Beverlie Baquir

Th1-Th17 Cells Mediate Protective Adaptive Immunity against Staphylococcus aureus and Candida albicans Infection in Mice

We sought to define protective mechanisms of immunity to Staphylococcus aureus and Candida albicans bloodstream infections in mice immunized with the recombinant N-terminus of Als3p (rAls3p-N) vaccine plus aluminum hydroxide (Al(OH3) adjuvant, or adjuvant controls. Deficiency of IFN-γ but not IL-17A enhanced susceptibility of control mice to both infections. However, vaccine-induced protective immunity against both infections required CD4+ T-cell-derived IFN-γ and IL-17A, and functional phagocytic effectors. Vaccination primed Th1, Th17, and Th1/17 lymphocytes, which produced pro-inflammatory cytokines that enhanced phagocytic killing of both organisms. Vaccinated, infected mice had increased IFN-γ, IL-17, and KC, increased neutrophil influx, and decreased organism burden in tissues. In summary, rAls3p-N vaccination induced a Th1/Th17 response, resulting in recruitment and activation of phagocytes at sites of infection, and more effective clearance of S. aureus and C. albicans from tissues. Thus, vaccine-mediated adaptive immunity can protect against both infections by targeting microbes for destruction by innate effectors.

Active and Passive Immunization Protects against Lethal, Extreme Drug Resistant-Acinetobacter baumannii Infection

Extreme-drug-resistant (XDR) Acinetobacter baumannii is a rapidly emerging pathogen causing infections with unacceptably high mortality rates due to inadequate available treatment. New methods to prevent and treat such infections are a critical unmet medical need. To conduct a rational vaccine discovery program, OmpA was identified as the primary target of humoral immune response after intravenous infection by A. baumannii in mice. OmpA was >99% conserved at the amino acid level across clinical isolates harvested between 1951 and 2009 from cerebrospinal fluid, blood, lung, and wound infections, including carbapenem-resistant isolates, and was ≥89% conserved among other sequenced strains, but had minimal homology to the human proteome. Vaccination of diabetic mice with recombinant OmpA (rOmpA) with aluminum hydroxide adjuvant markedly improved survival and reduced tissue bacterial burden in mice infected intravenously. Vaccination induced high titers of anti-OmpA antibodies, the levels of which correlated with survival in mice. Passive transfer with immune sera recapitulated protection. Immune sera did not enhance complement-mediated killing but did enhance opsonophagocytic killing of A. baumannii. These results define active and passive immunization strategies to prevent and treat highly lethal, XDR A. baumannii infections.

Inhibition of LpxC Protects Mice from Resistant Acinetobacter baumannii by Modulating Inflammation and Enhancing Phagocytosis

ABSTRACT New treatments are needed for extensively drug-resistant (XDR) Gram-negative bacilli (GNB), such as Acinetobacter baumannii. Toll-like receptor 4 (TLR4) was previously reported to enhance bacterial clearance of GNB, including A. baumannii. However, here we have shown that 100% of wild-type mice versus 0% of TLR4-deficient mice died of septic shock due to A. baumannii infection, despite having similar tissue bacterial burdens. The strain lipopolysaccharide (LPS) content and TLR4 activation by extracted LPS did not correlate with in vivo virulence, nor did colistin resistance due to LPS phosphoethanolamine modification. However, more-virulent strains shed more LPS during growth than less-virulent strains, resulting in enhanced TLR4 activation. Due to the role of LPS in A. baumannii virulence, an LpxC inhibitor (which affects lipid A biosynthesis) antibiotic was tested. The LpxC inhibitor did not inhibit growth of the bacterium (MIC > 512 µg/ml) but suppressed A. baumannii LPS-mediated activation of TLR4. Treatment of infected mice with the LpxC inhibitor enhanced clearance of the bacteria by enhancing opsonophagocytic killing, reduced serum LPS concentrations and inflammation, and completely protected the mice from lethal infection. These results identify a previously unappreciated potential for the new class of LpxC inhibitor antibiotics to treat XDR A. baumannii infections. Furthermore, they have far-reaching implications for pathogenesis and treatment of infections caused by GNB and for the discovery of novel antibiotics not detected by standard in vitro screens. IMPORTANCE Novel treatments are needed for infections caused by Acinetobacter baumannii, a Gram-negative bacterium that is extremely antibiotic resistant. The current study was undertaken to understand the immunopathogenesis of these infections, as a basis for defining novel treatments. The primary strain characteristic that differentiated virulent from less-virulent strains was shedding of Gram-negative lipopolysaccharide (LPS) during growth. A novel class of antibiotics, called LpxC inhibitors, block LPS synthesis, but these drugs do not demonstrate the ability to kill A. baumannii in vitro. We found that an LpxC inhibitor blocked the ability of bacteria to activate the sepsis cascade, enhanced opsonophagocytic killing of the bacteria, and protected mice from lethal infection. Thus, an entire new class of antibiotics which is already in development has heretofore-unrecognized potential to treat A. baumannii infections. Furthermore, standard antibiotic screens based on in vitro killing failed to detect this treatment potential of LpxC inhibitors for A. baumannii infections. Novel treatments are needed for infections caused by Acinetobacter baumannii, a Gram-negative bacterium that is extremely antibiotic resistant. The current study was undertaken to understand the immunopathogenesis of these infections, as a basis for defining novel treatments. The primary strain characteristic that differentiated virulent from less-virulent strains was shedding of Gram-negative lipopolysaccharide (LPS) during growth. A novel class of antibiotics, called LpxC inhibitors, block LPS synthesis, but these drugs do not demonstrate the ability to kill A. baumannii in vitro. We found that an LpxC inhibitor blocked the ability of bacteria to activate the sepsis cascade, enhanced opsonophagocytic killing of the bacteria, and protected mice from lethal infection. Thus, an entire new class of antibiotics which is already in development has heretofore-unrecognized potential to treat A. baumannii infections. Furthermore, standard antibiotic screens based on in vitro killing failed to detect this treatment potential of LpxC inhibitors for A. baumannii infections.

