Glycosyltransferases within the psrP Locus Facilitate Pneumococcal Virulence.

The pneumococcal serine-rich repeat protein (PsrP) is a high-molecular-weight, glycosylated adhesin that promotes the attachment of Streptococcus pneumoniae to host cells. PsrP, its associated glycosyltransferases (GTs), and dedicated secretion machinery are encoded in a 37-kb genomic island that is present in many invasive clinical isolates of S. pneumoniae PsrP has been implicated in establishment of lung infection in murine models, although specific roles of the PsrP glycans in disease progression or bacterial physiology have not been elucidated. Moreover, enzymatic specificities of associated glycosyltransferases are yet to be fully characterized. We hypothesized that the glycosyltransferases that modify PsrP are critical for the adhesion properties and infectivity of S. pneumoniae Here, we characterize the putative S. pneumoniaepsrP locus glycosyltransferases responsible for PsrP glycosylation. We also begin to elucidate their roles in S. pneumoniae virulence. We show that four glycosyltransferases within the psrP locus are indispensable for S. pneumoniae biofilm formation, lung epithelial cell adherence, and establishment of lung infection in a mouse model of pneumococcal pneumonia.IMPORTANCE PsrP has previously been identified as a necessary virulence factor for many serotypes of S. pneumoniae and studied as a surface glycoprotein. Thus, studying the effects on virulence of each glycosyltransferase (GT) that builds the PsrP glycan is of high importance. Our work elucidates the influence of GTs in vivo We have identified at least four GTs that are required for lung infection, an indication that it is worthwhile to consider glycosylated PsrP as a candidate for serotype-independent pneumococcal vaccine design.

Prevalence and Homology of the Pneumococcal Serine-Rich Repeat Protein at the Global Scale.

Pneumococcal pneumonia remains a WHO high-priority disease despite multivalent conjugate vaccines administered in clinical practice worldwide. A protein-based, serotype-independent vaccine has long-promised comprehensive coverage of most clinical isolates of the pneumococcus. Along with numerous pneumococcal surface protein immunogens, the pneumococcal serine-rich repeat protein (PsrP) has been investigated as a potential vaccine target due to its surface exposure and functions toward bacterial virulence and lung infection. Three critical criteria for its vaccine potential - the clinical prevalence, serotype distribution, and sequence homology of PsrP - have yet to be well characterized. Here, we used genomes of 13,454 clinically isolated pneumococci from the Global Pneumococcal Sequencing project to investigate PsrP presence among isolates, distribution among serotypes, and interrogate its homology as a protein across species. These isolates represent all age groups, countries worldwide, and types of pneumococcal infection. We found PsrP present in at least 50% of all isolates across all determined serotypes and nontypeable (NT) clinical isolates. Using a combination of peptide matching and HMM profiles built on full-length and individual PsrP domains, we identified novel variants that expand PsrP diversity and prevalence. We also observed sequence variability in its basic region (BR) between isolates and serotypes. PsrP has a strong vaccine potential due to its breadth of coverage, especially in nonvaccine serotypes (NVTs) when exploiting its regions of conservation in vaccine design. IMPORTANCE An updated outlook on PsrP prevalence and serotype distribution sheds new light on the comprehensiveness of a PsrP-based protein vaccine. The protein is present in all vaccine serotypes and highly present in the next wave of potentially disease-causing serotypes not included in the current multivalent conjugate vaccines. Furthermore, PsrP is strongly correlated with clinical isolates harboring pneumococcal disease as opposed to pneumococcal carriage. PsrP is also highly present in strains and serotypes from Africa, where the need for a protein-based vaccine is the greatest, giving new reasoning to pursue PsrP as a protein vaccine.

Pneumococcal Surface Proteins as Virulence Factors, Immunogens, and Conserved Vaccine Targets.

Streptococcus pneumoniae is an opportunistic pathogen that causes over 1 million deaths annually despite the availability of several multivalent pneumococcal conjugate vaccines (PCVs). Due to the limitations surrounding PCVs along with an evolutionary rise in antibiotic-resistant and unencapsulated strains, conserved immunogenic proteins as vaccine targets continue to be an important field of study for pneumococcal disease prevention. In this review, we provide an overview of multiple classes of conserved surface proteins that have been studied for their contribution to pneumococcal virulence. Furthermore, we discuss the immune responses observed in response to these proteins and their promise as vaccine targets.

Immunogenicity and protective efficacy of a prototype pneumococcal bioconjugate vaccine.

Capsular polysaccharides (CPSs), with which most pathogenic bacterial surfaces are decorated, have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide. Pneumococcal conjugate vaccines (PCVs) are administered globally to prevent invasive pneumococcal disease (IPD). While PCVs have played important roles in controlling IPD in all age groups, their empirical, and labor-intensive chemical conjugation yield poorly characterized, heterogeneous, and variably immunogenic vaccines, with poor immune responses in high-risk populations such as the elderly and patients with weak immune systems. We previously developed a method that bypasses the dependency of chemical conjugation and instead exploits prokaryotic glycosylation systems to produce pneumococcal conjugate vaccines. The bioconjugation platform relies on a conjugating enzyme to transfer a bacterial polysaccharide to an engineered carrier protein all within the lab safe bacterium E. coli. In these studies, we demonstrate that a serotype 8 pneumococcal bioconjugate vaccine is highly immunogenic and elicits functionally protective anti-serotype 8 antibody responses. Specifically, using multiple models we show that mice immunized with multiple doses of a serotype 8 bioconjugate vaccine elicit antibody responses that mediate opsonophagocytic killing, protect mice from systemic infection, and decrease the ability of serotype 8 pneumococci to colonize the nasopharynx and disseminate. Collectively, these studies demonstrate the utility of bioconjugation to produce efficacious pneumococcal conjugate vaccines.

Glycopeptide epitope facilitates HIV-1 envelope specific humoral immune responses by eliciting T cell help.

The inherent molecular complexity of human pathogens requires that mammals evolved an adaptive immune system equipped to handle presentation of non-conventional MHC ligands derived from disease-causing agents, such as HIV-1 envelope (Env) glycoprotein. Here, we report that a CD4+ T cell repertoire recognizes a glycopeptide epitope on gp120 presented by MHCII pathway. This glycopeptide is strongly immunogenic in eliciting glycan-dependent cellular and humoral immune responses. The glycopeptide specific CD4+ T cells display a prominent feature of Th2 and Th17 differentiation and exert high efficacy and potency to help Env trimer humoral immune responses. Glycopeptide-induced CD4+ T cell response prior to Env trimer immunization elicits neutralizing antibody development and production of antibodies facilitating uptake of immunogens by antigen-presenting cells. Our identification of gp120 glycopeptide-induced, T cell-specific immune responses offers a foundation for developing future knowledge-based vaccines that elicit strong and long-lasting protective immune responses against HIV-1 infection.