Human monoclonal antibodies protect against viral-mediated pneumococcal superinfection

Introduction Community-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. Methods We 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. Results The 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. Discussion Given 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.


Introduction
Infectious respiratory diseases continue to pose a significant and daunting threat to global public health.A variety of pathogens are responsible for respiratory illnesses, with Streptococcus pneumoniae (Spn) being a major contributor.This gram-positive bacterium causes an array of infections such as pneumonia, otitis media, sinusitis, and invasive diseases like bacteremia and meningitis (1).Additionally, respiratory viruses like influenza virus, respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) significantly contribute to the global burden of respiratory infections (2).Bacterial/viral co-infections have been a concerning medical phenomenon since the early 1900s when influenza and pneumonia were recognized as public health concerns (3,4).Spn is notorious for its involvement in co-infections with influenza A virus (IAV), RSV, and hMPV (5)(6)(7)(8)(9)(10).When these virally infected individuals are also infected with Spn, due to colonization or new infection, the resulting synergy often culminates in more severe and complex disease manifestations than single-pathogen infections (3,4).Research has shown that co-infections can amplify pathogen virulence, which worsens patient outcomes (3,11).This lethal synergy occurs through various mechanisms such as the destruction of epithelial tissue (12)(13)(14)(15)(16), dysregulated immune function, (17-23) and a modified microbial environment (24)(25)(26)(27).Thorough investigation into coinfections is crucial as they can lead to severe clinical manifestations across all age groups, but particularly in elderly individuals, infants, young children, and the immunocompromised (11,28).Although influenza vaccination has proven to decrease hospitalization rates for pneumonia patients (29), and immunization against Spn has greatly reduced the incidence of invasive pneumococcal disease (30,31), the vaccines for Spn and IAV offer protection against single infections however (32,33), they have shown limited protection against coinfection in mice (34)(35)(36).Present treatments for these co-infections consist of antibiotic therapy aimed at Spn and antiviral medications targeting viruses.Both approaches demonstrate some protective effects (37)(38)(39), however, due to the emergence of drug-resistant pathogens (40) and the declining efficacy of existing treatments, there is a pressing need for innovative approaches in tackling these coinfections.Furthermore, gaining an in-depth understanding of dual-pathogen infection mechanisms may pave the way for developing cutting-edge prevention and management strategies.
In our previous research, we discovered that the protective effect of the human mAb PhtD3 during pneumococcal infections was facilitated by macrophages and the complement system (41).Although this protection was significantly reduced in an IAV/Spn coinfection model, we managed to restore protection using a mAb cocktail of PhtD3 + 7 (41).In this study, we established additional lethal coinfection models for both RSV/Spn and hMPV/Spn, and we aimed to explore whether therapeutically targeting the virus with mAbs could offer protection against secondary pneumococcal infections.Our findings demonstrate that the PhtD3 + 7 mAb cocktail provides protection across all three coinfection models, with noticeably enhanced efficacy compared to antiviral mAbs alone.Our findings highlight the exciting potential of innovative therapeutics in tackling Spn coinfections.

Ethics statement
All animal studies performed were in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Georgia.

Bacterial strains and growth conditions
Bacterial colonies were grown on BD Trypticase Soy Agar II with 5% Sheep Blood (BD, Franklin Lakes NJ).Bacteria cultures were grown at 37°C in 5% CO2 in Todd-Hewitt broth (BD, Franklin Lakes NJ) supplemented with 0.5% yeast extract for 12 hrs.Cultures were frozen and stored at -80°C with 10% glycerol until used, and following thawing, cultures were washed twice with PBS before being used in experiments.The numbers of CFUs per milliliter of these stocks were determined by plating a single quick-thawed diluted aliquot on sheep's blood agar plates.The calculated number of CFUs was subsequently used to make dilutions for experiments from aliquots thawed at later times.In each experiment, the actual number of CFUs administered was determined by plating on blood agar at the time of the assay.

Antibody generation with CHO and 293 cells
mAbs PhtD3, PhtD7 and Ab6649 were generated as previously described in ExpiCHO cells (41,42).Briefly, ExpiCHO cells were grown in FreeStyleCHO media supplemented with L-glutamine.For transfections, cells were transferred to ExpiCHO media and transfected using the high-titer protocol according to the manufacturer's instructions.mAbs were purified from culture supernatants using Protein G columns (Cytiva) as previously described (41,42).mAbs MPV467 and D25 were expressed by transfecting Expi293 cells with HC/LC plasmids and purified from culture supernatant with a Protein G column (Cytiva) as previously described (43).

