Fengrong Zuo

Adults 65 Years Old and Older Have Reduced Numbers of Functional Memory T Cells to Respiratory Syncytial Virus Fusion Protein

ABSTRACT Respiratory syncytial virus (RSV) infects elderly (≥65 years) adults, causing medically attended illness and hospitalizations. While RSV neutralizing antibody levels correlate inversely with RSV-associated hospitalization in the elderly, the role of RSV-specific T cells in preventing disease in the elderly remains unclear. We examined RSV-specific humoral, mucosal, and cellular immune profiles in healthy elderly (65 to 85 years) and young (20 to 30 years) adults. RSV neutralization antibody titers in the elderly (10.5 ± 2.2 log2) and young (10.5 ± 2.1 log2) were similar. In contrast, levels of RSV F protein-specific gamma interferon (IFN-γ)-producing T cells were lower in elderly (180 ± 80 spot-forming cells [SFC]/106 peripheral blood mononuclear cells [PBMC]) than in young adults (1,250 ± 420 SFC/106 PBMC). Higher levels of interleukin-13 (IL-13; 3,000 ± 1,000 pg/ml) in cultured PBMC supernatants and lower frequency of RSV F-specific CD107a+ CD8+ T cells (3.0% ± 1.6% versus 5.0% ± 1.6%) were measured in PBMC from elderly than young adults. These results suggest that deficient RSV F-specific T cell responses contribute to susceptibility to severe RSV disease in elderly adults.

Safety and Immunogenicity of a Live Attenuated RSV Vaccine in Healthy RSV-Seronegative Children 5 to 24 Months of Age

Despite substantial morbidity associated with respiratory syncytial virus (RSV) infection, there is no licensed vaccine. MEDI-559 is a live attenuated intranasal vaccine candidate being developed for prevention of lower respiratory illness due to RSV in young children. This randomized, placebo-controlled study evaluated safety of MEDI-559 in healthy, RSV-seronegative children. MEDI-559 or placebo was administered on 3 occasions, 2 months apart. Primary safety was based on solicited symptoms (SSs) and adverse events (AEs) collected for 28 days after each dose. Nasal wash samples were collected 3 times after each dose (days 7-10, 12-18, 28-34) and at sick visits. Serum was collected for measuring antibody immune responses to RSV prior to first vaccination and 28 days post final dose. Long-term safety was monitored for 365 days from first dose. SSs were mild and frequent (MEDI-559 84%; placebo 91%); most common SSs were runny/stuffy nose, cough, and irritability/fussiness. AEs occurred in 67% MEDI-559 and 57% placebo recipients: most common AE was upper respiratory tract infection (MEDI-559 35%; placebo 23%). Higher incidence of medically attended lower respiratory illness within 28 days after dosing occurred in the MEDI-559 arm compared to placebo (none associated with vaccine virus shedding). There was no evidence of enhanced RSV disease. Vaccine virus was detected only in MEDI-559 recipients; shedding occurred in 56%subjects, primarily post dose 1. A functional immune response was observed in 59% and 9% MEDI-559 and placebo recipients, respectively, by an RSV microneutralization assay. Vaccine take, assessed by proportion that shed vaccine-type virus or had a seroresponse against RSV, was seen in 95% MEDI-559 subjects. MEDI-559 is therefore biologically active and immunogenic in this seronegative pediatric population. Although the frequency of SSs and AEs was not considered clinically significant, the increase in medically attended lower respiratory illnesses in the vaccine group warrants expanded safety studies. Trial Registration ClinicalTrials.gov NCT00767416

Implication of respiratory syncytial virus (RSV) F transgene sequence heterogeneity observed in Phase 1 evaluation of MEDI-534, a live attenuated parainfluenza type 3 vectored RSV vaccine.

