RESUMEN

The Quality of Medicines & HealthCare (EDQM, Council of Europe) and the European Union (EU) Commission to evaluate the reproducibility of clinical serology results for seasonal influenza vaccines and to assess the impact of technical differences between laboratories on the compliance with the Committee for Human Medicinal Products (CHMP) criteria set by the European Medicines Agency (EMA). The study was run in 2 phases. The present article reports the 1st phase of the study, which aimed at evaluating the variability of the results obtained by 11 laboratories (5 national control laboratories and 6 influenza vaccine manufacturers) using their routine haemagglutination inhibition (HI) assay to test a common panel of clinical trial sera. The results confirmed the limited inter-laboratory reproducibility of the HI testing of influenza vaccine clinical trial samples. In some cases a good agreement was found between laboratories, while a systematic bias or a random scatter of results was observed in other cases. Analysis of estimated systematic bias confirmed that differences between laboratories can be significant (up to 16-fold) in some cases. Correction for this bias resulted in limited improvement. Differences between laboratories were found to result in discrepant decisions on marketing acceptance of vaccines or to decisions based on compliance to different criteria. The study showed that the seroconversion (SC) and mean fold increase (MFI) criteria are more robust against systematic over- or under-estimation of titres whereas the protection rate (PR) is very sensitive to this effect. The fundamental issues with the PR criteria are discussed.

Asunto(s)

RESUMEN

A collaborative study was run by the Biological Standardisation Programme (BSP) under the aegis of the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) and the European Union (EU) Commission, to address the issue of the poor standardisation of serological assays used for the evaluation of seasonal influenza vaccines in Europe. The Phase 1 of the study focused on the compliance to Committee for Human Medicinal Products (CHMP) criteria by 6 manufacturers and 5 public laboratories. It confirmed the poor inter-laboratory correlation of haemagglutination inhibition (HI) test results. Phase 2 consisted in a reproducibility study examining the impact of extended method standardisation and the use of reference sera on inter-laboratory variation. Six manufacturers and 5 public laboratories contributed HI results, while the 5 public laboratories also performed single radial haemolysis (SRH) tests on the same sample panels. Results showed that method standardisation failed to significantly improve the inter-laboratory variation. Correction for pre-vaccination titres (Beyer correction) was found to have limited effect to improve the bias constituted by the Protection Rate (PR) criterion. The reasons underlying the difficulty in standardisation of HI and SRH tests are discussed and improved approaches for the compliance testing to CHMP criteria are suggested.

Asunto(s)

RESUMEN

Influenza vaccines are licensed in the European Union (EU) by means of the mutual recognition procedure, which includes a "fast track" variation for rapid approval of new vaccine strains. This "fast track" procedure involves an annual clinical trial and assessment of immunogenicity according to serological criteria published by the EU Committee for Proprietary Medicinal Products (CPMP). A survey of vaccine clinical trials in the elderly over the past four years has demonstrated that inter-laboratory differences in serology results can affect compliance with CPMP licensing criteria. Thus even well established immune correlates of influenza vaccine efficacy can be problematic. For the next generation of vaccines, however, where mucosal or cell-mediated immunity may contribute towards vaccine efficacy, the serological correlates may be inadequate.

Asunto(s)

RESUMEN

During normal interpandemic influenza seasons, immune responses to vaccines are quite predictable and meet the licensing criteria of the European Union (EU) Committee for Proprietary Medicinal Products (CPMP). In a pandemic situation, large sections, if not all of the community will be immunologically naïve and therefore new immunisation strategies will be needed. In 1976 and 1977 H1N1 vaccines were prepared and tested clinically. To stimulate 'protective' antibody responses, two doses of vaccine were needed in people below the age of 24 years (no previous experience of H1N1 virus), whereas one conventional dose was adequate in older people. In 1997, the highly pathogenic avian influenza H5N1 virus caused widespread concern when it infected man, with lethal effects. Due to safety concerns it was necessary to adopt new strategies for vaccine development and one such strategy was to produce vaccine from an avirulent H5N3 virus, A/Duck/Singapore-Q/F119-2/97. Clinical trials of a subunit vaccine prepared from A/Duck/Sing/97 virus revealed that even two doses of twice the normal vaccine concentration (i.e. 30 micro g haemagglutinin) were poorly immunogenic, whereas an H5N3 vaccine adjuvanted with microfluidised emulsion (MF) 59 stimulated antibody levels that complied with CPMP criteria after two half strength doses (i.e. 7.5 micro g haemagglutinin).

