Invasive pneumococcal disease in Canada 2010-2017: The role of current and next-generation higher-valent pneumococcal conjugate vaccines.

BACKGROUND: In 2010-2011, the 13-valent pneumococcal conjugate vaccine (PCV13) replaced the 7- or 10-valent vaccine (PCV7 and PCV10, respectively) in pediatric immunization programs across Canada. For adults aged ≥65 years, the 23-valent pneumococcal polysaccharide vaccine (PPSV23) has been publicly funded for several decades; PCV13 funding was not recommended in this population, partly due to expected ongoing vaccine-serotype disease decline stemming from herd effects of the pediatric program. Higher-valent PCVs (ie, 15- and 20-valent PCVs [PCV15 and PCV20, respectively]) currently in development may become available in Canada in the coming years. METHODS: Using the National Microbiology Laboratory surveillance reports, annual case counts and serotype distribution of invasive pneumococcal disease (IPD) from 2010 to 2017 in Canada were examined to assess the impact of existing programs on PCV13-serotype IPD and determine the proportion of IPD that can potentially be prevented by current and forthcoming higher-valent PCVs. RESULTS: The percentages of PCV13-serotype IPD decreased from 55% [1492/2708] in 2010 to 30% [902/3006] in 2017 in all age groups combined, including a decline from 67% [221/331] to 18% [40/219] in children aged <5 years and from 50% [487/967] to 23% [287/1238] in adults aged ≥65 years. Overall, IPD cases declined mainly before 2014 and have plateaued since then. In 2017, PCV15- and PCV20-serotypes (inclusive of PCV13 serotypes) accounted for 42% and 58% of IPD cases, respectively, in all ages. CONCLUSIONS: In Canada, publicly funded pediatric PCV13 use was associated with large declines in IPD due to vaccine serotypes. Substantial residual PCV13-serotype IPD proportions observed among all ages imply limits to indirect protection afforded by the pediatric PCV13 program at the current uptake level and suggest the adult PPSV23 program alone is insufficient. Higher-valent PCVs have the potential to address a substantial proportion of remaining IPD cases among all age groups.

The SAGA Deubiquitination Module Promotes DNA Repair and Class Switch Recombination through ATM and DNAPK-Mediated γH2AX Formation.

Class switch recombination (CSR) requires activation-induced deaminase (AID) to instigate double-stranded DNA breaks at the immunoglobulin locus. DNA breaks activate the DNA damage response (DDR) by inducing phosphorylation of histone H2AX followed by non-homologous end joining (NHEJ) repair. We carried out a genome-wide screen to identify CSR factors. We found that Usp22, Eny2, and Atxn7, members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitination module, are required for deubiquitination of H2BK120ub following DNA damage, are critical for CSR, and function downstream of AID. The SAGA deubiquitinase activity was required for optimal irradiation-induced γH2AX formation, and failure to remove H2BK120ub inhibits ATM- and DNAPK-induced γH2AX formation. Consistent with this effect, these proteins were found to function upstream of various double-stranded DNA repair pathways. This report demonstrates that deubiquitination of histone H2B impacts the early stages of the DDR and is required for the DNA repair phase of CSR.

Induction of apoptosis in Eµ-myc lymphoma cells in vitro and in vivo through calpain inhibition.

Calpains are cysteine proteases that have been implicated as both effectors and suppressors of apoptosis. Previously, we showed that c-myc transformation regulated calpain activity and sensitized cells to apoptosis induced by calpain inhibition. The objective of this study was to investigate the role of calpain in the Eµ-myc transgenic model of B-cell lymphoma. Calpain activity assays, apoptosis, cell cycle assays, and expression measurements were used to determine the activity and role of calpain in vitro and in vivo. We found that Eµ-myc transgenic cells have highly elevated calpain activity. Calpastatin, the negative calpain regulator, was expressed at much lower levels in Eµ-myc lymphoma cells compared to normal splenic B cells. The primary isoform in Eµ-myc lymphoma is calpain 1. Treatment of Eµ-myc lymphoma cells with the calpain inhibitors PD150606 or calpain inhibitor III induced caspase-3-dependent apoptosis in vitro. General caspase inhibitors or caspase-3/7 inhibitor protected cells from death induced by calpain inhibitor, whereas caspase-9 inhibitors failed to rescue cells. Human Burkitt's lymphoma (BL2) cells display a pattern of sensitivity and caspase-3 dependence similar to calpain inhibition. Treatment of Eµ-myc lymphoma-bearing mice with PD150606 inhibited calpain activity in vivo and induced cell death in these cells as determined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Multiple daily treatments resulted in reduced tumor load, particularly in combination with etoposide. In conclusion, calpain is highly elevated in the Eµ-myc lymphoma and calpain inhibition has therapeutic potential.