Co-infection studies
To establish the model of pneumococcal co-infection with hMPV or RSV, 5-7 week-old BALB/c mice (Jackson Laboratories) were anesthetized by inhalation of 5% isoflurane and intranasally challenged with 50 µL of 5x10 5 PFU of hMPV TN/93-32 or with 50 µL of 3-5x10 5 PFU of RSV A2 in PBS.Previous studies have shown this dose to be non-lethal but viral replication does occur (43), Five days post viral infection, mice were anesthetized by inhalation of 5% isoflurane and intranasally challenged with 40 µL of 10 4 , 10 5 , or 10 6 CFUs of pneumococcal strain WU2 serotype 3 in PBS.Based on these dosing experiments, we utilized 10 6 CFUs of Spn for the coinfection studies.On day 3 post viral infection, some mice were intraperitoneally inoculated with 10 mg/kg of mAb MPV467 (hMPV infection) or 10 mg/kg mAb D25 (RSV infection).Two hours prior to bacterial challenge, mice were intraperitoneally inoculated with 15 mg/kg of mAb (PhtD cocktail) or PBS.
For the IAV/Spn model, we utilized doses and timepoints as previously described (41).5-7 weeks old C57BL/6 mice (Charles River) were anesthetized by inhalation of 5% isoflurane and intranasally challenged with 100 FFU of H1N1 A/California/07/ 2009 in 40 µL of PBS.After 7 days, mice were anesthetized by inhalation of 5% isoflurane and intranasally challenged with 40 µL of 1x10 4 CFUs of pneumococcal strain WU2 in PBS.Twenty-four hours post viral infection, some mice were intraperitoneally inoculated with 10 mg/kg of mAb Ab6649.Two hours prior to bacterial challenge, mice were intraperitoneally inoculated with 15 mg/kg of mAb or PBS.In all studies above, mice were weighed and assessed daily and were euthanized when >30% of pre-infection body weight was lost, were nonresponsive to manual stimulation, and/or were exhibiting respiratory distress.

Lung viral titers
To determine viral titers of hMPV or RSV at the time of bacterial infection, mice were euthanized on day 5 post-viral challenge and lungs were collected and homogenized for virus titration as previously described (44).Briefly, RSV-challenged lung homogenates were plated on HEp-2 cells (Opti-MEM+2% FBS) while hMPV-challenged lung homogenates were plated on LLC-MK2 cells (Opti-MEM + 5 mg/mL trypsin-EDTA and 100 mg/ mL CaCl 2 ) in 24 well plates.After 4 days for RSV and 5 days for hMPV, the cells were fixed with 10% neutral buffered formalin.Cell monolayers were next blocked with block buffer comprising of 2% nonfat milk supplemented with 2% goat serum in PBS-T for 1 h.Next, the plates were washed three times with water, and 200 mL of MPV364 (for hMPV) or 101F (for RSV) (44) was added to a final concentration of 1 mg/mL (1:1,000 dilution) in blocking solution.The plates were then washed three times with water, and 200 mL of goat anti-human IgG HRP secondary antibody (Southern Biotech) diluted to a ratio of 1:2,000 in block buffer was added and incubated for 1 h at room temperature followed by 1 hr of incubation.Plates were washed again with water five times, and 200 mL of TrueBlue peroxidase substrate (SeraCare) was added to each well.The plates were incubated for 20-30 min until the plaques were clearly visible.Plaques were counted manually under a microscope.For influenza virus, lungs were collected and homogenized on day 7 post infection and viral titers were determined via plaque assay.Briefly, IAV challenged lung homogenates were serially diluted and plated on MDCK cells (2x overlay media MEM+2 mg/mL TPCK-treated trypsin+40mM HEPES+4mM L-glutamine+0.15% NaHCO 3 and 1.4% Avicel) in 12-well plates.After 3 days, cells were fixed with Acetone : Methanol (20:80) and then stained with crystal violet.Plaques were then counted, and titers were determined from the average of three replicates.

