Nucleic Acids Research

Cells and virus

Spodoptera frugiperda cells (Sf9, CRL 1711; ATCC, Rockville, MD, USA) were grown in IPL-41 medium (Sigma-Aldrich Chemical, Deisenhofen, Germany) containing yeastolate and a lipid/sterol cocktail with optional 3 or 10% fetal calf serum at 27°C using T-flasks or spinner flasks. Ac-omega and its derivatives were propagated in Sf9 cells. Viruses were isolated and plaque-purified by standard procedures (18).

DNA manipulations

DNA manipulations were carried out essentially as summarized by Sambrook et al. (19). Restriction enzymes, T4 DNA ligase, calf intestine phosphatase and Taq DNA polymerase were purchased from Boehringer Mannheim (Vienna, Austria) and used according to the manufacturer's recommendation. All primers were synthesized by Codon (Neusiedl, Austria). Polymerase chain reactions (PCRs) were carried out in a 50 or 100 µl reaction buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 5% dimethyl sulfoxide and 200 µM dNTP) using 20 pmol of each primer and 2.5 U of Taq DNA polymerase. Samples were subjected to 30 cycles (at 92°C, 55°C or 60°C and 72°C) on a Model 9600 Thermal Cycler (Perkin-Elmer, Norwalk, CT, USA). DNA sequence-analysis were carried out on an Applied Biosystems 373A DNA sequencer, using a Taq DyeDeoxy[trade] Termination Cycle Sequencing Kit from Applied Biosystems (Foster City, CA, USA).

Transfection efficiency of Ac-omega

For testing the transfection efficiency, the [beta]-galactosidase gene ([beta]-gal) and the green fluorescent protein gene (gfp) were PCR amplified using the primers [beta]-galSceback 5[prime]-gatcgcggatcctatattaccctgttatccctatgaccatgattacggattcactggccg-3[prime] and [beta]-galScefor 5[prime]-gatcgcggatccgctagggataacagggtaattattattatttttgacaccagaccaactgg-3[prime], and gfpSceback 5[prime]-cgcggatcctatattaccctgttatccctaacatgggtaaaggagaagaacttttc-3[prime] and gfpScefor 5[prime]-cgcggatccgctagggataacagggtaatttattatccggacttgtatagttcttcc-3[prime], respectively. The SceI treated reporter genes [beta]-gal and gfp were inserted into Ac-omega according to Table 1.

Table 1. Transfection efficiency of Ac-[Omega]^a

		DNA (ng) ratio	Molar	[beta]-gal/ gfp	[beta]-gal rec. plaques
Ac-[Omega]	(134 kb)	50	1
gfp	(0.7 kb)	270	1000
[beta]-gal	(3 kb)	1.25	1	1: 1000	+
Ac-[Omega]		50	1
gfp		270	1000
[beta]-gal		0.125	0.1	1: 10 000	+

^aViral Ac-omega (Ac-[Omega]) DNA was simultanously ligated with different ratios of two reporter genes, [beta]-gal and gfp. Sf9 cells were transfected with the ligation mixtures, incubated for 6 days and plaqued. The table indicates different molar ratios ([beta]-gal/gfp) from which [beta]-gal recombinant plaques could still be recovered.