Negative supercoiling creates single-stranded patches of DNA that are substrates for AID-mediated mutagenesis.

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates-which we found to be unique to actively transcribed genes-as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID.

The mismatch repair pathway functions normally at a non-AID target in germinal center B cells.

Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to an approximately 10-fold increase in the mutation frequency in most tissues. By contrast, Msh2 deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V region as a dU:dG mismatch produced by AID initiates modifications by MMR, resulting in mutations at nearby A:T base pairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene that is not mutated by AID. Strikingly, we found that GC B cells accumulate 5 times more mutations at a reporter gene than during the development of the mouse. Notably, the mutation frequency at this reporter gene was approximately 10 times greater in Msh2(-/-) compared with wild-type GC B cells cells. In contrast to the V region, the increased level of mutations at A:T base pairs in GC B cells was not caused by MMR. These results show that in GC B cells, (1) MMR functions normally at an AID-insensitive gene and (2) the frequency of background mutagenesis is greater in GC B cells than in their precursor follicular B cells.

The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination.

An effective immune response requires B cells to produce several classes of antibodies through the process of class switch recombination (CSR). Activation-induced cytidine deaminase initiates CSR by deaminating deoxycytidines at switch regions within the Ig locus. This activity leads to double-stranded DNA break formation at the donor and recipient switch regions that are subsequently synapsed and ligated in a 53BP1-dependent process that remains poorly understood. The DNA damage response E3 ubiquitin ligases RNF8 and RNF168 were recently shown to facilitate recruitment of 53BP1 to sites of DNA damage. Here we show that the ubiquitination pathway mediated by RNF8 and RNF168 plays an integral part in CSR. Using the CH12F3-2 mouse B cell line that undergoes CSR to IgA at high rates, we demonstrate that knockdown of RNF8, RNF168, and 53BP1 leads to a significant decrease in CSR. We also show that 53BP1-deficient CH12F3-2 cells are protected from apoptosis mediated by the MDM2 inhibitor Nutlin-3. In contrast, deficiency in either E3 ubiquitin ligase does not protect cells from Nutlin-3-mediated apoptosis, indicating that RNF8 and RNF168 do not regulate all functions of 53BP1.

Msh2-dependent DNA repair mitigates a unique susceptibility of B cell progenitors to c-Myc-induced lymphomas.

C-Myc is one of the most common targets of genetic alterations in human cancers. Although overexpression of c-Myc in the B cell compartment predisposes to lymphomas, secondary mutations are required for disease manifestation. In this article, we show that genetic deficiencies causing arrested B cell development and accumulation of B cell progenitors lead to accelerated lymphomagenesis in Emu c-myc transgenic mice. This result suggests that B cell progenitors are more prone than their mature counterparts to developing secondary oncogenic lesions that complement c-Myc in promoting transformation. To investigate the nature of these oncogenic lesions, we examined Emu c-myc mice deficient in mismatch repair function. We report that Msh2(-/-) Emu c-myc and Msh2(G674A/G674A) Emu c-myc mice rapidly succumb to pro-B cell stage lymphomas, indicating that Msh2-dependent mismatch repair function actively suppresses c-Myc-associated oncogenesis during early B cell development.

Cathepsin L maturation and activity is impaired in macrophages harboring M. avium and M. tuberculosis.

Mycobacterium tuberculosis-infected macrophages demonstrate diminished capacity to present antigens via class II MHC molecules. Since successful class II MHC-restricted antigen presentation relies on the actions of endocytic proteases, we asked whether the activities of cathepsins (Cat) B, S and L-three major lysosomal cysteine proteases-are modulated in macrophages infected with pathogenic Mycobacterium spp. Infection of murine bone marrow-derived macrophages with either Mycobacterium avium or M. tuberculosis had no obvious effect on Cat B or Cat S activity. In contrast, the activity of Cat L was altered in infected cells. Specifically, whereas the 24-kDa two-chain mature form of active Cat L predominated in uninfected cells, we observed an increase in the steady-state activity of the precursor single-chain (30 kDa) and 25-kDa two-chain forms of the enzyme in cells infected with either M. avium or M. tuberculosis. Pulse-chase analyses revealed that maturation of nascent, single-chain Cat L into the 25-kDa two-chain form was impaired in infected macrophages, and that maturation into the 24-kDa two-chain form did not occur. Consistent with these data, M. avium infection inhibited the IFNgamma-induced secretion of active two-chain Cat L by macrophages. Viable bacilli were not required to disrupt Cat L maturation, suggesting that a constitutively expressed mycobacterial component was responsible. The absence of the major active form of lysosomal Cat L in M. avium- and M. tuberculosis-infected macrophages may influence the types of T cell epitopes generated in these antigen-presenting cells, and/or the rate of class II MHC peptide loading.