The consistency approach for the substitution of in vivo testing for the quality control of established vaccines: practical considerations and progressive vision.

The aim of this letter is to share the discussions and proposals made by the VAC2VAC consortium on how to support the deployment of the "Consistency Approach" for quality control of established vaccines and thus facilitate the substitution of in vivo testing. This work answers specific questions about " what does a control strategy according to the consistency testing look like" and " how to submit a control strategy defined according to the consistency testing". Some topics were answered in a very straightforward manner. This was the case when the deployment of the consistency approach and the corresponding changes in vaccines control strategy was supported by the generic application of procedures already described in regulatory guidelines/requirements and related to the establishment or change in the control strategy of vaccines. The application of other procedures required more specific attention and some were deeply debated before reaching a proposal. The key outcomes of this work are that robust science must be used to develop a substitution strategy and generate supportive data packages. And this good science can best occur with good scientific collaboration between the different parties involved. Therefore, early interaction between manufacturers and competent authorities before and during dossier submission is critical to success. The consistency approach, when approved and in place, will ensure vaccine products of assured quality reach the patient in a more efficient manner than when relying on in vivo testing. Adapting the mindset was one of the major hurdles to a progressive vision but there is now consensus between manufacturers and competent authorities to foster the elimination of in vivo testing for routine vaccine release testing.

Decontamination efficacy of sodium hypochlorite solutions for poliovirus.

Effective decontamination procedures are critical to the successful manufacture and control of poliovirus vaccines to minimize the risk to personnel and the environment. Polio viruses have been reported to be more resistant to disinfectants than many other viruses. We assessed the efficacy of sodium hypochlorite-containing disinfectants for decontamination for three poliovirus serotypes to implement decontamination procedures that are fully compliant with the WHO GAP III and Health authorities' requirements. A 10.4 log reduction was observed with a 0.63% sodium hypochlorite solution in a suspension with high protein and high poliovirus concentrations diluted 10-fold compared with a 6 log reduction in an undiluted sample. Treatment efficacy increased with sodium hypochlorite content and decreased with sample protein content. The surface tests showed that two 1-min treatments, 5-min apart, with a 0.63% Chl sodium hypochlorite solution effectively reduced the concentration of all poliovirus serotypes by 10 log10, irrespective of the protein and virus concentration in the sample. Sodium hypochlorite solutions lower than 0.52% were less effective for complete inactivation of poliovirus. In conclusion, we demonstrated that a high level of virus reduction (>10 log10) can be achieved with sodium hypochlorite solutions with poliovirus in suspension and dried on surfaces.

Potency test to discriminate between differentially over-inactivated rabies vaccines: Agreement between the NIH assay and a G-protein based ELISA.

The NIH assay is used to assess the potency of rabies vaccine and is currently a key measure required for vaccine release. As this test involves immunization of mice and subsequent viral challenge, efforts are being made to develop alternative analytical methods that do not rely on animal testing. Sanofi Pasteur has reported the development of a G-protein specific ELISA assay that has shown agreement with the NIH test. In this study we have generated several non-conform vaccine lots by an excessive inactivation with ß-propiolactone (BPL) and assessed the capacity of both tests to detect the corresponding consequences. Excessive BPL inactivation causes G-protein unfolding, altering in turn viral morphology and the continuity of the G-protein layer in the viral particle. Both the NIH and the ELISA tests were able to monitor the consequences of excessive inactivation in a similar manner. Of note, the experimental error of the ELISA test was well below that of the NIH test. These results increase the prospect that the ELISA test could be considered a suitable candidate for the replacement of the NIH test.

Comparative analysis of the performance of residual host cell DNA assays for viral vaccines produced in Vero cells.

