centro biotecnologie avanzate

LABORATORY OF: Microbiology and Virology
CONTACT PERSON: Prof. Oliviero Varnier (University of Genova)
Phone +010 3537649 E-mail: oliviero.varnier@unige.it

Description of Laboratory and Expertise:

The laboratory of Microbiology and Virology has been organised respecting biosafety criteria which protect the operator and guarantee the efficiency of the result. The working atmosphere has been subdivided in 3 areas: a pre-amplification, a post-amplification and a cell culture area. The laboratory carries out quantitative and qualitative molecular analyses on different viruses (herpes-, adeno-, entero-, polyoma-, papilloma, parvo- and retro-viruses.) using the most recent technologies and is in continuous expansion. Molecular techniques are favourable when faced with pathogenic microbes which are difficult or dangerous to cultivate. Currently molecular tests can be performed for Toxoplasma, Mycobacteria, Borrelia, Leishmania and Pneumocystis.

Abstract of Activities:

The activities of the Laboratory of Microbiology and Virology includes the viral clearance evaluation: we are focused on the development of viral clearance models with the aim of providing evidence that GMP purification steps have inactivated/removed viruses.
The second research is the HIV p24 Antigen assay, which has been re-evaluated in the diagnosis of HIV infection in newborns and, in resource poor setting, novel features will be evaluated.
The third project will monitor JCV reactivation and shedding in immunocompromised patients and immunocompetent healthy individuals, and analyze JCV sequences to identify pathogenic JCV (see below) associated with PML.

Detailed Research Activities:

VIRAL CLEARANCE EVALUATION
The risk of virus contamination is a feature common to biopharmaceutical products such as recombinant monoclonal antibodies, vaccines, cytokines and human-derived proteins or cells. Viral contaminants can arise from the source cell line or tissue used for production, reagents used in purification processes or adventitious virus introduced during production or unknown accidental introduction.
Reasons for concerns are several: 1) bioproducts are frequently administered to bypass the body’s defences; 2) viral contamination poses very serious risks; 3) one infectious particle is sufficient to cause damage and, 4) virus detection and removal/ inactivation are not easy to perform.
History of viral contamination in Biopharmaceutical Products: in 1942 the Yellow fever vaccine was contaminated by the Hepatitis B Virus, which was present in the albumin used as stabilizer; in 1955, the polio vaccine was contaminated by the SV40 virus, which was present in the monkey cell line used for poliovirus propagation; in 1960 the rabies vaccine contained rabies virus because of incomplete inactivation; and in 1981, the Foot and Mouth Disease (FMD) vaccine was also found to contain alive FMD virus due to incomplete virus inactivation.
The capacity of the purification process (“Good Manufacturing Process” = GMP) to remove or inactivate viral contaminants must be evaluated to assure the absence of these viruses in the final product.
International process validation studies to determine the ability of the downstream process to remove and/or inactivate potential viral contamination must be performed prior to clinical trials. These validation studies use either specific or model viruses depending upon the cell substrate or the tissue used. By spiking the viruses at known titers directly into a down-scaled version of the purification step to be studied (figure 1 and 2), the effectiveness of its ability to inactivate or remove viruses is quantified by determining the log reductions factor. Examples of specific or model viruses routinely used for such studies include: MuLV (RNA virus, enveloped), PI3 (RNA virus, enveloped), REO 3 (RNA virus, non-enveloped), PRV (DNA virus, enveloped), PPV (DNA virus, non-enveloped), SV40 (DNA virus, non-enveloped), HIV (RNA virus, enveloped) and HAV (RNA virus, non-enveloped).
The effort of our team is focused on the development of viral clearance models with the aim of providing evidence that GMP purification steps have effectively inactivated/removed viruses, which are either known to contaminate the starting materials, or which could conceivably do so; to provide indirect evidence that the purification step might inactivate/remove novel or unpredictable virus contamination, and to assure the absence of these viruses in the final product.
The viral clearance evaluation is aimed at the assessment of viral removal and inactivation during the production processes examined, employing selected viruses of the four viral types classified according to physiochemical resistance (European Agency for the Evaluation of Medical Products, CPMP/BWP/268/95):

