Different pathogens share similar medical settings and rely on similar virulence strategies to cause infections. We have previously applied 3-D computational modeling and bioinformatics to discover novel antigens that target more than one human pathogen. Active and passive immunization with the recombinant N-terminus of Candida albicans Hyr1 (rHyr1p-N) protect mice against lethal candidemia. Here we determine that Hyr1p shares homology with cell surface proteins of the multidrug resistant Gram negative bacterium, Acinetobacter baumannii including hemagglutinin (FhaB) and outer membrane protein A (OmpA). The A. baumannii OmpA binds to C. albicans Hyr1p, leading to a mixed species biofilm. Deletion of HYR1, or blocking of Hyr1p using polyclonal antibodies, significantly reduce A. baumannii binding to C. albicans hyphae. Furthermore, active vaccination with rHyr1p-N or passive immunization with polyclonal antibodies raised against specific peptide motifs of rHyr1p-N markedly improve survival of diabetic or neutropenic mice infected with A. baumannii bacteremia or pneumonia. Antibody raised against one particular peptide of the rHyr1p-N sequence (peptide 5) confers majority of the protection through blocking A. baumannii invasion of host cells and inducing death of the bacterium by a putative iron starvation mechanism. Anti-Hyr1 peptide 5 antibodies also mitigate A. baumannii /C. albicans mixed biofilm formation in vitro. Consistent with our bioinformatic analysis and structural modeling of Hyr1p, anti-Hyr1p peptide 5 antibodies bound to A. baumannii FhaB, OmpA, and an outer membrane siderophore binding protein. Our studies highlight the concept of cross-kingdom vaccine protection against high priority human pathogens such as A. baumannii and C. albicans that share similar ecological niches in immunocompromised patients. Different pathogens share similar medical settings and rely on similar virulence strategies to cause infections. We have applied computational modeling and bioinformatics to discover novel antigens that target organisms sharing ecological niches in the intensive care units (ICUs): the fungus Candida albicans, and the multidrug resistant (MDR) Gram-negative bacterium, Acinetobacter baumannii. We identified that C. albicans hyphal wall protein Hyr1p shares significant structural homology to A. baumannii cell surface proteins, and is the receptor for A. baumannii binding to the fungus. Active vaccination (with rHyr1p) or passive immunization (anti-Hyr1p antibodies) protect mice from A. baumannii bacteremia and pneumonia. Anti-Hyr1p antibodies act synergistically with antibiotics in abrogating mixed species biofilms. Our groundbreaking studies reveal novel cross-kingdom immunotherapeutic strategies that target healthcare-associated MDR infections.

The fast development of multi-drug resistant (MDR) organisms increasingly threatens global health and well-being. Plant natural products have been known for centuries as alternative medicines that can possess pharmacological characteristics, including antimicrobial activities. The antimicrobial activities of essential oil (Calli oil) extracted from the Calligonum comosum plant by hydro-steam distillation was tested either alone or when combined with lawsone, a henna plant naphthoquinone, against MDR microbes. Lawsone showed significant antimicrobial activities against MDR pathogens in the range of 200–300 µg/mL. Furthermore, Calli oil showed significant antimicrobial activities against MDR bacteria in the range of 180–200 µg/mL, Candida at 220–240 µg/mL and spore-forming Rhizopus fungus at 250 µg/mL. Calli oil’s inhibition effect on Rhizopus, the major cause of the lethal infection mucormycosis, stands for 72 h, followed by an extended irreversible white sporulation effect. The combination of Calli oil with lawsone enhanced the antimicrobial activities of each individual alone by at least three-fold, while incorporation of both natural products in a liposome reduced their toxicity by four- to eight-fold, while maintaining the augmented efficacy of the combination treatment. We map the antimicrobial activity of Calli oil to its major component, a benzaldehyde derivative. The findings from this study demonstrate that formulations containing essential oils have the potential in the future to overcome antimicrobial resistance.

