Streptococcus pneumoniae (Spn) remains a serious health threat due to growing antibiotic resistance and serotype replacement. Monoclonal antibodies (MAbs) are an alternate approach for combating infections and can be engineered to drive specific effector functions for improved in vivo potency. Here, we characterize a DNA-encoded MAb targeting the conserved pneumococcal histidine triad protein D (PhtD3 DMAb) and evaluate efficacy in a lethal Spn murine infection model. Expression of MAb PhtD3 on DNA plasmid improved overall antibody durability; meanwhile, incorporation of the complement-enhancing E430G Fc-modification increased antibody affinity for C1q and was dose-sparing at challenge. Structural analysis by cryo-EM resolved the binding footprint of PhtD-PhtD3 MAb, suggesting cross-reactivity with other Pht-family proteins while providing rationale for the design of broadly targeting vaccines. We also demonstrate the breadth of MAb PhtD3 against a panel of Spn strains, including multiple drug-resistant isolates. These findings support DNA expression of MAbs and Fc-engineering as a valuable strategy to improve durability and efficacy of antibodies against lung bacterial pathogens.

IntroductionCommunity-acquired pneumonia (CAP) is a global health concern, with 25% of cases attributed to Streptococcus pneumoniae (Spn). Viral infections like influenza A virus (IAV), respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) increase the risk of Spn, leading to severe complications due to compromised host immunity.MethodsWe evaluated the efficacy of an anti-PhtD monoclonal antibody (mAb) cocktail therapy (PhtD3 + 7) in improving survival rates in three viral/bacterial coinfection models: IAV/Spn, hMPV/Spn, and RSV/Spn.ResultsThe PhtD3 + 7 mAb cocktail outperformed antiviral mAbs, resulting in prolonged survival. In the IAV/Spn model, it reduced bacterial titers in blood and lungs by 2-4 logs. In the hMPV/Spn model, PhtD3 + 7 provided greater protection than the hMPV-neutralizing mAb MPV467, significantly reducing bacterial titers. In the RSV/Spn model, PhtD3 + 7 offered slightly better protection than the antiviral mAb D25, uniquely decreasing bacterial titers in blood and lungs.DiscussionGiven the threat of antibiotic resistance, our findings highlight the potential of anti-PhtD mAb therapy as an effective option for treating viral and secondary pneumococcal coinfections.

Parainfluenza virus 3 (PIV3) infection poses a substantial risk to vulnerable groups including infants, the elderly, and immunocompromised individuals, and lacks effective treatments or vaccines. This study focuses on targeting the hemagglutinin-neuraminidase (HN) protein, a structural glycoprotein of PIV3 critical for viral infection and egress. With the objective of targeting these activities of HN, we identified eight neutralizing human monoclonal antibodies (mAbs) with potent effects on viral neutralization, cell-cell fusion inhibition, and complement deposition. Three epitopes on PIV3 HN were delineated and one epitope, Site 2, elicits a mAb with cross-neutralizing ability against PIV1 and PIV3. Cryo-EM revealed the cross-neutralizing mAb utilizes a long CDR3 loop to bind inside the pocket of the sialic acid binding site. Additionally, we resolved the structure of a non-protective mAb binding to Site 1 near the HN:F-interaction site. The potent Site 2-directed mAb demonstrated clinical efficacy in hamsters, reducing viral replication prophylactically and therapeutically. These findings advance our understanding of PIV3 immunity and underscore the significance of targeting HN for clinical therapeutic development against PIV3.

Bone marrow stromal cells (BMSCs) have immunomodulatory activities in numerous species and have been used in clinical trials. BMSCs also make antibacterial agents. Since hepcidin is known to have antimicrobial effects in fish, we wondered if it might also be used as an antimicrobial agent by mammalian BMSCs. In the present study, we show hepcidin expression in both mouse (mBMSC) and human BMSCs (hBMSC). We observed a hBMSC hepcidin-dependent degradation of ferroportin in HEK-293 reporter cells in vitro. In human and mouse bone marrows (BM) we detected hepcidin-positive BMSCs in close proximity to hematopoietic progenitors. The conditioned culture medium of hBMSCs significantly reduced bacterial proliferation that was partially blocked by a hepcidin-neutralizing antibody. Similarly, medium in which hepcidin-deficient (Hamp−/−) mouse BMSCs had been grown was significantly less effective in reducing bacterial counts than the medium of wild-type cells. In a zymosan-induced peritonitis mouse model we found that mBMSC-derived hepcidin reduced the number of invading polymorphonuclear (PMN) cells in the peritoneal cavity. Our results show that BMSC-derived hepcidin has antimicrobial properties in vitro and also reduces inflammation in vivo. We conclude that hepcidin should be added to the expanding arsenal of agents available to BMSCs to fight infections and inflammation.

