Respiratory Syncytial Virus Neutralizing Antibody Research Articles

RSV neutralizing antibodies in dried blood.

The key correlate of protection of respiratory syncytial virus (RSV) vaccines and monoclonal antibodies (mAb) is virus neutralization, measured using sera obtained through venipuncture. Dried blood obtained with a finger prick can simplify acquisition, processing, storage, and transport in trials, and thereby reduce costs. In this study we validate an assay to measure RSV neutralization in dried capillary blood. Functional antibodies were compared between matched serum and dried blood samples from a phase I trial with RSM01, an investigational anti-RSV Prefusion F mAb. Hep-2 cells were infected with a serial dilution of sample-virus mixture using RSV-A2-mKate to determine half-maximal inhibitory concentration. Stability of dried blood was evaluated over time and during temperature stress. Functional antibodies in dried blood were highly correlated with serum (R2 = 0.98, p < 0.0001). The precision of the assay for dried blood was similar to serum. The function of mAb remained stable for 9 months at room temperature and frozen dried blood samples. We demonstrated the feasibility of measuring RSV neutralization using dried blood as a patient-centered solution that may replace serology testing in trials against RSV or other viruses, such as influenza and SARS-CoV-2.

In-vivo and human evidence for potential efficacy of therapeutic polyclonal RSV neutralizing antibodies for palivizumab-resistant RSV infections

BackgroundMonoclonal antibody (palivizumab), intravenous immune globulin (IGIV), or respiratory syncytial virus (RSV)-polyclonal-hyperimmune-globulin (RSV-IG as Respigam®, RI-001, RI-002) are used with ribavirin in RSV-infected immunocompromised patients, with debated efficacy. Palivizumab-resistance (PR) can arise during treatment of persistent infections in this population. RSV-IG may confer benefit in PR-RSV infection. MethodsRSV-IG [RI-001] was provided for an immunocompromised infant with RSV-pneumonitis refractory to ribavirin and palivizumab. RSV-neutralizing antibody, respiratory RSV load (qPCR), and F-gene-sequence-detection of PR was determined. Prophylactic RSV-IG [RI-002] or palivizumab was administered in a cotton-rat model infected with wild-type and PR-RSV. Lung RSV load and neutralizing antibody were measured. ResultsAs protective RI-001-neutralizing antibody titers waned in the infant, a subpopulation of PR-escape mutants were detected with a fatal RSV-burden in the lungs. In PR-RSV-infected cotton rats, prophylactic RI-002 reduced RSV-load in the lungs (2.45 vs 0.28 log10 PFU/g lung-tissue reduction, respectively, p < 0.05) and provided protective RSV-neutralizing antibody. ConclusionsRSV-IG and ribavirin use in immunocompromised patients requires further study.

Virus-like particles containing a prefusion-stabilized F protein induce a balanced immune response and confer protection against respiratory syncytial virus infection in mice.

Respiratory syncytial virus (RSV) is a serious respiratory pathogen in infants and young children worldwide. Currently, no licensed RSV vaccines are available. In this study, we explored stable prefusion conformation virus-like particles (Pre-F VLPs) as RSV vaccine candidates. RSV fusion (F) protein mutants were constructed to form stabilized Pre-F or postfusion (Post-F) configurations. VLPs containing Pre-F or Post-F protein were generated using a recombinant baculovirus (rBV)-insect cell expression system. The assembly and immunological properties of Pre-F or Post-F VLPs were investigated. Pre-F and Post-F VLPs contained antigenic sites Ø and I of pre- and postfusion conformations, respectively. Compared with Post-F VLPs, immunization with Pre-F VLPs elicited upregulation of IFN-γ, IL-2 and IL-10 and downregulation of IL-4 and IL-5 cytokine production in mice. A high percentage of CD25+ Foxp3+ cells or a low percentage of IL-17A-producing cells among CD4+ T cells was observed in the lungs of mice vaccinated with Pre-F VLPs. Importantly, immunization with Pre-F VLPs induced a high level of RSV neutralizing antibody and a balanced immune response, which protected mice against RSV infection without evidence of immunopathology. Our results suggested that Pre-F VLPs generated from rBV-insect cells represent promising RSV vaccine candidates.

