Increased titers of neutralizing antibodies after immunization with both envelope proteins of the porcine endogenous retroviruses (PERVs)

In order to induce neutralizing antibodies, the transmembrane envelope protein p15E and the surface envelope protein gp70 of PERV (Figure 1A) were expressed in E. coli, purified (Figure 1B) and hamsters were immunized (Figure 1B). Specific binding antibodies were found using Western blot analysis and ELISA in all immune sera (Figure 1C, Figure 2). Although the animals in the group immunized with p15E alone and the animals immunized with p15E and gp70 together received identical amounts of p15E, it remains unknown why the response against p15E is higher in the first group. Investigations of the kinetics showed that already after the first immunization relatively high titers of binding antibodies were observed, which gradually increased after each immunization (Figure 2B).

In order to map the epitopes in p15E recognized by the immune sera, two different methods were performed. First, overlapping peptides corresponding to the entire p15E were immobilized on a membrane (Figure 3A), and second, a new method was applied using the same overlapping peptides but immobilized on a glass chip (Figure 3B). Both methods identified the same epitopes. In the case of the serum from animal 1/3 immunized with p15E alone, the only epitopes were localized in the MPER of p15E. The localization of the epitopes is similar to the localization described for epitopes induced by immunization with p15E in goats and rats [13]. One of the epitopes in the MPER (GWFEGWFNR) is similar in localization and sequence with the epitope of the monoclonal antibody 4E10 (NWFNIT, identical amino acids in bold) (Figure 3). 4E10 had been isolated from a HIV-1 infected individual and neutralizes up to 95% of all HIV-1 [14, 15]. Despite this limited sequence homology the anti-p15E antibodies and 4E10 did not cross-react and not cross-neutralize (not shown). A screening for binding antibodies using the peptides E1 and E2 (Figure 3C) was not succesful, obviously the peptides were too short to bind the antibodies.

To analyze the neutralizing activity of the sera a neutralization assay based on the measurement of viral DNA after transcription of the viral genomic RNA by the reverse transcriptase using a real-time PCR was performed and neutralizing activity was found in all immune sera, but not in the preimmune sera and in the sera from control animals immunized with adjuvant alone. The titers of neutralizing activity were higher in sera from animals immunized with gp70 when compared with sera from animals immunized with p15E and the best neutralization was achieved when both envelope protein were used simultaneously (Figure 4A, Figure 4B). Taking both experiments together, the differences were significant (p=0.004 for the difference p15E and gp70 plus p15E; p=0.019 for the difference gp70 and gp70 plus p15E). Investigation of the kinetics showed that the neutralizing activity was still low after two immunizations but increased significantly after the fourth immunization, indicating that multiple immunizations are required to achieve a strong neutralization (Figure 4C). Purified IgG from the immune sera had the same neutralizing effect as the whole serum, indicating that antibodies are responsible for it.

The ectodomain of p15E of PERV-A (amino acids 488–596, accession number HQ688786) and a recombinant protein corresponding to gp70 of PERV-A (amino acids 49–487, accession number HQ688785) (Figure 1A) were cloned into the pET-22b(+) expression vector (Novagen, San Diego, CA), expressed in E. coli BL21-CodonPlus(DE3)-RP (Stratagene, Amsterdam), and purified by metal chelating affinity chromatography using Ni-NTA (Qiagen) as described [12]. The cloned sequence of gp70 resembles the sequence of gp70 of FeLV, used as the commercial “Leucogen” for vaccination of cats containing a small part of p15E [31]. The p15E (LITGPQQLEKGLSNLHRIVTEDLQALEKSVSNLEESLTSLSEVVLQNRRGLDLLFLKEGGLCVALKEECCFYVDHSGAIRDSMSKLRERLEKRHKEKEAGQGWFEGWFN) is a fusion protein with the calmodulin binding protein (CBP) (MKRRWKKNFIAVSAANRFKKISSSGALLVPR). It theoretical molecular mass is 16.3 kDa, however it is always detected at 12 kDa (Figure 1C). The molecular mass of the recombinant gp70 is 54 kDa (Figure 1D).

