Review 1: "Semi-synthetic Glycoconjugate Vaccine Candidate Against Cryptococcus Neoformans"

RR:C19 Evidence Scale rating by reviewer:

• Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review: Cryptococcus neoformans is an opportunistic pathogen encased with thick capsular polysaccharide glucuronoxylomannan (GXM), paralleling some bacterial pathogens such as Pneumococcus for which effective vaccine/s is available. GXM of Crypto is highly diverse but has discrete repeats that can be targeted to elicit neutralizing antibodies. The authors (Crawford et al.) of this manuscript titled “Semi-synthetic glycoconjugate vaccine candidate against Cryptococcus neoformans evaluated the immunogenicity and efficacy of conjugate vaccine/s using semi-synthetic glycans and protein carriers CRM197 and Anthrax PA63. The authors hypothesize that semi-synthetic glycans help in the rational design of vaccines for better efficacy. Further, capsular polysaccharides may elicit higher antibody levels but are T-cell help-independent with poor affinity and longevity. The authors' basis for choosing semi-synthetic decasaccharide 15 glycan is that it mimics the M2 motif necessary for the conformation of GXM polysaccharide, and the pentasaccharide is too small to assume the conformation. Overall, the manuscript presents the data with appropriate interpretations suitable for this type of journal. Some of the comments are below:

1. Whether variable valency occupancy of 1-6 glycans/protein dictate variable/weaker neutralizing antibodies and whether uniform occupancy can be reached is unclear. A discussion is worthy.

2. Fig. 4 data may be misconstrued due to low immunogenicity in the PA63 carrier immunized group. It is unclear if an equal amount of serum was used in Fig. 2b and why ODs are higher in all isotypes except IgM despite the lower immunogenicity of the PA63 Group.

3. The number of replicates in Fig 5 is not clear. Are they mouse samples or replicates from a sample? The opsonization experiment should include PA63 group sera.

4. The stats/significance need to be included in Figure 6.

5. Check the abbreviation in Results Section 2. EPS/CPS.

6. Check to reference fig text (is it Fig 2a?) in the Figure 4 result section.

7. SI Fig. 4. Methods are not described.

8. The logic behind the loading ratio of 30:1 is not clear.