An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1.

Broadly HIV-1-neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1-infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1-infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient's plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells.

IGHV1-69 B cell chronic lymphocytic leukemia antibodies cross-react with HIV-1 and hepatitis C virus antigens as well as intestinal commensal bacteria.

B-cell chronic lymphocytic leukemia (B-CLL) patients expressing unmutated immunoglobulin heavy variable regions (IGHVs) use the IGHV1-69 B cell receptor (BCR) in 25% of cases. Since HIV-1 envelope gp41 antibodies also frequently use IGHV1-69 gene segments, we hypothesized that IGHV1-69 B-CLL precursors may contribute to the gp41 B cell response during HIV-1 infection. To test this hypothesis, we rescued 5 IGHV1-69 unmutated antibodies as heterohybridoma IgM paraproteins and as recombinant IgG1 antibodies from B-CLL patients, determined their antigenic specificities and analyzed BCR sequences. IGHV1-69 B-CLL antibodies were enriched for reactivity with HIV-1 envelope gp41, influenza, hepatitis C virus E2 protein and intestinal commensal bacteria. These IGHV1-69 B-CLL antibodies preferentially used IGHD3 and IGHJ6 gene segments and had long heavy chain complementary determining region 3s (HCDR3s) (≥21 aa). IGHV1-69 B-CLL BCRs exhibited a phenylalanine at position 54 (F54) of the HCDR2 as do rare HIV-1 gp41 and influenza hemagglutinin stem neutralizing antibodies, while IGHV1-69 gp41 antibodies induced by HIV-1 infection predominantly used leucine (L54) allelic variants. These results demonstrate that the B-CLL cell population is an expansion of members of the innate polyreactive B cell repertoire with reactivity to a number of infectious agent antigens including intestinal commensal bacteria. The B-CLL IGHV1-69 B cell usage of F54 allelic variants strongly suggests that IGHV1-69 B-CLL gp41 antibodies derive from a restricted B cell pool that also produces rare HIV-1 gp41 and influenza hemagglutinin stem antibodies.

siRNA-optimized Modifications for Enhanced In Vivo Activity.

Current modifications used in small interfering RNAs (siRNAs), such as 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), improve stability, specificity or immunogenic properties but do not improve potency. These modifications were previously designed for use in antisense and not siRNA. We show, for the first time, that the siRNA-optimized novel 2'-O modifications, 2'-O-benzyl, and 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), are tolerated at multiple positions on the guide strand of siRNA sequences in vivo. 2'-O-benzyl and 2'-O-CH2Py(4) modifications were tested at each position individually along the guide strand in five sequences to determine positions that tolerated the modifications. The positions were combined together and found to increase potency and duration of siRNAs in vivo compared to their unmodified counterparts when delivered using lipid nanoparticles. For 2'-O-benzyl, four incorporations were tolerated with similar activity to the unmodified siRNA in vivo, while for 2'-O-CH2Py(4) six incorporations were tolerated. Increased in vivo activity was observed when the modifications were combined at positions 8 and 15 on the guide strand. Understanding the optimal placement of siRNA-optimized modifications needed for maximal in vivo activity is necessary for development of RNA-based therapeutics.

Nucleoside optimization for RNAi: a high-throughput platform.

The RNA induced silencing complex (RISC) contains at its core the endonuclease Argonaute (Ago) that allows for guide strand (GS)-mediated sequence-specific cleavage of the target mRNA. Functionalization of the sugar/phosphodiester backbone of the GS, which is in direct contact with Ago, presents a logical opportunity to affect RISC's activity. A systematic evaluation of modified nucleosides requires the synthesis of phosphoramidites corresponding to all four canonical bases (A, U, C, and G) and their sequential evaluation at each position along the 21-nucleotide-long GS. With the use of a platform approach, the sequential replacement of canonical bases with inosine greatly simplifies the problem and defines a new activity baseline toward which the corresponding sugar-modified inosines are compared. This approach was validated using 2'-O-benzyl modification, which demonstrated that positions 5, 8, 15, and 19 can accommodate this large group. Application of this high-throughput methodology now allows for hypothesis-driven rational design of highly potent, immunologically silent and stable siRNAs suitable for therapeutic applications.

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and in vivo.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.