Unique molecular signatures in rebound viruses from antiretroviral drug and CRISPR-treated HIV-1-infected humanized mice

HIV-1 elimination from a subset of virus-infected humanized mice (hu-mice) is reported following sequential dual treatment with long-acting (LA) antiretroviral (ART) and CRISPR-Cas9 therapies. However, viral rebound is observed in > 50% of the dual-treated animals. The molecular signatures of the rebound virus, recovered from plasma, have now been determined. The LA-ART treatment contains nanoformulated dolutegravir, lamivudine, abacavir, and rilpivirine combinations, and the HIV-1 excision treatment is CRISPR-Cas9 targeting the HIV-1-LTR-gag. One-step reverse transcriptase polymerase chain reaction, which avoids spontaneous preparatory mutations, is performed on plasma-derived RNA. Sanger and Next-Generation Sequencing are employed targeting the HIV-1-gag, pol, and env genes. HIV-1 env shows the most divergence. LA-ART, with or without CRISPR, is responsible for the new mutations. The primary and accessory mutations are detected by deep sequencing. Viral evolution reflects changes in the virus as reported by ART-treated and HIV-1-infected patients. No major CRISPR-specific mutations are observed. The molecular viral signatures demonstrate an accelerated HIV-1 drug resistance escape from ART rather than from the generation of CRISPR mutants. These define viral rebound in the dual-treated hu-mice. The data underscores the limited role of CRISPR excision in generating these rebound HIV-1 mutants from dual-treated hu-mice.