Intranasal and intrapulmonary vaccination with an M protein-deficient respiratory syncytial virus (RSV) vaccine improves clinical signs and reduces viral replication in infant baboons after an RSV challenge infection

Respiratory syncytial virus (RSV) is the major viral respiratory pathogen for human infants and children. Despite a severe global burden incurred by annual RSV epidemics, there is no licensed RSV vaccine. We have developed an RSV vaccine from a human RSV strain from which the gene for the viral M protein has been deleted (“Mnull RSV”). RSV infects airway cells and produces each of its proteins. The M protein is responsible for reassembling the various other synthesized viral proteins into new, intact virus. In the absence of the M protein, therefore, reassembly does not occur, and the Mnull RSV does not replicate.We vaccinated 2-week old infant baboons with Mnull RSV either intranasally (IN) or directly into the lung (intratracheal, or IT), then infected these animals by inoculating human RSV directly into the lung. IN vaccination induced inconsistent serum RSV neutralizing antibody (NA) responses, but provided moderate reductions in respiratory rates, overall signs of illness and viral replication in bronchoalveolar lavage (BAL) fluid following infection. Intratracheal vaccination induced much stronger RSV NA responses, which persisted for at least 4–6 months. Following RSV infection, animals vaccinated by the IT route had much greater reductions in tachypnea and work of breathing than animals vaccinated IN, and had undetectable amounts of virus in BAL fluids. These results support the further development of IT Mnull RSV vaccination to reduce the impact of RSV infection in humans.

A phase 1, randomized, placebo-controlled study to evaluate the safety and immunogenicity of an mRNA-based RSV prefusion F protein vaccine in healthy younger and older adults

ABSTRACT Respiratory Syncytial Virus (RSV) causes lower respiratory tract infections that can be severe and sometimes fatal. The risk for severe RSV infection is highest in infants and older adults. A safe and effective RSV vaccine for older adults represents a serious unmet medical need due to higher morbidity and mortality in this age group. In this randomized, partially double-blind, placebo-controlled, phase 1 dose-escalation study, we evaluated the safety, tolerability and immunogenicity of an investigational messenger ribonucleic acid (mRNA) vaccine encoding the RSV fusion protein (F) stabilized in the prefusion conformation. The study was conducted in healthy younger adults (ages ≥18 and ≤49 years) and healthy older adults (ages ≥60 and ≤79 years). Participants received mRNA-1777 (V171) or placebo as a single intramuscular dose. For each dose level, three sentinel participants were administered open-label mRNA-1777 (V171). Seventy-two younger adults were randomized and administered 25, 100, or 200 µg mRNA-1777 (V171) or placebo, and 107 older adults were randomized and administered 25, 100, 200 or 300 µg mRNA-1777 (V171) or placebo. Primary objectives were safety and tolerability and secondary objectives included humoral and cell-mediated immunogenicity. All dose levels of mRNA-1777 (V171) were generally well tolerated and no serious adverse events related to the vaccine were reported. Immunization with mRNA-1777 (V171) elicited a humoral immune response as measured by increases in RSV neutralizing antibody titers, serum antibody titers to RSV prefusion F protein, D25 competing antibody titers to RSV prefusion F protein, and cell-mediated immune responses to RSV-F peptides.

Antigenicity and immunogenicity of unique prefusion-mimic F proteins presented on enveloped virus-like particles

Pre-fusion stabilizing mutations (DS-Cav1) in soluble fusion (F) proteins of human respiratory syncytial virus (RSV) were previously reported. Here we investigated the antigenic and immunogenic properties of pre-fusion like RSV F proteins on enveloped virus-like particles (VLP). Additional mutations were introduced to DS-Cav1 (F-dcmTM VLP); fusion peptide deletion and cleavage mutation site 1 (F1d-dcmTM VLP) or both sites (F12d-dcmTM VLP). F1d-dcmTM VLP and F12d-dcmTM VLP displayed higher reactivity against pre-fusion specific site Ø and antigenic site I and II specific monoclonal antibodies, compared to F-dcmTM VLP with DS-Cav1 only. Mice immunized with F1d-dcmTM VLP and F12d-dcmTM VLP induced higher levels of DS-Cav1 pre-fusion specific IgG antibodies, RSV neutralizing activity titers, and effective lung viral clearance after challenge. These results suggest that cleavage site mutations and fusion peptide deletion in addition to DS-Cav1 mutations have contributed to structural stabilization of pre-fusion like F conformation on enveloped VLP, capable of inducing high levels of pre-fusion F specific and RSV neutralizing antibodies.

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.

RSV Vaccine Based on Rhabdoviral Vector Protects after Single Immunization.

