Monoclonal IgM antibodies raised against Candida albicans Hyr1 provide cross-kingdom protection against Gram negative bacteria

Recent years have seen an unprecedented rise in the incidence of multidrug resistant (MDR) Gram negative bacteria (GNB) 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 GNB are urgently needed. Previously, we have reported that the C. albicans hyphal-regulated protein Hyr1 shares striking 3D structural homology with cell surface proteins of A. baumannii; and active or passive vaccination with rHyr1p-N or anti-Hyr1p polyclonal antibody, respectively; protect mice from Acinetobacter infections. Here, we show that monoclonal antibodies (mAb) generated against Hyr1p, bind to the surface of Acinetobacter as well as K. pneumoniae. The anti-Hyr1 mAb also block damage to primary endothelial cells by the bacteria, and protect mice from lethal pulmonary infections mediated by A. baumannii and K. pneumoniae. Our current studies emphasize the potential of harnessing Hyr1p mAb as a cross-kingdom immunotherapeutic strategy against MDR GNB.


Introduction
Infections caused by multidrug resistant organisms (MDRO) continue to pose a therapeutic challenge. In the past decade, A. baumannii has emerged as one of the most common MDRO of hospital acquired infections, causing a range of diseases from pneumonia to severe blood or wound infections [1][2][3][4][5][6] . Of concern is that 40-70% of A. baumannii isolates are now extensively drug resistant (XDR; i.e. resistant to all antibiotics except colistin and tigecycline), reflecting a >15-fold increase since 2000 1,6-8 . Likewise, the Enterobaceriaceae organism K. pneumoniae causes high rates of morbidity and mortality in critically-ill, hospitalized patients, and in recent years have developed antimicrobial resistance to almost all classes of antibacterial drugs including carbapenamases [9][10][11] . Together, Acinetobacter and carbapenam-resistant K.
pneumoniae (KPC) have been flagged by the CDC as two of the top "Serious Threat Level Pathogens" due to resistance, failure of current standard of treatment, and high mortality rates.
To make matters worse, the existing drug development pipeline against these bugs is unacceptably lean and it is almost certain that the organisms will develop resistance to any new approved antibiotics. Hence, novel strategies to prevent and treat life-threatening infections by the two species are urgently needed.
We have previously exploited innovative computational molecular modeling and bioinformatic strategies to discover novel vaccine and immunotherapy candidates that target more than one high priority pathogen. Our immunotherapeutics-discovery campaign culminated in the identification of Candida albicans Hyr1p, a hyphae regulated cell surface protein that helps the fungus to resist phagocyte killing. Mice vaccinated with Hyr1p are protected from C. albicans infections 12,13 .
Recently, we found that the Hyr1p protein shares striking 3D structural, and immunological homologies to antigens present on the Gram negative bacteria (GNB) A. baumannii, including with the putative hemagglutinin/hemolysin protein FhaB, and a number of siderophore-binding proteins 14 . Polyclonal antibodies (pAbs) against peptides derived from the Hyr1p N-terminus blocked A.
baumannii-mediated lung epithelial cell invasion, and killed the bacterium in vitro 14 . Importantly, anti-Hyr1p pAb completely protected mice from A. baumannii infections. These results provided proof of concept for targeting Hyr1p for developing immunotherapies against GNB, and laid a groundwork for generation and evaluation of the efficacy of anti-Hyr1p monoclonal antibodies against MDR GNB.
In the current study, we generated monoclonal antibodies (mAb) against Hyr1p and show that these mAb not only recognize different clinical isolates of A. baumannii, but also bind to drug resistant K. pneumoniae. We further demonstrate the efficacy of these targeted mAb to block bacterial invasion to host cells, and protect mice against lethal pulmonary infection by both MDR bacteria. Given the alarming rate at which MDRO's are growing as a global threat, a passive vaccination strategy using therapeutic mAbs serves as a highly desirable strategy to combat these difficult to treat infections either as a standalone or adjunctive therapies to antibiotics.

