Biology subjects

Levitz, S. M.

Publications and source records attributed to Levitz, S. M..

8 recordsLinked to original sources

bioRxiv2026

Identification of Cryptococcus neoformans capsule-associated protein antigens that mediate B cell-dependent vaccine protection in mice and elicit antibody responses in humans with cryptococcosis

Infection with the encapsulated fungal pathogen Cryptococcus neoformans causes substantial global disease burden in persons with compromised T cell function. Previously, we demonstrated that vaccines containing adjuvanted recombinant cryptococcal proteins protected mice against lethal infection by mechanisms dependent on CD4+ T cells. For the present study, we hypothesized that vaccines containing protein antigens naturally located on the surface of the cryptococcal capsule would contribute protective B cell responses. We found that B cell-deficient mice exhibited reduced survival following vaccination with any of four capsule-associated antigens: Blp4, Cpd1, Lhc1, or Krp1. To further elucidate the role of antibodies in protection, we tested serum from mice vaccinated with a live-attenuated strain (cda123) and then challenged with a virulent C. neoformans strain for IgG responses to a panel of 26 cryptococcal recombinant proteins. Strong reactivity was detected against Blp4, which we focused on because it is capsule-associated and protects mice when administered in adjuvanted subunit vaccines, by mechanisms partly dependent on B cells. Blp4 antibodies were consistently present in surviving mice, including those vaccinated with unrelated proteins or those challenged with diverse clinical isolates. Additionally, serum from Blp4-vaccinated mice promoted macrophage opsonophagocytosis of C. neoformans. Finally, IgG antibodies against Blp4 were detected in 20 of 21 serum samples from patients who survived cryptococcosis, compared with only 4 of 14 samples from healthy controls. Together, these data identify Blp4 as an antigen that elicits protective B cell responses and is a strong candidate for inclusion in subunit cryptococcal vaccines.

immunology↗

bioRxiv2026

A protective hybrid protein vaccine informed by mapping major histocompatibility class II epitopes in Cryptococcus neoformans chitin deacetylases that stimulate CD4+ T cell responses

Infections due to Cryptococcus species are estimated to cause over 100,000 deaths a year. No cryptococcal vaccines are approved for human use. We have shown that vaccine formulations consisting of C. neoformans chitin deacetylase (Cda)2 and Cda1 adjuvanted with Cationic Adjuvant Formulation 01 (CAF01) protect mice against experimental cryptococcosis by a mechanism dependent upon CD4+ T cells. Here, using overlapping peptide libraries, we mapped epitopes in Cda2 and Cda1 that stimulated interferon-gamma (IFN{gamma}) release from splenocytes of C57BL/6 and DR4 mice immunized with these proteins. DR4 mice lack mouse major histocompatibility class II (MHC-II) proteins and express chimeric MHC-II proteins with the specificity of the human HLA-DR allele, HLA-DRB1*04:01. Experimental results were then compared with MHC-II binding predictions using the Immune Epitope Database (IEDB) NetMHCIIpan 4.1 BA MHC-II epitope prediction tool. Unique epitopes in Cda2 and Cda1 were discovered for each mouse strain, some of which were not predicted to bind well to MHC-II. CD4+ T cells were responsible for cytokine release as IFN{gamma} production was lost if CD4+ T cells were depleted from the splenocyte population. Finally, we tested a hybrid recombinant protein that consisted of portions of Cda2 and Cda1 containing the MHC-II/CD4+ T cell epitopes which induce CD4+ T cells in DR4 mice. When administered as a CAF01-adjuvanted vaccine, the hybrid protein protected DR4 mice from an otherwise lethal C. neoformans pulmonary challenge. These results provide a proof of principle regarding the utility of MHC-II/CD4+ T cell epitope mapping in cryptococcal vaccine development.

immunology↗

bioRxiv2024

Immunological correlates of protection mediated by a whole organism Cryptococcus neoformans vaccine deficient in chitosan

