P4 is a 28-amino acid peptide fragment of the pneumococcal virulence factor PsaA. [276]. P4 contains a functional epitope for the adherence of PsaA to nasopharyngeal cells [276]. A 3D model of the P4 epitope within PsaA is shown in Figure 21. This epitope adheres to epithelial cells via a binding moiety on human E-Cadherin receptors (an important glycoprotein in calcium-dependent binding at tight junctions between epithelial cells) [277].
Figure 21 - Three-dimensional structure of Pneumococcal surface adhesin A (PsaA, Protein Database ID: 1PSZ) with the immunomodulating peptide fragment P4, highlighted in blue (Residues 251-278). N = N-terminal.
4.2.1 Structure
P4 peptide sequence is highly conserved within PsaA proteins from all pneumococcal serotypes and also amongst several other bacterial species. The LpeA protein from Listeria spp. in particular exhibits remarkable similarities both in structure and function to PsaA. LpeA has recently been shown to be used by Listeria spp. to invade macrophages through mechanisms still to be elucidated [278]. Table 3 (adapted from [279]) shows the sequence homologies
roles of binding and adhesion, other functional similarities remain to be determined for proteins other than LpeA.
Table 3 - P4 sequence homology with non-streptococcal proteins. Adapted from [279]
Source organism Putative function Sequences producing significant alignments*
S. pneumoniae P4 peptide sequence 251LFVESSVKRRPMKTVSQDTNIPIYAQIF278
S. pneumoniae PsaA 245LFVETSVDDRSMETVSKETNVPIAGTIF272
S. pneumoniae ABC transporter substrate- binding protein
251LFVETSVDDRSMETVSKETNVPIAGTIF278
Bacillus anthracis
Manganese binding protein and adhesin
257LFVETSVDRRSMETVSKETNVPIAGTIF284
B. cereus Adhesion lipoprotein 253LFVETSVDRRSMETVSKETNVPIAGTIF280
B. cereus E33L Adhesion lipoprotein 253LFVETSVDRRSMETVSKETNVPIAGTIF280
Listeria innocua Adhesion binding protein and ABC transporter
252LFVETSVDPRSMETVSKETNVPIFAKIF279
L.
monocytogenes
Adhesion binding protein and ABC transporter
252LFVETSVDPRSMETVSKETNVPIFAKIF279
Leuconostoc mesenteroides
ABC transport protein and adhesin
255LFVESSVSPKAMEKVSKETGLPIYSKIY282
4.2.2 Functions in vitro
P4 peptide has been shown to activate human nasopharyngeal cells, modulating their cytology and cytokine secretions thereby enhancing adherence and internalization of pneumococcal bacteria in vitro [279]. P4- mediated activation was also demonstrated with other eukaryotic cells, including mouse macrophages (RAW 261.4) and freshly isolated polymorphonuclear neutrophils from healthy human donors [279] [280]. When exposed to the peptide, HL-60 cells undergo various phenotypical changes such as increased pseudopods, foamy, granulated cytoplasm with
Figure 22 - HL-60 cells in a normal state (A) exposed to the P4 peptide (B). The arrows highlight increased intra-cellular granules present. Adapted from [279].
The increased activation seen in phagocytes following P4 exposure also leads to enhanced phagocytosis. In opsono-phagocytosis assays, in the presence of species specific antibody and complement, P4 is able to increase the uptake and subsequent killing of bacteria by >70%. This has been established in the HL60 cell line as well as mouse & human derived peripheral blood neutrophils.
4.2.3 Functions in vivo
Additionally, Rajam et al (280) were able to show the potential of using P4 mediated passive immunotherapy in vivo. In a fatal pneumococcal mouse model, P4 therapy was able to rescue 80% of moribund mice by giving them a combined treatment of P4 and pathogen specific antibodies. In contrast, only 10% of the control strain and 30% of the mice treated purely with antibodies and complement survived [280]. Both in vivo and in vitro, P4 shows no signs of toxicity.
Further research at the CDC has shown that enhanced phagocytosis in vitro confers therapeutic benefit in vivo [281]. Rajam et al. used reference in vitro
opsonophagocytic killing and uptake assays to show that P4 enhanced opsonophagocytosis of S. pneumoniae by human promyelocytic leukaemia cells (HL-60) differentiated into granulocytes [280]. In an in vivo extension of this study, mice were infected with S. pneumoniae, then injected with IVIG and/or P4 at 72 and 96 hours post-infection. Survival was 10% in untreated mice, ~30% in mice given IVIG or P4 alone, and 80% in those treated with both IVIG and P4.
Adding a low dose of ceftriaxone to P4 and immunoglobulin increased mouse survival to 100% [280]. P4-activation of in vitro opsonophagocytosis has also been demonstrated with Neisseria meningitidis and Staphylococcus aureus, in the presence of pathogen-specific antibodies [282]. In a murine model of overwhelming S. aureus infection, intravenous P4 with immunoglobulin rescued 70% of mice, compared with 0% survival of untreated controls [282].
Augmented passive immunotherapy combines commercially available pooled immunoglobulin G (IVIG) and the phagocyte stimulating peptide P4 to rapidly and effectively augment an immune response before the effect of antibiotic treatment would be apparent.
4.2.4 Summary of P4 publications
There are currently 7 publications related to P4 peptide. Their main findings are summarized in Table 4.
Table 4 – Main findings from previous P4 peptide publications.
Year Main findings Ref
2007 P4 treatment of epithelial cells leads to cytological changes allowing for increased binding and internalization of S. pneumoniae.
NP cells reduce their secretion of FGF-β, but increase their secretion of IL-6, IL-8 and VEGF cytokines following peptide treatment.
[279]
2008 P4 treatment of HL-60 neutrophil cell line leads to increased phagocytosis in a dose-dependent manner.
P4 and antibody treatment of S. pneumoniae infected mice increased survival from 10% to 80%.
[280]
2009 P4-antibody therapy as an adjunct to antibiotics reduced the need for dual administration and led to 100% survival (compared to 90% P4-antibody alone and 20% control).
Repeat therapy was effective following infection of previously rescued mice. Septic blood neutrophils from mice showed enhanced phagocytosis following peptide treatment.
[281]
2010 Peptide-antibody treatment of two strains of aged mice led to significantly increased survival.
[283] 2011 Survival was improved from 20% to 80% in mice with S.pneumoniae-influenzea
co-infection and treated with P4. Survival correlated with increased clearance of bacteria and virus and decreased lung consolidation.
[284]
2011 API increased survival of S. aureus infected mice infected mice from 20% to 70%
P4 stimulation led to enhanced phagocytosis of S. aureus by HL-60 neutrophil cells in vitro.
[282]
2012 Increased survival following API treatment of sepsis due to increased phagocytic activation and recruitment.
Intranasal administration of P4 to treat pneumococcal pneumonia.
Ex vivo and in vitro macrophages increase phagocytosis following peptide treatment.