Overview

The research aims of the unit are to use modern molecular biology expression techniques to produce pure protein antigens cloned from respiratory viruses [or other pathogens] for scientific studies as well as diagnostic tests. These expressed antigens can be used for the production of antibodies in whole animals or to screen recombinant bacteriophage libraries for the production of recombinant antibodies. Together these recombinant antigens and antibodies can be used to generate novel enzyme linked immuno-sorbent assays (ELISA) based detection systems for the detection of newly emerged viruses for which there are no current immunological-based detection systems. We have evaluated several expression systems for their ability to reliably produce large quantities of authentic antigen for these studies and produced several recombinant antibodies against hMPV and WU virus.

Recently, several newly described respiratory viruses have been detected overseas and within a short period of time these viruses have also been detected in Australia. These viruses include human metapneumoviruses (hMPV) of both “type A” and “type B” lineages and more recently, another respiratory virus, human Bocavirus (hBoV), have been detected in several patients with pneumonia in Australia. Two other newly described viruses implicated in respiratory disease are WU and KI viruses. These Parvoviruses cannot be propagated in tissue culture at present and thus we cannot generate virus to raise polyclonal or monoclonal antibodies for diagnostic tests. At the present time there are no functional monoclonal antibodies available against these human viruses, nor are the complete nucleotide sequences for some of these Australian viruses available for comparison with the overseas isolates.

The lack of serological tests is in part due to the use of RT-PCR reactions, which are currently used to detect these viruses in Australia, that have been developed using sequence data from overseas isolates. Only relatively small regions from defined gene segments have thus been compared for the Australian viruses. Approximately 30-50% of all moderate to severe respiratory cases admitted to Australian and overseas hospitals remain undiagnosed whether due to novel viral infections or gaps in the screening/detections systems employed. Worldwide hMPV is a significant cause of lower respiratory tract infections in infants, young children, the elderly and immuno-compromised patients resulting in bronchiolitis and pneumonia. Recent estimates of health costs due to this virus alone in Australia is in the order of several million dollars.

We aim to produce a suite of recombinant antibodies against the newly emerged Australian human metapneuomovirus and bocavirus. These will be used to develop immunological assays to compliment the current RT-PCR assays, increase the detection rate of these respiratory viruses and augment basic research studies on these viruses.

Principally we wish to develop a suite of recombinant antibodies against the Australian hBoV, WU, KI and hMPV isolates. Specifically, chickens were investigated as an mRNA source for the generation of scFv antibodies. mRNA coding for the VH and VL chains of antibody genes was amplified by PCR from chicken spleens of immunized animals. These amplicons were cloned into bacterial phage libraries and screened for reactive antibodies (scFv) binding to proteins translated from the viral genome(s) using ELISA and Western blotting. However this system was found to be less able to generate scFvs or the required diversity in our hands. Thus a naïve Llama single-domain antibody library [sdAb] expressed in bacteriophage was constructed and utilized to screen for binding and neutralising recombinant antibodies. Recombinant antibodies against conserved proteins such as the M and N proteins of hMPV and the VP1/VP2 of hBoV will also be of interest as these are generally the most highly conserved proteins within these viruses, respectively and useful for developing broadly reactive immunological tests. These proteins are currently being expressed in bacterial and yeast systems for eventual sdAb production. The use of conserved proteins for ELISA-based tests will overcome some of the shortcomings of PCR based tests wherein genome variability results in a false negative due to lack of oligomer binding in the PCR test. Antigen detections systems such as ELISA, blocking-ELISA as well as direct immuno-detection of viral antigens will be developed and evaluated for their sensitivity and specificity. The production of a suite of recombinant antibodies will also be extremely useful in epidemiological surveys of respiratory virus infections within Australia. The development of these new reagents and tests will be used in conjunction with existing RT-PCR and PCR diagnostic tests to synergistically enhance current molecular detection rates and research into respiratory viruses within Australia.

