. Plain Language Summary: RSV is a leading cause of respiratory disease in infants and the elderly. The only Food and Drug Administration-approved prophylactic treatment is limited to an anti-F protein monoclonal antibody (mAb), that is, palivizumab which has modest efficacy against RSV disease. Accumulating evidence suggests that targeting the RSV attachment (G) protein may provide improved protection from RSV disease. It is known that the G protein is an IFN antagonist, and IFN has been shown to be protective against RSV disease. In this study, we compared IFN responses in mouse lung epithelial (MLE-15) cells and in mice infected with RSV Line19 F treated with anti-G protein or anti-F protein mAbs. The levels of type I and III IFNs were determined. Anti-G protein mAbs improved the levels of IFNs compared with isotype-treated controls. These findings support the concept that anti-G protein mAbs mediate improved IFN responses against RSV disease, which may enable improved treatment of RSV infections.

Immune Prophylaxis Targeting the Respiratory Syncytial Virus (RSV) G Protein.

The respiratory syncytial virus (RSV) causes significant respiratory disease in young infants and the elderly. Immune prophylaxis in infants is currently limited to palivizumab, an anti-RSV fusion (F) protein monoclonal antibody (mAb). While anti-F protein mAbs neutralize RSV, they are unable to prevent aberrant pathogenic responses provoked by the RSV attachment (G) protein. Recently, the co-crystal structures of two high-affinity anti-G protein mAbs that bind the central conserved domain (CCD) at distinct non-overlapping epitopes were solved. mAbs 3D3 and 2D10 are broadly neutralizing and block G protein CX3C-mediated chemotaxis by binding antigenic sites γ1 and γ2, respectively, which is known to reduce RSV disease. Previous studies have established 3D3 as a potential immunoprophylactic and therapeutic; however, there has been no similar evaluation of 2D10 available. Here, we sought to determine the differences in neutralization and immunity to RSV Line19F infection which recapitulates human RSV infection in mouse models making it useful for therapeutic antibody studies. Prophylactic (24 h prior to infection) or therapeutic (72 h post-infection) treatment of mice with 3D3, 2D10, or palivizumab were compared to isotype control antibody treatment. The results show that 2D10 can neutralize RSV Line19F both prophylactically and therapeutically, and can reduce disease-causing immune responses in a prophylactic but not therapeutic context. In contrast, 3D3 was able to significantly (p < 0.05) reduce lung virus titers and IL-13 in a prophylactic and therapeutic regimen suggesting subtle but important differences in immune responses to RSV infection with mAbs that bind distinct epitopes.

Monoclonal Antibody Disrupts Biofilm Structure and Restores Antibiotic Susceptibility in an Orthopedic Implant Infection Model.

Bacterial biofilms on orthopedic implants are resistant to the host immune response and to traditional systemic antibiotics. Novel therapies are needed to improve patient outcomes. TRL1068 is a human monoclonal antibody (mAb) against a biofilm anchoring protein. For assessment of this agent in an orthopedic implant infection model, efficacy was measured by reduction in bacterial burden of Staphylococcus aureus, the most common pathogen for prosthetic joint infections (PJI). Systemic treatment with the biofilm disrupting mAb TRL1068 in conjunction with vancomycin eradicated S. aureus from steel pins implanted in the spine for 26 of 27 mice, significantly more than for vancomycin alone. The mechanism of action was elucidated by two microscopy studies. First, TRL1068 was localized to biofilm using a fluorescent antibody tag. Second, a qualitative effect on biofilm structure was observed using scanning electron microscopy (SEM) to examine steel pins that had been treated in vivo. SEM images of implants retrieved from control mice showed abundant three-dimensional biofilms, whereas those from mice treated with TRL1068 did not. Clinical Significance: TRL1068 binds at high affinity to S. aureus biofilms, thereby disrupting the three-dimensional structure and significantly reducing implant CFUs in a well-characterized orthopedic model for which prior tested agents have shown only partial efficacy. TRL1068 represents a promising systemic treatment for orthopedic implant infection.