Salmonella enterica sv. Typhimurium χ³⁹⁸⁷ (Galan et al., 1990) obtained from Roy Curtiss III was grown in Luria-Bertani broth (LB) in the presence of diaminopimelic acid (DAP) (100 μg/ml final concentration), Escherichia coli Top10 was grown in LB broth, and H. influenzae was grown in brain heart infusion broth (BHI) (Difco) supplemented with hemin (10 μg/ml) and nicotinamide adenine dinucleotide (NAD) (2 μg/ml). All bacteria were incubated at 37°C with aeration. Isolation of S. enterica ser. Typhimurium, E. coli, and H. influenzae strains carrying pBSKS::Φ6fm (Hin) was described previously (Piekarowicz et al., 2016). S. enterica ser. Typhimurium 3872 (pMPMT6::Φ6fmST) carrying plasmid pE1-HCV [E1 protein (NC004102) virus MycD Tagged ORF clone Origene, United States] was obtained by introduction of the plasmid DNA by electroporation into E. coli Top10, purifying the plasmid, and then using the isolated plasmid to transform S. enterica ser. Typhimurium #3872 (pMPMT6::Φ6fmST). For convenience, phagemids propagated in E. coli are designated as pBSKS::Φ6fm(Ec), in S. enterica ser. Typhimurium as pBSKS::Φ6fm(ST), and in H. influenzae as pBSKS::Φ6fm(Hin).

DNA and protein size markers were purchased from Fisher Scientific (Vilno). All chemicals used were of reagent grade or better and were obtained from Sigma-Aldrich (St. Louis, MO, United States), unless otherwise noted. Hepatitis C virus E1 recombinant protein was obtained from ProSci (United States).

To isolate phage and phagemid particles, overnight cultures were diluted 50-fold into 1,000 ml of an appropriate medium and grown overnight with shaking at 37°C. Bacteria were collected by centrifugation (20 min at ∼6,000 RCF). The supernatant was mixed with 1/5 volume of a solution containing 20% polyethylene glycol (PEG-8000) and 2.5 M NaCl and kept at 4°C overnight to precipitate the phage particles. The precipitate was collected by centrifugation (20 min ∼7,500 RCF), dissolved in 4 ml of phosphate-buffered saline (PBS), and centrifuged at 4,000 rpm in an SS34 rotor for 10 min. The phage particles were then purified by sequential centrifugation of the PBS phage suspension at ∼2,000 RCF for 10 min and ∼43,000 RCF for 120 min at 4°C. This procedure was repeated two times. The phagemid particles were further purified and quantified as described previously (Piekarowicz et al., 2020). Phagemid pBSKS::Φ6fm(ST) was additionally purified by CsCl gradient centrifugation as described by Sambrook and Russell (2001).

Samples of purified NgoΦ6 phagemid particles containing ∼200 μg of protein were sent to EUROGENTEC S.A., Liege, Belgium, for immunization of three rabbits according to their standard protocol (ref. no AS-PNOR-3MORAB). In this protocol, phage suspensions were introduced two times subcutaneously at days 0 and 21 in each animal without any adjuvant. Sera were collected at day 0 (before immunization) and at days 21 and 28. To determine the activity, these sera were pooled. All animal work performed at the EUROGENTEC S.A., Liege, Belgium, was carried out in accordance with the 2010/63/EU directive on the protection of animals used for scientific purposes. The protocols were approved under reference CE/SANTE/E/001 by the CER ethical licensing committee.

Determination of antibodies against phage particles was carried out as described previously (Piekarowicz et al., 2016) with modifications. Bacteria [3 μl of a suspension made of (2 × 10⁸ cells/ml)] diluted in PBS or 10 ng of EP1 protein (NC-004102) in carbonate buffer (50 mM sodium bicarbonate, 0.03 M sodium azide, pH 9.6) were spotted in triplicate onto a nitrocellulose membrane and dried at room temperature. After three washes with 20 ml of PBS buffer, the membranes were blocked with 1% alkaline casein (Sigma) in PBS at 25°C for 1 h. The membranes were then washed three times for 20 min with PBS. Following the washes, different dilutions of sera collected after 0 and 28 days in PBS with 1% alkaline casein were incubated at 25°C overnight. The membranes were washed three times with PBS and incubated with secondary antibodies at room temperature for 1 h [alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich, United States) diluted in PBS at 1:2,000]. The secondary antibody was removed, and the membranes were washed four times for 15 min each with PBS + 0.1% Tween-20 and once with PBS. Membranes were soaked in 20 ml of detection buffer (AP: 0.1 M Tris-HCl, pH 9.5; 0.1 M NaCl; 5 mM MgCl₂, pH 9.5) containing 20 μl of NBT BCIP (Sigma, United States) for 30 min at room temperature in darkness. The reaction was stopped by intensive washing of the membrane with distilled water and dried. The amount of protein contained in each spot was visualized on the membrane and quantified using the Quantity One BioRad program. The intensity of each spot was expressed as the increase of the intensity compared with the negative control, where spotting of phagemid or EP1 protein was omitted.

