Binding and Neutralizing Capacity of Respiratory Syncytial Virus (RSV)-Specific Recombinant IgG Against RSV in Human Milk, Gastric and Intestinal Fluids from Infants.

Oral administration of pathogen-specific recombinant antibodies may help to prevent infant gastrointestinal (GI) pathogen infection; however, to neutralize an infectious agent, these antibodies must resist degradation in the GI tract. Palivizumab, a recombinant antibody specific for the respiratory syncytial virus (RSV), was used as a model for pathogen-specific IgG in human milk. The aim was to compare the remaining binding capacity of palivizumab in milk between three mothers after exposure to an in vitro model of infant gastrointestinal digestion (gastric and duodenal fluids) using ELISA. The neutralizing capacity of palivizumab in pooled human milk, gastric contents, and stools from preterm infants was also evaluated for blocking RSV with green fluorescent protein (RSV-GFP) infection in Hep-2 cells using confocal and inverted microscopy and flow cytometry. The reduction of palivizumab binding capacity in human milk and digested samples was slightly different between mothers. Overall, palivizumab decreased 50% after simulated gastric digestion with pepsin and 62% after simulated intestinal digestion with pancreatin. Palivizumab (2-8 µg/mL) in human milk or stool samples blocked RSV (3.4 × 104 FFU/mL) infection (no syncytia formation on Hep-2 cells) by microscopy. Syncytia formation was detected on Hep-2 cells when RSV was incubated in gastric contents or virus medium with 2-4 µg/mL of palivizumab, but no infection was observed at 8 µg/mL. No fluorescence (absence of infected cells) was detected when palivizumab (100 µg/mL) was incubated in human milk or medium with RSV-GFP (1.1 × 105 FFU/mL), whereas fluorescence increased with the reduced concentration of palivizumab using flow cytometry. These results suggest that undigested and digested matrices could change the binding and neutralizing capacity of viral pathogen-specific antibodies.

Up-to-date role of biologics in the management of respiratory syncytial virus.

INTRODUCTION: Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in young children and a substantial contributor to respiratory tract disease throughout life. Despite RSV being a high priority for vaccine development, there is currently no safe and effective vaccine available. There are many challenges to developing an RSV vaccine and there are limited antiviral drugs or biologics available for the management of infection. In this article, we review the antiviral treatments, vaccination strategies along with alternative therapies for RSV. AREAS COVERED: This review is a summary of the current antiviral and RSV vaccination approaches noting strategies and alternative therapies that may prevent or decrease the disease severity in RSV susceptible populations. EXPERT OPINION: This review discusses anti-RSV strategies given that no safe and efficacious vaccines are available, and therapeutic treatments are limited. Various biologicals that target for RSV are considered for disease intervention, as it is likely that it may be necessary to develop separate vaccines or therapeutics for each at-risk population.

Estimating the impact of multiple immunization products on medically-attended respiratory syncytial virus (RSV) infections in infants.

BACKGROUND: Palivizumab, a monoclonal antibody and the only licensed immunization product for preventing respiratory syncytial virus (RSV) infection, is recommended for children with certain high-risk conditions. Other antibody products and maternal vaccines targeting young infants are in clinical development. Few studies have compared products closest to potential licensure and have primarily focused on the effects on hospitalizations only. Estimates of the impact of these products on medically-attended (MA) infections in a variety of healthcare settings are needed to assist with developing RSV immunization recommendations. METHODS: We developed a tool for practicing public health officials to estimate the impact of immunization strategies on RSV-associated MA lower respiratory tract infections (LRTIs) in various healthcare settings among infants <12 months. Users input RSV burden and seasonality and examine the influence of altering product efficacy and uptake assumptions. We used the tool to evaluate candidate products' impacts among a US birth cohort. RESULTS: We estimated without immunization, 407,360 (range: 339,650-475,980) LRTIs are attended annually in outpatient clinics, 147,240 (126,070-168,510) in emergency departments (EDs), and 33,180 (24,760-42,900) in hospitals. A passive antibody candidate targeting all infants prevented the most LRTIs: 196,470 (48% of visits without immunization) outpatient clinic visits (range: 163,810-229,650), 75,250 (51%) EDs visits (64,430-86,090), and 18,140 (55%) hospitalizations (13,770-23,160). A strategy combining maternal vaccine candidate and palivizumab prevented 58,210 (14% of visits without immunization) LRTIs in outpatient clinics (range: 48,520-67,970), 19,580 (13%) in EDs (16,760-22,400), and 8,190 (25%) hospitalizations (6,390-10,150). CONCLUSIONS: Results underscore the potential for anticipated products to reduce serious RSV illness. Our tool (provided to readers) can be used by different jurisdictions and accept updated data. Results can aid economic evaluations and public health decision-making regarding RSV immunization products.