Bacterial burden studies
To determine bacterial burden in the lungs and blood of coinfected animals, the protocols described above were used.For IAV/Spn co-infection, groups of mice were euthanized at 12-, 24and 36-hours post-infection mice, and blood and lungs were collected.For RSV/Spn and hMPV/Spn co-infections, groups of mice were euthanized on days 1, 2, and 3 post bacterial infection, and blood and lungs were collected.Blood was collected via cardiac puncture and was serially diluted and plated on TSA 5% sheep blood agar plates to determine bacterial titers.Lungs were extracted and homogenized in 1 mL of PBS, homogenates were then serially diluted and plated to determine bacterial titers as above.

Assessment of bacterial vs viral mAb efficacy against secondary pneumococcal infection
In our previous studies, we isolated protective anti-PhtD mAbs and elucidated the mechanism of protection for mAb PhtD3 (41).Through immune depletion studies, we found that protection by mAb PhtD3 is likely mediated by macrophages and the complement system (41).While single anti-PhtD mAb treatment did not confer robust protection in an IAV/Spn murine coinfection model, a cocktail of mAbs PhtD3 and PhtD7 (PhtD3+PhtD7) did provide protection (41).Prior influenza infection in mice dysregulates the immune system impairing the antibacterial function of alveolar macrophages in C57BL/6 mice and depleting alveolar macrophages in BALB/c mice (19,45).Other studies have shown that virus mediated damage and interactions of influenza virus and Spn also enhance the secondary bacterial infection (25,46,47).Due to virus mediated effects, we determined if mAbs targeting the virus would also confer protection upon secondary bacterial infection as the bacterial dose on its own is not lethal.Utilizing our previously established IAV/Spn coinfection model in C57BL/6 mice, we compared the protective efficacy of mAb Ab6649 to our mAb cocktail of PhtD3+PhtD7, which was previously shown to be protective against IAV/Spn coinfection using the H1N1 A/California/07/ 2009 (42) virus, as outlined in Figure 1A.In our first experiment, we administered 10 mg/kg of mAb Ab6649, which is a 3x higher dose than a protective dose previously shown (42), 24 hrs post IAV infection to one group, and 7.5 mg/kg each of mAb cocktail PhtD3 +PhtD7 to a different group 2 hrs prior to Spn infection on day 7 post IAV infection.Compared to the PBS control group (0%), the mAb PhtD3 + 7 and mAb Ab6649 treated groups had a significant increase in survival (58 and 41%, respectively) with no significant difference between the different mAb treatment groups (Figure 1B).We also collected lung homogenates of IAV infected, Ab6649 treated animals and found that the PBS control animals had residual levels of IAV still present (average 2.1 log 10 PFU/mL) whereas the Ab6649 treated animals had no detectable virus present in the lungs (Figure 1C).We next examined the kinetics and dissemination of bacterial growth after secondary pneumococcal infection in the IAV/Spn model.Utilizing the same infection and treatment plans as used in the survival study above, we collected the lungs and blood of mice at 12, 24 and 36 hrs post pneumococcal infection.At the first time point 12 hrs post Spn infection, we observed significantly reduced bacterial titers in the lungs of mAb PhtD3 + 7 and mAb Ab6649 treated mice compared to the PBS treated control group (average 4.18 versus 2.58 versus 6 log 10 CFU/mL, respectively) (Figure 2A).To determine dissemination of the bacteria from the airways to the blood stream, we analyzed blood collected via cardiac puncture.At the 12 hrs time point, only one PBS control mouse had detectable levels of Spn in the bloodstream (Figure 2B).After 24 hrs, we saw an increase in lung bacterial titers of both mAb PhtD3 + 7 and mAb Ab6649 treated animals from the 12 hr timepoint (average 5.05 versus 5.41 log 10 CFU/mL, respectively), however both groups were significantly lower than PBS control animals (average 7.5 log 10 CFU/mL) (Figure 2A).At this same time point, we observed a significant increase in blood bacterial titers in the PBS control animals compared to mAb PhtD3 + 7 and Ab6649 treated animals (average 3.13 versus 2.16 versus 2 log 10 CFU/mL, respectively) (Figure 2B).Finally at 36 hrs, we continued to see a significant decrease in lung bacterial titers in mAb PhtD3 + 7 and mAb Ab6649 treated animals compared to PBS treated animals (average 5.55 versus 3.95 versus 8.25 log 10 CFU/mL, respectively) (Figure 2A).In parallel, we saw a significant decrease in blood bacterial titers in the mAb PhtD3 + 7 and mAb Ab6649 control animals compared PBS to treated animals (average 2 versus 2 versus 4.64 log 10 CFU/mL, respectively) (Figure 2B).In conclusion, our experiments have determined that both mAb PhtD3 + 7 and mAb Ab6649 exhibit protective efficacy and reduce blood and lung titers in an IAV/Spn coinfection model.

