Streptococcus infantis, Streptococcus mitis, and Streptococcus oralis Strains With Highly Similar cps5 Loci and Antigenic Relatedness to Serotype 5 Pneumococci.

Streptococcus pneumoniae is a highly impactful bacterial pathogen on a global scale. The principal pneumococcal virulence factor and target of effective vaccines is its polysaccharide capsule, of which there are many structurally distinct forms. Here, we describe four distinct strains of three Mitis group commensal species (Streptococcus infantis, Streptococcus mitis, and Streptococcus oralis) recovered from upper respiratory tract specimens from adults in Kenya and the United States that were PCR-positive for the pneumococcal serotype 5 specific gene, wzy5. For each of the four strains, the 15 genes comprising the capsular polysaccharide biosynthetic gene cluster (cps5) shared the same order found in serotype 5 pneumococci, and each of the serotype 5-specific genes from the serotype 5 pneumococcal reference strain shared 76-99% sequence identity with the non-pneumococcal counterparts. Double-diffusion experiments demonstrated specific reactivity of the non-pneumococcal strains with pneumococcal serotype 5 typing sera. Antiserum raised against S. mitis strain KE67013 specifically reacted with serotype 5 pneumococci for a positive Quellung reaction and stimulated serotype 5 specific opsonophagocytic killing of pneumococci. Four additional commensal strains, identified using PCR serotyping assays on pharyngeal specimens, revealed loci highly homologous to those of pneumococci of serotypes 12F, 15A, 18C, and 33F. These data, in particular the species and strain diversity shown for serotype 5, highlight the existence of a broad non-pneumococcal species reservoir in the upper respiratory tract for the expression of capsular polysaccharides that are structurally related or identical to those corresponding to epidemiologically significant serotypes. Very little is known about the genetic and antigenic capsular diversity among the vast array of commensal streptococcal strains that represent multiple diverse species. The discovery of serotype 5 strains within three different commensal species suggests that extensive capsular serologic overlap exists between pneumococci and other members of the diverse Mitis group. These findings may have implications for our current understanding of naturally acquired immunity to S. pneumoniae and pneumococcal serotype distributions in different global regions. Further characterization of commensal strains carrying homologs of serotype-specific genes previously thought to be specific for pneumococci of known serotypes may shed light on the evolution of these important loci.

Enhancement of antibody functions through Fc multiplications.

Antibodies carry out a plethora of functions through their crystallizable fragment (Fc) regions, which can be naturally tuned by the adoption of several isotypes and post-translational modifications. Protein engineering enables further Fc function modulations through modifications of the interactions between the Fc and its functional partners, including FcγR, FcRn, complement complex, and additions of auxiliary functional units. Due to the many functions embedded within the confinement of an Fc, a suitable balance must be maintained for a therapeutic antibody to be effective and safe. The outcome of any Fc engineering depends on the interplay among all the effector molecules involved. In this report, we assessed the effects of Fc multiplication (or tandem Fc) on antibody functions. Using IgG1 as a test case, we found that, depending on the specifically designed linker, Fc multiplication led to differentially folded, stable molecules with unique pharmacokinetic profiles. Interestingly, the variants with 3 copies of Fc improved in vitro opsonophagocytic killing activity and displayed significantly improved protective efficacies in a Klebsiella pneumoniae mouse therapeutic model despite faster clearance compared with its IgG1 counterpart. There was no adverse effect observed or pro-inflammatory cytokine release when the Fc variants were administered to animals. We further elucidated that enhanced binding to various effector molecules by IgG-3Fc created a "sink" leading to the rapid clearance of the 3Fc variants, and identified the increased FcRn binding as one strategy to facilitate "sink" escape. These findings reveal new opportunities for novel Fc engineering to further expand our abilities to manipulate and improve antibody therapeutics.

Target-Agnostic Identification of Functional Monoclonal Antibodies Against Klebsiella pneumoniae Multimeric MrkA Fimbrial Subunit.

The increasing incidence of Klebsiella pneumoniae infections refractory to treatment with current broad-spectrum antibiotic classes warrants the exploration of alternative approaches, such as antibody therapy and/or vaccines, for prevention and treatment. However, the lack of validated targets shared by spectrums of clinical strains poses a significant challenge. We adopted a target-agnostic approach to identify protective antibodies against K. pneumoniae Several monoclonal antibodies were isolated from phage display and hybridoma platforms by functional screening for opsonophagocytic killing activity. We further identified their common target antigen to be MrkA, a major protein in the type III fimbriae complex, and showed that these serotype-independent anti-MrkA antibodies reduced biofilm formation in vitro and conferred protection in multiple murine pneumonia models. Importantly, mice immunized with purified MrkA proteins also showed reduced bacterial burden following K. pneumoniae challenge. Taken together, these results support MrkA as a promising target for K. pneumoniae antibody therapeutics and vaccines.