MEDI-534 is the first live vectored RSV vaccine candidate to be evaluated in seronegative children. It consists of the bovine parainfluenza virus type 3 (PIV3) genome with substituted human PIV3 F and HN glycoproteins engineered to express RSV F protein. A Phase 1 study of 49 healthy RSV and PIV3 seronegative children 6 to <24 months of age demonstrated an acceptable safety profile at the following doses: 10(4), 10(5) and 10(6)TCID50. After 3 doses of MEDI-534 at 10(6)TCID50, administered at 0, 2 and 4 month intervals, 100% of subjects seroresponded to PIV3, whereas only 50% seroresponded to RSV. To investigate the discordance in seroresponse rates, the RSV F transgene and its flanking non-coding nucleotides were sequenced from shed virus recovered from the nasal washes of 24 MEDI-534-vaccinated children. Eleven out of 24 samples contained no nucleotide changes in the analyzed region. The other 13 samples contained mixtures of variant subpopulations. Fifty-five percent exhibited changes in the transcription termination poly A gene sequences of the upstream bPIV3N gene while 21% had variant subpopulations in the RSV F open reading frame that resulted in pre-mature stop codons. Both types of changes are expected to reduce RSV F expression. Evaluation of the administered vaccine by dual immunofluorescence staining showed ~2.5% variants with low or no RSV F expression while single nucleotide primer extension detected ~1% variation at nucleotide 2045 that resulted in a pre-mature translational termination at codon 85. An association between shedding of variants and lower RSV F serological response was observed but it was not possible to establish a definitive clinical significance due to the small number of subjects in this study.

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.

Translational sciences approach to RSV vaccine development.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants and the elderly. Despite its relatively low degree of antigenic variation, it causes frequent reinfection throughout life. Clinical manifestations of RSV disease and the immune response to infection differ in infants and the elderly, suggesting that vaccines designed to protect these two populations may require different attributes. Here, the authors describe the translational approach of utilizing data from epidemiology studies performed in these populations, the use of RSV diagnostics in clinical practice, lessons learned from previous vaccine clinical trials and the success of palivizumab in prevention of RSV disease in premature and high-risk infants to aid the development of safe and effective RSV vaccines.

Identification of the critical attribute(s) of EBV gp350 antigen required for elicitation of a neutralizing antibody response in vivo.

Vaccine prophylaxis with EBV glycoprotein 350 (gp350) subunit plus adjuvant has been demonstrated clinically to protect individuals against infectious mononucleosis (IM), but the specifications of the antigen required to elicit this protection has remained largely theoretical. Previous studies have shown that antibodies to gp350 comprise the principle component of EBV-neutralizing sera. Further, a murine monoclonal antibody against gp350 (clone 72A1) is able to prevent infection by the virus both in vitro and in vivo. In the present study, we identify the 72A1 epitope on recombinant gp350 antigen as the site required for binding to CD21 on human B cells. We also identify the need for conformational-dependence of the antigen to generate EBV-neutralizing antibodies in vivo. Further, we have characterized the glycosylation status and antigenicity profiles of both native and denatured CHO-produced soluble gp350 as well as non-glycosylated protein produced in Escherichia coli. Collectively our in vitro and in vivo data demonstrate the requirement for a conformationally accessible 72A1 epitope on gp350 to elicit EBV-neutralizing responses, and establish this as a critical attribute of this vaccine antigen. These data provide direction for commercial vaccine development, as the absence of this epitope on either E. coli-expressed or denatured gp350, may limit production and purification options for the antigen.

Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized “gold standard” panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the “gold standard” diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.

Development of a robust, higher throughput green fluorescent protein (GFP)-based Epstein-Barr Virus (EBV) micro-neutralization assay

The goal of most prophylactic vaccines is to elicit robust and effective neutralizing antibodies against the human pathogen target. The titer of neutralizing antibodies to Epstein-Barr Virus (EBV) is a useful biomarker for evaluating EBV vaccines. Here, the development and optimization of a 96-well micro-neutralization fluorescent imaging assay (FIA) using an EBV virus-encoding green fluorescent protein (GFP) to infect adherent EBV recipient cells is reported. The conditions were optimized for generating reproducible EBV-GFP virus, for maintaining viral infectivity for months, and for efficient viral infection of recipient cell culture. The utility of the EBV-GFP FIA neutralization assay was demonstrated in a mouse study of an investigational adjuvanted EBV gp350 subunit vaccine. This assay confirmed the generation of high titers of anti-EBV-neutralizing antibodies which correlated well with the established Raji cell-based flow cytometry-based EBV neutralization assay, as well as with anti-gp350 IgG titers. In naturally infected EBV+ human serum samples, a good correlation between anti-gp350 IgG ELISA titer and EBV-GFP FIA neutralization antibody titer was also observed. Taken together, these results demonstrate the establishment of a scalable high throughput EBV-GFP FIA micro-neutralization assay suitable to measure humoral EBV vaccine response in a large-scale human trial.

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.