Expansion of the 1st WHO international standard for antiserum to respiratory syncytial virus to include neutralisation titres against RSV subtype B: An international collaborative study.

An International Standard to harmonise results from RSV subtype A neutralisation assays was generated and established by the World Health Organization in 2018. Here we report on a study to expand the use of that standard to include neutralisation assays using human sera against RSV subtype B and to test its ability to harmonise neutralisation titres from neutralisation assays including complement. The study included 11 laboratories from 6 countries. All participants used their own in-house virus neutralisation assay and their own virus stocks. The study samples comprised the current International Standard (16/284) and its potential replacement (16/322), individual sera from naturally infected humans, a monoclonal antibody to RSV (palivizumab) and samples from the BEI Resources panel of human antiserum and immune globulin to RSV. Of the 11 laboratories that took part in the study, 5 returned data from neutralisation assays with and without the inclusion of serum complement. The study showed that inter-laboratory variability in neutralisation titres was significantly reduced when values were expressed relative to 16/284 or 16/322. Complement did not affect the ability of the International Standard to decrease inter-laboratory variability as the standard was able to reduce the differences between titres from assays with and without complement. Based on these results, we will recommend to the WHO Expert Committee on Biological Standardisation (ECBS) that 16/284 and 16/322 be expanded in their use to include neutralisation assays against RSV/B.

Respiratory syncytial virus prefusogenic fusion (F) protein nanoparticle vaccine: Structure, antigenic profile, immunogenicity, and protection.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in the very young, elderly, and immunocompromised for which there is no vaccine. The surface exposed RSV fusion (F) glycoprotein is required for membrane fusion and infection and is a desirable vaccine candidate. RSV F glycoprotein structure is dynamic and undergoes significant rearrangements during virus assembly, fusion, and infection. We have previously described an RSV fusion-inactive prefusogenic F with a mutation of one of two furin cleavage sites resulting in the p27 region on the N-terminus of F1 with a truncated fusion peptide covalently linked to F2. A processing intermediate RSV prefusogenic F has been reported in infected cells, purified F, budded virus, and elicited a strong immune response against p27 in RSV infected young children. In this report, we demonstrate that prefusogenic F, when expressed on the cell surface of Sf9 insect and human 293T cells, binds monoclonal antibodies (mAbs) that target prefusion-specific antigenic sites Ø and VIII, and mAbs targeting epitopes common to pre- and postfusion F sites II and IV. Purified prefusogenic F bound prefusion F specific mAbs to antigenic sites Ø and VIII and mAbs targeting pre- and postfusion sites II, IV, and p27. Mice immunized with prefusogenic F antigen produced significantly higher levels of anti-F IgG and RSV neutralizing antibodies than prefusion or postfusion F antigens and induced antibodies competitive with mAbs to sites Ø, VIII, II, and IV. RSV prefusogenic F neutralization antibody responses were enhanced with aluminum phosphate adjuvant and significantly higher than prefusion F. Prefusogenic F vaccine protected cotton rats against upper and lower respiratory tract infection by RSV/A. For the first time, we present the structure, antigenic profile, immunogenicity, and protective efficacy of RSV prefusogenic F nanoparticle vaccine.

Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen.

Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies.

State of the Art in Respiratory Syncytial Virus Drug Discovery and Development.