Immunological surrogate marker of rAls3p-N vaccine-induced protection against Staphylococcus aureus

Dear Editor, We have reported that a vaccine based on the recombinant N-terminus of the candidal surface adhesin, Als3p (rAls3p-N), induced cross-kingdom protection against otherwise lethal intravenous infection caused by the bacterium, Staphylococcus aureus (Spellberg et al., 2008). Vaccine efficacy was mediated by CD3 lymphocytes, predominantly with a T helper type 1 (Th1) phenotype; humoral immunity was neither necessary nor sufficient to mediate protection. Nevertheless, because Th1 cells do provide help to B cells and stimulate production of antibody, and antibody titers are elevated even during polarized type 1 immune responses (Mosmann et al., 1986; Smith et al., 2000), we hypothesized that antibody titers could serve as surrogate markers of protection for the rAls3p-N vaccine, despite the fact that the vaccine mediates protection by inducing cell-mediated immunity. To determine the potential for antibody titers to serve as accurate surrogate markers of protection for our antiS. aureus vaccine, we studied serum banked from 32 mice vaccinated and infected in the previously published study (Spellberg et al., 2008). Antibody titers were determined by enzyme-linked immunosorbent assay, as previously described (Spellberg et al., 2005, 2006), and compared with the known time to death from infection. A single dose of vaccine plus 0.1% aluminum hydroxide adjuvant [Al(OH)3] resulted in a >20-fold increase in median titers of both total immunoglobulin G (IgG) and IgG2a antibodies compared with Al(OH)3 alone (IgG titers = 1 : 4800 vs. 1 : 200; IgG2a titers = 1 : 1600 vs. 1 : 50, P < 0.01 for both comparisons). Two doses of vaccine resulted in an additional 10-fold rise in median titers of IgG antibodies [IgG titers = 1 : 51 200 vs. 1 : 200; P < 0.01 vs. both Al(OH)3 alone and one dose of vaccine]. Surviving vaccinated and adjuvant control mice had higher anti-rAls3p-N IgG (12 800 vs. 200; P = 0.03) and IgG2a (3200 vs. 200; P = 0.002) titers than those that died from infection. However, IgG titers were similar between vaccinated mice (exclusive of adjuvant control mice) that did or did not survive subsequent infection (25 600 vs. 25 600; P = 0.9). Furthermore, there was no correlation between individual IgG antibody titers and time to death when comparing all mice (ρ = 0.2, P = 0.1), all vaccinated mice [including both one and two doses of rAls3p-N plus Al(OH)3] (ρ = 0.01, P = 0.7), or in mice vaccinated with one dose (ρ = 0.2, P = 0.4) or with two doses (ρ =−0.2, P = 0.99). In contrast, in vaccinated mice (exclusive of adjuvant control mice) anti-rAls3p-N IgG2a titers were higher in survivors than nonsurvivors (4800 vs. 800; P = 0.01). Furthermore, IgG2a titers correlated with time to death (ρ = 0.4, P = 0.02), even when excluding adjuvant control mice and analyzing only mice vaccinated with either one or two doses (ρ = 0.7, P = 0.003), or mice vaccinated with one dose (ρ = 0.7, P = 0.049), or with two doses (ρ = 0.8, P = 0.02) of rAls3p-N plus Al(OH)3. Similarly, the ratio of IgG2a/IgG antibody titers in individual mice vaccinated with rAls3p-N plus Al(OH)3 was higher in survivors than nonsurvivors (median ratio = 0.5 vs. 0.03; P = 0.049). IgG2a/IgG ratios correlated with time to death when analyzing just mice vaccinated with either one or two doses (ρ = 0.5, P = 0.02), or in mice vaccinated with one dose (ρ = 0.8, P = 0.03), or with two doses (ρ = 0.8, P = 0.02) of rAls3p-N plus Al(OH)3. No IgG titer had a positive or negative predictive value, or overall accuracy, of >70%, at predicting survival in vaccinated mice (exclusive of adjuvant control mice) (Table 1). In contrast, an IgG2a titer of ≥3200 had 86% positive and negative predictive values, and an overall accuracy of 86% for survival. An IgG2a titer of ≥1 : 6400 had 100% positive predictive value for survival, but the