Stacie L. Lambert

Molecular and Cellular Response Profiles Induced by the TLR4 Agonist-Based Adjuvant Glucopyranosyl Lipid A

Background Toll-like receptor (TLR)4 agonists are known potent immunostimulatory compounds. These compounds can be formulated as part of novel adjuvants to enhance vaccine medicated immune responses. However, the contribution of the formulation to the innate in vivo activity of TLR4 agonist compounds is not well understood. Methodology and Principal Findings We evaluated synthetic TLR4 agonist Glucopyranosyl Lipid A (GLA) for its effects on molecular and cellular innate immune responses in the murine model. Microarray techniques were used to compare the responses to GLA in an aqueous formulation or in an oil-in-water Stable Emulsion formulation (GLA-SE) versus either SE alone or the mineral salt aluminum hydroxide (alum) at the muscle injection site over multiple timepoints. In contrast to the minimal gene upregulation induced by SE and alum, both GLA and GLA-SE triggered MyD88- and TRIF-dependent gene expression. Genes for chemokines, cytokine receptors, signaling molecules, complement, and antigen presentation were also strongly upregulated by GLA and GLA-SE. These included chemokines for TH1-type T cells (CXCL9 and CXCL10) and mononuclear leukocytes (CCL2, CCL3) among others. GLA-SE induced stronger and more sustained gene upregulation than GLA in the muscle; GLA-SE induced genes were also detected in local draining lymph nodes and at lower levels in peripheral blood. Both GLA and GLA-SE resulted in increased cellular trafficking to the draining lymph nodes and upregulated MHC molecules and ICAM1 on local dendritic cells. GLA and GLA-SE transiently upregulated circulating MCP-1, TNFα, IFNγ and IP-10 in blood. Conclusions/Significance While GLA and GLA-SE activate a large number of shared innate genes and proteins, GLA-SE induces a quantitatively and qualitatively stronger response than GLA, SE or alum. The genes and proteins upregulated could be used to facilitate selection of appropriate adjuvant doses in vaccine formulations.

A novel respiratory syncytial virus (RSV) F subunit vaccine adjuvanted with GLA-SE elicits robust protective TH1-type humoral and cellular immunity in rodent models.

Background Illness associated with Respiratory Syncytial Virus (RSV) remains an unmet medical need in both full-term infants and older adults. The fusion glycoprotein (F) of RSV, which plays a key role in RSV infection and is a target of neutralizing antibodies, is an attractive vaccine target for inducing RSV-specific immunity. Methodology and Principal Findings BALB/c mice and cotton rats, two well-characterized rodent models of RSV infection, were used to evaluate the immunogenicity of intramuscularly administered RSV vaccine candidates consisting of purified soluble F (sF) protein formulated with TLR4 agonist glucopyranosyl lipid A (GLA), stable emulsion (SE), GLA-SE, or alum adjuvants. Protection from RSV challenge, serum RSV neutralizing responses, and anti-F IgG responses were induced by all of the tested adjuvanted RSV sF vaccine formulations. However, only RSV sF + GLA-SE induced robust F-specific TH1-biased humoral and cellular responses. In mice, these F-specific cellular responses include both CD4 and CD8 T cells, with F-specific polyfunctional CD8 T cells that traffic to the mouse lung following RSV challenge. This RSV sF + GLA-SE vaccine formulation can also induce robust RSV neutralizing titers and prime IFNγ-producing T cell responses in Sprague Dawley rats. Conclusions/Significance These studies indicate that a protein subunit vaccine consisting of RSV sF + GLA-SE can induce robust neutralizing antibody and T cell responses to RSV, enhancing viral clearance via a TH1 immune-mediated mechanism. This vaccine may benefit older populations at risk for RSV disease.