The global burden of infections due to the pathogenic fungus Cryptococcus is substantial in persons with low CD4+ T cell counts. Previously, we deleted three chitin deacetylase genes from C. neoformans to create a chitosan-deficient, avirulent strain, designated cda1{Delta}2{Delta}3{Delta} which, when used as a vaccine, protected mice from challenge with virulent C. neoformans strain KN99. Here, we explored the immunological basis for protection. Vaccine-mediated protection was maintained in mice lacking B cells or CD8+ T cells. In contrast, protection was lost in mice lacking /{beta} T cells or CD4+ T cells. Moreover, CD4+ T cells from vaccinated mice conferred protection upon adoptive transfer to naive mice. Importantly, while monoclonal antibody-mediated depletion of CD4+ T cells just prior to vaccination resulted in complete loss of protection, significant protection was retained in mice depleted of CD4+ T cells after vaccination, but prior to challenge. Vaccine-mediated protection was lost in mice genetically deficient in IFN{gamma}, TNF, or IL-23p19. A robust influx of leukocytes and IFN{gamma}- and TNF-expressing CD4+ T cells was seen in the lungs of vaccinated and challenged mice. Finally, a higher level of IFN{gamma} production by lung cells stimulated ex vivo correlated with lower fungal burden in the lungs. Thus, while B cells and CD8+ T cells are dispensable, IFN{gamma} and CD4+ T cells have overlapping roles in generating protective immunity prior to cda1{Delta}2{Delta}3{Delta} vaccination. However, once vaccinated, protection becomes less dependent on CD4+ T cells, suggesting a strategy for vaccinating HIV+ persons prior to loss of CD4+ T cells. ImportanceThe fungus Cryptococcus neoformans is responsible for >100,000 deaths annually, mostly in persons with impaired CD4+ T cell function such as AIDS. There are no approved human vaccines. We previously created a genetically engineered avirulent strain of C. neoformans, designated cda1{Delta}2{Delta}3{Delta}. When used as a vaccine, cda1{Delta}2{Delta}3{Delta} protects mice against a subsequent challenge with a virulent C. neoformans strain. Here, we defined components of the immune system responsible for vaccine-mediated protection. We found that while B cells and CD8+ T cells were dispensible, protection was lost in mice genetically deficient in CD4+ T cells, and the cytokines IFN{gamma}, TNF, or IL-23. A robust influx of cytokine-producing CD4+ T cells was seen in the lungs of vaccinated mice following infection. Importantly, protection was retained in mice depleted of CD4+ T cells following vaccination, suggesting a strategy to protect persons who are at risk for future CD4+ T cell dysfunction.

immunology↗

bioRxiv2024

Protection against experimental cryptococcosis elicited by Cationic Adjuvant Formulation 01-adjuvanted subunit vaccines

The fungal infection, cryptococcosis, is responsible for >100,000 deaths annually. No licensed vaccines are available. We explored the efficacy and immune responses of subunit cryptococcal vaccines adjuvanted with Cationic Adjuvant Formulation 01 (CAF01). CAF01 promotes humoral and T helper (Th) 1 and Th17 immune responses and has been safely used in human vaccine trials. Four subcutaneous vaccines, each containing single recombinant Cryptococcus neoformans protein antigens, partially protected mice from experimental cryptococcosis. Protection increased, up to 100%, in mice that received bivalent and quadrivalent vaccine formulations. Vaccinated mice that received a pulmonary challenge with C. neoformans had an influx of leukocytes into the lung including robust numbers of polyfunctional CD4+ T cells which produced Interferon gamma (IFN{gamma}), tumor necrosis factor alpha (TNF), and interleukin (IL)-17 upon ex vivo antigenic stimulation. Cytokine-producing lung CD8+ T cells were also found, albeit in lesser numbers. A significant, durable IFN{gamma} response was observed in the lungs, spleen, and blood. Moreover, IFN{gamma} secretion following ex vivo stimulation directly correlated with fungal clearance in the lungs. Thus, we have developed multivalent cryptococcal vaccines which protect mice from experimental cryptococcosis using an adjuvant which has been safely tested in humans. These preclinical studies suggest a path towards human cryptococcal vaccine trials. Author summaryCryptococcosis is a fungal infection that poses great challenges to public health, especially in resource-limited regions with high HIV prevalence. Despite the urgent need, no licensed vaccines are currently available. In this study, we used a lethal mouse model of cryptococcosis to explore protection and immune responses elicited by vaccines consisting of recombinant cryptococcal proteins formulated with CAF01, an adjuvant that has an established safety and immunogenicity profile in human clinical vaccine trials. We discovered that while vaccines containing a single protein partially protected mouse strains, the protection was greatly augmented when the mice received vaccines formulated with multiple antigens. The lungs of vaccinated and infected mice had a robust influx of CD4+ T cells, many of which made the cytokines IFN{gamma} and IL-17 when stimulated ex vivo. Moreover, we found the production of IFN{gamma} directly correlated with clearance of fungi from the lungs. Cytotoxic CD8+ T cell responses were also observed, albeit in lesser numbers. Our promising findings from this preclinical research paves the way for future human cryptococcal vaccine trials.

immunology↗

bioRxiv2023

Dysregulated Pulmonary Inflammatory Responses Exacerbate the Outcome of Secondary Aspergillosis Following Influenza

