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

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.

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).

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 5x10⁵ PFU of hMPV TN/93-32 or with 50 µL of 3-5x10⁵ 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 10⁴, 10⁵, or 10⁶ CFUs of pneumococcal strain WU2 serotype 3 in PBS. Based on these dosing experiments, we utilized 10⁶ CFUs of Spn for the co-infection 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 1x10⁴ 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.

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 μg/mL trypsin-EDTA and 100 μg/mL CaCl₂) 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 μL of MPV364 (for hMPV) or 101F (for RSV) (44) was added to a final concentration of 1 μg/mL (1:1,000 dilution) in blocking solution. The plates were then washed three times with water, and 200 μL 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 μL 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 μg/mL TPCK-treated trypsin+40mM HEPES+4mM L-glutamine+0.15% NaHCO₃ 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.

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-, 24- and 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.

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 log₁₀ 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 log₁₀ 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 log₁₀ CFU/mL, respectively), however both groups were significantly lower than PBS control animals (average 7.5 log₁₀ 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 log₁₀ 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 log₁₀ 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 log₁₀ 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.

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, 10⁷, 10⁶, 10⁵, 10⁴ and 10³ CFUs of serotype 3 WU2. Mice infected with 10⁵, 10⁴ and 10³ had 100% survival and 10⁶ and 10⁷ 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 5x10⁵ PFU of hMPV strain TN/93-32 (49). We first infected mice intranasally with 5x10⁵ PFU of hMPV TN/93-32, and 5 days later mice were infected with 3 different doses of Spn at 10⁶, 10⁵ and 10⁴ CFU/mouse. At 10⁴, 10⁵, and 10⁶ CFU/mouse we observed 0%, 20% and 60% mortality, respectively ( Figure 3B ). Based on these data, we utilized 10⁶ 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 log₁₀ 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 log₁₀ 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 log₁₀ CFU/mL, respectively) ( Figure 5A ). At 48 hrs we began to see a rise in blood bacterial titers; however, mice treated with mAb 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 log₁₀ 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 log₁₀ 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 log₁₀ 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.

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 3.5x10⁵ PFU/mouse (44). We first infected mice intranasally with 3.5x10⁵ PFU of RSV A2, and 5 days later we infected groups of mice with 4 different doses of Spn, 10⁷, 10⁶, 10⁵ and 10⁴ CFU/mouse. At 10⁴, 10⁵, 10⁶, 10⁷ CFU/mouse we observed 0%, 0%, 80% and 100% mortality, respectively ( Figure 6B ). Comparable to the hMPV/Spn infection, we used a 10⁶ 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 log₁₀ 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 versus 3.64 log₁₀ 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 log₁₀ 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 versus 4.51 log₁₀ CFU/mL, respectively) ( Figure 7A ). Furthermore, we detected no bacteria in the blood of mAb 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.