An adjuvanted respiratory syncytial virus fusion protein induces protection in aged BALB/c mice

BackgroundRespiratory Syncytial Virus (RSV) causes significant disease in the elderly, in part, because immunosenescence impairs protective immune responses to infection in this population. Despite previous and current efforts, there is no RSV vaccine currently licensed in infants or elderly adults. Adjuvanted RSV subunit vaccines have the potential to boost waning immune responses and reduce the burden of RSV disease in the elderly population.ResultsWe used an aged BALB/c mouse model to evaluate immune responses to RSV Fusion (F) protein in the absence and presence of an alum adjuvant. We demonstrate that aged BALB/c mice immunized with alum-adjuvanted RSV F protein had significantly reduced lung viral titers at day 4 following challenge with wild-type (wt) RSV. Serum neutralizing antibody titers measured on day 27 correlated with protection in both young and aged vaccinated mice, although the magnitude of antibody titers was lower in aged mice. Unlike young mice, in aged mice, alum-adjuvanted RSV F did not induce lung TH2-type cytokines or eosinophil infiltration compared to non-adjuvanted F protein following wt RSV challenge.ConclusionOur studies demonstrate that neutralizing anti-RSV antibody titers correlate with protection in both young and aged BALB/c mice vaccinated with RSV F protein vaccines. The F + alum formulation mediated greater protection compared to the non-adjuvanted F protein in both young and aged mice. However, while alum can boost F-specific antibody responses in aged mice, it does not completely overcome the reduced ability of a senescent immune system to respond to the RSV F antigen. Thus, our data suggest that a stronger adjuvant may be required for the prevention of RSV disease in immunosenescent populations, to achieve the appropriate balance of protective neutralizing antibodies and effective TH1-type cytokine response along with minimal lung immunopathology.

A phase 1a, first-in-human, randomized study of a respiratory syncytial virus F protein vaccine with and without a toll-like receptor-4 agonist and stable emulsion adjuvant.

BACKGROUND Respiratory syncytial virus (RSV) causes significant illness in older adults resulting in substantial health and economic impact. A successful vaccine would reduce morbidity in this growing segment of the population. METHODS In this double-blind phase 1 study, subjects 60 years of age and older were enrolled by cohort and randomized to receive vaccines containing escalating doses (20, 50, or 80μg) of soluble RSV fusion protein (sF) alone or adjuvanted with 2.5μg of glucopyranosyl lipid A, a toll-like receptor-4 agonist, in 2% stable emulsion (GLA-SE). Each cohort included 20 vaccine and 4 placebo recipients. Immune responses were evaluated using assays for RSV microneutralizing, anti-F IgG, and palivizumab competitive antibodies and for F-specific interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) responses. RESULTS The inclusion of adjuvant increased local reactogenicity, with the majority of subjects who received sF and adjuvant reporting low-grade injection site pain or tenderness. At all doses, the safety profile was acceptable for further development. Immune responses were antigen dose-dependent, and the inclusion of adjuvant increased both humoral and cellular immune responses, with responses statistically higher than for placebo recipients in all 4 assays. At the highest dosage level with adjuvant, half of the subjects had a ≥3-fold rise from day 0 in RSV neutralizing antibody titers, and all had a ≥3-fold rise in antibody levels by anti-F IgG and palivizumab competitive antibody assays on day 29. For the day 8 IFNγ ELISPOT assay, 74% of subjects in the highest dosing cohort had a ≥3-fold rise from baseline. CONCLUSIONS The safety and immunogenicity results from this study support inclusion of the GLA-SE adjuvant in this RSV vaccine for older adults and also support assessment of the efficacy of the vaccine in a larger clinical trial. Clinicaltrials.gov NCT02115815.

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.