negative predictive value fell to 64%, resulting in an overall accuracy of 73%. Table 1 Accuracy of antibody titer cut-offs for predicting survival in mice vaccinated with one or two doses of rAls3p-N+Al(OH)3 and infected with Staphylococcus aureus In this set of experiments, humoral immune responses served as accurate surrogate markers of anti-rAls3p-N vaccine protection even though the mechanism of the vaccine has been previously established to be independent of humoral immunity. In a recent review, Plotkin (2008) emphasized the difference between a ‘correlate’ and a ‘surrogate’ of vaccine protection. He defined a correlate as ‘A specific immune response to a vaccine that is closely related to protection against infection, disease, or other defined end point,’ while he defined a surrogate as ‘A quantified specific immune response to a vaccine that is not in itself protective but that substitutes for the true (perhaps unknown) correlate.’ Our data are concordant with Plotkin’s definition of a surrogate marker of vaccine protection, and underscore that a surrogate of protection in a set of experiments does not necessarily imply a connection between the immune assay read-out and the mechanism by which the vaccine induces protection. We analyzed IgG2a titers as a subset of IgG titers because interferon-γ(IFN-γ) induces class switching to IgG2a (Snapper et al., 1988; Stevens et al., 1988), and hence IgG2a antibodies are produced in the setting of type 1 immunity (Spellberg & Edwards, 2001). Although Al(OH)3 is not typically considered a type 1-polarizing adjuvant, we have previously reported that when administered with rAls3p-N, Al(OH)3 adjuvant resulted in strong priming of type 1 inflammatory cytokines in reactive murine splenocytes (Lin et al., 2008). Furthermore, a recent study reported that a major mechanism by which aluminum adjuvants enhance immune responses was by direct agonism of an intracellular pattern recognition receptor, Nalp3 (Eisenbarth et al., 2008). Activation of Nalp3 by aluminum particles resulted in expression of several cytokines, including interleukin-1β (IL-1β) and IL-18, which are proinflammatory. Indeed IL-18 is a powerful inducer of IFN-γ expression, and hence can direct subsequent Th1 cytokine production (Sugawara, 2000). These results suggested that aluminum adjuvants have the potential to promote both Th1 and Th2 immune responses, and are concordant with our finding of strong induction of IgG2a antibody titers in mice vaccinated with Al(OH)3 adjuvant. Our data support the concept that facile measurement of antibody titers, particularly of subsets induced by proinflammatory cytokines, may also serve as useful surrogate markers of protection even for vaccines that function by inducing cell-mediated, rather than humoral, immunity.

Macrophage killing of bacterial and fungal pathogens is not inhibited by intense intracellular accumulation of the lipoglycopeptide antibiotic oritavancin

Intact phagocytic effector function is fundamental to host defense against microbial pathogens. Concern has been raised regarding the potential that accumulation of certain agents, including cationic amphiphilic antibiotics, within macrophages could cause a mixed-lipid storage disorder, resulting in macrophage dysfunction in recipients. The ability of 2 macrophage cell lines (HL-60; RAW 264.7) to kill archetypal Gram-positive (Staphylococcus aureus), Gram-negative (Acinetobacter baumannii), and fungal (Candida albicans) pathogens was tested following exposure of the macrophages to the lipoglycopeptide antibiotic oritavancin. Oritavancin did not affect killing of C. albicans but markedly enhanced killing of S. aureus by both macrophages. Oritavancin modestly reduced killing of A. baumannii by HL-60 cells but not by RAW 264.7 cells. Thus, macrophage killing of microbes remains intact despite substantial intracellular accumulation with a lipoglycopeptide antibiotic.

