Valentina Avanesian

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.

Efficacy of the Anti-Candida rAls3p-N or rAls1p-N Vaccines against Disseminated and Mucosal Candidiasis

We have shown that vaccination with the recombinant N terminus of Als1p (rAls1p-N) protects mice against disseminated and oropharyngeal candidiasis. We now report that vaccination of mice with a related candidate, rAls3p-N, induces a broader antibody response than rAls1p-N and a similar cell-mediated immune response. The rAls3p-N vaccine was equally as effective as rAls1p-N against disseminated candidiasis but was more effective than rAls1p-N against oropharyngeal or vaginal candidiasis. Antibody titers did not correlate with protection against disseminated candidiasis, but delayed-type hypersensitivity did. The rAls3p-N vaccine is a promising new vaccine candidate for further exploration to prevent systemic and mucosal candidal infections.

The Antifungal Vaccine Derived from the Recombinant N Terminus of Als3p Protects Mice against the Bacterium Staphylococcus aureus

ABSTRACT Vaccination with the recombinant N terminus of the candidal adhesin Als3p (rAls3p-N) protects mice from lethal candidemia. Candidal Als3p also is structurally similar to the microbial surface components recognizing adhesive matrix molecule adhesin, clumping factor, from Staphylococcus aureus. To determine the potential for cross-kingdom vaccination, we immunized mice with rAls3p-N or negative control proteins and challenged them via the tail vein with S. aureus or other gram-positive or gram-negative pathogens. The rAls3p-N vaccine, but neither tetanus toxoid nor a related Als protein (Als5p), improved the survival of vaccinated mice subsequently infected with multiple clinical isolates of S. aureus, including methicillin-resistant strains. The rAls3p-N vaccine was effective against S. aureus when combined with aluminum hydroxide adjuvant. However, the vaccine did not improve the survival of mice infected with other bacterial pathogens. Vaccinated, infected mice mounted moderated type 1 immune responses. T lymphocyte-deficient mice were more susceptible to S. aureus infection, but B lymphocyte-deficient mice were not. Furthermore, T but not B lymphocytes from vaccinated mice mediated protection in adoptive transfer studies. The passive transfer of immune serum was not protective. These data provide the foundation for cross-kingdom vaccine development against S. aureus and Candida, which collectively cause 200,000 bloodstream infections resulting in ≥40,000 to 50,000 deaths annually in the United States alone.

Antibody Titer Threshold Predicts Anti-Candidal Vaccine Efficacy Even though the Mechanism of Protection Is Induction of Cell-Mediated Immunity

We previously reported that vaccination with Freunds adjuvant plus the recombinant N-terminus of the candidal adhesin, Als3p (rAls3p-N), protects mice from disseminated candidiasis. Here we report that the rAls3p-N vaccine is effective when combined with aluminum hydroxide adjuvant. Antibody titers of > or =1:6400 accurately predicted protection from infection. Nevertheless, neither B lymphocytes nor serum from immunized animals transferred protection to vaccine-naive animals. In contrast, CD3(+), CD4(+), or CD8(+) T lymphocytes from immunized animals transferred protection, and the vaccine was efficacious in IL-4-deficient mice but not in IFN-gamma-deficient mice. These data have significant implications for the development and interpretation of vaccine surrogate markers.

The Anti-Candida Vaccine Based on the Recombinant N-Terminal Domain of Als1p Is Broadly Active against Disseminated Candidiasis

ABSTRACT We have previously shown that vaccination with a vaccine based on the recombinant N-terminal domain of Als1p (rAls1p-N) protected BALB/c mice against disseminated infection caused by a single strain of Candida albicans (A. S. Ibrahim, B. J. Spellberg, V. Avenissian, Y. Fu, S. G. Filler, and J. E. Edwards, Jr., Infect. Immun. 73:999-1005, 2005, and B. J. Spellberg, A. S. Ibrahim, V. Avenissian, S. G. Filler, C. Myers, Y. Fu, and J. E. Edwards, Jr., Infect. Immun. 73:6191-6193, 2005). Here we show that the rAls1p-N vaccine also improves survival of outbred mice from disseminated candidiasis and that it is active against multiple virulent strains of C. albicans and non-C. albicans spp.

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.

Optimization of a myeloid cell transfusion strategy for infected neutropenic hosts

Although granulocyte transfusion is a logical, therapeutic option for neutropenic patients with refractory infections, significant technical barriers have prevented its widespread use. A novel phagocyte transfusion strategy has been developed based on activation of a human myeloid cell line HL‐60. To further define the potential for HL‐60 cells to recapitulate white cell transfusions, a shortened duration of activation was evaluated, facile quality control markers were defined, and the impact of low‐dose irradiation on cell function was determined. Three days of activation resulted in increased cell viability and in vitro candidacidal capacity but with slightly higher cell replication compared with 7 days of activation. Cell viability and several flow cytometric measurements were accurate, quality control markers for HL‐60 activation. In combination with activation, low‐dose irradiation abrogated replication while sparing the candidacidal effects of the HL‐60 cells. Infusion of irradiated, activated HL‐60 cells improved survival of neutropenic, candidemic mice significantly. In summary, activated, irradiated HL‐60 cells are microbicidal, have virtually no replicative capacity, and are safe and effective at protecting neutropenic mice against an otherwise 100% fatal candidal infection. With continued development, this strategy to recapitulate neutrophil functions has the potential to serve as an effective alternative to granulocyte transfusions.

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.