Construction of the library and vectors for surface display

The epitope library was constructed by annealing two synthetic oligonucleotides 5[prime]-cagtatcctgcagccataggggaaaaagcagtttttacagaaatttgctatggctgacgaag-3[prime] and 5[prime]-attggtaagctttgggtannnnnnagcccatttatcgagctcnnncttcttcgtcagccatagcaaatt-3[prime] encoding the epitope motive flanked by three randomly selected amino acids (XELDKWAXX). After annealing, the two oligonucleotides have been end filled with Klenow enzyme. This double stranded DNA-fragment was then treated with restriction endonucleases PstI and HindIII, and inserted into the plasmid pT3WSN-HAm1 (20) by replacing the wild-type fragment in this vector. In the resulting plasmid pHA-Lib, the epitope-motive is located at antigenic site B of influenza hemagglutinin. Construction of pHA-B5: the epitope sequence ELDKWAS was inserted into the plasmid pT3WSN-HAm1 (20) by replacing a PstI and HindIII fragment with a PCR product which was obtained using pT3WSN-HAm1 as template and the sense and antisense primers 5[prime]-cagtatcctgcagccata-3[prime] and 5[prime]-attggtaagctttgggtatgaatcgctagcccatttatcgagctcccccttcttcgtcagc-3[prime], respectively. Purified plasmids pHA-Lib and pHA-B5 were subjected to PCR using primers HASceback 5[prime]-cggatcctatattaccctgttatccctaacatgaaggcaaaactactggtcc-3[prime] and HAScefor 5[prime]-gcggatccgctagggataacagggtaattcagatgcatattctgcactgc-3[prime].

Direct gene insertion into Ac-omega

Generated PCR-fragments were gel purified, treated with SceI and ligated to 140 ng of purified, dephosphorylated viral Ac-omega DNA at a molar ratio of 1:40 as described previously (21). After incubating the ligation mixture at 16°C over night, Sf9 cells were tranfected by lipofection (22). Insertion of foreign sequences into Ac-omega were verified by PCR, amplifying a specific fragment of viral DNA with Ac-44back 5[prime]-tttactgttttcgtaacagttttg-3[prime] and Ac+778for 5[prime]-caacaacgcacagaatctagc-3[prime] primers. PCR products of individual viral clones were sequenced with primer B1antisense 5[prime]-ggagtaacagtatcatgctcccata-3[prime] to identify the amino acids flanking the epitope.

Transfection of the epitope library

For transfection,140 ng of ligated Ac-omega DNA containing the library insert, was added to 20 µl lipofectin, diluted in serum free medium. After 15 min this mixture was transfered to 2.5 × 10⁶ cells and after 6 h completed by the addition of serum containing medium. The transfection was incubated at 27°C for 6 days before the cells expressing the epitope library were analysed and sorted by FACS.

Fluorescent labelling of cells

Six days post-transfection or 48 h after infection, respectively, the cells were harvested and washed twice with phosphate-buffered saline (PBS). Subsequently, cells were incubated with 1.5 µg/ml hmAb 2F5 for 1 h, washed again and stained with anti-human IgG-FITC conjugate (Sigma, Germany) at a dilution of 1:40. After an additional 1 h, the labelled cells were pelleted and resuspended in PBS to give a final concentration of [sim]4 × 10⁶ cells/ml and subjected to FACS.

Forty-eight hours post-infection (h p.i.), virus infected cells were incubated with 1 µg/ml, 200, 40 and 8 ng/ml of hmAb 2F5 in order to determine a titration curve and treated in the same manner as described in the labelling protocol (see above).

For the competition assay virus infected cells (48 h p.i.) were preincubated with 1 µg/ml hmAb 2F5 for 15 min before addition of increasing concentrations (0.2, 1, 5, 25 and 125 molar ratios) of competitor ELDKWAS-alkaline phosphatase fusion protein (23). Staining of cells was carried out as specified above.

Flow cytometric analysis and sorting

Labelled Sf9 cells were analysed on a FACS-Vantage, equipped with a 5 W argon laser (Coherent) tuned to 488 nm, output power 250 mW (Becton Dickinson, San Jose, CA). The 0.5% of the cells with the highest fluorescence signal were expected to contain viral clones binding with high affinity to hmAb 2F5, and were separated from the remaining cells. This fraction, containing the enriched cellular population, was resuspended in 1 ml complete medium. A sample (0.5 ml) of this suspension was amplified before viral clones were isolated, the other 0.5 ml was directly subjected to a plaque assay following standard protocols (24) to allow isolation of individual viral clones.