Residual host cell DNA (rcDNA) from continuous cell lines used for manufacturing of biological medicinal products has been considered as safety risk. Historically, several analytical methods have been used for rcDNA quantitation including hybridization assay, Threshold® assay and quantitative polymerase chain reaction (qPCR). Sanofi Pasteur has a wealth of experience in the development of methods quantifying rcDNA in vaccines. Here, we compared the performance of our in-house assays for quantifying rcDNA in viral vaccines produced in Vero cells. Vero alpha-satellite sequence qPCR was compared with the hybridization and Threshold® assays in terms of specificity, sensitivity and precision. The impact of viral inactivation with ß-propiolactone (BPL) on rcDNA, within the vaccine production process, was also assessed. We demonstrate that the quantity of rcDNA measured is influenced by the analytical method used. Vero cell DNA-specific qPCR assay was shown to be robust with a large dynamic range and no matrix interference on a range of products. The qPCR assay demonstrated greater sensitivity and specificity versus the hybridization and Threshold® methods. Vero alpha-satellite sequence qPCR is a specific and sensitive method for the assessment of the quantity of Vero rcDNA in the highly purified vaccines.

Recommendations of the VAC2VAC workshop on the design of multi-centre validation studies.

Within the Innovative Medicines Initiative 2 (IMI 2) project VAC2VAC (Vaccine batch to vaccine batch comparison by consistency testing), a workshop has been organised to discuss ways of improving the design of multi-centre validation studies and use the data generated for product-specific validation purposes. Moreover, aspects of validation within the consistency approach context were addressed. This report summarises the discussions and outlines the conclusions and recommendations agreed on by the workshop participants.

Robust real-time cell analysis method for determining viral infectious titers during development of a viral vaccine production process.

The classical cell-culture methods, such as cell culture infectious dose 50% (CCID50) assays, are time-consuming, end-point assays currently used during the development of a viral vaccine production process to measure viral infectious titers. However, they are not suitable for handling the large number of tests required for high-throughput and large-scale screening analyses. Impedance-based bio-sensing techniques used in real-time cell analysis (RTCA) to assess cell layer biological status in vitro, provide real-time data. In this proof-of-concept study, we assessed the correlation between the results from CCID50 and RTCA assays and compared time and costs using monovalent and tetravalent chimeric yellow fever dengue (CYD) vaccine strains. For the RTCA assay, Vero cells were infected with the CYD sample and real-time impedance was recorded, using the dimensionless cell index (CI). The CI peaked just after infection and decreased as the viral cytopathic effect occurred in a dose-dependent manner. The time to the median CI (CITmed) was correlated with viral titers determined by CCID50 over a range of about 4-5log10 CCID50/ml. This in-house RTCA virus-titration assay was shown to be a robust method for determining real-time viral infectious titers, and could be an alternative to the classical CCID50 assay during the development of viral vaccine production process.

G-protein based ELISA as a potency test for rabies vaccines.

The NIH test is currently used to assess the potency of rabies vaccine, a key criterion for vaccine release. This test is based on mice immunization followed by intracerebral viral challenge. As part of global efforts to reduce animal experimentation and in the framework of the development of Sanofi Pasteur next generation, highly-purified vaccine, produced without any material of human or animal origin, we developed an ELISA as an alternative to the NIH test. This ELISA is based on monoclonal antibodies recognizing specifically the native form of the viral G-protein, the major antigen that induces neutralizing antibody response to rabies virus. We show here that our ELISA is able to distinguish between potent and different types of sub-potent vaccine lots. Satisfactory agreement was observed between the ELISA and the NIH test in the determination of the vaccine titer and their capacity to discern conform from non-conform batches. Our ELISA meets the criteria for a stability-indicating assay and has been successfully used to develop the new generation of rabies vaccine candidates. After an EPAA international pre-collaborative study, this ELISA was selected as the assay of choice for the EDQM collaborative study aimed at replacing the rabies vaccine NIH in vivo potency test.

Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA - Results of an international collaborative study.

Three different ELISAs quantifying rabies glycoprotein were evaluated as in vitro alternatives to the National Institutes of Health (NIH) in vivo potency test for batch release of human rabies vaccines. The evaluation was carried out as an international collaborative study supported by the European Partnership for Alternatives to Animal Testing (EPAA). This pre-validation study, the results of which are presented in this paper, compared three different ELISA designs, assessing their within- and between-laboratory precision. One of the ELISA designs was proposed to the European Directorate for the Quality of Medicines & HealthCare (EDQM) and accepted for an international collaborative study under the umbrella of the Biological Standardisation Programme.

