Conclusions Results show that the mutation at the N-linked glycosylation site N276D has a distinct influence on sensitivity to the HJ16 CD4bs neutralizing mAb. This mutation is quite unique and occurs very rarely in naturally infected patients rendering this epitope very suitable for further vaccine development. The impact of the N276D mutation on viral fitness still needs to be determined. "This research was conducted as part of the Collaboration for AIDS Vaccine Discovery with (partial) support from the Bill & Melinda Gates Foundation" HJ16 induced resistant virus displays rare natural N276D mutation highlighting the conserved nature of this new CD4bs epitope Institute of Tropical Medicine, Antwerp Nationalestraat 155, B-2000 Antwerp, Belgium Tel Fax Introduction Immunogen development for HIV-1 vaccines can be based on epitope identification of naturally occurring neutralizing antibodies (NAbs) in HIV-1 infected patients. A neutralizing monoclonal antibody (NAb) has been obtained at IRB as reported by Corti et al 1 from a clade C infected patient recruited at ITM which recognizes an epitope in the CD4bs and neutralized mostly strains that are not neutralized by b12. The b12 and HJ16 NAbs recognize related but non-overlapping CD4bs proximal epitopes and display a complementary non-overlapping pattern of reactivity with HJ16 being able to neutralize about one third of HIV-1 isolates tested across clades and which are known as tier 2 strains. Recent studies by Pietzsch et al 2 highlight the special features of HJ16 as a non-core antibody unlike the traditional ‘core’ antibodies such as b12. This new CD4bs epitope is not fully characterized yet but could be important for immunogen design. We therefore induced a neutralization sensitive virus into a resistant variant and after this resistance induction we compared the strains to map important regions for the HJ16 neutralizing activity. Materials and methods Results Sequence data Virus: VI1090 is a CRF02_AG CCR5 strain, isolated from a 38-year old male infected through heterosexual contact in Nigeria. Corresponding Env pseudovirus construct has been obtained by DNA amplification of the complete Env gene and subsequent cloning into the pcDNA4/TO expression vector (Heyndrickx et al 3 ). Antibody: HJ16 was isolated from the memory B cells of a 45-year old male visiting the Antwerp HIV clinic from Congo 12 years after diagnosis. TriMab is an equal mixture of the three Mabs IgG1b12, 2G12 and 2F5. HJ16 resistance induction: HJ16 resistance was induced by culturing the wildtype replication competent CRF02_AG strain VI1090 (VI1090_WT, IC 50 <0.15 µg/ml) in the presence of increasing amounts of HJ16 on freshly isolated donor PBMC until a resistant strain was obtained that was able to replicate in 200 µg/ml HJ16 (VI1090_resist). Neutralizing activity was measured using both TZMbl and PBMC neutralization assays. VI1090_sens was VI1090_WT cultured in parallel without HJ16 to exclude natural mutations. HIV-1 neutralization assays ¤ TZMbl based: - incubation NAb or plasma + virus 1 hr - absorption on TZMbl cells 2 hr - culture cells 2 days, luciferase readout ¤ PBMC based: - incubation NAb or plasma + virus 24 hr - absorption to PHA/IL-2 stimulated PBMC 1 hr, wash - culture cells 7 or 14 days, p24 ELISA readout Neutralization profile of original HJ16 patient plasma in PBMC and TZMbl neutralization assays Neutralization profile of HJ16 NAb in TZMbl and PBMC based neutralization assays Neutralization profile of HJ16 NAb against the different VI1090 strains in neutralization assays. - VI1090_WT: original stock is neutralized by HJ16 and TriMab - VI1090_sens: VI1090 cultured without HJ16 is still neutralized by HJ16 and TriMab - VI1090_resist: VI1090 cultured with HJ16 is resistent to neutralization by HJ16 and TriMab Sequencing of the HJ16 sensitive versus the resistant strain revealed a distinct point mutation where the neutral asparagine (N) was replaced by the negative charged aspartic acid (D). This N276D point mutation is only seen in 0.85% of the group M strains described in the Los Alamos database (total of 2447 viruses, 19 cases with N276D mutation). Sunita Balla-Jhagjhoorsingh 1, Betty Willems 1, Leo Heyndrickx 1, Katleen Vereecken 1, Katrijn Grupping 1, Davide Corti 2, David Davis 3, Guido Vanham 1 and The BMGF CAVD UCL Vaccine Discovery Consortium 1. Department of Virology, Institute of Tropical Medicine (ITM), Antwerp, Belgium; 2. Institute for Research in Biomedicine (IRB), Bellinzona, Switzerland; 3. Biomedical Primate Research Centre, Rijswijk, the Netherlands References 1.Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. Corti D et al, PLoS One Jan 20;5(1). 2.Human anti-HIV-neutralizing antibodies frequently target a conserved epitope essential for viral fitness. Pietzsch J et al, J Exp Med Aug 30;207(9). 3.Antiviral compounds show enhanced activity in HIV-1 single cycle pseudovirus assays as compared to classical PBMC assays. Heyndrickx L et al, J Virol Methods Mar;148(1-2). E: enhancement HJ16 NAb does not neutralize SF162 but displays potent neutralization of the CRF02_AG strain VI1090 (pseudoviruses as well as replicating viruses). AIDS Vaccine 2010, Atlanta, –