In vitro approaches for the evaluation of human vaccines

For many vaccines currently available, every newly produced batch has to be tested in vivo – on animals – to verify its potency. To avoid animal suffering and reduce the variability associated with these tests, we developed assays that could assess the vaccine quality in vitro – using cells. We showed that these assays can distinguish vaccine batches of high and low quality. The cell-based tests can therefore be used to approve the commercial use of a batch without animal testing. The methods we developed are also highly informative: they allowed us to understand the mechanism through which the investigated vaccines activate the human immune system. A better understanding of the immunological basis of protection can lead us to better vaccines. These in vitro approaches are therefore beneficial in multiple ways, since they can create a vaccine development and testing environment that is both more effective for humans and less harmful to animals

In vitro assessment of tick-borne encephalitis vaccine:Suitable human cell platforms and potential biomarkers

Tick-borne encephalitis (TBE) virus causes a severe disease that can lead to permanent neurological complications. The whole inactivated TBE vaccine is highly effective, as proven by high seroconversion rates and near eradication of the disease in countries where vaccination programs have been implemented. TBE vaccine potency testing currently requires the use of in vivo methods that present issues of reproducibility as well as animal discomfort. As an alternative, public and private entities are currently exploring a batch-to-batch consistency approach which would demonstrate conformity of a newly produced vaccine batch with a batch with proven in vivo efficacy with respect to a range of in vitro measurable quality parameters. For the identification of a suitable cellular platform to be used in a panel of in vitro batch-to-batch assessments for the TBE vaccine, we exposed human cell-based systems, both of primary origin and cell line-derived, to vaccine formulations of high and low quality. Following stimulation, cell responses were evaluated by assessing the expression of selected genes by qPCR. Our findings show that the expression of interferon-stimulated genes differed after treatment with non-adjuvanted vaccine batches of different quality in peripheral blood mononuclear cells (PBMCs) and in monocyte-derived dendritic cells, but not in monocyte-free PBMC suspensions nor in cell line-derived immune cells. These results indicate suitable platforms and potential biomarkers for a cell-based assay that, together with other immunochemical analyses, could serve for batch-to-batch assessment of the TBE vaccine, reducing (and eventually replacing) in vivo methods for potency testing

Inactivated or damaged? Comparing the effect of inactivation methods on influenza virions to optimize vaccine production

The vast majority of commercially available inactivated influenza vaccines are produced from egg-grown or cell-grown live influenza virus. The first step in the production process is virus inactivation with β-propiolactone (BPL) or formaldehyde (FA). Recommendations for production of inactivated vaccines merely define the maximal concentration for both reagents, leaving the optimization of the process to the manufacturers. We assessed the effect of inactivation with BPL and FA on 5 different influenza virus strains. The properties of the viral formulation, such as successful inactivation, preservation of hemagglutinin (HA) binding ability, fusion capacity and the potential to stimulate a Toll-like receptor 7 (TLR7) reporter cell line were then assessed and compared to the properties of the untreated virus. Inactivation with BPL resulted in undetectable infectivity levels, while FA-treated virus retained very low infectious titers. Hemagglutination and fusion ability were highly affected by those treatments that conferred higher inactivation, with BPL-treated virus binding and fusing at a lower degree compared to FA-inactivated samples. On the other hand, BPL-inactivated virus induced higher levels of activation of TLR7 than FA-inactivated virus. The alterations caused by BPL or FA treatments were virus strain dependent. This data shows that the inactivation procedures should be tailored on the virus strain, and that many other elements beside the concentration of the inactivating agent, such as incubation time and temperature, buffer and virus concentration, have to be defined to achieve a functional product