The amount of antibody induced was determined by comparing the optical density of specific anti-S. enterica ser. Typhimurium antibodies bound to the spots as presented in a standard curve obtained with known quantities of purified mouse IgG reference antibodies. Standard curves were prepared by the quantitative spot ELISA method. The amount of protein was quantified using GeneTools GBox (Syngen) program and expressed as the intensity of spot versus concentration of IgG protein. Since the measurement range of the ELISA dot was between dilution 2,000 and 5,000, the final determination of IgG concentration in all sera tested was based on the spot intensity values in this range.

Flow cytometry was performed according to Price et al. (2007) and described previously in detail (Piekarowicz et al., 2016).

For Western blot analysis, proteins were transferred to a positively charged nylon membrane (Roche), blocked with 4% (w/v) non-fat milk in TBS at 25°C for 16 h, and then incubated with various antibodies suspended in TBS at 16°C overnight. After three washes with 20 ml of TBS buffer, the membranes were incubated with secondary antibodies at room temperature for 1 h. Secondary antibody was removed, and the membranes were washed four times for 5 min with TBS + 0.1% Tween-20 and once with TBS. Membranes were then soaked in 20 ml of detection buffer (AP: 0.1 M Tris-HCl, pH 9.5; 0.1 M NaCl; 5 mM MgCl₂, pH 9.5) containing 20 μl of NBT BCIP for 30 min at room temperature in darkness. The reaction was stopped by washing the membrane with distilled water and dried. The following primary antibodies were used during studies: (1) (DYKDDDK) monoclonal antibodies (FG4R; Thermo Scientific), (2) monoclonal anti-E1 HCV protein (Clone BD198, OriGene), (3) antibodies obtained after subcutaneous vaccination of rabbit with phagemid particles pBSKS::Φ6fm(ST), (4) rabbit serum obtained after subcutaneous vaccination with phagemid particles pBSKS::Φ6fm(Hin), and (5) rabbit serum obtained after subcutaneous vaccination with phagemid particles pMPMT6::Φ6fm(ST) propagated in S. enterica ser. Typhimurium carrying plasmid encoding E1 HCV protein (all sera were used with 1:500 dilution). The following secondary antibodies were used: secondary mouse monoclonal 2A9 anti-rabbit IgG heavy β chain (alkaline phosphatase; Abcam; 1:2,000 dilution); and secondary goat anti-mouse IgG H&L (alkaline phosphatase; Abcam; 1:4,000 dilution).

Electrocompetent cells and transformation of Salmonella were carried out according to a procedure described previously (Sambrook and Russell, 2001) and stored at −80°C. Transformed bacteria were selected by plating onto LB agar plates containing appropriate antibiotics.

All statistical analysis was carried out using Student’s t-test using Social Science Statistic web service.¹ One-tailed P-values of < 0.05 were considered statistically significant.

Our discovery that proteins other than phage filament proteins copurified with bacteriophage particles suggested that these particles could serve as a source of host-cell antigens (Piekarowicz et al., 2020). To test this possibility, we used phagemids propagated in (a) S. enterica ser. Typhimurium carrying pBSKS::Φ6fm plasmid, (b) H. influenzae Rd carrying pBSKS::Φ6fm phagemid integrated into bacterial chromosome, and (C) strain of S. enterica ser. Typhimurium carrying pMPMT6::Φ6 phagemid and plasmid pE1 HCV encoding Hepatitis C virus envelope glycoprotein E1. Approximately 200 μg of protein in the form of phage preparation was used to subcutaneously immunize rabbits without any adjuvant. Quantitative spot ELISA results used for the determination of levels of IgG antibodies specific for S. enterica ser. Typhimurium cells elicited by immunization of rabbits with purified pBSKS::Φ6fm(ST) showed significant levels of antibody induction (Figure 1). The above results showed that phagemid pBSKS::Φ6fm(ST) particles induce after subcutaneous delivery a strong immunological response and elicit a large amount of anti-Salmonella antibodies.

