Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function.

Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.

Treating Cancer by Targeting Telomeres and Telomerase.

Telomerase is expressed in more than 85% of cancer cells. Tumor cells with metastatic potential may have a high telomerase activity, allowing cells to escape from the inhibition of cell proliferation due to shortened telomeres. Human telomerase primarily consists of two main components: hTERT, a catalytic subunit, and hTR, an RNA template whose sequence is complimentary to the telomeric 5'-dTTAGGG-3' repeat. In humans, telomerase activity is typically restricted to renewing tissues, such as germ cells and stem cells, and is generally absent in normal cells. While hTR is constitutively expressed in most tissue types, hTERT expression levels are low enough that telomere length cannot be maintained, which sets a proliferative lifespan on normal cells. However, in the majority of cancers, telomerase maintains stable telomere length, thereby conferring cell immortality. Levels of hTERT mRNA are directly related to telomerase activity, thereby making it a more suitable therapeutic target than hTR. Recent data suggests that stabilization of telomeric G-quadruplexes may act to indirectly inhibit telomerase action by blocking hTR binding. Telomeric DNA has the propensity to spontaneously form intramolecular G-quadruplexes, four-stranded DNA secondary structures that are stabilized by the stacking of guanine residues in a planar arrangement. The functional roles of telomeric G-quadruplexes are not completely understood, but recent evidence suggests that they can stall the replication fork during DNA synthesis and inhibit telomere replication by preventing telomerase and related proteins from binding to the telomere. Long-term treatment with G-quadruplex stabilizers induces a gradual reduction in the length of the G-rich 3' end of the telomere without a reduction of the total telomere length, suggesting that telomerase activity is inhibited. However, inhibition of telomerase, either directly or indirectly, has shown only moderate success in cancer patients. Another promising approach of targeting the telomere is the use of guanine-rich oligonucleotides (GROs) homologous to the 3' telomere overhang sequence (T-oligos). T-oligos, particularly a specific 11-base oligonucleotide (5'-dGTTAGGGTTAG-3') called T11, have been shown to induce DNA damage responses (DDRs) such as senescence, apoptosis, and cell cycle arrest in numerous cancer cell types with minimal or no cytostatic effects in normal, non-transformed cells. As a result, T-oligos and other GROs are being investigated as prospective anticancer therapeutics. Interestingly, the DDRs induced by T-oligos in cancer cells are similar to the effects seen after progressive telomere degradation in normal cells. The loss of telomeres is an important tumor suppressor mechanism that is commonly absent in transformed malignant cells, and hence, T-oligos have garnered significant interest as a novel strategy to combat cancer. However, little is known about their mechanism of action. In this review, we discuss the current understanding of how T-oligos exert their antiproliferative effects in cancer cells and their role in inhibition of telomerase. We also discuss the current understanding of telomerase in cancer and various therapeutic targets related to the telomeres and telomerase.