Hemadsorptionassay

Hemadsorption of recombinant HA-PVII clone was tested by adding human erythrocytes (1% in PBS) to infected insect cells. Sf9 cells were infected with recombinant HA-PVII and wild-type control Ac-omega and incubated for 48 h. Erythrocytes were added to the infected cells and incubated at room temperature. After 30 min the cells were examined under the microscope for the presence of bound erythrocytes.

Figure 1. The library was constructed using two synthetic oligonucleotides encoding the epitope sequence ELDKWA and three randomly selected, flanking amino acids. The mixture of generated epitope variants was inserted as PstI and HindIII fragment into antigenic site B of influenza virus hemagglutinin. Using SceI site containing primers, the pool of modified hemagglutinin genes was amplified by PCR and directly inserted into linearized Ac-omega viral DNA.

RESULTS AND DISCUSSION

Construction and characterization of an epitope library

The conserved linear epitope ELDKWA of HIV-1, specific for the hmAb 2F5 (16) had been shown to be capable of inducing HIV-1 neutralizing antibodies in mice, when these were immunized with the chimeric influenza virus presenting the epitope associated with the hemagglutinin on the viral surface (17). Previously the amino acid sequence ELDKWAS had been inserted into the antigenic site B of influenza virus hemagglutinin to mimic the antigenicity of the native 2F5 epitope on HIV-1 (17). Though the results obtained with the chimeric influenza virus were very encouraging, it was assumed that there was still potential for improved presentation of the epitope. To investigate the influence on immunogenicity of amino acids adjacent to the ELDKWA-motive, a baculovirus epitope library was constructed.

The size of a library is limited by the number of clones. To determine the efficiency of our direct cloning system (21) two reporter genes, gfp and [beta]-gal, were simultanously ligated into the viral genome of Ac-omega (Table 1). The amount of the gfp-DNA was kept constant while the [beta]-gal-DNA was serially diluted by a factor of ten (Table 1). At a 10 000-fold molar excess of gfp-DNA, relative to [beta]-gal-DNA, we were still able to recover [beta]-gal recombinant plaques from the transfection which was performed with 50 ng viral Ac-omega DNA. Results from this experiment indicate that 1 µg of Ac-omega DNA yields 2 × 10⁵ recombinant plaques.

A synthetic oligonucleotide encoding the sequence ELDKWA, flanked by one random amino acid N-terminally and two randomly selected amino acids C-terminally (XELDKWAXX) was inserted into the antigenic site B of the hemagglutinin gene of influenza virus A (Fig. 1). The generated pool of DNA plasmids was subjected to PCR, amplifying the entire, modified hemagglutinin sequence, using SceI recognition site containing primers. The pool of generated PCR-fragments was treated with the restriction enzyme SceI and directly ligated into the linearized viral DNA of Ac-omega (21). Insect cells were transfected with the ligation mix by lipofection and incubated for 6 days. The derived baculovirus expression library contained various viral clones, each of which displaying the epitope ELDKWA in a slightly different conformation and therefore differing in binding characteristics to hmAb 2F5.

Twenty individual plaques of the initial library were randomly picked, isolated from the transfection supernatant and subjected to PCR and sequence analysis. No contaminating wild-type virus could be detected. All viral clones contained the heterologous DNA insert in the proper orientation (due to the non-palindromic SceI restriction site), confirming the method of direct cloning to be efficient for the generation of recombinant viral clones (21). Sequence analysis revealed the required variability of the epitope library (Fig. 2). The viral clone HA-B5 was included in this study, containing an ELDKWAS-motive, flanked by the native hemagglutinin wild-type amino acids. This construct also exists as chimeric influenza virus and it's potential to induce HIV-1 neutralizing antibodies had been investigated by Muster and coworkers (17). The binding capacity of this clone to hmAb 2F5 served as an internal standard for binding requirements in this experiment. We intended to select for a hemagglutinin construct with higher, or at least comparable binding characteristics relative to the HA-B5 clone by screening this baculovirus expression library.