In Vitro Characterization of the Innate Immune Pathways Engaged by Live and Inactivated Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) infection can lead to inflammation of the central nervous system. The disease can be effectively prevented by whole inactivated virus vaccines. Here, we investigated the innate immune profile induced in vitro by the antigen component of the vaccines, inactivated TBEV (I-TBEV), to gain insights into the mechanism of action of the TBE vaccine as compared to the live virus. To this end, we exposed human peripheral blood mononuclear cells (PBMCs) to inactivated and live TBEV and assessed cellular responses by RNA sequencing. Both inactivated and live TBEV significantly induced an interferon-dominated gene signature and an increased RIG-I-like receptor (RLR) expression. Using pathway-specific inhibitors, we assessed the involvement of pattern recognition receptors in the sensing of inactivated or live TBEV. Only RLR pathway inhibition significantly suppressed the downstream cascade induced by I-TBEV, while responses to the replicating virus were impacted by the inhibition of RIG-I-like, as well as Toll-like, receptors. Our results show that inactivated and live TBEV predominantly engaged an interferon response in our in vitro PBMC platform, and indicate RLRs as the main pattern recognition receptors involved in I-TBEV sensing

Human plasmacytoid dendritic cells at the crossroad of type I interferon-regulated B cell differentiation and antiviral response to tick-borne encephalitis virus

The Tick-borne encephalitis virus (TBEV) causes different disease symptoms varying from asymptomatic infection to severe encephalitis and meningitis suggesting a crucial role of the human host immune system in determining the fate of the infection. There is a need to understand the mechanisms underpinning TBEV-host interactions leading to protective immunity. To this aim, we studied the response of human peripheral blood mononuclear cells (PBMC) to the whole formaldehyde inactivated TBEV (I-TBEV), the drug substance of Encepur, one of the five commercially available vaccine. Immunophenotyping, transcriptome and cytokine profiling of PBMC revealed that I-TBEV generates differentiation of a sub-population of plasmacytoid dendritic cells (pDC) that is specialized in type I interferon (IFN) production. In contrast, likely due to the presence of aluminum hydroxide, Encepur vaccine was a poor pDC stimulus. We demonstrated I-TBEV-induced type I IFN together with Interleukin 6 and BAFF to be critical for B cell differentiation to plasmablasts as measured by immunophenotyping and immunoglobulin production. Robust type I IFN secretion was induced by pDC with the concerted action of both viral E glycoprotein and RNA mirroring previous data on dual stimulation of pDC by both S. aureus and influenza virus protein and nucleic acid that leads to a type I IFN-mediated sustained immune response. E glycoprotein neutralization or high temperature denaturation and inhibition of Toll-like receptor 7 signalling confirmed the importance of preserving the functional integrity of these key viral molecules during the inactivation procedure and manufacturing process to produce a vaccine able to stimulate strong immune responses.</p

Human plasmacytoid dendritic cells at the crossroad of type I interferon-regulated B cell differentiation and antiviral response to tick-borne encephalitis virus

The Tick-borne encephalitis virus (TBEV) causes different disease symptoms varying from asymptomatic infection to severe encephalitis and meningitis suggesting a crucial role of the human host immune system in determining the fate of the infection. There is a need to understand the mechanisms underpinning TBEV-host interactions leading to protective immunity. To this aim, we studied the response of human peripheral blood mononuclear cells (PBMC) to the whole formaldehyde inactivated TBEV (I-TBEV), the drug substance of Encepur, one of the five commercially available vaccine. Immunophenotyping, transcriptome and cytokine profiling of PBMC revealed that I-TBEV generates differentiation of a sub-population of plasmacytoid dendritic cells (pDC) that is specialized in type I interferon (IFN) production. In contrast, likely due to the presence of aluminum hydroxide, Encepur vaccine was a poor pDC stimulus. We demonstrated I-TBEV-induced type I IFN together with Interleukin 6 and BAFF to be critical for B cell differentiation to plasmablasts as measured by immunophenotyping and immunoglobulin production. Robust type I IFN secretion was induced by pDC with the concerted action of both viral E glycoprotein and RNA mirroring previous data on dual stimulation of pDC by both S. aureus and influenza virus protein and nucleic acid that leads to a type I IFN-mediated sustained immune response. E glycoprotein neutralization or high temperature denaturation and inhibition of Toll-like receptor 7 signalling confirmed the importance of preserving the functional integrity of these key viral molecules during the inactivation procedure and manufacturing process to produce a vaccine able to stimulate strong immune responses