Review 3: "Semi-synthetic Glycoconjugate Vaccine Candidate Against Cryptococcus Neoformans"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: The manuscript by Crawford et al. entitled “Semi-synthetic glycoconjugate vaccine candidate against Cryptococcus neoformans” describes the creation of a series of synthetic saccharides mimicking the structure of the capsular glucuronoxylomannan (GXM) molecule found on C. neoformans and investigates its potential for vaccine development. The study is well-designed and clearly communicates its purpose throughout. The study rational is well supported by several evidence that GXM is essential for fungal virulence it exhibits immunogenic properties, and the present work shows that the M2 motif is the largest of all and is required to adopt the conformation of GXM polysaccharides. Based on this the authors proposed that although GXM polysaccharide does not have a well-defined repeating unit it does have repeating motifs that can potentially be used for antigenic purposes.

The study effectively utilizes GXM microarrays, molecular modeling, and NMR to develop saccharides of varying sizes, eventually identifying a decasaccharide (15) with promising antigenic potential. Interestingly, the authors explore the immunogenicity of these saccharides conjugated to two carrier proteins, CRM197 and PA63. Initial results suggest the DECA-CRM197 conjugate may be more immunogenic.

In summary, the results presented here are of unquestionable importance and relevance to the fungal vaccine development field, even though there is still a long way to go for developing an efficient vaccine candidate. Few minor points are below:

1. While the in vitro data strongly suggests the vaccine candidate's potential, the in vivo results in mice are less conclusive. Survival rates between vaccinated and unvaccinated animals show minimal difference. The observed effect might be primarily due to the protein carrier itself, requiring further investigation. Overall, the study presents a promising approach to vaccine development. However, further optimization is necessary to achieve statistically significant survival benefits in animal models. One suggestion at this point would be to optimize the glycan-protein loading, formulation, and immunization schedules with DECA-CRM197 (as the authors suggest) before investing in more complex approaches. Optimizing these aspects could potentially extend the observed survival difference and provide better guidance for the next steps in the study. Do the authors have any optimization data relevant to this point that could be included in the manuscript?

2. Another issue concerns the use of C. neoformans strain 24067 (serotype D) instead of strain H99 (serotype A) for the phagocytic evaluation in Figure 5, since H99 was consistently used in this study. Is there a rationale behind using a different serotype for this experiment? As the motif distribution can vary significantly among serotypes, it's unclear if this switch might affect the internalization of cryptococcal cells by macrophages in the presence of serum from immunized mice. Adding some clarification on this topic would be beneficial.

3. Additionally, could a control with serum (1:50 dilution) from non-immunized mice be included in this experiment?