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, and the primate foamy virus (PFV) (our unpublished data) immunizing with the transmembrane envelope protein also failed.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.