Figure 2. Viral clones were sequenced at antigenic site B to varify the insertion of the ELDKWA-motive. HA-B5 was included in this study, containing an ELDKWAS-motive, flanked by the native hemagglutinin wild-type amino acids.

Affinity selection by FACS

The binding properties of expressed proteins on the surface of infected cells, rather than virus particles itself, were analysed, since insect cells can directly be sorted by FACS, while virus particles give too low fluorescent signals, and are too small for the cell sorter to select. This technique was used to sort a pool of initially transfected cells. Sf9 cells, transfected with the recombinant hemagglutinin constructs, were treated with hmAb 2F5 and anti-human IgG FITC-conjugate. Analysis of 20 different single clones of the library pool revealed that the fluorescence signal intensity spanned a range of one to two orders of magnitude. After a 6 day incubation period the initial pool of transfected cells was analysed and sorted by FACS (Fig. 3). Detected binding capacity of the infected cell-pool to hmAb 2F5 showed a quite homogenic distribution of the fluorescence signal-intensity. Only a small number of individual cells was considered to contain high affinity binders. The gate for FACS-sorting, therefore, was set to include <0.5% of the total cell population. This selected cellular fraction was enriched by re-infection of cells or alternatively directly subjected to a plaque assay to isolate individual viral clones.

Figure 3. FACS-affinity selection was carried out from thepopulation of initially transfected cells, representing the entire epitope-library (red graph). Less than 0.5% (M 1) of this initial pool of clones was selected. Analysis of sorted fraction, 48 h post-infection showed a markedly increase in binding capacity to mAb 2F5 (green line).

Analysis of this enriched fraction showed an increase in binding capacity to hmAb 2F5 of about two orders of magnitude compared to the signal-intensity of the initial cell pool, which was derived after transfection of the library-DNA (Fig. 3). Subsequently 18 randomly picked plaques of the selected population were sequence analysed. All viral clones were found to contain three proline residues flanking the epitope-motive (P-ELDKWA-PP). Apparently, the chosen 0.5% gate represented only one clone (HA-PVII). Counting the particles by FACS might not give realistic numbers of living cells, also the survival rate during and after the process of preparation and sorting is not known. Presumably only a small number of viable virus particles were selected. However, by a one-step sorting procedure, a viral clone could be identified out of a pool of 8000 different variants (three randomized amino acids account for 20³ = 8000), that showed a markedly increased affinity to hmAb 2F5 compared to the initial expression library (Fig. 4).

Figure 4. Results of FACS-analysis are shown of four representative clones (including the HA-B5 construct), defining the range of signal intensity from randomly selected clones out of the initial pool. The green graph represents the signal of the selected HA-PVII, containing the three proline residues flanking the epitope. It's binding capacity to mAb 2F5 shows higher signal intensity compared to viral isolates from the initial library.

Figure 5. The binding capacity of HA-B5 and HA-PVII was evaluated by FACS, diluting hmAb 2F5 to determine individual reduction of signal intensity with descending concentrations of hmAb 2F5.

Characterization of selected clone

The newly selected construct HA-PVII was compared to initially characterized clones, in terms of its binding profile to hmAb 2F5. The fluorescent signal given by HA-PVII is clearly higher than the signal of the HA-B5 construct or of other library clones (Fig. 4), demonstrating that screening this baculovirus expression library was successful in identifying a molecule of higher binding capacity to hmAb 2F5.

For additional characterization of the binding profiles of selected clones, a titration curve was performed (Fig. 5). We investigated the influence of decending concentrations of hmAb 2F5, ranging from 1 µg/ml down to 8 ng/ml, on the reduction of signal intensity of HA-PVII and HA-B5, respectively. A more rapid decrease in loss of binding capacity was observed for HA-B5. The reactivity of both clones is expressed as relative values on a percentage basis (100% binding at 1 µg/ml 2F5). At a concentration of 8 ng/ml hmAb 2F5 a plateau was reached where the intensity of the signal approached background levels measured for the HA-wild-type construct.