Anti-HYR1 mAbs bind to Gram negative bacteria
We have previously reported that pAb raised against a 14 amino acid peptide of Hyr1p (LKNAVTYDGPVPNN; also called peptide#5) blocked virulence traits of A. baumannii in vitro, and completely protected diabetic and neutropenic mice from Acinetobacter bacteremia and pulmonary infection 14 . Encouraged by these results, and to enhance the therapeutic potential of the antibodies, we developed monoclonal antibodies (mAb) against the same surface-exposed and immunodominant peptide. The mAbs (all produced antibodies belonged to the IgM isotype) were tested for their binding abilities to C. albicans and also the GNB A. baumannii, and K.
pneumoniae. Four individual FITC labeled mAb clones (H1, H2, H3 and H4) at 100 µg/ml were tested against three MDR GNB: K. pneumoniae-RM (KPC-RM, carbapenam-resistant isolate), K. pneumoniae-QR (KP-QR, MDR strain sensitive to carbapenam), and MDR A. baumannii (HUMC-1), and the extent of mAb binding to the bacterial surface was quantified by flow cytometry. The results were compared to isotype matching nonspecific control antibodies. Out of the four clones of mAb tested, H3 and H4 displayed the highest levels of binding to all tested GNB with at least 30 to 300-fold increase compared to the isotype matching control IgM ( Fig   1A). Binding potential was also visualized by a shift in the peaks of the anti-Hyr1 IgM binding versus the isotype matching control antibodies (Fig 1B). The right shift in the peaks of individual mAb also correlated with their respective increase in mean fluorescence of the cells.
Next, we tested the sensitivity of the clones to recognize the GNB. Clone H3 bound to the three organisms even at low mAbs concentrations. Specifically, 30 µg/ml of H3 displayed 50% of binding to both the KP-QR and A. baumannii MDR strain HUMC1, while 3 µg/ml of the mAb bound to at least 10% of the total cells, which was significantly higher than the isotype-matching control IgM (Fig 2). We further evaluated the binding affinity of the two clones H3 and H4 against other drug resistant clinical isolates of Acinetobacter and K. pneumoniae (KPC). The mAbs bound KPC-6, KPC-8, HUMC-6 and HUMC-12 at significantly high affinity compared to the control IgM (S1). These results indicate that binding of mAbs to surface of the tested GNB is not isolate specific.

mAbs protect host cells from damage by GNB
Our previous studies demonstrated that anti-Hyr1 pAbs could not only bind to, but also inhibit A.
baumannii's ability to damage mammalian cells 14  μ g/ml of mAbs prevented 70% of damage to A549 cells by the KPC (Fig 3A). The two mAb also protected the lung cell line from damage by other clinical isolates of GNB such as, HUMC-6 and KPC-8 (S2). Consistent with these results, both mAb at 15 µg/ml resulted in ~40-70% inhibition of A. baumannii HUMC1-or KP-QR-mediated damage to the primary human vascular endothelial cells (HUVEC), respectively (Fig 3B). However, it took a higher mAb concentration, 30 µg/ml, to protect HUVECs from KPC-RM. The mAbs were also found to be active in protecting HUVEC cells from two other clinical isolates, A. baumannii HUMC-6 and KPC-8 (Fig S2). Furthermore, we found that mAb H3 was capable of directly killing KP-QR and HUMC1, but not another drug resistant GNB Pseudomonas aeruginosa (PA) which does not have surface proteins homologous to Hyr1p (Fig 3C). Interestingly, KPC-RM was completely resistant to direct killing by the both the mAb clones H3 and H4 (S3A). Overall, these results show that mAbs raised against Hyr1p peptide 5 bind to MDR A. baumannii and K. pneumoniae strains, block their ability to damage host cells, and directly kill the bacteria, in vitro.

Anti-Hyr1 mAbs protect mice from pulmonary infection caused by K. pneumoniae and A.
baumannii Given that the mAbs bound to GNB and neutralized their ability to damage host cells in vitro, we tested their ability in protecting mice from GNB infections. Pneumonia is a major manifestation of the disease caused by both K. pneumoniae and A. baumannii [15][16][17][18] .
Thus, we evaluated H3 and H4 for their potential to protect against pulmonary disease caused by A. baumannii HUMC1. Although benign in immunocompetent individuals, A. baumannii can cause life-threatening pneumonia in immunosuppressed hospitalized patients 18 . Thus, we utilized a neutropenic mouse model to infect mice with HUMC1, via inhalation. The mAb were administered on Day +1, and +4, relative to infection mice at a dose of 30 μ g administered intraperitoneally. Placebo mice were treated similarly with an isotype matched control IgM.
Compared to the placebo-treated mice, mAb H4 resulted in a high 70% overall survival versus 20% overall survival for placebo mice. Impressively, complete protection (100% survival) was elicited in mice receiving H3 mAb (Fig 4A). Surviving mice appeared healthy on Day +21 when the experiment was terminated.