Research Projects

Cloning, sequencing and expression of viral genes hMPV, WUV and KIV

We have been able to design primers and amplify full-length copies of the genes encoding the VP1,
VP2 and VP3 capsid proteins of the WU and KI viruses. To date these genes have been cloned into
bacterial expression vectors (pGEX) as well as baculovirus expression vectors (pFastBac HT A). Sequence analysis of the cloned genes has shown them to be authentic copies of the genes with open reading frames (Kattenbelt and Gould, unpublished). We have previously used these systems as well as the pMAL (bacterial fusion expression utilizing the maltose binding protein, Fig 1) and yeast expression system  (K. lactis, Fig 2) to express hMPV viral proteins of interest for the generation of recombinant proteins as well as constructs of the hepatitis core surface antigen using these bacterial and yeast expression systems, however these proteins were observed to be produced in either low yields or prone to degradation (Tindle, Kattenbelt and Gould, Figs 1 & 2; unpublished). Never-the-less, expression of parvovirus B19 VP1/VP2 empty capsids was previously achieved using a similar approach by Brown et al, 28 using yeast expression. However, we have now moved our constructs into the pFastBac HT A baculovirus expression system exclusively as we have found this system to produce higher yields of protein (Figs.1 and 2) which are suitable for purification using Talon® columns via a His6 ‘tag’ added to the amino-terminus of the expressed proteins (Fig. 3). We have expressed hMPV N,M and F proteins as well as WUV VP1, VP2 and VP3 as well as KIV VP1, VP2 and VP3 in high yields (mg quantities). Results from baculovirus expression of WUV VP1 have shown that this protein can accumulate in the external medium of the spf9 cells and can be observed as a discrete band of protein in a 20-60% sucrose gradient (which consists of entirely WUV VP1 Protein, not shown). Electron micrographs of these cells (Fig. 4) show accumulation of electron dense protein at the cell membrane and budding/shedding of particulate matter from the cell surface. It is assumed that this process represents the production of virus-like particles of WUV. This is currently being investigated.

Western blot using anti-H6-his antibody. Mr=Molecular weight standards; M= Matrix protein hMPV; N= nucleocapsid protein hMPV, C=Control plasmid. Fig.2: Western blot using anti-H6-his antibody. Mr= Molecular weight standards; F= Fusion protein hMPV; VP2=VP2 of WU virus; VP3=VP3 WU virus. Fig.3: Coomassie blue stained SDS gel analysis of WU virus VP1 purification from baculovirus-infected sf9cells. 1, Molecular weight standards; 2, Soluble lysed material from sf9 cells; 3, insoluble lysed material; 4, Flow through from Talon® column; 5, Wash from Talon® column; 6, Elution 1 of WU virus VP1 from Talon® column; 7, Elution 2 of WU virus VP1 from Talon® column. Fig. 4: Electron micrographs showing the cell surface of (A) Spodoptera frugiperda (sf9) cells infected with pFastBac HT A vector containing  no insert, (B) sf9 cells infected with pFastBac HT A vector containing WU VP1 construct.

It should be stressed that since these human viruses cannot be propagated in tissue culture the genetic material that was used as source material to amplify these gene was obtained from clinical samples (nasopharangeal swabs) from Australian respiratory patients. To date we have been unable to propagate these viruses in tissue culture, in line with overseas observations. At the present time there are no monoclonal antibodies available for the recently described human bocavirus, WUV or KIV. For the common respiratory viruses, the only monoclonal antibodies available are against human RSV or hMPV are limited (i.e. a single mAb against the M protein of hMPV) and have been imported from overseas for research purposes. Also to date the complete sequence of the Australian isolate of hMPV is not available for comparison with the overseas isolates. Similarly, the RT-PCR reactions which have been developed have used sequence data from these overseas isolates to develop the Australian tests and are only able to compare relatively small regions of defined gene segments. Thus it is envisaged that ELISA-based diagnostic tests based on protein sequences of highly conserved motifs will be more reliable in detecting related viral isolates within a group than the RT-PCR based tests.

sdAb isolated from a naïve llama phage library

We have been able to establish the relevant technology to express, pan and produce in bacteria sdAbs from the “Nomad1” library of Hayhurst. As a test system, we utilized bovine serum albumin to probe the system before utilizing it against expressed recombinant antigens as it was easily available in a pure form. A number of ELISA positive bacteriophage isolates expressing sdAb reactive to BSA were isolated and sequenced to demonstrate individual differences between their heavy chain complementarity determining regions (CDR1, CDR2 and CDR3) which in turn differ from those for vaccinia virus and lysozyme 36.  Using these procedures, we have isolated several sdAb candidates by panning against purified hMPV (Fig. 6)

