Neutralisation with Antisera

1. Evolution of HIV-1 and Concomitant Antibody Responses in HIV-1 Seroconverters Rachel P.J. Lai, David Bonsall, Anna Helander, Jonathan N. Weber, Myra O. McClure and SPARTAC Trial Steering Committee R2 = 0.992 Section of Infectious Diseases, Wright Fleming Institute, Imperial College London, St Mary’s Hospital, London W2 1PG, United Kingdom Neutralisation of B-clade isolates with NAbs Evolutionary History of the Primary Isolates Abstract Background: The Short Pulse Antiretroviral Therapy at Seroconversion (SPARTAC) clinical trial recruited 371 patients with acute HIV-1 infection from centres world-wide. The patients were randomised to one of three arms: a short course of antiretroviral therapy (ART) for 12 weeks, or a long course of ART for 48 weeks, or no therapy. This investigation aims to examine the development neutralising antibody (NAb) responses in association with viral diversity and evolution. Methods: A panel of 18 patients (6 with clade-B and 12 with clade-C virus) were selected blind for detailed study. Full-length HIV-1 envelope (env) cassettes , constructed from all patients from samples taken within six months of seroconversion and one year later, were co-transfected with a laboratory-strain (HXB2) backbone into 293T cells to produce pseudovirions. Neutralisation of pseudovirions by Nabs: b12, 2F5, 2G12 or heterologous and autologous sera was assayed. Clonal sequencing of the env V1-V3 regions was carried-out and viral diversity and evolution analysed in relation to baseline viral load were analysed. Result: The clade-B viruses taken at week 52 were equally or more readily neutralised by b12 and 2F5, but were more resistant to 2G12 neutralisation compared to their baseline counterparts. While the baseline antisera were poorly neutralising, antisera taken at week 52 neutralised baseline isolates (1/125) and, to a lesser extent (1/50), isolates from week 52. Figures in parenthesis represent reciprocal dilutions of sera effecting 90% neutralisation. Viral load did not correlate with neutralisation by NAbs. However, antisera from patients with low viral load neutralised early viruses. A direct correlation between viral load and viral variation was found. Conclusion: This study confirms that virus isolated at the time of acute infection is resistant to neutralisation and appeared to have developed early escape mechanisms. Moreover, high viral load at seroconversion may enhance viral diversification and contribute to the weak neutralising responses. Left: Phylogenetic tree of env isolated from B-clade primary isolates. Except for patient H, the env gene for all other patients have evolved little over 52 weeks. Of note, patient F has env sequence highly similar to laboratory-adapted strains HXB2 and NL.671. Right: A preliminary phylogenetic tree of env isolated from C-clade primary isolates. Pseudoviruses containing primary env,isolated from B-clade patients at baseline and at week 52, are subjected to neutralisation by 50μg/ml of 2F5 (top left), b12 (top right) and 2G12 (bottom). Three env clones were assayed per patient and the average % residual infectivity is shown. Left: For B-clade isolates, intra-patient env variation at baseline directly correlates with its corresponding viral load. Right: Except for patient H (green dot), there is direct correlation between baseline viral load and evolutionary distance of env over 52 weeks. Conclusion Patients Neutralisation with Antisera • The B-clade primary isolates from week 52 are equally or more neutralisable by 2F5 and b12, but more resistant to 2G12, than their baseline counterparts, suggesting that NAbs targeting epitopes on the glycans may be more effective in neutralising viruses in vivo than those targeting gp120 or gp41. • We have identified a primary isolate (from patient F) with env sequence highly similar to a laboratory-adapted strain, which is neutralised well by heterologous antisera. Potential use as an immunogen? • The antisera taken from C-clade patients can only neutralise viruses isolated from the same geographic location. • There is a direct correlation between baseline viral load and env variation. Is high initial viral load a driving force behind viral diversity? Patient Country Subtype Co-receptor Usage Baseline Viral Load (copies/ml) Baseline 1° Isolate (3 clones) Control (negative pooled sera) Baseline Sera Week 52 Sera Week 52 virus (3 clones) Control (negative pooled sera) Baseline Sera Week 52 Sera Wk 52 Serum Du 156.12 Du 172.17 Du 422.1 