The respiratory syncytial virus (RSV) is one major cause of lower respiratory tract infections in childhood and an effective vaccine is still not available. We previously described a new rhabdoviral vector vaccine, VSV-GP, a variant of the vesicular stomatitis virus (VSV), where the VSV glycoprotein G is exchanged by the glycoprotein GP of the lymphocytic choriomeningitis virus. Here, we evaluated VSV-GP as vaccine vector for RSV with the aim to induce RSV neutralizing antibodies. Wild-type F (Fwt) or a codon optimized version (Fsyn) were introduced at position 5 into the VSV-GP genome. Both F versions were efficiently expressed in VSV-GP-F infected cells and incorporated into VSV-GP particles. In mice, high titers of RSV neutralizing antibodies were induced already after prime and subsequently boosted by a second immunization. After challenge with RSV, viral loads in the lungs of immunized mice were reduced by 2–3 logs with no signs of an enhanced disease induced by the vaccination. Even a single intranasal immunization significantly reduced viral load by a factor of more than 100-fold. RSV neutralizing antibodies were long lasting and mice were still protected when challenged 20 weeks after the boost. Therefore, VSV-GP is a promising candidate for an effective RSV vaccine.

Improving ability of RSV microneutralization assay to detect G-specific and cross-reactive neutralizing antibodies through immortalized cell line selection

Respiratory syncytial virus (RSV) is a significant cause of bronchiolitis and pneumonia. Protection against RSV is associated with neutralizing antibodies against the fusion (F) and attachment (G) glycoproteins. Several RSV vaccine candidates are in development, but their immunogenicity is hard to compare due to the little-understood differences between multiple RSV neutralizing antibody assays used. Existing assays utilize primarily Vero or HEp-2 cells, but their ability to detect G-neutralizing antibodies or antibodies against specific RSV strains is unclear. In this work, we developed an RSV microneutralization assay (MNA) using unmodified RSV and immortalized cell line derived from human airway epithelial cells (A549). Performance of A549-, HEp-2- and Vero-based MNA was compared under the same assay conditions (fixed amount of virus and cells) with regards to detection of neutralizing antibodies against RSV A or B viruses, G-reactive neutralizing antibodies, and effect of complement. Our results indicate that A549 cells yield the highest MNA titers, particularly in the RSV A/A2 MNA, are least susceptible to complement-enhancing effect of neutralizing titer readout and are superior to Vero or HEp-2 MNA at recognizing G-reactive neutralizing antibodies when no complement is used. Vero cells, however, can be more consistent at recognizing neutralizing antibodies against multiple RSV strains. The choice of substrate cells thus affects the outcome of MNA, as some immortalized cells better support detection of broader range of neutralizing antibodies, while others facilitate detection of G-targeting neutralizing antibodies, a long-thought prerogative of primary airway epithelial cells.

Comparison of Palivizumab-Like Antibody Binding to Different Conformations of the RSV F Protein in RSV-Infected Adult Hematopoietic Cell Transplant Recipients.

Most respiratory syncytial virus (RSV) vaccine candidates include fusion (F) protein in different conformations. Antigenic site II found in the different F conformations is the target of palivizumab, the only US Food and Drug Administration approved monoclonal antibody (mAb). Serum palivizumab-like antibody (PLA) is a potential serologic correlate of immunity. Our objective was to determine if different conformations of F protein in a palivizumab competitive antibody (PCA) assay affect the PLA concentrations. Four PCA assays were standardized using mAbs. Each contained prefusion, postfusion, or intermediate F forms. PLA concentrations were measured in acute and convalescent sera from 22 RSV/A and 18 RSV/B-infected adult hematopoietic cell transplant (HCT) recipients. PLA concentrations were calculated using a 4-parameter logistic regression model and analyzed for statistical significance. PCA assays revealed significantly greater PLA concentrations in convalescent sera; comparable increases in PLA concentration in RSV/A and RSV/B-infected HCT recipients; and significantly reduced PLA concentrations in HCT recipients who shed RSV ≥14 days. A significant positive correlation was observed between PCA assays and RSV neutralizing antibody titers. F protein conformation does not appear to have a measurable impact on PCA assays for measuring PLA induced by RSV/A or RSV/B infection.

