Increased titers of neutralizing antibodies after immunization with both envelope proteins of the porcine endogenous retroviruses (PERVs)
© Denner et al.; licensee BioMed Central Ltd. 2012
Received: 28 May 2012
Accepted: 31 October 2012
Published: 5 November 2012
Despite enormous difficulties to induce antibodies neutralizing HIV-1, especially broadly neutralizing antibodies directed against the conserved membrane proximal external region (MPER) of the transmembrane envelope protein, such antibodies can be easily induced in the case of gammaretroviruses, among them the porcine endogenous retroviruses (PERVs). In addition to neutralizing antibodies directed against the transmembrane envelope protein p15E, neutralizing antibodies were also induced by immunization with the surface envelope protein gp70. PERVs represent a special risk for xenotransplantation using pig tissues or organs since they are integrated in the genome of all pigs and infect human cells and a vaccine may protect from transmission to the recipient. To investigate the effect of simultaneous immunization with both proteins in detail, a study was performed in hamsters. Gp70 and p15E of PERV were produced in E. coli, purified and used for immunization. All animals developed binding antibodies against the antigens used for immunization. Sera from animals immunized with p15E recognized epitopes in the MPER and the fusion peptide proximal region (FPPR) of p15E. One MPER epitope showed a sequence homology to an epitope in the MPER of gp41 of HIV-1 recognized by broadly neutralizing antibodies found in HIV infected individuals. Neutralizing antibodies were detected in all sera. Most importantly, sera from animals immunized with gp70 had a higher neutralizing activity when compared with the sera from animals immunized with p15E and sera from animals immunized with gp70 together with p15E had a higher neutralizing activity compared with sera from animals immunized with each antigen alone. These immunization studies are important for the development of vaccines against other retroviruses including the human immunodeficiency virus HIV-1.
A vaccine is the best protection from an infection with retroviruses including the human immunodeficiency virus HIV-1. Since retroviruses integrate a DNA copy of their genome into the genome of the target cell, were they may persist undetected from the immune system if not expressed, only vaccines based on neutralizing antibodies can prevent virus infection and integration. Neutralizing antibodies have been found in HIV-1 infected individuals, most of them are directed against the surface envelope protein gp120, and only some against the transmembrane envelope protein [1, 2]. Two monoclonal antibodies binding to a highly conserved domain in the membrane proximal external region (MPER) of gp41, designated 2F5 and 4E10, have been shown to neutralize up 95% of HIV, however, all attempts to induce such broadly neutralizing antibodies failed until now (for review see ). In contrast to HIV-1 it seems easy to induce MPER-specific neutralizing antibodies against gammaretroviruses such as the porcine endogenous retroviruses (PERVs), the feline leukemia virus (FeLV), and the Koala retrovirus, KoRV [4–9].
PERVs pose a special risk when xenotransplantation using pig cells, tissues or organs will be performed in order to overcome the shortage of human allotransplants . PERVs are present in the genome of all pigs, can be released by normal pig cells and infect human cells in vitro (for review see ). Although in all preclinical and clinical xenotransplantations as well as in infection experiments, no transmission of PERV was observed, additional safety strategies are under consideration among them vaccination of the human recipient . We recently reported neutralizing antibodies after immunization with the transmembrane envelope protein p15E of PERV in goats  and rats . The sera recognized epitopes in the fusion peptide proximal region (FPPR) and the MPER of p15E. When immunizing with the surface envelope protein gp70 much higher titers of neutralizing antibodies were induced compared with p15E . Since studies in rats were hampered by the high prevalence of preexisting antibodies against p15E, hamsters without such preexisting antibodies against p15E were immunized and the effect of simultaneous immunization with p15E and gp70 was analyzed. Immunizing hamsters with p15E resulted in neutralizing antibodies showing a similar epitope pattern as described when immunizing other species. Immunizing hamsters with gp70 also induced higher levels of neutralizing antibodies when compared with the immunization with p15E and immunizing with both envelope proteins resulted in higher titers of neutralizing antibodies compared with the immunization with one antigen alone.