PhtD3 + 7 mAb cocktail protects mice against hMPV/Spn coinfection
Primary hMPV infection can also lead to secondary pneumococcal infection resulting in increased morbidity and mortality (7).hMPV-infected mice showed impaired recruitment of airway neutrophils, possibly leading to delayed bacterial clearance and exacerbated pulmonary inflammation after secondary infection with Spn (48).Since hMPV does not replicate well in C57BL/6 mice, we used BALB/c mice for the hMPV/Spn studies, as these mice are more susceptible to hMPV infection (49).Before testing the mAb PhtD3 + 7 cocktail, we established a hMPV/ Spn coinfection model similar to a previous study (48).While BALB/c mice are more susceptible to hMPV infection, they are more resistant to Spn infection, so first we established a new dosing standard to determine the optimal dose of Spn.We infected 6-8 week old male BALB/c mice with 5 different doses, 10 7 , 10 6 , 10 5 , 10 4 and 10 3 CFUs of serotype 3 WU2.Mice infected with 10 5 , 10 4 and 10 3 had 100% survival and 10 6 and 10 7 had 80% and 0% survival, respectively (Figure 3A).Our previous studies have demonstrated that hMPV infection does not cause substantial disease in BALB/c mice using 5x10 5 PFU of hMPV strain TN/93-32 (49).We first infected mice intranasally with 5x10 5 PFU of hMPV TN/93-32, and 5 days later mice were infected with 3 different doses of Spn at 10 6 , 10 5 and 10 4 CFU/mouse.At 10 4 , 10 5 , and 10 6 CFU/mouse we observed 0%, 20% and 60% mortality, respectively (Figure 3B).
Based on these we utilized 10 6 CFUs of Spn for subsequent studies due to the limited mortality seen in our singly infected animals.We then tested the mAb PhtD3 + 7 cocktail in the newly established coinfection model.On day 5 post hMPV infection, we administered 7.5 mg/kg each of mAb PhtD3 and mAb PhtD7 2 hrs prior to Spn infection.mAb PhtD3 + 7 showed a significant protective effect when compared to the isotype mAb and PBS control groups (100% versus 8.3% versus 16.6%, respectively) (Figure 3C).We next compared the protective effects of the PhtD3 + 7 mAb cocktail to a protective hMPV antibody, MPV467 (43).We observed 100% survival in both of the singly infected controls, Spn and hMPV only (Figure 3C).We utilized the previously published mAb MPV467, which targets the F protein of the virus and has been shown to neutralize hMPV in vivo (43).We followed the same experimental timeline as above, but added a group that was treated with MPV467 (10 mg/kg (43)) on day 3 post hMPV infection, as outlined in Figure 4A.mAb PhtD3 + 7 treatment was found to be the most protective compared to MPV467 treatment and the PBS control (83.3% versus 41.6% versus 15.3%, respectively) (Figure 4B).On day 5 post infection, we measured the lung viral titers of MPV467 treated mice compared to PBS treated animals and found a significant decrease with no detectable virus in MPV467 treated animals (average 1.3 versus 1.96 log 10 PFU/mL, respectively) (Figure 4C).To better understand the dynamics of bacterial dissemination, we measured lung and blood bacterial titers at 24 hrs, 48 hrs and 72 hrs post Spn infection.At 24 hrs, we saw a significant reduction in lung bacterial titers in both the mAb PhtD3 + 7 and mAb MPV467 treated groups compared to PBS treated groups (average 3.95 versus 4.3 versus 6.08 log 10 CFU/mL, respectively) (Figure 5A).Additionally, we found no detectable bacteria in the blood of mice (Figure 5B).At 48 hrs, we observed a significant reduction in lung bacterial titers in both the mAb PhtD3 + 7 and mAb MPV467 groups compared to PBS treated groups (average 3.2 versus 5 versus 6.3 log 10 CFU/mL, respectively) (Figure 5A).At