Immunological Reactivity of Blood from Healthy Humans to the rAls3p-N Vaccine Protein

We determined reactivity of human blood to a vaccine based on the recombinant N-terminus of candidal Als3p (rAls3p-N) in preparation for future clinical trials. Healthy donor plasma had high immunoglobulin G titers (median, 1:51,200) and lower immunoglobulin A (median, 1:3,200) and immunoglobulin E (median, 1:128) titers to rAls3p-N by enzyme-linked immunosorbent assay. rAls3p-N stimulated interferon gamma (IFN-gamma) and interleukin (IL)-17, but not IL-4, from donor lymphocytes by enzyme-linked immunosorbent spot assay and IL-12 p70, IFN-gamma, IL-17, and IL-10 by cytometric bead array. Donors reacted to diverse immunodominant epitopes. Thus, facile humoral and cellular assays can monitor immune responses to the rAls3p-N vaccine in planned clinical trials.

Safety and Efficacy of Activated Transfected Killer Cells for Neutropenic Fungal Infections

BACKGROUND Invasive fungal infections cause considerable morbidity and mortality in neutropenic patients. White blood cell transfusions are a promising treatment for such infections, but technical barriers have prevented their widespread use. METHODS To recapitulate white blood cell transfusions, we are developing a cell-based immunotherapy using a phagocytic cell line, HL-60. We sought to stably transfect HL-60 cells with a suicide trap (herpes simplex virus thymidine kinase), to enable purging of the cells when desired, and a bioluminescence marker, to track the cells in vivo in mice. RESULTS Transfection was stable despite 20 months of continuous culture or storage in liquid nitrogen. Activation of these transfected cells with retinoic acid and dimethyl sulfamethoxazole enhanced their microbicidal effects. Activated transfected killer (ATAK) cells were completely eliminated after exposure to ganciclovir, confirming function of the suicide trap. ATAK cells improved the survival of neutropenic mice with lethal disseminated candidiasis and inhalational aspergillosis. Bioluminescence and histopathologic analysis confirmed that the cells were purged from surviving mice after ganciclovir treatment. Comprehensive necropsy, histopathology, and metabolomic analysis revealed no toxicity of the cells. CONCLUSIONS These results lay the groundwork for continued translational development of this promising, novel technology for the treatment of refractory infections in neutropenic hosts.

Considerable Differences in Vaccine Immunogenicities and Efficacies Related to the Diluent Used for Aluminum Hydroxide Adjuvant

ABSTRACT We are developing an anticandidal vaccine using the recombinant N terminus of Als3p (rAls3p-N). We report that although more rAls3p-N was bound by aluminum hydroxide diluted in saline than by aluminum hydroxide diluted in phosphate-buffered saline (PBS), its immunogenicity and efficacy were superior in PBS. Thus, protein binding, by itself, may not predict the efficacy of some vaccines with aluminum adjuvants.

Luminescent-Activated Transfected Killer Cells to Monitor Leukocyte Trafficking During Systemic Bacterial and Fungal Infection

BACKGROUND Activated transfected killer (ATAK) cells are immortal phagocytes transfected with a luminescence reporter that effectively treat lethal infections in neutropenic mice. Their in vivo trafficking, lifespan, and immunogenicity are unknown. METHODS Mice were made neutropenic; infected or not with Staphylococcus aureus, Acinetobacter baumannii, Candida albicans, or Aspergillus fumigatus; and treated intraperitoneally with ATAK cells. Cell trafficking and lifespan were assessed by in vivo imaging and reverse transcription-polymerase chain reaction. RESULTS In uninfected neutropenic mice, ATAK cells spread from the mesentery into visceral organs on days 1-3. Splenic accumulation of ATAK cells increased at day 1 after infection with S. aureus and A. baumannii, and kidney accumulation increased in mice infected with C. albicans. Lung accumulation was seen at day 3 in mice infected by inhalation with A. fumigatus. By day 8, coincident with increasing anti-ATAK antibodies, luminescence signal was lost and there was no detectable mRNA transcription from ATAK cells. CONCLUSIONS ATAK cells accumulated in target organs with distinct profiles, depending on the microbial etiology of infection. Finally, generation of an anti-ATAK immune response may provide an important safety mechanism that helps clear the cells from the host as the marrow recovers.