FamilyVirusesTypeSize (nm)Physiochemical ResistanceRetrovirusMurine Leukaemia VirusesRNA enveloped80-110LowPicornavirusPoliovirus Sabin type 3RNA non-enveloped25-30MediumHerpesvirusHerpes simplex virus type 1/2DNA enveloped120-200MediumPapovavirusSV40 PoliomavirusDNA non-enveloped40-50High
For each selected type, frozen aliquots of infectious virus are used for the viral clearance evaluation. The product purification procedure is done in reduced-scale in the biosafety controlled laboratories of CBA by trained personnel. This procedure might include single or multiple steps of liquid chromatography, filtration, and other preparative techniques:
one aliquot is kept frozen as virus load control;
one aliquot of infectious virus is added to the product at the beginning of each step of the purification process. The processed product is collected at the end of the purification step and stored at -70°C;
The viral clearance evaluation includes the testing of all the collected viral aliquots for in vitro infectivity assays to determine viral inactivation, and molecular quantification of the viral genome to determine virion removal. The clearance evaluation of retroviruses can be determined as described below.
Clearance Evaluation of Retroviruses (Murine Leukemia Virus, MuLV)
In vitro Infectivity assay
One set of all the collected vials is stored at -70°C. The titer of the infectious MuLV contained in all samples is determined using an infectious assay in Mink Lung (ML) cell cultures. Briefly, 1x10 ML cells are plated into 6, 24 or 48 well plate dishes in RPMI growth Medium containing 10% foetal calf serum, 10% glutamine and 20% penicillin/streptomycin (5,000 U/ml and 5 mg/ml, respectively) and incubated at 37°C in 5% CO atmosphere. After 24 hours, triplicate ML cell cultures are inoculated with 10 up to 10 dilutions of each sample. The inoculum volume is 1/10 of the growth medium volume. After two days, medium is changed and supernatants are collected from all plates at day 3 and frozen at -70°C. The titer of the infectious replicating MuLV is determined by molecular amplification of the viral genome in the supernatant samples.
Molecular Quantification of Viral Genome
Supernatant samples are processed by the MagNA Pure to obtain purified genomic RNA. Briefly, RNA is extracted from 0.2 ml of supernatant collected from cell cultures inoculated with the tested vials using the RNA Isolation High Performance kit and the MagNa Pure automatic Instrument. RNA is eluted in 50 mL (final volume) and RNA eluates are stored at -70°C until tested.
RNA samples are amplified using the Real Time LighCycler Technology using the MuLV env R and MuLV S1 primers. The LightCycler system is a microvolume fluorimeter integrated with a thermalcycler that combines rapid-cycle PCR with fluorescence monitoring. Two specific fluorophore-labelled probes hybridize in close proximity to the newly synthesized DNA strand allowing a fluorescence resonance energy transfer (FRET) to occur which generates a fluorescent signal. Fluorescence is monitored during the PCR at the end of each annealing step and increases in proportion to the amount of product produced and is dependent on the initial template concentration. Accurate quantification is performed using an external standard curve of serial dilutions of MuLV with a dynamic range of 7 logs.
The safety of human therapeutical compounds can be only be rationally guaranteed by the analysis of viral inactivation and removal during the GMP production process. A core facility for viral clearance evaluation at the CBA would be instrumental for drug production and the use of biological products for human therapy.

HIV p24 ANTIGEN ASSAY
For the last 20 years we have carried out research and diagnostic studies of HIV infection with advanced expertise in the detection of HIV p24 antigen. As members of the HIV Forum, an international HIV expert group, we are currently performing investigation on different applications of the HIV p24 Antigen assay in the diagnosis of HIV infection in newborns and its use for the quantitation of HIV viremia in resource poor settings. Recently, we have set up this assay at the Alli Causai Hospital in Ambato, Ecuador. Innovations are foreseen and they will be investigated in collaboration with Perkin Elmer.