Asunto(s)

RESUMEN

In response to the pandemic warning provided by the highly pathogenic H5N1 influenza virus infections in Hong Kong, there were world-wide attempts to develop vaccines. Three strategies were followed and although each was associated with some success, there were also some problems. Pre-clinical vaccine efficacy results are presented from one such strategy, that of using an apathogenic H5N3 avian strain for vaccine production.

Asunto(s)

RESUMEN

Conventional influenza vaccines are standardised using the single-radial-immunodiffusion (SRD) test where reagents are produced from egg-grown viruses. It is important to ensure homology between SRD antigen reagents and test vaccines. There was concern that cell-grown vaccines may differ antigenically from corresponding egg-grown vaccines, which may in turn affect vaccine standardisation. In an examination of five cell-grown vaccines from two companies, only one vaccine was affected by the specificity of the SRD test. Options for standardisation of cell-grown vaccines are considered and recommendations are made for further studies.

Asunto(s)

Asunto(s)

RESUMEN

Steadfast progress has been made from biopsy to surgery with interventional MRI (iMRI). Such image-guided interventions require specialized instrumentation due to the unusual elements of the MR environment. Suppliers/manufacturers of MR-compatible instrumentation were few in 1994, but now there are more than 50. We present fundamental issues of MR compatibility and a list of known suppliers/manufacturers.

Asunto(s)

Asunto(s)

RESUMEN

PURPOSE: To describe new techniques for percutaneous biopsy with use of an open-configuration magnetic resonance (MR) imaging system with integrated frameless stereotaxic guidance tools. MATERIALS AND METHODS: In 28 patients, biopsy was performed in which the image plane was interactively controlled by the position of a hand-held probe attached to the biopsy needle. An icon integrated into the image was used to guide needle advancement in three planes orthogonal to the needle. In vitro measurements of spatial accuracy were also performed. RESULTS: Diagnostic tissue was retrieved in 25 of 28 patients. The system was most accurate near the isocenter with a maximum measured error of 3.1 mm within a sphere of radius 2.5 cm about the isocenter. CONCLUSION: MR-guided biopsy with a frameless stereotaxic technique is safe and accurate. Image feedback is near real time, and the procedure is interactive. These techniques may be used to perform MR-guided biopsies and to place probes for MR-guided therapies.

Asunto(s)

RESUMEN

PURPOSE: To evaluate the usefulness of a magnetic resonance (MR) imaging-guided system for localization and biopsy of mammographically and clinically occult breast lesions. MATERIALS AND METHODS: The 11 needle localizations and one cyst aspiration were performed with MR imaging guidance in 11 patients with breast lesions. Two MR systems were tested; both required the patient to lie prone with the breast compressed between medial and lateral plates. One system used a grid with 18-gauge holes placed at 5-mm intervals and two reference markers to position the needle; the other, a stereotaxic external needle guide and a software system to calculate coordinates. RESULTS: Fibroadenoma, ductal carcinoma in situ, invasive ductal carcinoma, atypical ductal hyperplasia, fat necrosis, and sclerosing adenosis were found at histologic examination. In two cases, biopsies revealed multi-focal breast cancer where mammographic and clinical findings had indicated a single lesion. CONCLUSION: MR imaging-guided needle localizations may be performed in a clinical setting with the systems described. Accurate localization of mammographically and clinically occult lesions will allow MR imaging to achieve a clinically significant role.

Asunto(s)

RESUMEN

Inactivated subunit vaccines were prepared from high-growth reassortants derived from two separate egg isolates from a single clinical specimen of influenza A (H1N1) virus. One of these reassortants, NIB-14, was antigenically indistinguishable from isolates made in tissue culture, while the other, NIB-17, was antigenically different and typical of egg isolates. The viruses differed by three amino acid residues in the haemagglutinin (HA) molecule and the anti-HA serological response induced was studied in animal models and human volunteers. In the volunteer groups both vaccines induced very high levels of circulating haemagglutination inhibition antibodies but with different serological specificities. Both NIB-14 and NIB-17 vaccines induced high levels of cross-reactive antibodies capable of reacting with both strains, but only NIB-14 vaccine induced significant levels of strain-specific antibodies capable of reacting exclusively with the homologous strain. Antisera containing only cross-reactive antibodies proved as capable of virus neutralization as antisera containing high levels of strain-specific antibodies. We extended the argument that epidemic strains are antigenically more closely related to tissue culture isolates and established that viruses which differ by only single amino acids at critical points in the HA structure can induce a significantly different immune response when used as inactivated vaccines.