Enzyme-Linked Immunospot Assay for Detection of Human Respiratory Syncytial Virus F Protein-Specific Gamma Interferon-Producing T Cells

ABSTRACT Respiratory syncytial virus (RSV) causes significant disease in elderly adults, and we have previously reported that individuals 65 years of age and older have reduced RSV F protein-specific gamma interferon (IFN-γ)-producing T cells compared to healthy younger adults. To measure RSV F-specific memory T cell responses in the elderly following infection or vaccination, we optimized and qualified an IFN-γ enzyme-linked immunospot (ELISPOT) assay. Since peripheral blood mononuclear cells (PBMC) from the elderly could be more fragile, we established optimal cryopreservation techniques and minimal viability acceptance criteria. The number of cells per well, types and concentrations of stimulation antigens, and incubation times were evaluated to maximize assay sensitivity and precision. The optimized assay uses 300,000 cells/well, 2 μg/ml of an RSV F peptide pool (RSV Fpp), and incubation for 22 ± 2 h in serum-free CTL-Test medium. The assay was qualified by 3 analysts using 3 RSV F-responding donor PBMC samples (high, medium, and low responders) tested on 5 different assay days. The assay sensitivity or limit of detection (LOD) was determined to be 21 spot-forming cells (SFC) per 106 PBMC, and the lower limit of quantitation (LLOQ) was estimated to be 63 SFC/106 PBMC. The intra- and interassay percent coefficients of variation (CV) were <10.5% and <31%, respectively. The results of the qualification study demonstrate that a robust, precise, and sensitive IFN-γ ELISPOT assay has been developed that is fit for measuring RSV F-specific IFN-γ T cell responses in subjects enrolled in a vaccine clinical trial or in epidemiology studies.

Dose selection for an adjuvanted respiratory syncytial virus F protein vaccine for older adults based on humoral and cellular immune responses

ABSTRACT This is the second phase 1 study of a respiratory syncytial virus (RSV) vaccine containing RSV fusion protein (sF) adjuvanted with glucopyranosyl lipid A (GLA) in a squalene-based 2% stable emulsion (GLA-SE). In this randomized, double-blind study, 261 subjects aged ≥60 years received inactivated influenza vaccine (IIV), a vaccine containing 120 μg sF with escalating doses of GLA (1, 2.5, or 5 μg) in SE, or a vaccine containing 80 μg sF with 2.5 μg GLA in SE. Subjects receiving 120 μg sF with 2.5 or 5 μg GLA were also randomized to receive IIV or placebo. Immunity to RSV was assessed by detection of microneutralizing, anti-F immunoglobulin G, and palivizumab-competitive antibodies and F-specific gamma interferon enzyme-linked immunosorbent spot assay T-cell responses. Higher adjuvant doses increased injection site discomfort, but at the highest dose, the reactogenicity was similar to that of IIV. Significant humoral and cellular immune responses were observed. The 120 μg sF plus 5.0 μg GLA formulation resulted in the highest responses in all subjects and in older subjects. These results confirm previous observations of vaccine tolerability, safety, and immunogenicity and were used to select the 120 μg sF plus 5.0 μg GLA formulation for phase 2 evaluation. (This study has been registered at ClinicalTrials.gov under registration no. NCT02289820.)

Production of cytomegalovirus dense bodies by scalable bioprocess methods maintains immunogenicity and improves neutralizing antibody titers.

ABSTRACT With the goal of developing a virus-like particle-based vaccine based on dense bodies (DB) produced by human cytomegalovirus (HCMV) infections, we evaluated scalable culture, isolation, and inactivation methods and applied technically advanced assays to determine the relative purity, composition, and immunogenicity of DB particles. Our results increase our understanding of the benefits and disadvantages of methods to recover immunogenic DB and inactivate contaminating viral particles. Our results indicate that (i) HCMV strain Towne replicates in MRC-5 fibroblasts grown on microcarriers, (ii) DB particles recovered from 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB)-treated cultures and purified by tangential flow filtration (TFF-DB) or glycerol tartrate gradient sedimentation (GT-DB) constitute 92% or 98%, respectively, of all particles in the final product, (iii) epithelial cell-tropic DB particles are recovered from a single round of coinfection by AD169 and Towne strain viruses, consistent with complementation between the UL130 and UL131A expressed by these strains and restoration of gH/gL/UL128-UL131A (gH pentamer), (iv) equivalent neutralizing antibody titers are induced in mice following immunization with epithelial cell-tropic DB or gH pentamer-deficient DB preparations, (v) UV-inactivated residual virus in GT-DB or TFF-DB preparations retained immunogenicity and induced neutralizing antibody, preventing viral entry into epithelial cells, and (vi) GT-DB and TFF-DB induced cellular immune responses to multiple HCMV peptides. Collectively, this work provides a foundation for future development of DB as an HCMV-based particle vaccine. IMPORTANCE Development of a vaccine to prevent congenital HCMV infection remains a high priority. Vaccination with human cytomegalovirus-derived noninfectious particles, or dense bodies, may constitute a safe vaccination strategy that mimics natural infection. The standard approach for purification of virus particles has been to use a multiple-step, complex gradient that presents a potential barrier to production scale-up and commercialization. In the study described here, we employed an approach that combines treatment with an antiviral terminase inhibitor and purification by a simplified process to produce a vaccine candidate providing broad antiviral humoral and cellular immunity as a foundation for future development.