Streptococcus pneumoniae persists as a leading cause of bacterial pneumonia despite the widespread use of polysaccharide-based vaccines. The limited serotype coverage of current vaccines has led to increased incidence of nonvaccine serotypes, as well as an increase in antibiotic resistance among these serotypes. Pneumococcal infection often follows a primary viral infection such as influenza virus, which hinders host defense and results in bacterial spread to the lungs. We previously isolated human monoclonal Abs (mAbs) against the conserved surface Ag pneumococcal histidine triad protein D (PhtD), and we demonstrated that mAbs to this Ag are protective against lethal pneumococcal challenge prophylactically and therapeutically. In this study, we elucidated the mechanism of protection of a protective anti-pneumococcal human mAb, PhtD3, which is mediated by the presence of complement and macrophages in a mouse model of pneumococcal infection. Treatment with mAb PhtD3 reduced blood and lung bacterial burden in mice, and mAb PhtD3 is able to bind to bacteria in the presence of the capsular polysaccharide, indicating exposure of surface PhtD on encapsulated bacteria. In a mouse model of secondary pneumococcal infection, protection mediated by mAb PhtD3 and another mAb targeting a different epitope, PhtD7, was reduced; however, robust protection was restored by combining mAb PhtD3 with mAb PhtD7, indicating a synergistic effect. Overall, these studies provide new insights into anti-pneumococcal mAb protection and demonstrate, to our knowledge, for the first time, that mAbs to pneumococcal surface proteins can protect against secondary pneumococcal infection in the mouse model.

Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 hrs before or 72 hrs after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 Å, which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal new insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a new mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a unique pre-fusion-specific epitope on the hMPV F protein.

The gram-positive bacterium Streptococcus pneumoniae is a leading cause of pneumonia, otitis media, septicemia, and meningitis in children and adults. Current prevention and treatment efforts are primarily pneumococcal conjugate vaccines that target the bacterial capsule polysaccharide, as well as antibiotics for pathogen clearance. While these methods have been enormously effective at disease prevention and treatment, there has been an emergence of non-vaccine serotypes, termed serotype replacement, and increasing antibiotic resistance among these serotypes. To combat S. pneumoniae , the immune system must deploy an arsenal of antimicrobial functions. However, S. pneumoniae has evolved a repertoire of evasion techniques and is able to modulate the host immune system. Antibodies are a key component of pneumococcal immunity, targeting both the capsule polysaccharide and protein antigens on the surface of the bacterium. These antibodies have been shown to play a variety of roles including increasing opsonophagocytic activity, enzymatic and toxin neutralization, reducing bacterial adherence, and altering bacterial gene expression. In this review, we describe targets of anti-pneumococcal antibodies and describe antibody functions and effectiveness against S. pneumoniae .

Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 h before or 72 h after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 Å, which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a prefusion-specific epitope on the hMPV F protein.

Computationally optimized broadly reactive Ag (COBRA) hemagglutinin (HA) immunogens have previously been generated for several influenza subtypes to improve vaccine-elicited Ab breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA Ags. Cross-reactivity between wild-type HA and H1 COBRA HA proteins P1, X6, and Y2 were observed for isolated mAbs. The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 HA head and stem domains, and most mAbs had hemagglutination inhibition and neutralizing activity against 2009 pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had hemagglutination inhibition and neutralizing activity against a prepandemic H1 strain. One mAb, P1-05, targeted the stem region of H1 HA, but did not compete with a known stemtargeting H1 mAb. We determined that mAb P1-05 recognizes a recently discovered HA epitope, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by mAb P1-05, suggesting the importance of protein design for vaccine formulations. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HA-reactive B cells that target head, central stalk, and anchor epitopes, and they demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes.

Streptococcus pneumoniae remains a leading cause of bacterial pneumonia despite the widespread introduction of vaccines for disease prevention. While vaccines have been effective at reducing the incidence of most vaccine-included serotypes, a rise in infection due to non-vaccine serotypes, and moderate efficacy against some vaccine included serotypes have contributed to high disease incidence, particularly in the elderly. Additionally, numerous isolates of S. pneumoniae are antibiotic resistant or multi-drug resistant. Several highly conserved pneumococcal proteins that are prevalent in the majority of serotypes have been examined and tested as potential vaccines in preclinical and clinical trials. We isolated the first human monoclonal antibodies (mAbs) (PhtD3, PhtD6, PhtD7, PhtD8, PspA16) against the pneumococcal histidine triad protein (PhtD), and the pneumococcal surface protein A (PspA), two conserved and protective antigens. mAbs to PhtD target diverse epitopes spanning the entire PhtD protein, and mAb PspA16 targets the N-terminal segment of PspA. The PhtD-specific mAbs were found to bind to multiple serotypes, while PspA16 serotype breadth was limited. In addition, we examined the prophylactic and therapeutic efficacy of mAb PhtD3 in several mouse models of pneumococcal pneumonia and sepsis. mAb PhtD3 prolonged the survival of infected mice when administered 2 hours before infection or 24 hours after infection. Additionally, mAb PhtD3 was effective against pneumococcal serotypes 4 and 3, the latter of which is a leading cause of invasive pneumococcal disease. Overall, our results provide new therapeutic reagents for disease prevention, and identify regions on PhtD and PspA recognized by human B cells.