Respiratory syncytial virus (RSV) is a globally prevalent viral infection with limited treatment options which hospitalizes millions each year. Treatment options have been limited to palivizumab, a monoclonal antibody, approved for prophylaxis in high-risk infants and ribavirin with very limited efficacy and significant safety concerns. This Perspective surveys the range of direct acting antiviral agents (DAAs) that target key steps in the viral life cycle. A number of approaches to DAAs have produced landmark clinical studies over the past few years, notably in fusion and nucleoside inhibitors, and an update of the clinical status of these compounds is provided. Non-nucleoside inhibitors of replication are reviewed in addition to inhibitors of other mechanisms, notably the RSV N and G proteins. This article will provide an informative perspective of the current status of drug discovery targeted at providing an effective therapy for RSV infection.

Engineered mRNA-expressed antibodies prevent respiratory syncytial virus infection.

The lung is a critical prophylaxis target for clinically important infectious agents, including human respiratory syncytial virus (RSV) and influenza. Here, we develop a modular, synthetic mRNA-based approach to express neutralizing antibodies directly in the lung via aerosol, to prevent RSV infections. First, we express palivizumab, which reduces RSV F copies by 90.8%. Second, we express engineered, membrane-anchored palivizumab, which prevents detectable infection in transfected cells, reducing in vitro titer and in vivo RSV F copies by 99.7% and 89.6%, respectively. Finally, we express an anchored or secreted high-affinity, anti-RSV F, camelid antibody (RSV aVHH and sVHH). We demonstrate that RSV aVHH, but not RSV sVHH, significantly inhibits RSV 7 days post transfection, and we show that RSV aVHH is present in the lung for at least 28 days. Overall, our data suggests that expressing membrane-anchored broadly neutralizing antibodies in the lungs could potentially be a promising pulmonary prophylaxis approach.

Human respiratory syncytial virus: pathogenesis, immune responses, and current vaccine approaches.

Respiratory syncytial virus continues to pose a serious threat to the pediatric populations worldwide. With a genomic makeup of 15,200 nucleotides, the virus encodes for 11 proteins serving as envelope spikes, inner envelope proteins, and non-structural and ribonucleocapsid complexes. The fusion (F) and attachment (G) surface glycoproteins are the key targets for neutralizing antibodies. The highly variable G with altered glycosylations and the conformational alternations of F create challenges for vaccine development. The metastable F protein is responsible for RSV-host cell fusion and thus infectivity. Novel antigenic sites were identified on this form following its stabilization and solving its crystal structure. Importantly, site ø displays neutralizing activity exceeding those of post-F-specific and shared antigenic sites, such as site II which is the target for Palivizumab therapeutic antibody. Induction of high neutralizing antibody responses by pre-F immunization in animal models promoted it as a major vaccine candidate. Since RSV infection is more serious at age extremities and in individuals with undermining health conditions, vaccines are being developed to target these populations. Infants below three months of age have a suppressive immune system, making vaccines' immunogenicity weak. Therefore, a suggested strategy to protect newborns from RSV infection would be through passive immunity of maternal antibodies. Hence, pregnant women at their third trimester have been selected as an ideal target for vaccination with RSV pre-F vaccine. This review summarizes the different modes of RSV pathogenesis and host's immune response to the infection, and illustrates on the latest updates of vaccine development and vaccination approaches.

Reformatting palivizumab and motavizumab from IgG to human IgA impairs their efficacy against RSV infection in vitro and in vivo.

Respiratory syncytial virus (RSV) infection is a leading cause of hospitalization and mortality in young children. Protective therapy options are limited. Currently, palivizumab, a monoclonal IgG1 antibody, is the only licensed drug for RSV prophylaxis, although other IgG antibody candidates are being evaluated. However, at the respiratory mucosa, IgA antibodies are most abundant and act as the first line of defense against invading pathogens. Therefore, it would be logical to explore the potential of recombinant human IgA antibodies to protect against viral respiratory infection, but very little research on the topic has been published. Moreover, it is unknown whether human antibodies of the IgA isotype are better suited than those of the IgG isotype as antiviral drugs to combat respiratory infections. To address this, we generated various human IgA antibody formats of palivizumab and motavizumab, two well-characterized human IgG1 anti-RSV antibodies. We evaluated their efficacy to prevent RSV infection in vitro and in vivo and found similar, but somewhat decreased efficacy for different IgA subclasses and formats. Thus, reformatting palivizumab or motavizumab into IgA reduces the antiviral potency of either antibody. Moreover, our results indicate that the efficacy of intranasal IgA prophylaxis against RSV infection in human FcαRI transgenic mice is independent of Fc receptor expression.