Binding antibodies after immunization with p15E and gp70 of PERV
Analyzes of the epitopes recognized by antibodies specific for p15E of PERV
Neutralizing antibodies after immunization with p15E and gp70 of PERV
Immunizing for the first time hamsters with the transmembrane envelope protein p15E of PERV resulted in neutralizing antibodies recognizing epitopes in the MPER. These data show that immunization with p15E in all species tested so far resulted in neutralizing antibodies and in antibodies recognizing similar epitopes [4, 12]. Neutralizing antibodies and antibodies recognizing the MPER and FPPR had been also induced immunizing with p15E of the feline leukemia virus (FeLV) [5–8], and the koala retrovirus (KoRV) . In the case of the FeLV it was shown that these antibodies protected cats in vivo from antigenemia . Whereas in the case of these gammaretroviruses neutralizing antibodies against the transmembrane envelope protein could be easily induced, all attempts to obtain antibodies such as 2F5 and 4E10 broadly neutralizing HIV-1 failed [1–3, 16]. In addition, attempts to induce neutralizing antibodies against HIV-2 , the feline foamyvirus (FFV) , and the primate foamy virus (PFV) (our unpublished data) immunizing with the transmembrane envelope protein also failed. There are some major differences between the transmembrane envelope proteins p15E of the gammaretroviruses and those of the lenti- and foamyviruses. The p15Es are not glycosylated whereas the transmembrane envelope proteins gp41 of HIV-1, gp36 of HIV-2, and gp48 of the foamyviruses are all glycosylated. Whether glycosylation is important for the interaction of the MPER and the FPPR when the N-terminal helical region (NHR) and the C-terminal helical region CHR of the transmembrane envelope proteins of lenti- and foamyviruses interact during infection remains unclear. There is evidence that in the case of HIV-1 MPER and FPPR are in closed proximity at certain moments of the infection process [19–21] and that the presence of a peptide corresponding to the FPPR increases the binding of 2F5 to a peptide containing its epitopes .
The neutralization assay used is based on real-time PCR measuring viral DNA in the cells. This assay has several advantages: First, it uses the property of retroviruses to transcribe the viral RNA genome into proviral DNA by the viral reverse transcriptase and measures therefore activity of this enzyme. Second, it measures infection, proviral DNA exists only in the cell. Than higher the ct values then less provirus and then better the neutralizing serum worked. Therefore we suggest that this assay is robust. We used the same assay to measure infection by HIV-1 . This neutralization assay is very sensitive and can be used with low-titer viruses such as PERV. To establish an alternative method, e.g. using an ELISA for viral proteins the virus titer is not high enough to quantify virus infection in 96 well plates. Measuring in parallel GAPDH allows screening of the cell viability.
Hamsters have been chosen for several reason: First to analyze the immune response to p15E in a new species, second to use a larger animal than mice to derive more serum for analysis, and third, to avoid the presence of preexisting antibodies against p15E which were observed for a long time in the preimmune serum of rats used for immunization. Obviously these preexisting antibodies were directed against an endogenous rat gammaretrovirus which is closely related to PERV and we assume that the antibodies were cross-reacting. The endogenous retroviruses of the rat are not well studied , but a strong homology with murine and feline leukemia viruses and PERV may be expected. Expression of endogenous retroviruses has been described in numerous species under physiological (e.g., immune responses [23–26]) or pathological conditions (e.g., in tumors of animals  and man ). Since in hamsters no antibodies cross-reacting with PERV proteins were found, these immunization studies could be performed.
When immunizing with gp70 the neutralizing activity is much higher compared to an immunization with p15E alone and immunization with both envelope proteins induced higher titers of neutralizing antibodies (Figure 4). The same observation was made when immunizing rats with the transmembrane envelope protein of FeLV and gp70 of FeLV .
Since there are other strategies under development to prevent transmission of PERVs during xenotransplantation such as inhibition of PERV expression by RNA interference [29, 30], it is unlikely that a vaccine against PERV will be required. However, immunization with the transmembrane envelope proteins of gammaretroviruses may help to understand the mechanism of neutralization by MPER-specific antibodies, which is still unclear. The neutralizing antibodies may prevent interaction with the lipids in the membrane or – most likely - conformational changes. The data shows that the MPER is important for the infection of all retroviruses and antibodies against the MPER prevent a crucial step in the infection process. In addition, the data suggests that the use of both envelope proteins may be of advantage despite the fact that the surface envelope protein gp120 of HIV-1 is – in contrast to that of the gammaretroviruses – highly variable. Furthermore, the data shows that two or more immunizations may be required to obtain neutralizing antibodies.
The induction of PERV-specific neutralizing antibodies in different species including hamster suggests that such antibodies may also be induced in primates including man. Since MPER-specific antibodies were found to neutralize HIV-1 and other retroviruses, these studies may be useful to understand the mechanism how these antibodies neutralize and how to induce such MPER-specific broadly neutralizing antibodies. This data also indicate that the MPER is a highly vulnerable target for the neutralization of retroviruses in general.
Cloning and purification of antigens p15E and gp70
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 . 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 . 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, Western blot, and ELISA
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  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).
Primers and probes
This work was supported by the Deutsche Forschungsgemeinschaft (DE 729/4-3).
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