SignificanceInfluenza infections kill millions of people worldwide. Current prophylactic treatment options are limited due to viral strain–specific vaccinations and emerging drug resistance. It is important to discover new immune mechanisms that can fight the influenza virus. Our work presented here identifies such a mechanism. The Duox1 protein helps the airways to clear influenza virus and to reduce infection-related death and sickness in an animal model. Duox1 delays the infection process by directly targeting the virus. Unlike current vaccines, this mechanism is effective against several influenza strains. Our study identifies the function of theDuox1gene and suggests that it has a therapeutic potential against influenza and potentially other respiratory pathogens, worth exploring in the future.

2021 (received for review August 12, 2020) Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre- ssed in respiratory epithelial cells and produces H 2 O 2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN − ) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1 − / − mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1 β , IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell re- cruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN − is generated by LPO using DUOX1-derived H 2 O 2 and inactivates several influenza strains in vitro. We also show that OSCN − diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H 2 O 2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN − in an H 2 O 2 -dependent manner in vitro. OSCN − does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN − , and proposes that boosting this mechanism in vivo can

Streptococcus pneumoniae remains a leading cause of bacterial pneumonia despite the widespread use of vaccines. While vaccines are effective at reducing the incidence of most serotypes included in vaccines, a rise in infection due to nonvaccine serotypes and moderate efficacy against some vaccine serotypes have contributed to high disease incidence.

For individuals who are immunocompromised, the opportunistic fungal pathogen Pneumocystis jirovecii is capable of causing life-threatening pneumonia as the causative agent of Pneumocystis pneumonia (PCP). PCP remains an acquired immunodeficiency disease (AIDS)-defining illness in the era of antiretroviral therapy. In addition, a rise in non-human immunodeficiency virus (HIV)-associated PCP has been observed due to increased usage of immunosuppressive and immunomodulating therapies. With the persistence of HIV-related PCP cases and associated morbidity and mortality, as well as difficult to diagnose non-HIV-related PCP cases, an improvement over current treatment and prevention standards is warranted. Current therapeutic strategies have primarily focused on the administration of trimethoprim-sulfamethoxazole, which is effective at disease prevention. However, current treatments are inadequate for treatment of PCP and prevention of PCP-related death, as evidenced by consistently high mortality rates for those hospitalized with PCP. There are no vaccines in clinical trials for the prevention of PCP, and significant obstacles exist that have slowed development, including host range specificity, and the inability to culture Pneumocystis spp. in vitro. In this review, we overview the immune response to Pneumocystis spp., and discuss current progress on novel vaccines and therapies currently in the preclinical and clinical pipeline.

Streptococcus pneumoniae (Pneumococcus) infections affect millions of people worldwide, cause serious mortality and represent a major economic burden. Despite recent successes due to pneumococcal vaccination and antibiotic use, Pneumococcus remains a significant medical problem. Airway epithelial cells, the primary responders to pneumococcal infection, orchestrate an extracellular antimicrobial system consisting of lactoperoxidase (LPO), thiocyanate anion and hydrogen peroxide (H2O2). LPO oxidizes thiocyanate using H2O2 into the final product hypothiocyanite that has antimicrobial effects against a wide range of microorganisms. However, hypothiocyanite’s effect on Pneumococcus has never been studied. Our aim was to determine whether hypothiocyanite can kill S. pneumoniae. Bactericidal activity was measured in a cell-free in vitro system by determining the number of surviving pneumococci via colony forming units on agar plates, while bacteriostatic activity was assessed by measuring optical density of bacteria in liquid cultures. Our results indicate that hypothiocyanite generated by LPO exerted robust killing of both encapsulated and nonencapsulated pneumococcal strains. Killing of S. pneumoniae by a commercially available hypothiocyanite-generating product was even more pronounced than that achieved with laboratory reagents. Catalase, an H2O2 scavenger, inhibited killing of pneumococcal by hypothiocyanite under all circumstances. Furthermore, the presence of the bacterial capsule or lytA-dependent autolysis had no effect on hypothiocyanite-mediated killing of pneumococci. On the contrary, a pneumococcal mutant deficient in pyruvate oxidase (main bacterial H2O2 source) had enhanced susceptibility to hypothiocyanite compared to its wild-type strain. Overall, results shown here indicate that numerous pneumococcal strains are susceptible to LPO-generated hypothiocyanite.