Candida auris is a multi-drug-resistant fungal pathogen that can survive outside the host and can easily spread and colonize the healthcare environment, medical devices, and human skin. C. auris causes serious life-threatening infections (up to 60% mortality) in immunosuppressed patients staying in such contaminated healthcare facilities. Some isolates of C. auris are resistant to virtually all clinically available antifungal drugs. Therefore, alternative therapeutic approaches are urgently needed. Using in silico protein modeling and analysis, we identified a highly immunogenic and surface-exposed epitope that is conserved between C. albicans hyphal-regulated protein (Cal-Hyr1p) and Hyr1p/Iff-like proteins in C. auris (Cau-HILp). We generated monoclonal antibodies (MAb) against this Cal-Hyr1p epitope, which recognized several clinical isolates of C. auris representing all four clades. An anti-Hyr1p MAb prevented biofilm formation and enhanced opsonophagocytic killing of C. auris by macrophages. When tested for in vivo efficacy, anti-Hyr1p MAb protected 55% of mice against lethal systemic C. auris infection and showed significantly less fungal burden. Our study is highly clinically relevant and provides an effective alternative therapeutic option to treat infections due to MDR C. auris.

Recent years have seen an unprecedented rise in the incidence of multidrug-resistant (MDR) Gram-negative bacteria (GNBs) such as Acinetobacter and Klebsiella species. In view of the shortage of novel drugs in the pipeline, alternative strategies to prevent, and treat infections by GNBs are urgently needed. Previously, we have reported that the Candida albicans hypha-regulated protein Hyr1 shares striking three-dimensional structural homology with cell surface proteins of Acinetobacter baumannii. Moreover, active vaccination with rHyr1p-N or passive immunization with anti-Hyr1p polyclonal antibody protects mice from Acinetobacter infection. In the present study, we use molecular modeling to guide design of monoclonal antibodies (mAbs) generated against Hyr1p and show them to bind to priority surface antigens of Acinetobacter and Klebsiella pneumoniae. The anti-Hyr1 mAbs block damage to primary endothelial cells induced by the bacteria and protect mice from lethal pulmonary infections mediated by A. baumannii or K. pneumoniae. Our current studies emphasize the potential of harnessing Hyr1p mAbs as a cross-kingdom immunotherapeutic strategy against MDR GNBs.

Mucormycosis is a fungal infection caused by Mucorales fungi that cause severe disease and fatality, especially in immunocompromised individuals. Although vaccines and immunotherapeutics have been successful in combating viral and bacterial infections, approved antifungal immunotherapies are yet to be realized. To address this gap, monoclonal antibodies targeting invasive fungal infections have emerged as a promising approach, particularly for immunocompromised patients who are unlikely to maximally benefit from vaccines. The Mucorales spore coat (CotH) proteins have been identified as crucial fungal invasins that bind to glucose-regulated protein 78 (GRP78) and integrins of host barrier cells. Previously, we described a murine monoclonal antibody, anti-CotH C2, which protected diabetic ketoacidosis (DKA) and neutropenic mice from mucormycosis. Here, we advanced the development of the C2 immunoglobulin G1 (IgG1) by humanizing it, establishing a stable Chinese hamster ovary cell line producing the antibody at commercial yields, and carried out optimization of the upstream and downstream manufacturing processes. The resultant humanized IgG1 (VX-01) exhibited a 10-fold increase in binding affinity to CotH proteins and conferred comparable in vitro and in vivo efficacy when compared to C2 antibody. The mechanism of protection was reliant on prevention of angioinvasion and enhancing opsonophagocytic killing. VX-01 demonstrated acceptable safety profiles with no detectable damage to host cells in vitro and weak or moderate binding to only cytoplasmic proteins in ex vivo good laboratory practice–human tissue cross-reactivity studies. Our studies warrant continued development of VX-01 as a promising adjunctive immunotherapy.