Evaluating the impact of immunization with the "pan-fungal" vaccine, NXT-2, on the gut mycobiome and microbiome in non-human primates (NHPs).

Fungal infections remain a significant public health concern with high mortality, morbidity, and increasing associated health costs. This burden is projected to rise due to expansion of at-risk populations, limited therapeutics, increasing drug resistance, and the emergence of new fungal pathogens. Even with these challenges, there are currently no approved vaccines. We previously developed a "pan-fungal" vaccine candidate, NXT-2, that confers protection against multiple invasive fungal infections such as pulmonary aspergillosis, pneumocystosis, and invasive candidiasis, as well as non-invasive vulvovaginal candidiasis. NXT-2 is a 90 amino acid consensus peptide designed from a conserved region of the fungal antigen (KEX1). We assessed the effect of NXT-2 immunization on gut microbial diversity, composition, and functional capacity in non-human primates. To do this, we monitored changes in the gut mycobiome and microbiome pre- and post-vaccination using ITS2 and metagenomic sequencing, respectively, in Japanese and rhesus macaque cohorts. NXT-2 elicited a robust antibody response without disrupting the gut microbial communities in both macaque species. The mycobiome exhibited stability with no significant changes in alpha and beta diversity, taxonomic composition, or functional guild distributions. The relative abundance of gut resident Candida and Aspergillus species remained stable and was not significantly altered following vaccination. The microbiome showed stability with preserved alpha and beta diversities, taxonomic composition, and functional capacity. Results from this study show the first cross-kingdom analysis demonstrating that antifungal vaccination can achieve protective immunity without perturbing gut microbial communities. This establishes a framework for microbiome-informed vaccine assessment beyond conventional immunogenicity and adverse effect monitoring.IMPORTANCEFungal infections cause millions of deaths annually, yet no vaccines are approved despite growing drug resistance and limited treatment options. NXT-2 is a pan-fungal vaccine that protects against multiple fungal infections such as pneumocystosis, candidiasis, and aspergillosis. Here, we demonstrate in NHPs that NXT-2 elicits robust protective antibody responses without altering gut bacterial or fungal communities. This is the first study to assess antifungal vaccination across both microbial kingdoms and establish that protective antifungal immunity can be achieved while preserving resident microbiota. This work provides a framework for incorporating microbiome assessment into vaccine development beyond conventional immunogenicity and adverse event monitoring.