Streptococcus pneumoniae (pneumococcus) infections affect millions of people worldwide, cause serious mortality and represent a major economic burden. Airway epithelial cells, the primary responders to pneumococcal infection, orchestrate an extracellular antimicrobial system consisting of lactoperoxidase, thiocyanate anion and hydrogen peroxide (H2O2). Lactoperoxidase oxidizes thiocyanate using H2O2 into the final product hypothiocyanite. While hypothiocyanite has known antimicrobial effects against a wide range of microorganisms, its effect on pneumococcus has never been studied. To test this, pneumococci were exposed to hypothiocyanite generated enzymatically in a cell-free in vitro system. Bacterial killing was determined by colony forming units on agar plates while bacteriostatic effects were measured by changes in optical density of liquid cultures. We demonstrate hypothiocyanite exerted robust killing of several pneumococcal strains. Nonencapsulated pneumococcal mutants were killed to the same extent as their parental strains, indicating no role of the capsule in protection against hypothiocyanite. Catalase, an H2O2 scavenger, inhibited pneumococcal killing by hypothiocyanite under all circumstances. Interestingly, a pneumococcal mutant deficient in pyruvate oxidase, the main bacterial H2O2 source, had enhanced susceptibility to hypothiocyanite indicating the role of this gene in partial protection of the bacterium against this oxidizing agent. Overall, numerous pneumococcal strains were found to be susceptible to lactoperoxidase-generated hypothiocyanite in vitro, which presents an opportunity to explore hypothiocyanite as a potential novel anti-pneumococcal therapy.

Influenza virus infections cause severe morbidity and mortality, especially in high-risk patient populations. Bronchoepithelial cells orchestrate an oxidative antimicrobial system present in the airway liquid consisting of lactoperoxidase, the thiocyanate ion, and hydrogen peroxide. Dual Oxidase 1 (Duox1), an enzyme highly expressed in these cells, is the source of hydrogen peroxide. While in vitro studies suggest an anti-influenza, and a general antimicrobial role of Duox1, its antiviral role in vivo has not been addressed so far. We hypothesized that Duox1 has a protective effect in vivo against influenza. To test this hypothesis, we infected wild-type C57BL/6 and Duox1-deficient mice intranasally with the mouse-adapted A/Puerto Rico/8/1934 H1N1 (PR8) influenza virus strain. To evaluate the clinical and immunological significance of Duox1 in influenza infection, multicolor flow cytometry, multiplex ELISA, viral titration, histology and immunostaining techniques were used. Our results indicate that Duox1-deficient mice have increased mortality following influenza infection. Weight loss of Duox1-deficient mice post-infection was also significantly higher than in Duox1-expressing animals. Duox1 also limited influenza virus replication in the lung. Characterization of infiltrated leukocyte subsets following influenza infection revealed impaired natural killer cell recruitment in Duox1-deficient animals while other lymphoid or myeloid leukocyte subsets were not affected In summary, our results provide the first evidence for the in vivo antiviral role of Duox1, and also implicate that targeting the Duox1-based system has the potential to provide novel treatment or prophylactic strategies against influenza.

The NOX/DUOX family of NADPH oxidases are transmembrane proteins generating reactive oxygen species as their primary enzymatic products. NADPH oxidase (NOX) 1-5 and Dual oxidase (DUOX) 1 and 2 are members of this family. These enzymes have several biological functions including immune defense, hormone biosynthesis, fertilization, cell proliferation and differentiation, extracellular matrix formation and vascular regulation. They are found in a variety of tissues such as the airways, salivary glands, colon, thyroid gland and lymphoid organs. The discovery of NADPH oxidases has drastically transformed our view of the biology of reactive oxygen species and oxidative stress. Roles of several isoforms including DUOX1 and DUOX2 in host innate immune defense have been implicated and are still being uncovered. DUOX enzymes highly expressed in the respiratory and salivary gland epithelium have been proposed as the major sources of hydrogen peroxide supporting mucosal oxidative antimicrobial defenses. In this review, we shortly present data on DUOX discovery, structure and function, and provide a detailed, up-to-date summary of discoveries regarding antibacterial, antiviral, antifungal, and antiparasitic functions of DUOX enzymes. We also present all the literature describing the immune functions of lactoperoxidase, an enzyme working in partnership with DUOX to produce antimicrobial substances.