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Coinfection with sublethal dose of hMPV followed by Spn causes mortality in mice.48 hrs we began to see a rise in blood bacterial titers; however, mice treated with PhtD3 + 7 still showed no detectable bacteria in the blood with MPV 467 and PBS treated showing a slight increase in titers (average 2 versus 2.23 versus 2.38 log 10 CFU/mL, respectively) (Figure 5B).Finally, at 72 hrs post Spn infection we saw no detectable bacteria in the lungs of mAb PhtD3 + 7 treated animals; however, we saw a further increase in titers of both MPV467 treated and PBS treated groups (average 2 versus 5.7 versus 6.6 log 10 CFU/mL, respectively) (Figure 5A).Continuing the trend from the lungs, we detected no bacteria in the blood of PhtD3 + 7 animals but an increase in titers for MPV467 and PBS (average 2 versus 2.48 versus 3.03 log 10 CFU/mL, respectively) (Figure 5B).In conclusion, the hMPV/Spn coinfection model revealed that the PhtD3 + 7 mAb cocktail substantially enhanced survival rates and decreased lung and blood bacterial levels, while mAb MPV467 provided moderate protection.

PhtD3 + 7 mAb cocktail protects mice against RSV/Spn coinfection
Primary RSV infection has also been shown to lead to an increased incidence and severity of secondary pneumococcal infection (2,6,50).Similar to hMPV, we established a coinfection model in BALB/c mice to allow for sufficient replication of the RSV in the lungs of the mice, as outlined in Figure 6A.Our previous studies have demonstrated that RSV A2 does not cause disease at a dose of 3.5x10 5 PFU/mouse (44).We first infected mice intranasally with 3.5x10 5 PFU of RSV A2, and 5 days later we infected groups of mice with 4 different doses of Spn, 10 7 , 10 6 , 10 5 and 10 4 CFU/ mouse.At 10 4 , 10 5 , 10 6 , 10 7 CFU/mouse we observed 0%, 0%, 80% and 100% mortality, respectively (Figure 6B).Comparable to the hMPV/Spn infection, we used a 10 6 dose Spn in the RSV/Spn model.We utilized mAb D25, which had previously been shown to target the F protein of RSV and was neutralizing in vivo (51).mAb D25 is the precursor to the recently approved RSV prophylactic mAb nirsevimab (51).We first performed a survival study to compare mAb PhtD3 + 7 to mAb D25 treatment.We observed a significant protective effect in both mAb PhtD3 + 7 and mAb D25 treated mice compared to PBS treated control (93.3% versus 73.3% versus 40%, respectively) (Figure 6C).Additionally, our singly infected animals showed 100% survival as expected.We then measured lung viral titers on day 5 post RSV infection.Mice administered mAb D25 on day 3 post RSV infection had a significant decrease in detectable virus titers compared to PBS (average 1.64 versus 4.7 log 10 PFU/mL, respectively) (Figure 6D).We next measured lung and blood bacterial titers at 24 hrs, 48 hrs and 72 hrs post Spn infection.At 24 hrs, we saw a significant reduction in lung bacterial titers in both the mAb PhtD3 + 7 and mAb D25 treated groups compared to the PBS treated group (average 2 versus 3.48 3.64 log 10 CFU/mL, respectively) with no bacteria detected in the PhtD3 + 7 group (Figure 7A).Additionally, we found no detectable bacteria in the blood of any groups (Figure 7B).At 48 hrs, we observed a significant reduction in lung bacterial titers in the mAb PhtD3 + 7 treated group, with no detectable bacteria compared to the mAb D25 and PBS treated groups (average 2 versus 3.78 versus 3.48 log 10 CFU/mL, respectively) (Figure 7A).We did detect bacteria in the blood of two animals in the mAb D25 treated group (Figure 7B).Lastly, at 72 hrs post bacterial infection, we observed only two animals with bacteria in the lungs in the mAb PhtD3 + 7 treated animals; however, we observed a further increase in bacterial titers in both mAb D25 and PBS treated groups (average 2.47 versus 4.55 Coinfection with sublethal RSV followed by Spn causes mortality in mice.versus 4.51 log 10 CFU/mL, respectively) (Figure 7A).Furthermore, we detected no bacteria in the blood of PhtD3 + 7 treated animals, but one animal in each mAb D25 and PBS treated groups had detectable bacteria (Figure 7B).In conclusion, the RSV/Spn coinfection study demonstrated the exceptional protective qualities and effectiveness of PhtD3 + 7 in significantly reducing lung and blood bacterial titers, and while mAb D25 treatment did improve survival, it failed to exhibit a similar reduction in bacterial titers.