The binding activity was further analyzed by flow cytometry. The data in Figure 2B show a significant shift in the binding profile. Using the gating shown in the figure, 65% of the cell population bound significant levels of IgG present in immunized sera (Figure 2B), demonstrating that the elicited antibody was able to bind to intact cells. Preimmunized sera provided minimal binding (Figure 2A).

The DNA sequence of pBSΦ6 is integrated into the chromosome of H. influenzae Rd30 cells (Piekarowicz et al., 2016). Lysogenic cells release phagemid particles that contain host cellular proteins (Piekarowicz et al., 2020). We used the same approach as described above to determine if these phagemid particles elicited antibodies against H. influenzae. Rabbit sera were obtained from animals immunized with purified pBSKS::Φ6fm(Hin) propagated in H. influenzae Rd30. Quantitative spot ELISA results again demonstrated significant levels of IgG antibodies specific for H. influenzae cells elicited by immunization of rabbits with purified pBKS::Φ6fm(Hin) (Figure 3). Flow cytometry analysis (Figure 4) shows a significant shift in the binding profile, again demonstrating that the elicited antibody bound intact cells.

We have previously shown that NgoPhi6 phagemid particles incorporate into/associate with the filament of not only phage structural proteins and host outer membrane proteins but also proteins produced by the expression of foreign genes (Piekarowicz et al., 2020). Purified pMPMT6::Φ6fm phagemid particles grown in S. enterica ser. Typhimurium cells expressing the HCV E1 gene incorporate protein E1 into phagemid particles shown by the reactivity of this protein with anti-Myc DDK antibodies. Testing the presence of E1 HCV protein in these phagemid particles showed that the E1 protein is present not only as native 25 kDa protein but also as smaller degradation products. This was also observed for the presence of Salmonella flagella protein incorporated into pBSKS::Φ6fm particles propagated in Salmonella cells (Piekarowicz et al., 2020). Purified phagemid pMPMT6::Φ6fm particles grown in S. enterica ser. Typhimurium cells expressing the HCV E1 protein were used for the immunization of rabbits, and the elicited antibodies were tested for the reactivity with protein E1 HCV. The spot ELISA results used for the determination of levels of IgG-specific HCV E1 protein showed a very high level of antibodies (Figure 5).

The amount of antibody induced by various vaccine constructs was determined by comparing the optical density of specific anti-target protein antibodies bound to the spots of a standard curve obtained with known quantities of purified mouse IgG reference antibodies. Standard curves were prepared by the quantitative spot ELISA method. Since the measurement range of the ELISA dot was between dilution 2,000 and 5,000, the final determination of IgG concentration in all sera tested was based on the spot intensity values in this range. The amount of specific anti-S. enterica ser. Typhimurium, anti-H. influenzae, and anti-E1 HCV protein was 170 μg/ml for anti-Salmonella, 80 μg/ml for anti-H. influenzae, and 65 μg/ml for anti-E1 HCV protein.

The data demonstrate that phagemid particles propagated in either S. enterica sr. Typhimurium, H. influenzae, or S. enterica sr. Typhimurium carrying pE1 plasmid encoding for HSV E1 protein in fact elicit a high level of IgG antibodies. To demonstrate that these antibodies bind to specific cellular proteins present in bacterial cells, several Western blots experiments were performed. We previously showed that flagellin proteins of 50 and 52 kDa molecular size or their degradation or precursor products are the predominant host proteins present in pBSKS::Φ6fm(ST) particles (Piekarowicz et al., 2020). Western blotting was carried out to detect whether they represent also a majority of elicited antibodies in rabbits immunized subcutaneously with pBSKS::Φ6(ST) phagemid particles. The data presented in Figure 6 show reactivity with several proteins, among them proteins in the 50 kDa range (Figure 6A, lane 1), when probed against S. enterica ser. Typhimurium cells without phagemids. The outer membrane protein P2 is a predominant host protein associated with pBS::Φ6fm(Hin) phagemid particles (Piekarowicz et al., 2020). Western blotting demonstrated that the phagemid isolated from pBS::Φ6fm(Hin) generated antibodies against both phagemid proteins and several host proteins, one with a mobility similar in mass to the P2 protein (molecular mass of ∼37 kDal) (Figure 6B). Finally, Western blotting demonstrated that phagemids isolated from a strain that also expressed the HCV E1 protein-induced antibody were able to recognize this protein (Figure 6C). From these data, we concluded that phages derived from NgoΦ6 induce immune responses that reflect the host from which they were isolated.