We next evaluated the efficacy of mAb in a similar pulmonary mouse model of K. pneumoniae infection. Our in vivo optimization studies have shown that KP-QR exhibits pronounced lethality even in healthy non-immunosuppressed mice, while KPC-RM are avirulent despite high cell numbers used for infection (S3B). Thus, we evaluated the protective effect of mAb against the KP-QR-mediated pneumonia in mice. Immunocompetent mice were infected intratracheally with KP-QR, and therapeutically treated twice (1 day and 4 days after infection) with mAbs, or isotype matching control IgM. Almost 60% of the mice treated with H4 survived the pulmonary infection by KP-QR (p<0.06), while yet again, H3 showed a significantly improved protection (trending to 100% survival in mice) (Fig 4B). Surviving mice appeared healthy at day 21 when the experiment was terminated.
At different time points during infection (+2 days for KP-QR and +4 days for HUMC1), lungs from H3 vaccinated mice were harvested for measurement of bacterial burden. Corroborating the survival data, in comparison to treatment with isotype matching IgM controls, H3 resulted in 1.5-and 3-log reduction in lung bacterial burden of HUMC1 and KP-QR, respectively (P<0.01) ( Fig 4C).
Together, these results demonstrate that therapeutic mAbs exhibit translational potential as a novel treatment option for GNB-associated pneumonia in different hosts (immunocompetent as well as neutropenic).

Discussion
Different pathogens inhabit common settings in the host and depend on similar strategies for adaptation to stress, to proliferate, and to cause pathogenesis. Indeed the fungus C. albicans and certain GNB, such as A. baumannii and K. pneumoniae infect the same patient populations, encompassing those who are immunocompromised and hospitalized, or patients suffering from burn and surgical wounds 7,19,20 . In fact, Candida species airway colonization together with A.
baumannii, during ventilator-associated pneumonia (VAP), are common among ICU patients, and identified as an independent risk factor for development of A. baumannii VAP 21 . Similarly, Candida and Klebsiella are the most frequent pathogens of the respiratory tract of patients with chronic obstructive pulmonary disease (COPD) 22,23 . Above and beyond their association with each other, Candida and GNB also individually cause healthcare-associated infections, often leading to life threatening diseases. The GNB, in particular, have evolved over the years into multidrug resistant entities causing infections that are often incurable 24 . In the last two decades, the incidence of MDR GNB including A. baumannii and K. pneumoniae has been so high, that the standard of public health in many parts of the world is considered equivalent to the pre-antibiotic era 24 . Hence, newer approaches that go further than simply the discovery of new antibiotics are needed to combat the crisis of drug resistance.
Our group uses advanced computational molecular modeling and bioinformatic approaches to discover novel vaccine antigen candidates that target more than one high priority human pathogens 14,25,26 . This strategy, known as unnatural or heterologous immunity, has been previously applied in the development of viral and bacterial vaccines in which an antigen protects against another pathogen from the same or from a different kingdom 25 . We have previously validated this approach by demonstrating cross kingdom immuno-protection against C. albicans and S. aureus, in which the C. albicans cell surface adhesin/invasion proteins (Als family of proteins) share structural and functional homology with MSCRAMMs of S. aureus (e.g. clumping factor A) 27 . A recombinant form of N-terminus of the Als3p (rAls3p-N) elicits robust Tand B-cell responses and protects mice from both Candida and MRSA infections 26,[28][29][30][31][32][33] . Most recently, we reported that another hyphal cell surface protein of C. albicans, Hyr1p, has 3-D structural homologies with candidate antigens of the MDR GNB A. baumannii 14 . Indeed, using different mouse models, active or passive immunization (with pAb) targeting either Als3p or Hyr1p, protected mice from S. aureus or A. baumannii infections, respectively 14,26,28 . In particular, antibodies against one specific surface exposed and highly antigenic15-mer peptide of Hyr1p offered the highest protection to host cells from A. baumannii both in vitro and in vivo 14 .
Encouraged by the potential of the pAb, and to further the clinical scope of our studies, we produced and developed mAbs against this highly antigenic peptide of Hyr1. Similar to pAbs, this study showed that mAbs blocked the pathogenesis of both A. baumannii-and K.
pneumoniae-mediated host cell damage and protected mice from pulmonary infections caused by MDR A. baumannii or K. pneumoniae. Initial functional assays revealed that four different clones of mAbs (H1-H4) recognized the two genera of bacteria in in vitro binding assays, with high sensitivity. This binding capacity was extended also to include several MDR clinical isolates of A. baumannii and K. pneumoniae.