and concentrated our preliminary efforts on one designated JAK-A8 which shows strong binding to hMPV in ELISA. The gene coding for JAK-A8 sdAb was transferred into the Pecan-22 expression plasmid, expressed and purified protein isolated from the periplasm of E. coli cells (not shown). Thus we have demonstrated our ability to clone, express and purify viral proteins of interest from human MPV, BoV, KIV and WUV in the baculovirus expression system resulting in milligram quantities for panning and ELISA tests. We have likewise been able to demonstrate the ability to pan and select from a Llama bacteriophage sdAb library, antibodies that bind to hMPV, to transfer these sdAbs to an E. coli expression system and to isolate and purify sdAb antibody for testing in ELISA. We are presently analyzing hMPV sdAbs to determine which bind to which protein. We have also constructed a Pecan16 vector which secretes sdAb fused to alkaline phosphatase as a reporter group, to be used in ELISA.
We are now in a position to commence immunization and panning of expressed antigens to generate recombinant antibodies against a range of important Australian respiratory viruses. To this end we have produced milligram amounts of highly purified WU VP1 and VP3 proteins to be used for immunological screening by Ass. Prof Theo Sloots’ research group to determine the serological presence of this virus in the human population.
We are also currently investigating the possibility of deriving a naïve sdAb library from Cammelids. It has been observed that the likelyhood of isolating a high affinity, high specificity sdAb is low from a naïve library but considerably enhanced from an immunocompetant animal (pers. comms.).

Cloning and expression of novel proteins.

We are at present investigating the use of our cloning systems to generate novel proteins which are improved antigen delivery systems or proteins that have proven difficult or impossible to purify or express by conventional means.
Preliminary cloning of other genes for expression studies of self-polymerising systems is currently being done with the eventual aim of using these as a particulate protein/antigen delivery system. One polymerising protein has been cloned from an animal virus and the E7 protein from human papilloma virus attached to the carboxyl terminus. This hybrid gene has been cloned and expressed in baculovirus, forms long polymerized tubules of protein and is currently being evaluated as an immunogen in mice.
Another system being evaluated is to use plant pathogens for carrying RNAi molecules to inhibit the expression of genes in mammalian systems. At present several constructs have been made and tested both in vitro and in vivo. This work involves a collaboration with the University of Western Australia and University of Adelaide.

Use of novel oligonucleotides primers for the identification of DNA sequences in respiratory diseases.

The first year of this project has been dedicated to setting up and validating methods for identifying novel viruses in respiratory samples. Overall, we have been successfully implemented an established detection tool, and have also developed novel tools that have considerable potential for novel respiratory virus investigations.

1  To date, we have been successful in identifying characterized viruses, including Adenoviruses, WU and KI viruses from control specimens using the previously described board range amplification and sequencing molecular technique outlined in our proposal. In addition, we have also identified a “sequence of unknown origin” in a respiratory sample using this technique. Analysis of this sequence is ongoing. 
2  While investigating the above established technique we have become increasingly aware of its limitations; the main one being the need for a large number of cloning and sequencing reactions – making the technique overly laborious and unnecessarily expensive. To circumvent this problem we have developed a novel (and potentially patentable) strategy for novel virus investigations. The new Random Amplified Fingerprint (RAF) strategy still uses random amplification, however the process uses a gel-based fingerprinting method to distinguish products that are “of interest” from those may be attributed to human genomic DNA. Notably, the RAF technique; utilises a novel primer design for radom amplification, reduces the need for cloning and the number of sequencing reactions, and may have other applications, including “genome walking” of viruses (currently being done with characterised viruses). Validation of the RAF technique is ongoing. 
3  During these studies we also became involved in the response to the novel influenza AH1N1 (swine flu) virus outbreak, by developing a novel positive-control tool for rare or emerging viruses.
 
When responding to outbreaks of rare or emerging viruses, laboratories are often faced with the problem of not having a ready source of positive control material for diagnostic assay development. The novel influenza AH1N1 (swine flu) virus pandemic was a very good example of this problem. Sequence data for the influenza A(H1N1) virus was promptly available from the Centres for Disease Control and Prevention (CDC, USA), allowing oligonucleotide primer and probes for RT-PCR assays to be developed. However, many diagnostic laboratories, ours (Pathology Queensland) included, were struggling to find a source of positive control material. To circumvent this problem, we have developed a new universal method for preparing PCR positive controls (Uni-Control) for rare or emerging viruses, which simply requires two synthetic control oligonucleotides and viral nucleic acid as an initiator template. So far, we have developed Uni-Controls for novel influenza A(H1N1) and human metapneumovirus PCR assays. Our results show the Uni-Control method provides appropriate quality control of critical processes in these PCR assays, including reverse transcription, primer and probe annealing and PCR amplification, that the method is versatile (can readily be adapted to almost any viral template) and most importantly can be rapidly introduced for rare or emerging viruses in the absence of wild-type control material. The details of the method have been submitted to Clinical Chemistry for publication.