Yellow fever vaccine: comparison of the neurovirulence of new 17D-204 Stamaril™ seed lots and RK 168-73 strain.

The neurovirulence of two new candidate 17D-204 Stamaril™ working seed lots and that of two reference preparations were compared. The Stamaril™ working seed lots have been used for more than twenty years for the manufacturing of vaccines of acceptable safety and efficacy. The preparation designated RK 168-73 and provided by the Robert Koch Institute was used as a reference. It was confirmed that RK 168-73 strain was not a good virus control in our study because it has a very low neurovirulence regarding both the clinical and histopathological scores in comparison with Stamaril™ strain and is not representative of a vaccine known to be satisfactory in use. The results were reinforced by the phenotypic characterization by plaque assay demonstrating that RK 168-73 was very different from the Stamaril™ vaccine, and by sequencing results showing 4 mutations between Stamaril™ and RK 168-73 viruses leading to amino acid differences in the NS4B and envelop proteins.

Detection of avian retroviruses in vaccines by amplification on DF-1 cells with immunostaining and fluorescent product-enhanced reverse transcriptase endpoint methods.

In order to ensure the safety of vaccines produced on avian cells, rigorous testing for the absence of avian retroviruses must be performed. Current methods used to detect avian retroviruses often exhibit a high invalid-test/false-positive rate, rely on hard-to-secure reagents, and/or have readouts that are difficult to standardize. Herein, we describe the development and validation of two consistent and sensitive methods for the detection of avian retroviruses in vaccines: viral amplification on DF-1 cells followed by immunostaining for the detection of avian leukosis virus (ALV) and viral amplification on DF-1 cells followed by fluorescent product-enhanced reverse transcriptase (F-PERT) for the detection of all avian retroviruses. Both assays share an infectivity stage on DF-1 cells followed by a different endpoint readout depending on the retrovirus to be detected. Validation studies demonstrated a limit of detection of one 50% cell culture infectious dose (CCID(50))/ml for retrovirus in a 30-ml test inoculum volume for both methods, which was as sensitive as a classical method used in the vaccine industry, namely, viral amplification on primary chicken embryo fibroblasts followed by the complement fixation test for avian leukosis virus (COFAL). Furthermore, viral amplification on DF-1 cells followed by either immunostaining or F-PERT demonstrated a sensitivity that exceeds the regulatory requirements for detection of ALV strains. A head-to-head comparison of the two endpoint methods showed that viral amplification on DF-1 cells followed by F-PERT is a suitable method to be used as a stand-alone test to ensure that vaccine preparations are free from infectious avian retroviruses.

Inactivation of hTERT transcription by Tax.

Telomerase expression is the hallmark of tumor cells in which this ribonucleoprotein complex preserves chromosome integrity by maintaining telomere length and thereby prevents cell death. However, recent data support a role of the combination of p53 and telomerase inactivation in initiating genetic instability that promotes malignant transformation. Through its pleiotropic effects on infected T-cell metabolism, the human T-cell leukemia virus type 1 (HTLV-1) oncoprotein Tax plays a central role in leukemogenesis. Here, we show that Tax inhibits human telomerase reverse transcriptase (hTERT) transcription, which is the rate-limiting factor of telomerase activity. This inhibitory effect, that occurs in competition with c-Myc through a canonical c-Myc binding site within the hTERT promoter, results in a decreased telomerase activity of Tax-expressing cells. This is the first demonstration of hTERT inhibition by an oncogene. Tax, which is only expressed in preleukemic cells, triggers infected T-cell cycle and keeps these cells cycling while inactivating p53. We propose that, in combination with these effects, hTERT repression by Tax at an early phase of carcinogenesis might contribute to the massive ploidy changes associated with the development of HTLV-1-associated malignancies.