Discussion
Taken together, this study highlights the potent protective capacity of the anti-pneumococcal human mAb cocktail, PhtD3+PhtD7, in combating coinfections involving IAV/Spn, hMPV/Spn, and RSV/Spn.The PhtD3 + 7 mAb cocktail outperformed individual human mAbs targeting each specific virus.Moreover, the PhtD3 + 7 mAb cocktail reduced lung bacterial titers and prevented bacterial dissemination into the bloodstream, surpassing the effectiveness of our antiviral mAbs.Building upon our past investigations (41,52), this study reaffirms the protective capabilities of anti-PhtD mAbs.
Primary influenza virus infection has been shown to predispose hosts to synergistic secondary bacterial infections leading to increased morbidity and mortality (45,53).Influenza virus achieves this through a variety of mechanisms.The airways can become compromised leading to a decrease in mucocilliary clearance, destruction of tight junctions, increase in available nutrients for the bacteria, and exposure of host receptors that Spn can utilize to bind to host cells (12,24,45,54).In addition to these physical phenomena, immune dysregulation occurs on multiple fronts.The dysregulation of alveolar macrophages is an important aspect as macrophages are essential in host defense against Spn (45).Neutrophils are also susceptible to viral priming, further impairing the innate immune response to secondary bacterial infection (55).This immune dysfunction is driven by IFN-g and its down streaming signaling impairing phagocytic cells, leading to a more inflammatory state (17,22,56).Based on the body of literature and our previous study (41) demonstrating the protective efficacy our PhtD mAbs in an IAV/Spn coinfection model, we wanted to determine if targeting the virus prior to infection with Spn would also be protective.Utilizing the previously described mAb Ab6649 (42) 24 hrs post viral infection, we found similar levels of protection between Ab6649 and the PhtD3 + 7 mAb cocktail.Upon titering of mouse lungs at day 7, we found that treatment with Ab6649 led to no detectable virus in the lungs at the point of challenge with Spn, but low levels of virus were present in the PBS control mice.To further dive into the kinetics of infection, we measured lung and blood titers at 12, 24 and 36 hrs post Spn infection.Both antibodies significantly reduced bacterial lung and blood titers compared to our PBS control.our results suggest that our monoclonal antibodies (mAbs) effectively halted the bacteria's invasion into the bloodstream, within the time points we've evaluated.Resolution of the viral infection by day 7 did show positive protective effects; however, both approaches targeting the virus or the bacteria yielded positive clinical outcomes, but seemingly through different mechanisms as they have different targets.It is likely that the Ab6649 treatment prevented widespread damage to the epithelium by neutralizing the virus and preventing an excessive inflammatory response, thereby allowing the innate immune system to keep Spn under control.The ability of PhtD3 + 7 to remain protective in a dysregulated immune state.As mentioned before, primary viral infections dysregulate macrophages antimicrobial abilities (19,45), and we have shown mAb PhtD3 requires macrophages to be protective (41).Further studies are needed to decipher the exact protective mechanism in the context of IAV/ Spn coinfections.