Passive and active immunization against respiratory syncytial virus for the young and old.

INTRODUCTION: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants worldwide and also causes significant disease in the elderly. Despite 60 years of RSV research and vaccine development, there is only one approved medicine to prevent RSV infections. Palivizumab, a monoclonal antibody (mAb) against the RSV fusion (F) protein, is indicated for preterm infants and children at high-risk for RSV infections. It is an active time in RSV vaccine and mAb development with 14 vaccines and 2 mAbs currently being tested in clinical trials as of 13 February 2017. Active vaccination of women in the third trimester or passive immunization of infants with a mAb are particularly attractive approaches as the most severe disease occurs within the first 6 months of life. Areas covered: Here, we review current approaches for preventing RSV in the young and old, describe proposed clinical endpoints for studies in pediatric and adult clinical trials and highlight results from recent and ongoing clinical studies. Expert commentary: With 16 candidates in clinical development, approval of the first RSV vaccine or mAb for the prevention of RSV in all infants or the elderly is likely to occur in the next five years.

Impact of the use of immunoglobulin palivizumab in the State of São Paulo: a cohort study / Impacto do uso da imunoglobulina palivizumabe no Estado de São Paulo: estudo de coorte / Impacto del uso de la inmunoglobulina palivizumab en el Estado (provincia) de São Paulo: estudio de cohorte

ABSTRACT Introduction: the use of palivizumab as prophylaxis of the respiratory syncytial virus is not a consensus. In Brazil, it is a public health program, but other countries do not consider it cost-effective. Objective: to identify the rate of hospitalization in Intensive Care Unit for respiratory illness or symptoms among children who received the immunoglobulin palivizumab, the proportion of children who failed to take any of the recommended doses and the impact of that failure on hospitalization. Method: cohort study conducted with 693 children enrolled in the palivizumab program in 2014 (85.1% of the population), with monthly assessment from April to September through a telephone call to the mothers or caregiver. The probability of hospitalization in the Intensive Care Unit related to failure in taking the palivizumab, was analyzed through multiple logistic regression, with p<0,05. Results: the hospitalization rate was 18.2%; 2.3% of the children did not receive all the recommended immunoglobulin doses; the probability of hospitalization for respiratory illness or symptoms increased by an average of 29% at each missed dose (p=0.007; OR=1.29, CI=1.07-1.56). Conclusion: the increase in the chance of hospitalization related to missed immunoglobulin doses indicates the need to implement health education actions and active search for absent children by the health services.

RESUMO Introdução: o uso da palivizumabe como profilaxia do vírus sincicial respiratório não é consenso. No Brasil, constitui programa de saúde pública, mas outros países não a consideram custo-efetiva. Objetivo: identificar a taxa de hospitalização em Unidade de Terapia Intensiva por doença ou sintomatologia respiratória entre crianças que receberam imunoglobulina palivizumabe, a proporção de crianças que falharam na tomada de alguma das doses indicadas e o impacto dessa falha na hospitalização. Método: estudo de coorte, incluídas 693 crianças inscritas no programa em 2014 (85,1% da população), com seguimento mensal de abril a setembro, por ligação telefônica às mães/responsáveis. A chance de hospitalização, em Unidade de Terapia Intensiva, em função da falha, foi avaliada por regressão logística múltipla, adotando-se p crítico <0,05. Resultados: a taxa de hospitalização foi de 18,2%; não receberam todas as doses da imunoglobulina 2,3% das crianças; a chance de hospitalização por doença ou sintomatologia respiratória aumentou, em média, 29% a cada falha na tomada de alguma dose (p=0,007; OR=1,29, IC=1,07-1,56). Conclusão: o aumento da chance de hospitalização na vigência de falha na tomada de alguma dose da imunoglobulina indica a necessidade de implementação de ações de educação em saúde e busca ativa de crianças faltosas pelos serviços de saúde.