Preclinical assessment of safety of maternal vaccination against respiratory syncytial virus (RSV) in cotton rats

Maternal immunization directed to control RSV infection in newborns and infants is an appealing vaccination strategy currently under development. In this work we have modeled maternal vaccination against RSV in cotton rats (CR) to answer two fundamental questions on maternal vaccine safety. We tested (i), whether a known, unsafe RSV vaccine (i.e., FI-RSV Lot 100 vaccine) induces vaccine enhanced disease in the presence of passively transferred, RSV maternal immunity, and (ii) whether the same FI-RSV vaccine could induce vaccine enhanced disease in CR litters when used to immunize their RSV-primed mothers. Our data show that FI-RSV immunization of pups with subsequent RSV infection results in vaccine-enhanced disease independent of whether the pups were born to RSV-seropositive or RSV-seronegative mothers, and that FI-RSV immunization of RSV-seropositive mothers does not present a health risk to either the mother or the infant. Our study also raises a novel concern regarding infant immunization, namely that “safe” RSV vaccines (e.g., live RSV administered intramuscularly) may induce vaccine-enhanced disease in RSV-infected pups born to seropositive mothers. Finally, we describe for the first time a sharp decrease in RSV neutralizing antibody titers in immunized seropositive CR at the time of delivery. This decline may reflect maternal immune suppression, potentially pinpointing a window of increased vulnerability to RSV infection that could be alleviated by effective immunization of expectant mothers.

A multi-laboratory study of diverse RSV neutralization assays indicates feasibility for harmonization with an international standard

A current barrier to the standardized evaluation of respiratory syncytial virus (RSV) vaccine candidates is the wide variety of virus neutralization assay formats currently in use for assessing immunogenicity. Assay formats vary widely in labor intensiveness, duration, and sample throughput. Furthermore, the cell lines and virus strains used are not consistent among formats. The purpose of this multi-laboratory study was to assess the variability across a diverse array of assay formats that quantitate RSV neutralizing antibodies. Using a common specimen panel, the degree of overall agreement among existing assays was evaluated to inform on the need for harmonization of assay results. A total of 12 laboratories participated in the blinded survey study by testing a panel comprised of 57 samples chosen to span the reportable titer range of the assays. An independent statistical analysis was conducted to measure overall agreement of assay results. This analysis showed that precision was consistently high, whereas agreement varied widely among assays. To examine whether agreement could be improved, we conducted a harmonization exercise using a variety of sample types as pseudo standards. The results showed that the level of agreement could be improved, and provided information on the suitability of samples for developing an international standard.

Baculovirus vectors expressing F proteins in combination with virus-induced signaling adaptor (VISA) molecules confer protection against respiratory syncytial virus infection

Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV.

An Epitope‐Specific Respiratory Syncytial Virus Vaccine Based on an Antibody Scaffold

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in children. We have generated an epitope-specific RSV vaccine by grafting a neutralizing epitope (F-epitope) in its native conformation into an immunoglobulin scaffold. The resulting antibody fusion exhibited strong binding affinity to Motavizumab, an RSV neutralizing antibody, and effectively induced potent neutralizing antibodies in mice. This work illustrates the potential of the immunoglobulin molecule as a scaffold to present conformationally constrained B-cell epitopes.

Virus-Like Particle Vaccine Containing the F Protein of Respiratory Syncytial Virus Confers Protection without Pulmonary Disease by Modulating Specific Subsets of Dendritic Cells and Effector T Cells.

There is no licensed vaccine against respiratory syncytial virus (RSV) since the failure of formalin-inactivated RSV (FI-RSV) due to its vaccine-enhanced disease. We investigated immune correlates conferring protection without causing disease after intranasal immunization with virus-like particle vaccine containing the RSV fusion protein (F VLP) in comparison to FI-RSV and live RSV. Upon RSV challenge, FI-RSV immune mice showed severe weight loss, eosinophilia, and histopathology, and RSV reinfection also caused substantial RSV disease despite their viral clearance. In contrast, F VLP immune mice showed least weight loss and no sign of histopathology and eosinophilia. High levels of interleukin-4-positive (IL-4(+)) and tumor necrosis factor alpha-positive (TNF-α(+)) CD4(+) T cells were found in FI-RSV immune mice, whereas gamma interferon-positive (IFN-γ(+)) and TNF-α(+) CD4(+) T cells were predominantly detected in live RSV-infected mice. More importantly, in contrast to FI-RSV and live RSV that induced higher levels of CD11b(+) dendritic cells, F VLP immunization induced CD8α(+) and CD103(+) dendritic cells, as well as F-specific IFN-γ(+) and TNF-α(+) CD8(+) T cells. These results suggest that F VLP can induce protection without causing pulmonary RSV disease by inducing RSV neutralizing antibodies, as well as modulating specific subsets of dendritic cells and CD8 T cell immunity. It has been a difficult challenge to develop an effective and safe vaccine against respiratory syncytial virus (RSV), a leading cause of respiratory disease. Immune correlates conferring protection but preventing vaccine-enhanced disease remain poorly understood. RSV F virus-like particle (VLP) would be an efficient vaccine platform conferring protection. Here, we investigated the protective immune correlates without causing disease after intranasal immunization with RSV F VLP in comparison to FI-RSV and live RSV. In addition to inducing RSV neutralizing antibodies responsible for clearing lung viral loads, we show that modulation of specific subsets of dendritic cells and CD8 T cells producing T helper type 1 cytokines are important immune correlates conferring protection but not causing vaccine-enhanced disease.