ZM197M.PB7 ZM214M.PL15 ZM233M.PB6 ZM249M.PL1 ZM53M.PB12 ZM109F.PB4 CAP45.2.00.G3 CAP210.2.00.E8 C F H J M X C F H J M X C F H J M X C F H J M X C6 20 50 20 20 20 50 20 <50 50 50 50 50 50 C11 20 20 20 20 20 20 20 50 20 50 20 <20 50 C United Kingdom B R5 74,321 SA SA SA Zambia Zambia Zambia Zambia Zambia Zambia SA SA C20 20 50 20 20 20 <20 20 125 <50 50 50 50 50 C16 20 20 20 20 20 20 20 50 <20 20 <20 50 50 F R5 421,500 C1 312.5 781.25 125 125 20 50 20 20 20 312.5 312.5 C28 20 <20 20 20 20 <20 20 125 50 50 50 50 50 C21 20 20 20 20 20 20 20 20 20 <20 50 20 20 H R5 54,780 F4 20 20 20 20 20 20 20 50 125 <20 50 <20 <50 C2 781.25 781.25 125 781.25 20 312.5 20 20 125 50 50 F17 20 20 20 20 <20 <20 20 125 50 50 125 50 125 J R5 315,764 F6 20 20 20 20 20 20 20 50 <50 50 50 50 <50 F23 20 20 20 20 20 20 20 50 50 50 125 20 50 C3 781.25 312.5 20 312.5 20 50 20 20 20 125 125 M X4 44,100 F8 20 50 20 20 20 <20 20 50 50 <20 50 50 <50 F35 20 20 20 20 20 20 20 20 50 20 125 50 50 C4 781.25 312.5 50 781.25 20 312.5 50 20 50 50 50 H7 20 50 20 20 20 <20 <20 50 20 <50 50 50 125 X R5 267,362 H11 20 20 20 20 20 20 20 20 20 50 <20 20 <20 H9 20 50 20 20 20 50 20 <20 <20 <50 <20 50 50 H24 20 20 20 20 20 20 20 20 20 50 50 20 <20 C5 312.5 781.25 50 781.25 50 50 20 20 50 125 50 1 South Africa C R5 98,100 H21 20 <20 20 50 20 50 20 50 <20 <50 50 <20 50 H25 20 20 20 20 20 20 20 20 20 50 50 50 50 2 R5 315,000 C6 781.25 125 50 781.25 50 50 50 50 50 50 312.5 J33 20 50 20 20 20 50 20 50 <20 50 125 <20 50 J2 20 20 20 20 20 20 20 20 20 20 50 50 50 3 R5 27,100 C7 781.25 125 20 125 <20 20 20 50 20 50 50 J42 20 20 20 20 20 50 20 50 50 50 125 50 50 J16 20 20 20 20 20 20 20 20 20 20 <20 20 <20 4 R5 122,000 J50 20 50 20 20 20 50 <20 <20 50 50 <125 50 <20 J40 20 20 20 20 20 20 20 20 20 20 50 20 50 C8 312.5 312.5 20 312.5 <20 125 20 20 20 50 50 5 R5 294,000 M2 20 50 20 20 20 50 20 50 <50 50 50 125 50 M5 20 <20 20 20 20 <20 20 50 20 50 <20 50 50 C9 312.5 125 20 125 20 125 20 20 50 50 50 M14 20 50 20 20 20 50 20 50 50 50 50 125 50 6 R5 494,000 M9 20 20 20 20 20 <20 20 50 20 <20 20 50 50 C10 125 50 20 312.5 <20 20 20 20 20 <20 20 Reference M17 20 50 20 20 20 50 20 50 50 <50 50 125 50 M43 20 20 20 20 20 20 20 50 20 <20 50 50 50 Kaufmann GR, Zaunders JJ, Cunningham P, Kelleher AD, Grey P, Smith D, Carr A, Cooper DA. 2000. Rapid restoration of CD4 T cell subsets in subjects receiving antiretroviral therapy during primary HIV-1 infection. AIDS 14:2643-51. Fidler S, Oxenius A, Brady M, Clarke J, Cropley I, Babiker A, Zhang HT, Price D, Phillips R, Weber J. 2002. Virological and immunological effects of short-course antiretroviral therapy in primary HIV infection. AIDS 16:2049-54. 7 R5 222,000 X10 20 50 20 20 20 50 20 50 20 50 50 50 50 X4 20 20 20 20 20 20 20 50 20 50 50 50 <50 C11 312.5 312.5 20 312.5 <20 20 20 20 20 125 20 8 R5 79,200 X27 20 <20 20 20 20 50 20 50 50 50 50 50 50 X10 20 20 20 20 20 20 20 50 50 <50 50 50 50 9 R5 104,000 C12 312.5 50 20 312.5 20 50 20 20 20 20 50 X29 20 <20 20 20 20 <20 <20 50 50 50 50 50 50 X18 20 20 20 20 20 20 <20 50 50 50 50 50 50 10 R5 168,000 The neutralisation sensitivity of B-clade primary isolates (3 clones each), isolated at baseline (table on the left) and at week 52 (table on the right), by autologous and heterologous antisera is shown. Primary isolates isolated at baseline are readily neutralisable by their week 52 autologous sera (reciprocal dilution of 50 or greater to achieve IC90). However, primary isolates isolated at week 52 are resistant to neutralisation by their week 52 antisera, suggesting that the viruses developed escape mechanism rapidly. The neutralisation sensitivity of a standard panel of C-clade isolates against the C-clade antisera taken from SPARTAC patients at week 52 is shown. The Zambian isolates (5 out of 6) are neutralised poorly by the antisera (minimum reciprocal dilution of 50 to achieve IC50), while the South African isolates are more sensitive to neutralisation by antisera which are taken from South African patients. 11 R5 275,000 12 R5 150,000 Acknowledgement This study selected 18 patients from the SPARTAC cohort. Six are UK patients with B-clade virus and 12 of the patients are from S.A. with C-clade virus. Patient M from the U.K. has X4-tropic virus and all others are R5-tropic. We would like to thank the Wellcome Trust for funding SPARTAC and its sub-studies. All the NAbs are obtained from the Centre for AIDS Reagents of NIBSC. The reference panel of C-clade virus is obtained from the AIDS Research and Reference Program of the NIH.