Genetic Engineering of Klebsiella pneumoniae ATCC 25955 for Bioconjugate Vaccine Applications

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Comment

General

Is there any evidence that antibodies against the LPS O-chain are protective against disease?  Please include a reference for this. Previous vaccines have targeted the CPS, not the LPS O-chain.

General

The focus of this paper is on the genetic changes to produce a specific product, however there is insufficicient information to prove that they have really made what they claim to have made: only a Western Blot to show the presence of the glycan and a DLS method [not described in the Materials and Methods] for homogeneity of molecular size. The molecular size is not calibrated to provide an estimate of molecular weight.

General

The Materials and Methods Section is incomplete, with little or no information describing the methods used: see below.

General

Normal expectations for a glycoconjugate vaccine would include the average chain lengths of the glycan chains, polysaccharide-protein ratio, number of glycan chains per carrier protein etc. Fig. 3 implies a single glycan chain per carrier protein with a single repeat unit. But no experimental evidence to support this. It is inconsistent with the claim of a ladder structure in Western Blot experiments. Some text suggests that there is a significant amount of unconjugated protein present, but no methodology to quantify this is reported, and it would not be detected by Western blotting targetting the glycan chain.

General

No evidence of immunogenicity, which is critical for further development of a glycoconjugate antigen into a vaccine, is presented.

18, 83

What is a "nanovaccine"? The paper would benefit from a clear definition of this term.

47

Jain and Ravinder papers [Refs. 10 and 12] recognised by first author, but not Ref. 11, which is linked to a different author. It seems curious way to do this, and the authors should be consistent in their usage.

36-51 and 63-67

All the papers cited report results with capsular polysaccharide immunogens [free or conjugated], but the authors here describe LPS O-chain conjugates. Is there any evidence antibodies against LPS O-chain are protective?

Materials and Methods

There is lack of description in the methodology, apart from Western blotting, seems to imply that no significant characterisation of the glycoconjugate product was carried out. No information about the DLS or electron microscopy work. There is no information about how carbohydrate catabolism was quantified. Analytical methods should not be described in Figure Legends. There is no information on protein or glycan quantification. The methods used to monitor elution from columns used in protein purification [and identification of required fractions] are not described. The authors should expand this section so that every technique and analytical procedure is adequately described.

Materials and Methods

The authors provide no information about the scale at which the cells were grown [which affects the size of columns used in purification], or the yield of protein and glycan that they achieved. If this material is to become a vaccine manufacturing platform, this information is critical.

205-206

No information in Materials and Methods as to the equipment and methodology used for these experiments. This should be included.

Legend to Fig. 2, Fig. 4, Fig. 5

This information should be in Materials and Methods section.

219-220

What is SC4573 - sequence, any info on previous use as a carrier protein etc.? If the information is in the Supplementary informatrion, then this should be explicit in the text.

Figure 3

The presence of strong bands from anti-His antibodies quite distinct from the anti-LPS bands seems to suggest that a relatively small proportion of the protein produced was glycosylated. Quantification of these two types of material is highly relevant to the reader's interpretation of this work.

292-293

"…. which have been described in our previous work…". The papers should be cited to allow readers to access the information.

Fig. 6

This shows results from dynamic light scattering experiments, but there is no information about the equipment, calibration and methodology in the Materials and Methods Section. The diagrams for Fig. 6(c) imply that there are six glycan chains per carrier protein. Is there any evidence for this, or is it just something to give a pretty picture? Was any molecular sizing carried out, by size exclusion chromatography [SEC], SEC-MALLS or mass spectrometry? FIgure 6(b) shows the separation of the the VLPs on SDS-PAGE. In my experience, SDS treatment leads to disaggregation of non-covalently associated protein aggregates. The claimed stability of the VLPs to SDS treatment deserves comment.

297-298

".. Still some unbound protein nanoparticles after size-exclusion chromatography…." Not bound to what? The authors provide to information. Or do they means not carrying glycan chains? The authors should clarify exactly what they mean.

330

" … did not lead to the expected increase in glycoprotein production…." There is no method reported in the Materials and Method Section to quantify the glycoprotein production. This should be included. Since this was measured, quantitative information in the manuscript would be of interest to the reader.