Dysregulated pulmonary inflammatory responses exacerbate the outcome of secondary aspergillosis following influenza.

IMPORTANCE: Severe influenza is a risk factor for fatal invasive pulmonary aspergillosis; however, the mechanistic basis for the lethality is unclear. Utilizing an influenza-associated pulmonary aspergillosis (IAPA) model, we found that mice infected with influenza A virus followed by Aspergillus fumigatus had 100% mortality when superinfected during the early stages of influenza but survived at later stages. While superinfected mice had dysregulated pulmonary inflammatory responses compared to controls, they had neither increased inflammation nor extensive fungal growth. Although influenza-infected mice had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus, influenza did not affect the ability of neutrophils to clear the fungi. Our data suggest that the lethality seen in our model of IAPA is multifactorial with dysregulated inflammation being a greater contributor than uncontrollable microbial growth. If confirmed in humans, our findings provide a rationale for clinical studies of adjuvant anti-inflammatory agents in the treatment of IAPA.

Protection of Mice against Experimental Cryptococcosis by Synthesized Peptides Delivered in Glucan Particles.

The high global burden of cryptococcosis has made development of a protective vaccine a public health priority. We previously demonstrated that a vaccine composed of recombinant Cryptococcus neoformans chitin deacetylase 2 (Cda2) delivered in glucan particles (GPs) protects BALB/c and C57BL/6 mice from an otherwise lethal challenge with a highly virulent C. neoformans strain. An immunoinformatic analysis of Cda2 revealed a peptide sequence predicted to have strong binding to the major histocompatibility complex class II (MHC II) H2-IAd allele found in BALB/c mice. BALB/c mice vaccinated with GPs containing a 32-amino-acid peptide (Cda2-Pep1) that included this strong binding region were protected from cryptococcosis. Protection was lost with GP-based vaccines containing versions of recombinant Cda2 protein and Cda2-Pep1 with mutations predicted to greatly diminish MHC II binding. Cda2 has homology to the three other C. neoformans chitin deacetylases, Cda1, Cda3, and Fpd1, in the high-MHC II-binding region. GPs loaded with homologous peptides of Cda1, Cda3, and Fpd1 protected BALB/c mice from experimental cryptococcosis, albeit not as robustly as the Cda2-Pep1 vaccine. Finally, seven other peptides were synthesized based on regions in Cda2 predicted to contain promising CD4+ T cell epitopes in BALB/c or C57BL/6 mice. While five peptide vaccines significantly protected BALB/c mice, only one protected C57BL/6 mice. Thus, GP-based vaccines containing a single peptide can protect mice against cryptococcosis. However, given the diversity of human MHC II alleles, a peptide-based Cryptococcus vaccine for use in humans would be challenging and likely need to contain multiple peptide sequences. IMPORTANCE Cryptococcosis, due to infection by fungi of the Cryptococcus neoformans species complex, is responsible for substantial morbidity and mortality in immunocompromised persons, particularly those with AIDS. Cryptococcal vaccines are a public health priority yet are not available for human use. We previously demonstrated mice could be protected from experimental cryptococcosis with vaccines composed of recombinant cryptococcal proteins encased in hollow highly purified yeast cell walls (glucan particles). In this study, we examined one such protective protein, Cda2, and using bioinformatics, we identified a region predicted to stimulate strong T cell responses. A peptide containing this region formulated in glucan particle-based vaccines protected mice as well as the recombinant protein. Other peptide vaccines also protected, including peptides containing sequences from proteins homologous to Cda2. These preclinical mouse studies provide a proof of principle that peptides can be effective as vaccines to protect against cryptococcosis and that bioinformatic approaches can guide peptide selection.