Evaluation of a test for its suitability in the diagnosis of variant Creutzfeldt-Jakob disease.

BACKGROUND AND OBJECTIVES: Evaluation of variant Creutzfeldt-Jakob disease (vCJD) diagnostic/donor screening tests is made complicated by the very limited supply of blood samples from clinically confirmed cases of vCJD. To determine appropriate access for test developers to rare Creutzfeldt-Jakob disease (CJD) blood samples, the oversight committee of the NIBSC CJD Resource Centre has developed a process and protocols detailing minimum requirements for both test sensitivity and specificity. This protocol is broadly similar to that outlined in the common technical specification (European Directive 98/79/EC). MATERIALS AND METHODS: Tests are subjected to a stepwise evaluation (step 1). vCJD tissue homogenates spiked into pooled human plasma (step 2). Blood samples from animals known to be incubating (Transmissible spongiform encephalopathy) TSE disease (scrapie/Bovine Spongiform encephalopathy (BSE)-infected sheep, BSE-infected primates) and appropriate controls (step 3). Fresh or frozen plasma from normal UK blood donors and (step 4). Plasma samples from individuals with confirmed clinical stage variant CJD (transfusion transmission) or sporadic CJD (no evidence of blood transmission). RESULTS: The assay evaluated performed with good sensitivity with vCJD-spiked tissue homogenates, poor sensitivity for ovine TSE-infected blood samples and failed with plasma from BSE-infected non-human primates and with true vCJD clinical samples. CONCLUSIONS: The test evaluated here is currently unsuitable for use in blood donor screening or diagnosis using blood.

Poliovirus-specific CD4+ Th1 clones with both cytotoxic and helper activity mediate protective humoral immunity against a lethal poliovirus infection in transgenic mice expressing the human poliovirus receptor.

The current understanding of the function of CD4+ T helper (Th) cells in immunity to infectious diseases is that Th1 cells, which secrete interleukin (IL)-2 and interferon-gamma, induce cellular immune responses, whereas Th2 cells, which secrete IL-4, IL-5, IL-6, and IL-10, provide helper function for humoral immunity. We have used a panel of poliovirus-specific murine CD4+ T cell clones and mice transgenic for the human poliovirus receptor to evaluate the role of Th cell subpopulations in protective immunity to poliovirus. The majority of T cell clones, as well as polyclonal T cells generated from mice infected or immunized with poliovirus, secreted IL-2 and interferon-gamma, but not IL-4, IL-5, or IL-10, a profile typical of Th1 cells. The Th1 clones displayed major histocompatibility complex class II-restricted cytotoxic T lymphocyte activity against specific poliovirus peptide-pulsed target cells, but also provided help for antipoliovirus neutralizing antibody production. To examine the mechanism of immunity in vivo, we have used poliovirus receptor-transgenic mice on a BALB/c (H-2d) background. These animals developed a poliomyelitis-like disease when challenged intravenously with a virulent wild-type strain of poliovirus, but not with an attenuated vaccine strain. Furthermore, mice immunized with the vaccine strain were protected against a subsequent challenge with wild-type virus. Using an adoptive transfer technique, we demonstrated that it was possible to confer protection with primed B cells in the presence of polyclonal poliovirus-specific T cells, but not when transgenic mice received either B cells or T cells alone. Furthermore, protection was observed when mice received primed B cells in the presence of a VP4-specific Th1 clone. The findings demonstrate that Th1 cells can mediate a protective immune response against poliovirus infection in vivo through helper activity for humoral immunity and that CD4+ T cells, specific for the internal poliovirus capsid protein, VP4, can provide effective help for a protective antibody response directed against surface capsid proteins.

Poliovirus biology.

Poliomyelitis has been largely controlled by the use of attenuated live vaccine strains. The molecular basis of attenuation is beginning to be understood, but the interactions between the virus and its human host remain mysterious.

The 5' noncoding region and virulence of poliovirus vaccine strains.