The ability of the mAbs (H3 and H4) to block GNB-mediated damage of host cells, was more pronounced in A. baumannii HUMC1, A. baumannii HUMC6, and K. pneumoniae KP-QR, rather than KPC-RM or KPC-8. Four factors, capsule, lipopolysaccharide, fimbriae, and siderophores, have been identified as important for pathogenesis, and resistance of hypervirulent KP strains to antibiotics 34 . Thus, resistance of KPC to killing by mAb could be due to the difference in the capsule structure, or their reported ability to produce a larger repertoire of siderophores 34 .
Indeed, our recent report does emphasize the importance of anti-Hyr1p peptide 5 polyclonal antibodies in blocking iron uptake, leading to killing of the GNB A. baumannii 14 .
Nevertheless, almost a 70% protection from cellular damage was provided by the mAbs in all GNB tested, making mAb a potential therapeutic agent with capacity to block virulence of GNB.
This point becomes even more important considering the mAb (just like the pAb 14 ) can directly kill Acinetobacter and Klebsiella, but not another GNB Pseudomonas whose proteins were not identified to be homologous to Hyr1p 14 . Our recent report on bioinformatic, homology and energy-based modeling strategies described that C. albicans Hyr1p shares striking similarity to A. baumannii FhaB protein, and anti-peptide #5 pAb could bind to FhaB as well as two other proteins on A. baumannii on 2 dimensional Western blotting assays 14 . The other two proteins included the outer membrane protein OmpA, and a ferric siderophore outer membrane binding protein (TonB) 14 . Not surprisingly, the three proteins are conserved in K. pneumoniae displaying >60% homologies to their Acinetobacter counterparts (protein sequence NCBI blast alignment) (S4). Whether these proteins have a significant role in virulence or nutrient uptake -and hence blocking their function would contribute the killing mechanisms afforded by the mAbs, is the subject of ongoing research by our group.
Because the mAbs significantly blocked the capacity of GNB to damage host cells, we further evaluated their potential to protect against pulmonary infections caused by the two respective bacteria. H4 protected >60% of mice from succumbing to pulmonary infection by KP-QR compared to placebo that only had a 20% survival rate. In fact, the mAb H3 provided total (100%) protection to mice from A. baumannii HUMC1 infection, similar to that conferred by pAb 14 . This indicates that mAb likely recognize specific targets on the bacterium that when neutralized, attenuate its ability to survive in the host and cause disease. Certainly, therapeutic treatment by mAbs interfered significantly with the dissemination of KP-QR and HUMC1 to their target organs within the first 2 to 4 days of treatment, versus those treated with control antibodies. This is an evidence for the robustness of the antibodies in abrogating pathogenesis, early into the onset of infection. In summary, we have demonstrated that mAbs raised against anti-peptide 5 of Hyr1p, specifically recognize A. baumannii and K. pneumoniae, and disrupt their ability to cause damage to host cells. Importantly, these mAbs protect mice from lethal pulmonary infections by these two GNB, and have the potential to be used as prophylactic or adjunctive therapy to Cell damage assay: The in vitro ability of mAbs to protect either A549 cells or HUVEC from damage caused by direct contact with bacteria was measured using 51 Cr release assay, modified from previous method 37 . Isolation of HUVEC cells were performed in the lab under a protocol approved by institutional IRB. Because umbilical cords are collected without donor identifiers, the IRB considers them medical waste not subject to informed consent.
Lung A549 cells and HUVEC cells were incubated overnight in 24-well plates with F-12K or RPMI medium containing 6 μ Ci/well of Na 2 51 CrO 4 (ICN Biomedicals, Irvine, CA). The next day, unincorporated tracer was aspirated and the wells were rinsed three times with warm HBSS.
One milliliter of HBSS containing HUMC1 or KP-QR was added to each well at an MOI of 1:100 (cells to bacteria), and the plate was incubated for 3 h at 37 o C in 5% CO 2 . At the end of the incubation, 0.5 ml of medium was gently aspirated from each well, after which the endothelial cells were lysed by the addition of 0.5 ml of 6 N NaOH. The lysed cells were aspirated, and the wells were rinsed twice with RadioWash (Atomic Products, Inc., Shirley, N.Y.). These rinses were added to the lysed cells, and the 51 Cr activity of the medium and the cell lysates was determined. Control wells containing HBSS but no organisms were processed in parallel to measure the spontaneous release of 51Cr. After corrections were made for the differences in the incorporation of 51Cr in each well, the specific release of 51Cr was calculated by the following formula: (2 X experimental release -2 X spontaneous release)/ (total incorporation -2 X spontaneous release).