Alternatives to HIST for acellular pertussis vaccines: progress and challenges in replacement.

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: Progress and Challenges in the Replacement of HIST' was held on 24 August 2014, in Prague, Czech Republic, as a satellite meeting to the 9th World Congress on Alternatives and Animal Use in the Life Sciences. Participants discussed the progress and challenges associated with the development, validation, and implementation of in vitro assays as replacements for the histamine sensitisation test (HIST) for acellular pertussis vaccines. Discussions focused on the consistency approach, the necessary framework for regulatory acceptance of a harmonised method, and recent international efforts towards the development of in vitro assays to replace the HIST. Workshop participants agreed that acceptable alternatives to the HIST should be based on ADP ribosylation-mediated cell intoxication and therefore that the CHO cell clustering assay, which measures cell intoxication, should be further pursued and developed as a possible replacement for the HIST. Participants also agreed to continue ongoing multinational discussions involving national and international standardisation authorities to reach consensus and to organise collaborative studies in this context for assay characterisation and calibration of reference materials.

In search of acceptable alternatives to the murine histamine sensitisation test (HIST): what is possible and practical?

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: In Search of Acceptable Alternatives to the Murine Histamine Sensitization Test (HIST): What is Possible and Practical?' was held on 4 and 5 March 2015 in London, United Kingdom. Participants discussed the results of the data generated from an international collaborative study (BSP114 Phase 2) sponsored by the European Directorate for the Quality of Medicines & Health Care (EDQM) to determine if a modified Chinese hamster ovary (CHO) cell-based clustering assay is a suitable alternative to replace HIST. Workshop participants agreed that protocol transferability demonstrated in the collaborative study indicates that a standardised CHO cell assay is adequate for measuring pure PTx in reference preparations. However, vaccine manufacturers would still need to demonstrate that the method is valid to detect or measure residual PTx in their specific adjuvanted products. The 2 modified CHO cell protocols included in the study (the Direct and the Indirect Methods) deserve further consideration as alternatives to HIST. Using the CHO cell assay, an in vitro alternative, for acellular pertussis (aP) vaccine batch release testing would reduce the number of animals used for aP vaccine safety testing. A strategic, stepwise adoption plan was proposed, in which the alternative test would be used for release purposes first, and then, once sufficient confidence in its suitable performance has been gained, its use would be extended to stability testing.

The vaccines consistency approach project: an EPAA initiative.

The consistency approach for release testing of established vaccines promotes the use of in vitro, analytical, non-animal based systems allowing the monitoring of quality parameters during the whole production process. By using highly sensitive non-animal methods, the consistency approach has the potential to improve the quality of testing and to foster the 3Rs (replacement, refinement and reduction of animal use) for quality control of established vaccines. This concept offers an alternative to the current quality control strategy which often requires large numbers of laboratory animals. In order to facilitate the introduction of the consistency approach for established human and veterinary vaccine quality control, the European Partnership for Alternatives to Animal Testing (EPAA) initiated a project, the "Vaccines Consistency Approach Project", aiming at developing and validating the consistency approach with stakeholders from academia, regulators, OMCLs, EDQM, European Commission and industry. This report summarises progress since the project's inception.

Comparison of the ability of formalin-inactivated respiratory syncytial virus, immunopurified F, G and N proteins and cell lysate to enhance pulmonary changes in Balb/c mice.

Formalin-inactivated respiratory syncytial virus (FI-RSV), a lysate of HEp-2 cells and proteins F, G and N, immunopurified from infected cell cultures, were compared for their ability to prevent infection and to enhance changes in lung cytology associated with RSV challenge. Mice were immunized at three weekly intervals with serial dilutions of the preparations and challenged by the nasal route 1 week after the last injection; their lungs were analysed 4 days later. The concentration of the immunogens was adjusted to test at least a range of 2 to 500 ng of proteins per injection. The dose of FI-RSV used for immunization influenced both the protection against infection and the potentiation of lung histopathology. There was a strong correlation between the lesion scores and the proportion of larger cells recovered in bronchoalveolar lavage fluid. We therefore used cytological changes as an index of lung alterations in further experiments. Glycoproteins F and G but not protein N were protective against challenge infection. Potentiation was observed in mice immunized with minute amounts (2 ng per injection) of F, G or N. HEp-2 cell lysate also caused potentiation but this required greater than 125 ng of proteins per injection.

Characterization of epitopes on native and denatured forms of herpes simplex virus glycoprotein B.

Herpes simplex virus 1 glycoprotein B (gB) is an envelope glycoprotein which promotes fusion of virions with the cell membrane. This report characterizes the epitopes on native, disulfide-linked dimers of gB and monomeric forms of the glycoprotein using a panel of monoclonal antibodies. The antibodies were divided into groups, based on immune reactions with denatured or native forms of gB. The first group reacted with discontinuous epitopes assembled on gB dimers but failed to detect native or denatured monomers. In contrast, the second group reacted with denatured gB recognizing continuous epitopes on the parent oligomer and monomeric forms. Comparison of gB dimers formed by HFEM and tsB5 revealed that mutant forms specified altered immunological properties. Analysis of gB made in Vero cells showed that discontinuous epitopes were retained whereas a subset of continuous ones were lost on the cleavage products. Results of this study indicate that more than half of the epitopes on gB are generated by juxtaposing amino acids from one or more gB subunits and differ from continuous epitopes present on both forms of the molecule.

Antigenic structure of transmissible gastroenteritis virus. I. Properties of monoclonal antibodies directed against virion proteins.

Thirty-two hybridoma cell lines producing monoclonal antibodies (MAbs) against the three major structural proteins of transmissible gastroenteritis virus (TGEV) have been isolated. Radioimmunoprecipitation of intracellular viral polypeptides showed that 17 hybridomas recognized both the peplomer protein [E2, 220 X 10(3) mol. wt. (220K)] and a lower mol. wt. species (E'2, 175K), which was characterized as a precursor of E2. Six MAbs selectively immunoprecipitated the E'2 protein. Four hybridomas were directed against the low mol. wt. envelope protein (E1, 29K), and three against the nucleoprotein (N, 47K). All major neutralization-mediating determinants were found to be carried by the peplomers. Several anti-E2 MAbs displayed an intrinsic neutralizing activity close to that of the most potent anti-TGEV polyclonal reagents tested (including ascitic fluid of feline infectious peritonitis virus-infected cats). None of the anti-E'2 MAbs induced significant neutralization, although this protein might be incorporated to some extent into the virions. Immunofluorescence patterns obtained with MAbs directed against either the envelope glycoproteins or the nucleocapsid revealed distinctly different distributions of these antigens within the cells. Comparison of nine TGEV strains using our panel of MAbs confirmed their close antigenic relationship, but revealed the occurrence of distinct antigenic differences.