All three live, attenuated vaccine strains of poliovirus contain important attenuation determinants in a short conserved sequence in the 5' noncoding region. Evidence suggests these act by weakening a secondary-structural element critical for the unusual mechanism of translational initiation of picornaviruses, in which ribosomes bind directly to a site far downstream of the 5' end. Understanding the molecular basis of attenuation may allow novel vaccine strains to be designed.

New model for the secondary structure of the 5' non-coding RNA of poliovirus is supported by biochemical and genetic data that also show that RNA secondary structure is important in neurovirulence.

A secondary structure model for the 5' non-coding RNA of poliovirus has been derived by comparing computer-generated folding patterns of equivalent sequences from a number of related enteroviruses and rhinoviruses and identifying compensating mutations that suggest conservation of a common secondary structure. Although certain elements are similar, the new model differs considerably from a previously published minimal energy structure and is consistent with the observed sensitivity of in vitro RNA transcripts of infectious poliovirus cDNA to RNases and modifying chemicals. The sequence of a neurovirulent revertant of an attenuated mutant provides additional evidence for an interaction between a region known to be important for neurovirulence, sequence 471-483, and nucleotides 528 to 538.

Assessment of mumps virus growth on various continuous cell lines by virological, immunological, molecular and morphological investigations.

Vero cells have been used as a convenient laboratory substrate for the isolation of mumps virus but may not be very sensitive and may select for particular adapted variants from clinical specimens. Continuous cell lines were evaluated for their ability to support the replication of mumps virus. Criteria included the production of infectious virus, detection of intracellular mumps proteins by immunofluorescence and electron microscopy and detection of specific nucleic acid by RT-PCR. Of the cells tested, CaCo-2, PLC/PRF/5, and Vero cells produced infectious virus, with Vero and CaCo-2 being the most permissive. The other substrates tested included cells of murine, canine and human origin showed signs of intracellular proteins and RNA but the amounts produced were much lower, and no infectious virus was detected in some cases. The virus use was a low passage of a Vero derived wild type strain, and it will ultimately be necessary to continue the studies with an unpassaged clinical specimen to identify a cell line able to isolate mumps virus at high efficiency and in unmodified form.

Recognition of poliovirus/HIV chimaeras by antisera from individuals with HIV infection.

The neutralization of five poliovirus/HIV chimaeras by serum from HIV-infected individuals was examined to evaluate the presentation of HIV envelope sequences, to assess the immune response of individuals to specific epitopes, and to relate it to the stage of disease. The sera were unable to differentiate between four of the chimaeras and the Sabin vaccine strain. With a fifth construct containing an immunodominant gp41 sequence, significant differential recognition was observed in approximately 67% of individuals with asymptomatic HIV infection [groups II and III of the Centers for Disease Control (CDC) classification of HIV infection] and 37% of patients with symptomatic disease (CDC group IV). Furthermore, among patients with CDC stage IV disease antibody levels against this construct and the titre achieved decreased with progression to further disease from approximately 40% in AIDS-related complex (ARC) patients (CDC group IVA and IVC-2 to 14% in those with AIDS (other group IV diseases). Loss of antibody to this construct did not result from a reduction in the anti-polio or anti-envelope response, but from a decline in antibody levels to the HIV sequence inserted in antigenic site 1.

Ensuring safety and consistency in cell culture production processes: viral screening and inactivation.

The history of the use of biological products is littered with examples of the iatrogenic transmission of viruses. A major effort of the biotechnology industry is directed towards the elimination of any risk of infection from the products of cell culture. This article summarizes the overall strategy that has been followed, so far with great success, in that there have, as yet, been no reported cases of viral transmission by the products of biotechnology.

Poliovirus vaccination: current understanding of poliovirus interactions in humans and implications for the eradication of poliomyelitis.