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Bacterial burden in blood and lungs of RSV/Spn coinfected mice.hMPV and RSV, both members of the Pneumoviridae family, are two major viral causes of secondary bacterial coinfections; however, these viral infections cannot be modeled using C57BL/6 mice.We established a model in BALB/c mice to enable viral replication, despite their enhanced resistance to Spn infection.It has been shown that C57BL/6 mice have more efficient Alveolar macrophage mediated bacterial clearance (57), whereas BALB/c mice have a more robust neutrophil response (58).C57BL/6 also demonstrate a greater inflammatory response compared to BALB/c mice (59).We found that for both coinfections, day 5 post viral infection was the optimal time to infect with Spn.It has been shown that a preceding hMPV infection makes the host more susceptible to Spn adherence (48).In RSV/Spn coinfections, studies show that the lethal synergy occurs via the formation of RSV-Spn complexes and expression of viral glycoproteins on the surface of infected cells, leading to increased attachment and adherence (60, 61).Due to these direct interactions between hMPV or RSV and Spn, we hypothesized that a mAb neutralizing these viruses would have therapeutic potential.Similar to our IAV coinfection, we showed that in PBS treated animals, there were low levels of virus present in the lungs, but upon antiviral mAb treatment, MPV467 (hMPV) and D25 (RSV), virus was not detectable in MPV467-treated animals.We saw significant protection with our PhtD3 + 7 mAb cocktail in both coinfection models and this protection was greater than our antiviral mAbs.Our data indicates that using antiviral mAbs to neutralize the virus prior to Spn infection is not as effective as using mAb PhtD3 + 7 for protection.We believe the lethal synergy between hMPV or RSV and Spn stems from immune dysfunction, not direct interactions between the virus and bacteria.Due to the less severe nature of these coinfections, we extended the time points for measuring bacterial titers up to 72 hrs post Spn infection.In the hMPV/Spn coinfection model, we saw a decrease in lung bacterial titers in our PhtD3 + 7 group with no bacteria detected at 72 hrs post Spn infection.Interestingly, in our RSV coinfection model, while D25 treatment did increase survival, there was no decrease in lung titers compared to our PBS control, whereas PhtD3 + 7 mAb cocktail effectively decreased lung titers below the limit of detection.It is possible that D25 treatment prevents robust immune dysfunction, which would explain the protective effect seen, but the lack of reduction in bacterial titers.Once again mAb PhtD3 + 7 effectively prevented detectable levels of bacteria from entering the bloodstream, which likely points to a key protective feature.
Prior research has highlighted the significance of physical damage and immune dysfunction resulting from primary viral infections, which can lead to subsequent bacterial infections (54).While we have previously pinpointed the protective role of PhtD3 in vivo, driven by macrophages, the protective mechanism of mAb PhtD7 still requires further investigation.Our ongoing research aims to solidify the notion that targeting conserved surface antigens is a fruitful approach not only for individual infections, but also for co-infections.To our knowledge, this is the first study to demonstrate mAbs to be effective across three distinct coinfection models.Future studies will delve deeper into how PhtD3 + 7 overcomes phagocytic deficiencies and will ultimately determine if a combination of antiviral and antibacterial monoclonal antibodies offers the most promising therapeutic solution for viral/bacterial co-infections.

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FIGURE 1 Evaluation of protective efficacy of PhtD3 + 7 cocktail and mAb Ab6649.(A) Timeline of IAV/Spn coinfection and data collection timepoints.*Each group of mice either received the Abb6649 mAb at Day 1 after IAV infection OR the PhtD3/7 mAb cocktail 2 hours prior to Spn infection.(B) Protective efficacy of mAb PhtD3 + 7 or Ab6649 in an IAV/Spn coinfection model.Mice were infected with H1N1 A/California/07/2009 at day 0 and with Spn serotype 3 (WU2) bacteria at day 7 (dotted line).Treatment with mAb Ab6649 occurred 24 hrs post IAV infection.Treatment with mAb PhtD3 + 7 occurred 2 hrs prior to pneumococcal infection.***p = 0.0005, ****p < 0.0001 via log-rank (Mantel Cox) test compared with the PBS control group.n = 12 mice/group; IAV-and Spn infected groups had n = 5 mice/group.(C) Viral burden in the lungs after mAb Ab6649 treatment.Viral burden was assessed on 7 d after IAV infection.Treatment with mAb Ab6649 occurred 24 hrs post IAV infection.***p = 0.0005 via unpaired t test.IAV, Influenza A Virus; Spn, S. pneumoniae; LOD, Limit of detection.

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FIGURE 4 Coinfection with sublethal hMPV followed by Spn causes mortality in mice and is improved by PhtD3 + 7 mAb treatment.(A) Timeline of hMPV/Spn coinfection and data collection timepoints.*Each group of mice either received the MPV467 mAb at day 3 after hMPV infection OR the PhtD3/7 mAb cocktail 2 hours prior to Spn infection.(B) Protective efficacy of PhtD3 + 7 or MPV467 mAbs in a hMPV/Spn coinfection model.Mice were infected with hMPV TN/93-32 at day 0 and Spn serotype 3 (WU2) bacteria at day 5 (dotted line).Treatment with MPV467 occurred 72 hrs post hMPV infection.Treatment with mAb PhtD3 + 7 occurred 2 hrs prior to Spn infection.***p < 0.0005 via log-rank (Mantel Cox) test compared with the PBS control group.Coinfected groups had n =12 mice/group; hMPV-and Spn infected groups had n = 5 mice/group.(C) Viral burden was assessed on day 5 after hMPV infection.Treatment with mAb MPV467 occurred day 3 post hMPV infection.**p < 0.01 via unpaired t test.LOD, Limit of detection.