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Background We previously reported developing 2 anticapsular monoclonal antibodies (mAbs) as a novel therapy for Acinetobacter baumannii infections. We sought to determine whether a bispecific mAb (bsAb) could improve avidity and efficacy while maximizing strain coverage in one molecule. Methods Humanized mAb 65 was cloned into a single-chain variable fragment and attached to humanized mAb C8, combining their paratopes into a single bsAb (C73). We tested bsAb C73’s strain coverage, binding affinity, ex vivo opsonic activity, and in vivo efficacy compared to each mAb alone and combined. Results The bsAb demonstrated strain coverage, binding affinity, opsonization, and in vivo efficacy superior to either original mAb alone or combined. Conclusions A humanized bsAb targeting distinct A. baumannii capsule moieties enabled potent and effective coverage of disparate A. baumannii clinical isolates. The bsAb enhances feasibility of development by minimizing the number of components of a promising novel therapeutic for these difficult-to-treat infections.

Acinetobacter baumannii is an extremely drug-resistant pathogen necessitating the development of new therapies. We seek to generate a cocktail of monoclonal antibodies (MAbs) that can target the full diversity of A. baumannii isolates.

Invasive pulmonary aspergillosis caused by the ubiquitous mold Aspergillus fumigatus is a major threat to immunocompromised patients, causing unacceptably high mortality despite standard of care treatment, and costing an estimated $1.2 billion annually. Treatment for this disease has been complicated by the emergence of azole resistant strains of A. fumigatus, rendering first-line antifungal therapy ineffective. The difficulties in treating infected patients using currently available drugs make immunotherapeutic vaccination an attractive option. Here, we demonstrate the efficacy of VesiVax® adjuvant liposomes, consisting of a combination of two individual liposome preparations, to which two recombinant A. fumigatus surface antigens, Asp f 3 and Asp f 9 (VesiVax® Af3/9), have been chemically conjugated. Using a murine model, we demonstrate that VesiVax® Af3/9 is protective against infection by azole resistant strains of A. fumigatus in both steroid-suppressed and neutropenic mice as quantified by improved survival and reduced fungal burden in the lungs. This protection correlates with upregulation of IL-4 produced by splenocytes, and the presence of Asp f 3 and Asp f 9 specific IgG2a antibodies in the serum of mice given VesiVax® Af3/9. Furthermore, mice given VesiVax® Af3/9 with a subsequent course of liposomal amphotericin B (AmBisome®) had improved survival over those given either treatment alone, indicating a benefit to VesiVax® Af3/9 vaccination even in the case of infections that require follow-up antifungal treatment. These data demonstrate that prophylactic vaccination with VesiVax® Af3/9 is a promising method of protection against invasive pulmonary aspergillosis even as the changing face of the disease renders current therapies ineffective.

Extremely drug-resistant (XDR) Acinetobacter baumannii is a notorious and frequently encountered pathogen demanding novel therapeutic interventions. An initial monoclonal antibody (MAb), C8, raised against A. baumannii capsule, proved a highly effective treatment against a minority of clinical isolates. To overcome this limitation, we broadened coverage by developing a second antibody for use in a combination regimen. We sought to develop an additional anti-A. baumannii MAb through hybridoma technology by immunizing mice with sublethal inocula of virulent, XDR clinical isolates not bound by MAb C8. We identified a new antibacterial MAb, 65, which bound to strains in a pattern distinct from and complementary to that of MAb C8. MAb 65 enhanced macrophage opsonophagocytosis of targeted strains and markedly improved survival in lethal bacteremic sepsis and aspiration pneumonia murine models of A. baumannii infection. MAb 65 was also synergistic with colistin, substantially enhancing protection compared to monotherapy. Treatment with MAb 65 significantly reduced blood bacterial density, ameliorated cytokine production (interleukin-1β [IL-1β], IL-6, IL-10, and tumor necrosis factor), and sepsis biomarkers. We describe a novel MAb targeting A. baumannii that broadens immunotherapeutic strain coverage, is highly potent and effective, and synergistically improves outcomes in combination with antibiotics.

Monoclonal antibodies (mAbs) are gaining significant momentum as novel therapeutics for infections caused by antibiotic-resistant bacteria. We evaluated the mechanism by which antibacterial mAb therapy protects against Acinetobacter baumannii infections. Anticapsular mAb enhanced macrophage opsonophagocytosis and rescued mice from lethal infections by harnessing complement, macrophages, and neutrophils; however, the degree of bacterial burden did not correlate with survival. Furthermore, mAb therapy reduced proinflammatory (interleukin-1β [IL-1β], IL-6, tumor necrosis factor-α [TNF-α]) and anti-inflammatory (IL-10) cytokines, which correlated inversely with survival. Although disrupting IL-10 abrogated the survival advantage conferred by the mAb, IL-10–knockout mice treated with mAb could still survive if TNF-α production was suppressed directly (via anti–TNF-α neutralizing antibody) or indirectly (via macrophage depletion). Thus, even for a mAb that enhances microbial clearance via opsonophagocytosis, clinical efficacy required modulation of pro- and anti-inflammatory cytokines. These findings may inform future mAb development targeting bacteria that trigger the sepsis cascade.

Background Antifungal treatment for pulmonary aspergillosis is more difficult if the fungal strain causing the infection is azole resistant. To investigate this problem, we used a murine model of pulmonary aspergillosis caused by azole-resistant Aspergillus fumigatus strains V29 and V45, and compared treatment with voriconazole (Vr, oral 40 mg/kg, bid) or liposomal amphotericin B (L-AmB, 5 mg/kg, IV) used alone or in combination. Methods Mice (n = 14/gp) were immunosuppressed with 24 mg/kg triamcinolone acetonide, IP, d-3, d-1, and d+1 relative to fungal challenge (d0). For 2 groups, Vr was given prophylactically (proph) d-3, d-2, d-1 followed by L-AmB or buffer, d+1, d+2, and d+3. The other groups were given Vr, L-AmB, Vr+L-AmB, or buffer d+1, d+2, and d+3. On d0, mice were given 1.3 to 1.6 × 107A. fumigatus spores intranasally (Vr MIC = 64 μg/mL, V29; Vr MIC = 8 μg/mL, V45). On d+3, lungs were collected from 7 mice/gp and fungal burden determined by plating for colony forming units; 7 mice/gp were then monitored for morbidity to d+21. Results Optimum treatment was observed when Vr was given proph, followed by L-AmB post-challenge (Vr/L-AmB), with better survival (100%) for both fungal strains vs. buffer or Vr post-challenge (P ≤ 0.04); for V29, significantly better survival was also seen with Vr/L-AmB vs. L-AmB or Vr+L-AmB post-challenge (P ≤ 0.01). For strain V45, lung fungal burden was significantly lower for Vr/L-AmB versus all other treatments (P ≤ 0.04), while for strain V29, fungal burden was lower for the Vf/L-AmB and L-AmB post-challenge groups versus the other groups, but the differences were not significant. Notably, although the lung fungal burden with Vr proph and Vr postchallenge were both similar to the buffer control, Vr proph yielded significantly better survival than Vr post-challenge (P ≤ 0.001). Conclusion These preclinical observations demonstrate that combining L-AmB with Vr for the postchallenge treatment of pulmonary aspergillosis caused by azole-resistant strains is not an effective therapeutic option. However, the results do show that Vr proph, but not Vr postchallenge, can have some limited antifungal activity, and can significantly enhance the antifungal effects of post-challenge L-AmB. This regimen could be considered in areas where there is a high incidence of azole-resistant A. fumigatus. Disclosures All authors: No reported disclosures.