Inhalation of airborne conidia of the ubiquitous fungus Aspergillus fumigatus commonly occurs but invasive aspergillosis is rare except in profoundly immunocompromised persons. Severe influenza predisposes patients to invasive pulmonary aspergillosis by mechanisms that are poorly defined. Using a post-influenza aspergillosis model, we found that superinfected mice had 100% mortality when challenged with A. fumigatus conidia on days 2 and 5 (early stages) of influenza A virus infection but 100% survival when challenged on days 8 and 14 (late stages). Influenza-infected mice superinfected with A. fumigatus had increased levels of the pro-inflammatory cytokines and chemokines IL-6, TNF, IFN{beta}, IL-12p70, IL-1, IL-1{beta}, CXCL1, G-CSF, MIP-1, MIP-1{beta}, RANTES and MCP-1. Surprisingly, on histopathological analysis, superinfected mice did not have greater lung inflammation compared with mice infected with influenza alone. Mice infected with influenza had dampened neutrophil recruitment to the lungs following subsequent challenge with A. fumigatus, but only if the fungal challenge was executed during the early stages of influenza infection. However, influenza infection did not have a major effect on neutrophil phagocytosis and killing of A. fumigatus conidia. Moreover, minimal germination of conidia was seen on histopathology even in the superinfected mice. Taken together, our data suggest that the high mortality rate seen in mice during the early stages of influenza-associated pulmonary aspergillosis is multifactorial, with a greater contribution from dysregulated inflammation than microbial growth. ImportanceSevere 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 A. 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 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.

immunology↗

bioRxiv2022

Human and murine Cryptococcus neoformans infection selects for common genomic changes in an environmental isolate.

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from the cerebral spinal fluid (CSF) of an immunocompromised patient with cryptococcosis based on the molecular analyses available in 2000. Here we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MAT strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including 8 nonsynonymous changes involving 7 genes. To ascertain whether changes in these genes are selected during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frame-shift mutation in one of the seven genes altered in the human sample, a gene predicted to encode a SWI-SNF chromatin-remodeling complex protein. Both cockatoo and patient strains as well as mouse passaged isolates obtained from brain tissue had a premature stop codon in a homolog of ZFC3, a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and appears to have reverted to a full-length protein in the mouse passaged isolates obtained from lung tissue. The patient strain and mouse passaged isolates show variability in the expression of virulence factors, with differences in capsule size, melanization, and rates on non-lytic expulsion from macrophages observed. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

genomics↗

bioRxiv2021

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 MHC 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. ImportanceCryptococcosis, 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). Here, we examined one such protective protein, Cda2, and using bioinformatics, 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.

immunology↗

bioRxiv2019

Chitosan biosynthesis and virulence in the human fungal pathogen Cryptococcus gattii

Cryptococcus gattii R265 is a hyper-virulent fungal strain responsible for the major outbreak of cryptococcosis in Vancouver Island of British Columbia in 1999. It differs significantly from C. neoformans in its natural environment, its preferred site in the mammalian host, and in the nature and mode of pathogenesis. Our previous studies in C. neoformans have shown that the presence of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in the host, while its absence induces robust immune responses, which in turn facilitate clearance of the fungus and induces a protective response. The results of the present investigation reveal that the cell wall of C. gattii R265 contains 2-3-fold higher amount of chitosan compared to that of C. neoformans. The genes responsible for the biosynthesis of chitosan are highly conserved in the R265 genome; the roles of the three chitin deacetylases (CDA) have however, been modified. To deduce their roles, single, double and a triple CDA deletion strains were constructed in a R265 background and were subjected to mammalian infection studies. Unlike C. neoformans where Cda1 has a discernible role in fungal pathogenesis, in R265 Cda3 is critical for virulence. Deletion of either CDA3 alone (cda3{Delta}) or in combination with either CDA1 (cda1{Delta}3{Delta}) or CDA2 (cda2{Delta}3{Delta}) or both (cda1{Delta}2{Delta}3{Delta}) rendered the yeast cells avirulent and were cleared from the infected host. Moreover, the cda1{Delta}2{Delta}3{Delta} strain of R265 induced a protective response to a subsequent infection with R265. These studies shed more light into the regulation of chitosan biosynthesis of C. gattii and its subsequent effect on fungal virulence.\n\nImportanceThe fungal cell wall is an essential organelle whose components provide the first line of defense against host-induced antifungal activity. Chitosan is one of the carbohydrate polymers in the cell wall that significantly affects the outcome of host-pathogen interaction. Chitosan-deficient strains are avirulent, implicating chitosan as a critical virulence factor. C. gattii R265 is an important fungal pathogen of concern due to its ability to cause infections in individuals with no apparent immune dysfunction and an increasing geographical distribution. Characterization of the fungal cell wall and understanding the contribution of individual molecules of the cell wall matrix to fungal pathogenesis offers new therapeutic avenues for intervention. In this report, we show that the C. gattii R265 strain has evolved alternate regulation of chitosan biosynthesis under both laboratory growth conditions and during mammalian infection compared to that of C. neoformans.

microbiology↗