Hamsters (Charles River) were immunized with 300 μg of p15E, gp70 or both. In the last case, gp70 and p15E were immunized in different parts of the body. The proteins were emulsified in complete Freund’s adjuvant intramuscularly and subcutaneously (i.m. 50μl, s.c. 700μl). The control animals were immunized with adjuvant and PBS. The immune response was boosted by second and third immunizations using incomplete Freund’s adjuvant (Figure 1B). IgGs were concentrated using Vivapure Q Mini spin columns (Vivascience). Control animals were immunized with adjuvant only.

Peptides E1(484–505) GTAALITGPQQLEKGLSNLHRI and E2(583–604) EREADQGWFEGWFNRSPWMTTL (Figure 3C) were synthesized by Gene Cust, Dulange, Luxembourg. Western blot and ELISA were performed as described before [12] using the recombinant proteins gp70 and p15E. 0.2 μg/well recombinant proteins were used for ELISA; sera were diluted 1:200 to 1:25600. A secondary antibody labeled with HRP was used for ECL detection. Each serum was titrated and the mean of each group is shown in Figure 1A, B, C).

The entire p15E of PERV (130 amino acids) was synthesized as a cellulose-adsorbed peptide spot library of 15-mer peptides overlapping by 12 amino acids or a glass based chip with the same peptides (JPT Peptide Technologies, Germany) using standard protocols of the supplier. Sera were diluted 1:1000 and binding was detected using a chemiluminescence detection solution (ECL, Amersham Pharmacia Biotech) or bound antibodies were detected using a DyLight 649 conjugated AffiniPure goat anti-rat IgG antibody and read at a wavelength of 635 nm in a GenePix 4000 microarray scanner (Molecular Devices, USA). The data were analyzed using the GenePix Pro software. Epitopes were defined as central amino acids shared by all peptides recognized by the antiserum (see Figure 3A, B).

The neutralization assays were performed as described using virus-containing cell-free supernatants produced by human embryonic kidney 293 cells infected with PERV/5° [12]. This virus is a recombinant human-tropic PERV-A/C repeatedly passaged on human cells which was associated with increased titers and genetic alterations in its long terminal repeats (LTR) [32]. 100 μl uninfected 293 cell (1.5 × 10⁵/ml) were seeded in 96 well plates and incubated for 4 h at 37°C in 5% (v/v) CO₂. Sera were decomplemented by heat inactivation (30 min at 56°C) and 20 μl were mixed with 80 μl of a PERV dilution, incubated for 30 min at 37°C and added to the 293 cells. Virus dilutions resulting in stable Ct values (25 to 27) were considered as optimal for the neutralization assay. After incubation for 72 h at 37°C cells were examined by light microscopy for viability and the medium was removed. Cells were lysed by heating at 95°C for 30 min, freezing at −20°C for 6 h, and incubation with lysis buffer (nuclease free water containing 0.2 mg/ml proteinase K and 10% (v/v) 10×PCR-buffer) at 60°C for at least 3 h. Proteinase K was heat inactivated (30 min at 95°C). For quantification of PERV proviral DNA the primers gag-for and gag-rev located in the gag gene and a specific PERV-gag probe (Table 1) were used in a duplex real-time PCR. The reference gene GAPDH was amplified with the primers GAPDH-for and GAPDH-rev and quantified using a GAPDH-probe (Table 1). The 22 μl reaction mixture consisted of 1x PCR buffer with 1.5 mM MgCl₂, 0.5 μM each of dATP, dCTP, dGTP, dTTP, 5 pmol of each primer, 5 pmol of probe, 1.25 U AmpliTaq Gold^® polymerase and 3 μl lysate. The thermal cycling conditions used were 10 min at 95°C followed by 50 cycles of 1 min at 95°C, 1 min at 59°C and 30 s at 72°C in a Stratagene MX4000 machine. Neutralization was defined as reduction of provirus integration in the presence of immune serum and calculated as (ct value of PERV - ct value of GAPDH) in the presence of serum - (ct value of PERV - ct value of GAPDH) in the absence of serum. The ct values of GAPDH were identical in all samples, indicating absence of toxic effects of the sera. In addition the delta delta ct values were determined as described [33].