Potent BCN antibodies targeting a quaternary neutralizing epitope involving the V2 region (31), equivalent to the PG9 and PG16 MAbs (15). Fine mapping showed that the CAP256 BCN antibody was dependent on residues F159, N160, L165, R166, D167, K169, and K171 (forming the FN/LRDK-K motif). Interestingly, the fine specificity of the evolving antibody response changed more than time, like dependence around the glycan at position 160 which is critical for recognition by PG9 and PG16 (31). Here, we’ve examined viral populations in CAP256 and characterized unique pathways of neutralization escape from autologous antibodies. We show that the early strain-specific response, like the BCN response, targets the V1V2 region of each the key infecting (PI) virus as well as the superinfecting (SU) virus. Nonetheless, the response was largely directed in the superinfecting virus with very higher titers, reminiscent of CAP256 heterologous neutralization. We further showed that inside the context of substantial recombination, neutralization escape occurred through amino acid substitutions at residues 166 and 169 in the FN/LRDK-K motif. These data present insights into how HIV evades neutralizing antibodies that target the V2 area.Materials AND METHODSCAPRISA participant CAP256. CAP256 was enrolled in to the CAPRISA Acute Infection study (32) that was established in 2004 in KwaZulu-Natal, South Africa. This study was reviewed and authorized by the investigation ethics committees of the University of KwaZulu-Natal (E013/04), the Universityof Cape Town (025/2004), and also the University in the Witwatersrand (MM040202). CAP256 offered written informed consent for study participation. The kinetics and specificity of BCN antibodies in CAP256 has been described in detail elsewhere (11, 31). SGA and sequencing of CAP256 envelope genes. HIV-1 RNA was purified from plasma utilizing the Qiagen Viral RNA kit and reverse transcribed to cDNA utilizing Superscript III Reverse Transcriptase (Invitrogen, CA).CNTF Protein, Human The env genes have been amplified from single-genome templates as described previously (33).Abacavir sulfate Amplicons have been straight sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Prepared Reaction kit (Applied Biosystems, Foster City, CA) and resolved on an ABI 3100 automated genetic analyzer.PMID:24059181 The full-length env sequences had been assembled and edited applying Sequencher v.four.0 software (Genecodes, Ann Arbor, MI). For strain-specific single-genome amplification (SGA) of your CAP256 superinfecting virus, reverse transcription was performed making use of a primer distinct for the superinfecting variant (256spR; 5=-CTCCCTCTGCTGTTGG CTGCGCTCGCGC-3=; HXB2 positions 8856 to 8884; Nef). SGA was performed as described above, making use of the strain-specific primer because the antisense primer in both rounds of amplification. Multiple-sequence alignments had been performed applying Clustal X (version 1.83) and edited with BioEdit (version five.0.9). Sequence alignments were visualized making use of Highlighter for Amino Acid Sequences v1.1.0 (beta) (http://www.hiv.lanl.gov/content/sequence /HIGHLIGHT/HIGHLIGHT_POSTSCRIPT/highlighter.html). Recombination was assessed employing the RIP tool (http://www.hiv.lanl.gov/content /sequence/RIP/RIP.html). Pairwise DNA distances and neighbor-joining trees had been computed employing MEGA 4 (34). Three genotypic coreceptor prediction algorithms, namely, C-PSSM (35), geno2pheno (36), as well as the 11/25 rule (37), have been applied to predict the coreceptor use of each and every envelope sequence. Cloning CAP256 envelopes and production of pseudoviruses. Se.