Recombinant low-seroprevalent adenoviral vectors Ad26 and Ad35 expressing the respiratory syncytial virus (RSV) fusion protein induce protective immunity against RSV infection in cotton rats

RSV is an important cause of lower respiratory tract infections in children, the elderly and in those with underlying medical conditions. Although the high disease burden indicates an urgent need for a vaccine against RSV, no licensed RSV vaccine is currently available. We developed an RSV vaccine candidate based on the low-seroprevalent human adenovirus serotypes 26 and 35 (Ad26 and Ad35) encoding the RSV fusion (F) gene. Single immunization of mice with either one of these vectors induced high titers of RSV neutralizing antibodies and high levels of F specific interferon-gamma-producing T cells. A Th1-type immune response was indicated by a high IgG2a/IgG1 ratio of RSV-specific antibodies, strong induction of RSV-specific interferon-gamma and tumor necrosis factor-alpha cytokine producing CD8 Tcells, and low RSV-specific CD4 T-cell induction. Both humoral and cellular responses were increased upon a boost with RSV-F expressing heterologous adenovirus vector (Ad35 boost after Ad26 prime or vice versa). Both single immunization and prime-boost immunization of cotton rats induced high and long-lasting RSV neutralizing antibody titers and protective immunity against lung and nasal RSV A2 virus load up to at least 30 weeks after immunization. Cotton rats were also completely protected against challenge with a RSV B strain (B15/97) after heterologous prime-boost immunization. Lungs from vaccinated animals showed minimal damage or inflammatory infiltrates post-challenge, in contrast to animals vaccinated with formalin-inactivated virus. Our results suggest that recombinant human adenoviral Ad26 and Ad35 vectors encoding the RSV F gene have the potential to provide broad and durable protection against RSV in humans, and appear safe to be investigated in infants.

Impact of Placental Malaria and Hypergammaglobulinemia on Transplacental Transfer of Respiratory Syncytial Virus Antibody in Papua New Guinea.

Passively acquired respiratory syncytial virus (RSV) neutralizing antibody protects against RSV-associated lower respiratory infections, but placental malaria (PM) and maternal hypergammaglobulinemia might interfere with transplacental immunoglobulin transport. We measured RSV plaque-reduction neutralization (PRN) antibody in 300 full-term maternal/cord serum pairs in 2 cohorts in malaria-endemic Papua New Guinea: Alexishafen (2005-2008) and the Fetal Immunity Study (FIS) (2011-2013). We defined impaired transport as a cord-to-maternal titer ratio <1.0 and a protective RSV PRN titer (PRNT) ≥1:200. PM and hypergammaglobulinemia occurred in 60% and 54% of Alexishafen mothers versus 8% and 9% of FIS mothers, respectively. 34% of Alexishafen and 32% of FIS pairs demonstrated impaired transport. Multivariate modeling revealed significant associations between increasing maternal IgG (log2) and impaired transport (adjusted OR, Alexishafen: 2.68 [1.17-6.14], FIS: 6.94 [1.94-24.8]) but no association with PM. 34% of Alexishafen and 31% of FIS cord PRNTs were <1:200. Impaired RSV antibody transport was observed in approximately one-third of maternal/cord pairs. Hypergammaglobulinemia, but not PM, was associated with impaired transport, particularly among women with low RSV PRNT. Detection of RSV PRNT <1:200 in one-third of cord sera confirms the need to increase levels of RSV neutralizing antibody in pregnant women through maternal immunization.