RESUMEN Introducción: el uso de la palivizumab como profilaxis del virus sincitial respiratorio no es consenso a nivel mundial. En el Brasil, está incluido en el programa de salud pública, pero en otros países no se lo considera costo-efectivo. Objetivo: identificar el porcentaje de hospitalización en Unidades de Terapia Intensiva por enfermedad o sintomatología respiratoria entre niños que recibieron inmunoglobulina palivizumab, determinar la proporción de niños que no tomaron alguna de las dosis indicadas y el impacto de dicha falla en la hospitalización. Método: estudio de cohorte, realizado entre 693 niños inscriptos en el programa en 2014 (85,1% de la población), con seguimiento mensual de abril a septiembre, a través de pláticas telefónicas con las madres/responsables. La probabilidad de hospitalización en una Unidad de Terapia Intensiva, en función de la falla, se evaluó por regresión logística múltiple, adoptando el p crítico <0,05. Resultados: la tasa de hospitalización fue del 18,2%; el 2,3% de los niños no recibió todas las dosis de la inmunoglobulina; la probabilidad de hospitalización por enfermedad o sintomatología respiratoria aumentó en un promedio del 29% a cada falla en la toma de alguna dosis (p = 0,007; OR = 1,29, IC = 1,07-1,56). Conclusión: el aumento de la probabilidad de hospitalización cuando hay falla en la ingestión de alguna dosis de la inmunoglobulina indica la necesidad de implantar acciones educativas en salud y que los servicios de salud se ocupen eficazmente de buscar a los niños faltantes.

RSV neutralization by palivizumab, but not by monoclonal antibodies targeting other epitopes, is augmented by Fc gamma receptors.

Palivizumab efficiently blocks respiratory syncytial virus (RSV) infection in vitro. However, virus neutralization assays generally omit Fc region-mediated effects. We investigated the neutralization activity of RSV-specific monoclonal antibodies on cells with Fc receptors. Subneutralizing concentrations of antibodies resulted in antibody-dependent enhancement of RSV infection in monocytic cells. Contrary to antibodies targeting other epitopes, the neutralization by palivizumab was augmented in cells with Fc receptors. This unrecognized characteristic of palivizumab may be relevant for its performance in vivo.

Experimental animal model for analyzing immunobiological responses following vaccination with formalin-inactivated respiratory syncytial virus.

Formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine was developed in the 1960s. However, this vaccine does not prevent infection in RSV-naïve recipients and has the paradoxical effect of increasing the severity of RSV illness following natural infection, which has been a major obstacle to developing RSV vaccines. Several experimental animal models for determining the cause of the severe symptoms in FI-RSV recipients have been developed. In the present study, cotton rats immunized with FI-RSV were challenged with RSV and histopathological findings and recovery of infectious virus were studied. Copy numbers of mRNA of Th1 and Th2 cytokines were measured in lung tissues to gain better understanding of their immune responses. Infiltration of inflammatory cells and prominent interstitial pneumonitis were observed in the FI-RSV group, as was induction of mRNA of Th2 cytokines such as IL-4, IL-10, IL-13 and RANTES. Rats immunized with recombinant measles virus expressing the RSV F protein (MVAIK/RSV/F) and those treated with anti-RSV mAb (palivizumab) showed very mild interstitial pneumonitis. Amounts of mRNA of IL-1ß, IFN-γ and IL-4 were higher in the MVAIK/RSV/F group. Administration of palivizumab before RSV challenge decreased the severity of interstitial pneumonitis in the FI-RSV group. FI-RSV induced skewed Th2 responses, resulting in severe inflammatory responses.

Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated in vitro neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.

Prevention of Respiratory Syncytial Virus Infection: From Vaccine to Antibody.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children. Initial efforts to develop a vaccine to prevent RSV lower respiratory tract disease in children were halted because of serious adverse events that occurred when children were infected with RSV following vaccination, including vaccine-related deaths. Subsequently, a major focus for researchers was to understand what led to these adverse events. Investment in a vaccine for RSV continues, and new strategies are under development. Success to prevent RSV disease was met by the development of immunoprophylaxis, first with intravenous immunoglobulin and then with recombinant monoclonal antibody. The story of immunoprophylaxis for RSV includes the first-in-class use of antibody technology for infectious disease, and palivizumab currently remains the only way to prevent serious lower respiratory tract disease due to RSV infection.