Figure 6. Competition assay was performed by FACS using increasing concentrations of ELDKWAS-alkaline phosphatase fusion protein as competitor.

Competition of hmAb 2F5 with recombinant epitope

The binding capacity of HA-PVII was further analysed by a competition assay (Fig. 6). To achieve 50% reduction in specific binding, a 2-fold molar excess of the competing ELDKWAS-fusion protein was required for HA-B5, whereas the same level of reduction was obtained not before a 40-fold molar excess of the competitor was reached in the case of HA-PVII. These results indicate a clear difference in resistence to competition, suggesting a better presentation of the epitope within the selected construct. Also the titration curve of hmAb 2F5, shown for both clones in Figure 5, confirms that enhanced binding characteristics of HA-PVII seem to be caused by the proline residues flanking the epitope. This effect could be attributed to higher concentration/stability of molecules available for 2F5-binding at the cell surface. Thus, a clone with increased binding properties was identified in only one step of sorting, making this procedure highly useful for identifying eukaryotic surface-exposed molecules with desired functions by simple means of selection.

Hemadsorption assay

HA-PVII infected insect cells were examined to test whether the functionality of the hemagglutinin had been maintained, since antigenic site B is located close to the receptor binding site of this protein. Generation of intact chimeric influenza virus, expressing this modified hemagglutinin, is dependent on preserving the authentic structure. Sf9 cells were infected with recombinant HA-PVII clone, and 2 days post-infection the cells were treated with human erythrocytes. Adsorption of erythrocytes to the cell surface confirmed that the biological function of the altered hemagglutinin of HA-PVII was not impaired by inserting the epitope structure into this antigenic site. The next steps will be to generate chimeric influenza viruses, containing the HA-PVII hemagglutinin to study the potential ability of this construct to induce HIV-1 neutralizing antibodies in an animal model.

CONCLUSION

We could demonstrate that the baculovirus insect cell system is highly useful for constructing and screening of surface display libraries, which are of complex, eukaryotic origin. Usually such expression libraries are limited by transfection efficiency and frequency of recombinants. We found the method of directly ligating the genes of interest into the baculoviral genome to provide sufficient transfection as well as recombination rates (minimum of 2 × 10⁵/µg DNA). Analysing the binding properties of foreign proteins on the surface of infected cells rather than monitouring virus particles directly, seemed to be advantageous in this study. By choosing a very small gate of selection (<0.5%), infected cells could directly be sorted by FACS, yielding a high factor of enrichment. By one step of selection we were able to identify a single viral clone out of 8000 variants, that presented a specific epitope of increased binding capacity to hmAb 2F5, compared to the HA-B5 construct or randomly chosen clones from the initial library. Results from competition assays suggest an improved presentation of the epitope within the selected construct. Also the titration curve of hmAb 2F5 confirmed enhanced binding characteristics to the epitope present in selected clone HA-PVII.

The baculovirus expression system offers eukaryotic protein processing and fullfills the requirements for constructing representative libraries (cloning and transfection efficiency). Surface targeting can be achieved by foreign, membrane associated proteins, such as the influenza virus hemagglutinin, or by expressing fusion proteins with the baculovirus major coat protein gp64 (4-6). Given the power of surface display libraries as a major tool in molecular biology, we consider the expression of complex proteins on the surface of baculovirus particles to be the most useful strategy for eukaryotic ligand technology. So far we demonstrated the successful screening of a eukaryotic epitope library, expressed on the surface of infected insect cells, however, our future work will include the optimisation of selection at the level of the actual recombinant virus particle.

ACKNOWLEDGEMENT

This project was funded by the Fonds zur Förderung der Wissenschaftlichen Forschung, project number P 11.523 MOB.

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*To whom correspondence should be addressed. Tel: +43 1 360066242; Fax: +43 1 3697615; Email: r.grabberr@iam.boku.ac.at

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