Enhanced immunogenicity of a respiratory syncytial virus (RSV) F subunit vaccine formulated with the adjuvant GLA-SE in cynomolgus macaques

Respiratory syncytial virus (RSV) causes significant disease in elderly adults, but an effective vaccine is not yet available. We have previously reported that vaccines consisting of engineered respiratory syncytial virus soluble fusion protein (RSV sF) adjuvanted with glucopyranosyl lipid A (GLA) in an oil-in-water emulsion (stable emulsion [SE]) induce RSV F-specific T and B cell responses in mice and rats that protect from viral challenge. Here, we evaluated the immunogenicity of GLA-SE adjuvanted RSV sF vs unadjuvanted RSV sF vaccines in cynomolgus macaques (Macaca fascicularis). RSV F-specific IgG, RSV neutralizing antibodies, and RSV F-specific T cell IFNγ ELISPOT responses induced by GLA-SE adjuvanted RSV sF peaked at week 6 at significantly higher levels than achieved by unadjuvanted RSV sF and remained detectable through week 24, demonstrating response longevity. Two weeks after a week 24 booster immunization, humoral and cellular responses reached levels similar to those seen at the earlier peak response. Importantly, the GLA-SE adjuvanted RSV sF vaccine induced cross-neutralizing antibodies to other RSV A and B strains as well as F-specific IgA and IgG memory B cells. GLA-SE adjuvanted RSV sF was also demonstrated to drive a Th1-biased response characterized by more IFNγ than IL-4. This study indicates that a GLA-SE adjuvanted RSV sF vaccine induces robust humoral and Th1-biased cellular immunity in non-human primates and may benefit human populations at risk for RSV disease.

Fc receptor γ chain plays a critical role in lung viral clearance and reducing vaccine-enhanced disease in mice immunized with respiratory syncytial virus vaccines (VAC9P.1063)

Abstract Vaccine-enhanced respiratory disease (ERD) is well-known for respiratory syncytial virus (RSV) after vaccination with formalin inactivated RSV (FI-RSV). Induction of Th2 type immune responses and deposition of immune complexes have been suggested to be possible mechanisms. We hypothesized that Fc receptor γ chain (FcRγ) would play a role in conferring protection against RSV after vaccination and that priming with Th1 inducing vaccine would prevent ERD. Virus-like particles presenting RSV fusion protein (F VLP) were found to induce Th1 type immune responses. Wild type (WT) mice and FcRγ KO mice were immunized with FI-RSV or primed with F VLP followed by FI-RSV boost (F VLP/FI-RSV). RSV specific serum IgG and isotype antibody levels were similar in both WT and FcRγ KO mice after RSV vaccination. However, F VLP/FI-RSV FcRγ KO mice showed lower levels of RSV neutralizing antibody titers and IFN-γ secreting CD8+ T cells, higher lung viral titers and more body weight loss compared to F VLP/FI-RSV WT mice. Heterologous F VLP/FI-RSV immunization induced Th1 type humoral and cellular immune responses, no overt sign of ERD in the bronchoalveolar lavage airways in both WT and FcRγ KO mice compared to homologous FI-RSV immunization. The results in this study support a critical role of FcRγ in lung viral clearance and diminishing ERD. More importantly, a strategy of priming with Th1 inducing vaccine might be a promising approach to induce protection against RSV and to prevent ERD.

Characterization of neutralizing affinity‐matured human respiratory syncytial virus F binding antibodies in the sub‐picomolar affinity range

In the human adaptation and optimization of a mouse anti-human respiratory syncytial virus neutralizing antibody, affinity assessment was crucial to distinguish among potential candidates and to evaluate whether this correlated with function in vitro and in vivo. This affinity assessment was complicated by the trimeric nature of the antigen target, respiratory syncytial virus F (RSV-F) glycoprotein. In the initial affinity screen, surface plasmon resonance was used to determine the intrinsic binding affinities of anti-RSV-F Fab and immunoglobulin G (IgG) to the extracellular domain of RSV-F. This assessment required minimal biotinylation of the RSV-F protein and design of a capture strategy to minimize avidity effects. Approximately 30 Fabs were selected from three optimization phage display libraries on the basis of an initial ELISA screen. Surface plasmon resonance analysis demonstrated the success of optimization with some candidates from the screened libraries having low picomolar dissociation constants, more than 700-fold tighter than the parental monoclonal antibody (B21M). The affinities of these antibodies were further evaluated by a kinetic exclusion assay, a solution binding technology. One IgG (monoclonal antibody 029) displayed a low picomolar K(D) comparable with that of motavizumab, an RSV antibody in clinical study. Kinetic exclusion assay showed that two other of the matured IgGs (011 and 019) had sub-picomolar dissociation constants that could not be resolved further. We discuss the relevance of these interaction analysis results in the light of recently published data on the mechanism of F-driven viral fusion during paramyxoviral infection and 101F epitope conservation revealed from the recent crystal structure of RSV-F in the post-fusion state.