Asunto(s)

RESUMEN

The immunogenicity and protective efficacy of formalin-inactivated vaccines prepared from influenza A (H1N1) viruses grown in MDCK cells and in eggs was compared in animal models. The A/Chr/157/83 virus grown in MDCK cells (157M) differed by two amino acid substitutions in the HA molecule from the corresponding virus grown in eggs (157E) and the two viruses could be distinguished antigenically by monoclonal and polyclonal antibodies. Following two intramuscular injections of vaccine in ferrets, guinea pigs, and hamsters, both vaccines were equally immunogenic when antibody was analyzed by hemagglutination inhibition using homologous virus. However, single radial hemolysis analysis following antibody cross-adsorption showed that antibody stimulated by 157E vaccine was exclusively strain specific whereas that produced by the 157M vaccine was more broadly reactive. When immunized hamsters were challenged with virus cultivated on mammalian (MDCK) cells, the homologous vaccine induced a higher degree of protection than the corresponding egg-grown vaccine.

Asunto(s)

RESUMEN

Pairs of influenza A(H1N1) viruses cultivated from the same clinical specimen in canine kidney (MDCK) cells or in embryonated hens' eggs can frequently be distinguished by their reactions with monoclonal antibodies to haemagglutinin and with antibodies in ferret or human sera. Egg-adapted virus, further passaged in MDCK cultures remained "egg-like" in serological characteristics indicating that the differences in their serological reactions were not a direct result of host cell-dependent glycosylation of the haemagglutinin. Haemagglutination-inhibiting (HI) or virus neutralizing antibodies in human sera can be detected more frequently, and to higher titre, in tests employing virus grown exclusively in MDCK cells than in tests with virus adapted to growth in embryonated eggs. Striking differences were detected in the serological reactions in HI tests when sera from ferrets infected with egg-grown virus were tested against a series of strains of influenza A(H1N1) virus isolated in 1983 and adapted to growth in eggs. In contrast, sera from ferrets infected with MDCK-derived virus failed to distinguish serologically between the same viruses that had been passaged exclusively in MDCK cells and also revealed relatively small differences between their egg-adapted counterparts.It was concluded that the cell substrate used for virus isolation and cultivation is a factor that should be considered when interpreting the results of strain characterization of influenza A(H1N1) isolates and in sero-surveys using these viruses.

Asunto(s)

Asunto(s)

RESUMEN

Single-radial-immunodiffusion (SRD) assays were used for measuring the haemagglutinin (HA) antigen content of equine influenza vaccines containing the virus strains A/equine/Prague/56 (H7N7) and A/equine/Miami/63 (H3N8). Three bivalent aqueous vaccines and one bivalent adjuvanted vaccine were standardized by SRD to contain graded amounts of HA antigen activity. The SRD reaction was influenza subtype specific and was not influenced by the presence of adjuvant in vaccine.

Asunto(s)

RESUMEN

Influenza A virus strains isolated in the United States of America from ring-billed and Franklin gulls (Larus delawarensis, L. pipixcan) were found to possess a haemagglutinin (HA) antigen distinct from those of the twelve previously designated haemagglutinin subtypes of influenza A virus. Serological assays with antisera to reference strains representing the HA subtypes 1-12 and to a gull isolate, A/gull/Maryland/704/77, showed that the haemagglutinin of the gull virus was not related antigenically to the previously designated subtypes. In addition, comparison of the nucleotide sequences, and deduced amino acid sequences, of the 3' region of the RNA genes coding for haemagglutinin indicated that the gull viruses represent a genetically distinct group. We propose that this new HA antigen, which has so far been detected only in gulls, be designated H13 and that A/gull/Maryland/704/77 (H13N6) be designated the reference strain for this subtype.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

A novel radial-diffusion technique in agarose gels is described which is applicable to the assay of small concentrations of influenza haemagglutinin (HA) antigen. The method is based on single-radial diffusion zone size enhancement (ZE) using autoradiography to demonstrate antigen--antibody reactions. The ZE technique is capable of reliably assaying concentrations of haemagglutinin as low as 0.1 microgram/ml, which represents a 20- to 40-fold increase in sensitivity over conventional single-radial-diffusion techniques. The ZE response is dependent upon the antigenic identity of the 125I-labelled 'marker' HA and the HA antigen being assayed. Haemagglutinins showing variation within a subtype (e.g. H3) show reduced ZE responses and thus the method has potential value for distinguishing between closely related HA antigens.

Asunto(s)