DYNAMICS OF JC POLYOMAVIRUS REACTIVATION AND SHEDDING
JC polyomavirus (JCV) is an unenveloped DNA virus that is widespread in human populations, with 50–70% of individuals having antibodies to the virus by the age of 65 years. JCV has not been associated with symptomatic clinical illness, and the distribution of the virus in systemic organs and/or the brain during primary infection is not known. Following initial infection, however, the virus persists in renal tubular epithelial cells, and excretion of JCV has been documented in immunologically normal older individuals, as well as during pregnancy or immunosuppression.
Progressive multifocal leukoencephalopathy (PML) was initially described in patients immunosuppressed because of malignant disease or iatrogenic immunosuppression. The aetiological agent of PML, JCV, was isolated in 1971. PML remained an extremely rare condition until the advent of AIDS in the 1980s, when it was identified as the cause of death in 4% of HIV-infected patients. Until 2005, PML had not been reported in patients with MS. PML has been thought to arise when reactivation of latent JCV in the kidneys results in a viremia, in which virus in blood or B-lymphocytes enters the brain to cause disease. It is also of note that JCV DNA was detected within the brains of random patients coming to autopsy, none of whom had PML. In addition meticulous studies of post-mortem material detected JCV DNA in almost 50% of brains from immunologically normal individuals. JCV early-region DNA sequences have been identified in 50% of brains obtained from individuals without PML or known immune deficiency. These data suggest that JCV may also persist within the central nervous system (CNS) of some individuals. Since the human immune system is normally capable of controlling latent or persistent viruses, diseases such as viral induced PML could be due to peculiar defects of humoral and/or cellular immunity. JCV persisting in kidneys contains a regulatory region without deletions or duplications. This strain of JCV is termed ‘archetypal’ and is the viral form believed to be transmitted between individuals. PML isolates, on the other hand, are almost always ’variant’ strains, which contain duplications or deletions within the enhancer region of the viral genome. The association of variant JCV strains with PML suggests that these viral genomic changes may have a not yet confirmed role in pathogenesis. The question is whether some individuals may carry potentially pathogenic JCV strains in their kidneys or brain, and whether such individuals might be at higher risk of developing PML under conditions of altered T-cell response.
Natalizumab is a humanized monoclonal antibody, which is reactive with the a-4 subunit of the integrin adhesion molecule expressed on the surface of all circulating leucocytes except neutrophils. Natalizumab is thought to block leukocyte migration across endothelial cells, and was designed to inhibit the recruitment of immune cells into the brain. The agent was demonstrated to have significant clinical efficacy for MS in two Phase III trials, which compared natalizumab with placebo or permitted the concurrent use of beta interferon. Natalizumab has been reintroduced under a careful risk management programme. The immunodeficiency acquired during HIV infection or caused by therapeutic regimens or organ and cell allograft leaves three important questions: the first of these concerns the effects on host immune surveillance. These studies suggest that the effects of natalizumab on human immune function may be quite complex and variable. The second question relates to the extent to which viral as well as host factors might have played a role in the onset of PML in three affected MS patients; whether some immunologically normal individuals may be persistently infected, either in their kidneys, B cells or brain, with JCV strains known to be associated with PML; and whether such individuals might be at greater risk of developing PML during the iatrogenic immunosuppression. The third question involves early detection of PML in immunosuppressed patients. This task, even with the new guidelines in place, may prove dauntingly difficult. The detection of JCV DNA in blood might conceivably be used to identify patients at risk, since the patient with Crohn’s disease and PML was found, retrospectively, to have JCV DNA present in blood collected prior to the onset of neurological signs. However, JCV DNA was also detected in blood from a natalizumab-treated patient in whom PML did not occur, although PCR analysis of CSF from 396 patients treated with natalizumab did not detect JCV DNA in any sample. Most important data regarding the presence of JCV DNA in blood from other asymptomatic individuals are not yet available.
The main objectives of this research, which is supported by Biogen Dompé, are to monitor the patterns of JCV reactivation and shedding in immunocompromised patients and immunocompetent healthy individuals over time; to assess whether the level of immunosuppression might be associated with JCV reactivation, and to analyze JCV sequences in order to identify potentially pathogenic JCV strains which might be associated with a higher risk of developing PML.

Applications and Developments:

The Laboratory of Microbiology and Virology has developed expertise and experience in Viral Clearance Evaluation to qualify as a provider of virological studies needed for GMP
biotech products and drugs.
Long term research is a prerequisite for R&D investigations and clinical trials of new reagents for infectious diseases.

Ongoing collaborations:

Philogen s.p.a, an Italian company engaged in the research & development of diagnostic and therapeutic products; Siena, Italy
Perkin Elmer and Analitical Sciences, a company engaged in the research & development and sale of diagnostic and biotechnology products; Turku, Finland
Biogen Dompé, a company engaged in the research and development of high biotech products and sale of highly therapeutic drugs; Milano, Italy
Alli Causai Hospital, Ambato, Ecuador

Most recent and significant publications:

Respess R.A., Cachafeiro A., Withum D., Fiscus S.A., Newman D., Branson B., Varnier O.E., Lewis K., Dondero T.J. Evaluation of an ultrasensitive p24 antigen assay as a potential alternative to human immunodeficiency virus type 1 RNA viral load assay in resource-limited settings. J Clin Microbiol. 2005 Jan;43(1):506-8.

Viscoli C., Varnier OE, Machetti M. Infections in patients with febrile neutropenia: epidemiology, microbiology, and risk stratification. Clin Infect Dis. 2005 Apr 1;40 Suppl 4:S240-5.

McDermott JL, Martini I, Ferrari D, Bertolotti F, Giacomazzi C, Murdaca G, Puppo F, Indiveri F, and Varnier OE. Decay of human immunodeficiency virus type 1 unintegrated DNA containing two long terminal repeats in infected individuals after 3 to 8 years of sustained control of viremia. J Clin Microbiol. 2005 Oct;43(10):5272-4.

Jennings C, Fiscus SA, Crowe SM, Danilovic AD, Morack RJ, Scianna S, Cachafeiro A, Brambilla DJ, Schupbach J, Stevens W, Respess R, Varnier OE, Corrigan GE, Gronowitz JS, Ussery MA, and Bremer JW. Comparison of two human immunodeficiency virus (HIV) RNA surrogate assays to the standard HIV RNA assay. J Clin Microbiol. 2005 Dec;43(12):5950-6.

Fiscus SA, Cheng B, Crowe SM, Demeter L, Jennings C, Miller V, Respess R, Stevens W and the Forum for Collaborative HIV Research Alternative Viral Load Assay Working Group (Varnier OE). HIV-1 viral load assays for resource-limited settings. PLoS Med. 2006 Oct;3(10):e417

Righi E., Giacomazzi C., Bisio F., Soro O., Ratto S., Varnier O., Bassetti M., Viscoli C. A case report of Absidia corymbifera soft-tissue infection with kidney involvement in a AIDS patient. Medical Mycology2007, 45, 637-640,.

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