Poliomyelitis is a paralytic disease of the motor neurones of the central nervous system, which is caused by poliovirus. The virus is transmitted by the faecal-oral route, and if virus replication is confined to the gut, it is harmless. Poliomyelitis is an ancient human disease, but was rare until the beginning of the 20th century, when children began to be exposed to the virus at older ages and were, therefore, no longer protected by maternal antibody, which had already been lost. Inactivated polio vaccines are increasingly being used in those countries in which poliomyelitis has been brought under control; however, live vaccines are still the most widely used types and the World Health Organization (WHO) have set the goal of using such vaccines to eliminate the wild-type virus throughout the world by the year 2000. Substantial progress has been made to this end; however, the strains of poliovirus that are used as vaccines are able to adapt rapidly to the human gut, losing their attenuated (weakened) character within a few weeks. Currently, there is urgent debate about the best method of stopping vaccination against poliomyelitis once the wild-type poliovirus has been eliminated completely, so that the vaccine-strain virus will also be eliminated. Proposed strategies include the abrupt cessation of vaccination with the live virus worldwide, followed by the optional use of inactivated vaccines for an appropriate period. Further information about both the epidemiology and the pathogenesis of the disease is required before an informed choice can be made. The topics covered in this article include a brief history of studies of the disease, its pathogenesis and its control by vaccination, the molecular biology of the live vaccines, which have been extremely successful in controlling poliomyelitis so far, and the concerns that are raised as the eradication of the wild-type virus approaches.

Monoclonal antibodies which block cellular receptors of poliovirus.

The isolation and properties of monoclonal antibodies which specifically inhibit the binding of poliovirus types 1, 2 and 3 to cells are described. The antibodies were of the IgG class and blocked infection of cells by all strains of the three poliovirus serotypes, but by none of a wide range of other viruses examined, including nine human enteroviruses. The antibodies prevented poliovirus growth in all susceptible human and primate cells tested. We conclude that the antibodies are directed against the receptor site for poliovirus which is distinct from those required by other picornaviruses, and which seems to be antigenically well conserved between cells of human and primate origin.

Resorbable lamellar particles of polylactide as adjuvants for influenza virus vaccines.

Lamellar particles and microspheres were produced by precipitation from solutions of resorbable, biocompatible, semi-crystalline poly(L-lactide)[PLA] and amorphous poly(DL lactide co-glycolide)[PLG] copolymer, respectively, to investigate their adjuvanticity towards adsorbed influenza virus. Both types of substrate were capable of adsorbing large quantities of virus (> 15% w/w) and retaining virus (> 60% of the initial load) over an 8 week time scale in-vitro. Potent immune responses were obtained in mice after the intra-muscular injection of adsorbed vaccine systems. The response to virus adsorbed on PLA lamellar particles was almost five times that obtained using PLG microspheres and fourteen times that using aqueous vaccine. The lamellar forms of PLA may function as an immunomodulator enhancing phagocytic activity due to their irregular shape and may be useful in improving the immune response to a variety of protein and viral antigens.

Assay of humoral immunity to mumps virus.

One hundred randomly chosen sera from blood donors from North London were assayed for antibodies to mumps virus by plaque reduction and microtitre neutralisation assay, haemagglutination inhibition and in-house ELISA. The assay reproducibility was determined, and there was reasonable agreement between antibody levels measured by the two neutralising methods. Neutralising antibody levels measured by either method were low but the strain used in the assay had a large effect on the antibody titres observed. Titres measured by neutralisation assay, HI assay and ELISA did not correlate well. Assessment of immunity to mumps virus remains problematical.

A novel cytopathic microtitre plate assay for hepatitis A virus and anti-hepatitis A neutralizing antibodies.

The slow growth of hepatitis A virus (HAV) has made tissue culture assay for infectious virus difficult. Strains of the virus of greater cytopathogenicity have been selected for use in plaque cytopathic assays. However, in our hands, this assay has been difficult to reproduce consistently due to problems in maintaining intact cell monolayers over the long incubations involved. This report describes the development of a cytopathic TCID50 assay for HAV. From the results of repeated assay of one preparation of the virus, the coefficient of variation of the assay was calculated to be 4%. This assay has also been adapted to quantitate antibodies to HAV. Initial results of assaying the WHO standard immune serum globulin are comparable with the titre obtained by the radioimmunofocus inhibition test. Antibody titres in human and mouse sera could also be quantitated. The cytopathic TCID50 assay and the adapted inhibition assay described, may prove useful for the development and control of HAV vaccines and the validation of viral inactivation protocols.

