Antibodies in lymphocyte supernatants can distinguish between neutralising antibodies induced by RSV vaccination and pre-existing antibodies induced by natural infection

Abstract

Introduction: Respiratory syncytial virus (RSV) is the single most important cause of severe respiratory illness in infants. There is no effective vaccine and the only effective treatment available is the monoclonal antibody palivizumab which reduces the risk of severe RSV disease in prematurely born infants. However, palivizumab is too costly to allow for wide implementation and thus treatment is restricted to supportive care. Despite extensive efforts to develop a vaccine, progress has been hindered by the difficulty in measuring and assessing immunological correlates of RSV vaccine efficacy in the presence of high levels of pre-existing RSV antibodies.

Methods: Here we describe a new method for measuring the functional activity of antibodies induced by vaccination distinct from pre-existing antibodies. Antibodies in lymphocyte supernatants (ALS) from the cultured peripheral blood mononuclear cells (PBMCs) of young adults who had recently been vaccinated with a novel RSV candidate vaccine were directly assayed for virus neutralising activity. An ELISA method was used to measure antibodies in nasal and serum samples and then compared with the adapted ALS based method.

Results: There was a wide background distribution of RSV-specific antibodies in serum and nasal samples that obscured vaccine-specific responses measured two weeks after vaccination. No RSV-specific antibodies were observed at baseline in ALS samples, but a clear vaccine-specific antibody response was observed in ALS seven days after the administration of each dose of vaccine. These vaccine-specific antibodies in ALS displayed functional activity in vitro, and quantification of this functional activity was unperturbed by pre-existing antibodies from natural exposure. The results demonstrate a promising new approach for assessing functional immune responses attributed to RSV vaccines.

Keywords: Antibodies; Antibodies in lymphocyte supernatant; Plasmablasts; Respiratory syncytial virus.

Fig. 1

An overview of the vaccination scheme followed in the phase I RSV clinical trial: Each participant was assigned to one of four groups. Each group received the boost vaccine eight weeks post prime vaccination, except for group two (*) which received the booster four weeks post prime. Number of participants in each group: n = 11 for group 1; n = 10 for group 2; n = 10 for group 3; n = 11 for group 4 (n = 10 for all groups after boost due to 2 withdrawals and replacement, per protocol).

Fig. 2

Antibody responses in nasal and serum samples. The relative distributions of RSV F-protein specific IgA and IgG in serum and nasal samples are shown at different time points. The error bars represent the mean antibody level with the 95% confidence intervals (CI). *ns – not significant.

Fig. 3

ALS IgG and IgA levels before and after priming and booster vaccinations are shown. Dotted lines represent a response threshold (mean of baseline antibody levels + 3 standard deviations) determined to differentiate between actual responses and background signals. The error bars represent the mean with the 95% CI.

Fig. 4

The magnitude of the IgG and IgA response to vaccination was expressed as fold change in RSV-specific antibody level, one (ALS) or two (serum/nasal samples) weeks after the priming and booster doses of vaccine. The dotted line represent a four- fold changes in antibody level. The numbers and corresponding proportions of volunteers who seroconverted are shown above each group.

Fig. 5

The functional activity of antibodies in ALS was quantified using a modified plaque reduction neutralization assay. ALS was used to neutralize RSV in an in vitro HEp-2 cell culture. Each plaque (red spot) represents un-neutralised virus. (a) ALS obtained from a patient vaccinated with PanAd3-RSV was diluted over an 8 series dilution range from 1:2 to 1:256 and each dilution was mixed with ~25 plaque forming units per well of RSV. Lower dilutions of ALS mediated potent neutralization of RSV, while at higher dilutions, this neutralization effect was diminished in proportion to the dilution. (b) The functional antibody response to vaccination was analysed in individuals vaccinated with PanAd3-RSV IM/MVA-RSV IM. At the pre-prime, pre-boost baseline and one week post boost time points, no significant in-vitro neutralization of RSV was observed. However, two weeks after PanAd3-RSV IM, a number of individuals had significant neutralizing antibody responses. The mean and 95% confidence limits are shown on each group. The dotted line is the mean of the pre-vaccination baseline plus three standard deviations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)