0264410X(95)000585 Vaccine, Vol. 13, No. 13. pp. 1233-1239, 1995 Comriaht 0 1995 Elsevier Science Ltd Printei k&eat Britain. All rights reserved 0264-410)(/95 $lO+O.OO zyxwvuts A new epitope presenting system displays a HIV-l V3 loop sequence and induces neutralizing antibodies Eduardo A. Scodeller, Sergio G. Tisminetzky, Fabiola Porro, Monica Schiappacassi, Anita De Rossi*, Luigi Chiecco-Bianchi* and Francisco E. Barallet zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA The principal neutralizing domain, IGPGRAF sequence, from the V3-loop of HIV-I was inserted in two positions on the surface of the protein that makes up the capside shell of the insect Flock House Virus. The hybrid proteins were expressed in insect cells via recombinant baculoviruses. Three d@erent hybrids were used as immunogens: two with a single copy’ of the insert in dtflerent positions of the carrier protein and a third with two copies of the insert at the same positions as before. All hybrid proteins induced strong and broad spectfic immune response in guinea pigs against d@erent V3-loop sequences. However, only one of the hybridproteins was able to induce a strong neutralizing response against MN and IIIB HIV-l isolates. Our results demonstrate that a very short peptide sequence of HIV-l can constitute a valuable immunogen able to induce a neutralizing response tfpresented to the immune system in the context of the FHV capsomer structure. zyxwvutsrqponmlkjihgfedcba Keywords: Flock House Virus: HIV-l V3 loop epitope; neutralizing antibodies The principal neutralizing domain (PND) of HIV-l lies within the loop forming the third hypervariable region (V3 loop) of the glycoprotein gp120’. A relation- ship between anti-V3 titer and virus neutralization capacityZm7 has been well established for immunized animals and human vaccines. Although the V3 loop had been originally defined as hypervariable, further analysis of a large number of isolates has recently revealed that two PND consensus sequences (IGPGRA and GP- GRAF) are present in approximately 60% of the viruses isolated in North Americas. These sequences seem to be extremely important for the antigenic structure of the V3 loop. In fact, human monoclonal antibodies (HuMAb) displaying a broad neutralization activity against several divergent strains of HIV-I bind to this region’.“. Fur- thermore, it has been shown that a peptide consisting of a trimer of the sequence GPGRAF induced broadly reactive antibodies when injected to animals”. Despite its immunogenicity, this region has been reported not to induce high titres of antibodies in infected patients’“. The immunogenicity of a peptide is known to depend not only on its sequence but also on the way it is presented to the immune system. It has been shown that the properties of a given epitope genetically introduced International Centre for Genetic Engineering and Biotech- noloav. Padriciano 99. 34012 Trieste. Italv. *Institute of Oncology, University oi Padova, Via Gattamklata, 6435128 Padova, Italy. tcorresponding author. (Received 8 September 1994; revised 2 March 1995; accepted 22 March 1995) in a particulate carrier moiety, such as the chimeric virus-like particles (VLP) based on the hepatitis B core antigen, are influenced by its position within that car- rier”. We have attempted to enhance the immunogenic- ity of the GPGRAF sequence found at the most external part of the HIV-l V3 loop by displaying it in a new carrier system developed in our laboratory. A number of proteins have been recently genetically modified to be used as carriers of heterologous sequences”-“, but the number of positions for the insertion of short heterologous epitopes is limited. We have now devel- oped a system that uses for the first time a modified capside precursor protein of the Flock House Virus (FHV) (Nodaviridae family) as an immunogen. This system allows the presentation of foreign epitopes in multiple internal sites. Its genome consists of two single- stranded positive-sense RNA molecules, both of which are encapsidated in the same particle19.20. RNA1 (3.1 kb) encodes proteins required for viral RNA replication and RNA2 (1.4 kb) encodes protein alpha (43 kDa), the precursor of the coat protein”-“. The structure of the protein shell of mature FHV has been elucidated by X-ray crystallographyZ4. The predominant folding feature is an eight-stranded antiparallel P-barrel which is very similar to the wedge-shaped core subunit seen in other plant and animal virus structures (Figure IA). This is a definite advantage over most of the carrier systems used up to now, whose precise 3D structure is not known and consequently the configur- ation of the epitopes introduced cannot be accurately predicted. Vaccine 1995 Volume 13 Number 13 1233