Primer extension dideoxy chain termination nucleotide sequencing of partially purified RNA virus genomes: a technique for investigating low titre viruses with extensive genome secondary structure.

A modified version of the primer extension dideoxy chain termination nucleotide sequencing technique (Sanger et al., 1977) is described. This method has advantages over existing molecular cloning and primer extension techniques in that it allows the genome of RNA viruses to be directly sequenced from partially purified RNA preparations. Thus, viruses growing at unacceptably low titres in tissue culture can now be partially purified from infected mouse brain and sequenced. The technique also incorporates steps for the denaturation of secondary structure which has previously provided difficulties for primer extension sequencing.

Studies on the attenuation of the Sabin type 3 oral polio vaccine.

The genetic basis of attenuation of the poliovirus type 3 vaccine strain P3/Leon 12a1b has been investigated by comparing the nucleotide sequence of this strain with that of its neurovirulent progenitor P3/Leon/37 and by constructing recombinants between these two viruses using infectious cDNAs. Preliminary results suggest that attenuation is caused by just two point mutations, one occurring in the 5' non-coding region and the other causing an amino acid change in coat protein VP3.

An improved immunoblotting procedure for the detection of antibodies against HIV.

Immunoblotting ('Western blotting') is routinely used for detection of antibodies against HIV in the diagnosis of HIV infection. We describe an improved procedure, which does not require virus purification and is easy to control for 'false-positive' results. The technique also does not produce erroneous results due to reactivity of the developing system with residual cellular proteins or viral antigens and does not give high nonspecific background staining. The technique can be applied to the detection of antibodies to HIV in serum, plasma, and blood products.

Growth and characterization of poliovirus antigen chimeras.

Chapter 22 outlines the construction of engineered, full-length poliovirus cDNAs in which the region encoding a well-characterized antigenic site has been replaced by sequences of choice. This chapter briefly describes the methods used to generate, maintain, and characterize infectious chimeric viruses. These techniques include several developed during the course of poliovirus study that have recently been published (1). This overview describes modifications to these techniques where relevant, but concentrates on methods not detailed by Minor (1). The production and analysis of poliovirus antigen chimeras can be readily subdivided into three discrete areas.

Virus validation procedures: practical aspects.

The Ad Hoc Working Party on Biotechnology and Pharmacy of the Committee for Proprietary Medicinal Products has developed a number of guidance notes for manufacturers intending to submit applications for market authorization in the European Union which includes one dealing specifically with the validation of production processes for the removal or inactivation of viruses. The strategies used to minimize the risk of viral transmission by biological products are: screening the source materials for viral contamination; examining the ability of the production process to remove or inactivate viruses; and examining the final product for evidence of viral contamination. It is concluded that validation studies provide a significant assurance of viral safety when properly carried out, using appropriate relevant and model viruses.

Virological issues in the use of cell therapies.

The testing of cells and starting materials for biological medicinal products made from human sources has tended to focus on blood-borne viruses and those with a tropism for B lymphocytes, probably arising from the existing guidance on human monoclonal antibodies and experience with proteins fractionated from human blood. This may not be the best course although blood may contaminate starting materials. Other agents may be more likely contaminants, and it is essential to apply sound virological principles to ensure product safety, particularly in instances such as cell therapy where the product is a living cell which is intended to persist in the recipient for some time.

Beyond HIV, HBV and HCV--how to deal with other viruses?

Transmission of viruses such as HBV, HIV and HCV by blood components and protein fractions are well documented and precautions based on donor selection and screening as well as processing are well established and effective. Other viruses, including small non-enveloped viruses, pose a greater challenge for removal, but are considered less hazardous clinically. While cell associated viruses such as HTLVI and CMV pose a risk to certain kinds of recipient of blood components the biggest single current issue is that of vCJD following the BSE epidemic in the United Kingdom.