Development of a High-Throughput Respiratory Syncytial Virus Fluorescent Focus-Based Microneutralization Assay

ABSTRACT Neutralizing antibodies specific for respiratory syncytial virus (RSV) represent a major protective mechanism against RSV infection, as demonstrated by the efficacy of the immune-prophylactic monoclonal antibody palivizumab in preventing RSV-associated lower respiratory tract infections in premature infants. Accordingly, the RSV neutralization assay has become a key functional method to assess the neutralizing activity of serum antibodies in preclinical animal models, epidemiology studies, and clinical trials. In this study, we qualified a 24-h, fluorescent focus-based microneutralization (RSVA FFA-MN) method that requires no medium exchange or pre- or postinfection processing to detect green fluorescent protein-expressing RSV strain A2 (RSVA-GFP)-infected cells, using a high-content imaging system for automated image acquisition and focus enumeration. The RSVA FFA-MN method was shown to be sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1:10, or 3.32 log2; linear over a range of 4.27 to 9.65 log2 50% inhibitory concentration (IC50); and precise, with intra- and interassay coefficients of variation of <21%. This precision allowed the choice of a statistically justified 3-fold-rise seroresponse cutoff criterion. The repeatability and robustness of this method were demonstrated by including a pooled human serum sample in every assay as a positive control (PC). Over 3 years of testing between two laboratories, this PC generated data falling within 2.5 standard deviations of the mean 98.7% of the time (n = 1,720). This high-throughput and reliable RSV microneutralization assay has proven useful for testing sera from preclinical vaccine candidate evaluation studies, epidemiology studies, and both pediatric and adult vaccine clinical trials.

Baseline immune profile by CyTOF can predict response to an investigational adjuvanted vaccine in elderly adults

BackgroundMass cytometry, or CyTOF (Cytometry by Time-of-Flight), permits the simultaneous detection of over 40 phenotypic and functional immune markers in individual cells without the issues of spectral overlap seen in traditional flow cytometry.MethodsIn this study, we applied CyTOF to comprehensively characterize the circulating immune cell populations in elderly individuals both before and after administration of an investigational adjuvanted protein vaccine against respiratory syncytial virus (RSV) in a Phase 1a trial. Antigen-specific T cell responses to RSV by IFNγ ELISPOT had been observed in most but not all recipients in the highest dose cohort in this trial. Here, CyTOF was used to characterize the cellular response profile of ELISPOT responders and non-responders in this vaccine dose cohort.ResultsBoth CD4+ and CD8+ T cell antigen-specific IFNγ responses were observed. Principal components analysis revealed baseline differences between responders and non-responders, including differences in activated (HLA-DR+) CD4+ and CD8+ T cells, which were higher in non-responders versus responders. Using viSNE to analyze RSV-responsive CD4+ and CD8+ T cells, we also found increased expression of HLA-DR, CCR7, CD127 and CD69 in non-responders versus responders.ConclusionsHigh parameter CyTOF can help profile immune components associated with differential vaccine responsiveness.