Nebulized fusion inhibitory peptide protects cynomolgus macaques from measles virus infection

Measles is the most contagious airborne viral infection and the leading cause of child death among vaccine-preventable diseases. We show here that aerosolized lipopeptide fusion inhibitors, derived from heptad-repeat regions of the measles virus (MeV) fusion protein, block respiratory MeV infection in a non-human primate model, the cynomolgus macaque. We used a custom-designed mesh nebulizer to ensure efficient aerosol delivery of peptides to the respiratory tract and demonstrated the absence of adverse effects and lung pathology in macaques. The nebulized peptide efficiently prevented MeV infection, resulting in the complete absence of MeV RNA, MeV-infected cells, and MeV-specific humoral responses in treated animals. This strategy provides an additional shield which complements vaccination to fight against respiratory infection, presenting a proof-of-concept for the aerosol delivery of fusion inhibitory peptides to protect against measles and other airborne viruses, including SARS-CoV-2, in case of high-risk exposure, that can be readily translated to human trials.

Measles virus (MeV), a member of the Paramyxoviridae family of single-stranded negative sense 57 RNA viruses, is one of the most infectious microorganisms worldwide, with a primary 58 reproduction rate of 12-18 1 . Despite the availability of a safe and effective vaccine, measles causes 59 3 to 4 million cases annually, claimed 207.500 lives in 2019, and remains a leading cause of 60 childhood death from vaccine-preventable diseases in many developing countries 2 . Although 61 incidence has decreased considerably from 2000 to 2016 (from 145 to 18 per million), measles has 62 increased since 2017 2 and is expected to further increase in incidence as a result of the SARS-63 CoV-2 pandemic and the intercurrent delays in childhood immunization programs and resultant 64 "immunity gaps" in the population 3-6 . In addition, in developed countries, imported outbreaks 65 pose a significant risk for immunocompromised people who rely on herd immunity and cannot 66 receive the current live vaccine 7 . 67 MeV is an airborne pathogen, transmitted by inhalation of respiratory droplets and smaller 68 aerosol. Initial infection targets susceptible cells in the respiratory tract 89 . After an incubation 69 period of 7 to 10 days, the acute phase is characterized by fever, oculo-respiratory inflammation, 70 cough, and Koplick spots 10 . The characteristic erythematous skin rash occurs around 14 days after 71 infection 11 when MeV infects cells in the epidermis 11,12 . MeV is amplified in regional lymphoid corresponding to the HRC region, conjugated to a cholesterol moiety (referred to as "HRC4" 94 peptide, Fig. 1D), inhibited the fusion process in cell culture and in organotypic brain cultures 24 . 95 HRC4 peptide administered intranasally to cotton rats and to humanized transgenic mouse models 96 of lethal measles disease led to reduction of the viral titer in cotton rat lungs and a significant 97 increase in survival of mice 2425 . 98 Advantages of inhaled protein therapeutics include the non-invasive needle-free drug 99 delivery route, and the ease of depositing drugs directly in the lungs while limiting systemic 100 toxicity 26 . Since the approval of inhaled Dornase alfa for treating pulmonary disease in cystic 101 fibrosis, several peptides have been under clinical development for inhaled delivery 27 . Nebulizers 102 can be used for high dose delivery with limited drug formulation development [26][27][28] . In the present 103 study, a mesh nebulizer was used to deliver MeV fusion inhibitory peptides to nonhuman primates 104 (NHPs) -cynomolgus macaques -a well-characterized model that recapitulates measles infection 105 in humans 29 . The mesh nebulizer applied in this study uses a piezo-electric generator to push the 106 drug solution through a micro-perforated metal sieve, allowing a fast and silent drug delivery 28 . 107 Small diameters of the sieve pores generate aerosols smaller than 5 µm, enabling efficient 108 pulmonary drug delivery 27 . Using this mesh nebulizer for respiratory administration of fusion

Treatment with HRC4 lipopeptide does not promote selection of drug-resistant variants 117
Generation of escape variants is a concern with any antiviral 30 , we initially tested for emergence 118 of peptide resistant MeV variants in cell culture. Recombinant MeV IC323-eGFP 31 was grown on 119 Vero-hSLAM cells in the presence of either 1µM HRC4 peptide or another fusion inhibitory 120 peptide (FIP) carbobenzoxy-(Z)-D-Phe-L-Phe-Gly peptide 32 , -[FIP-PEG4]2-Chol-which was 121 dimerized and coupled to cholesterol like HRC4 33 . Viruses were sequenced after eight passages 122 (Fig. 1a). In the [FIP-PEG4]2-Chol-treated cells, two mutations in MeV HRC domain were 123 identified, in the same residues as described previously under the selective pressure of the 124 unconjugated FIP, V459 and N462 34 (Fig. 1a, b). However, no mutations were identified in the 125 HRC4-treated MeV. The FIP resistant MeV variants were susceptible to inhibition by HRC4 126 peptide, as determined using a quantitative fusion assay (Fig. 1c). Fusion between cells expressing 127 wt or variant MeV glycoproteins and cells expressing hSLAM was measured by β-galactosidase 128 complementation in the presence of 5 µM of FIP or HRC4. FIP inhibited membrane fusion 129 mediated by the wild-type F and F-V459I but did not affect the fusion mediated by F-N462S or F-130 N462S/V459I mutated proteins. In contrast, HRC4 inhibited membrane fusion mediated by the wild-131 type and mutant F proteins (Fig. 1c). These data demonstrated the absence of HRC4-resistant MeV 132 mutants following multiple viral passages and strengthen the selection of HRC4 lipopeptide ( the treatment with the HRC4 (1 mg/kg) six and twenty-four hours before infection led to a 142 significantly higher survival rate (p <0.0001, Mantel-Cox test) (Fig. S1). 143 Based on these results and previous work 25,37 , we selected for further studies a 4 mg/kg 144 dose, given 3 times by nebulization, 24 h and 6 h before infection, and 24 h after infection, to 145 optimize the antiviral effect of HRC4 in primates which are highly susceptible to MeV, considering 146 the possible loss of peptide delivered through nebulization. This choice was also driven by a 147 recently published study of the therapeutic three-time nebulization of antiviral compound in 148 respiratory syncytial virus (RSV)-infected children 38 . 149 150 Characterization of an aerosol device for lipopeptide delivery into the lung alveoli 151 MeV infection of the respiratory tract targets the lung alveoli 8 , we therefore engineered an inhaled 152 strategy and used a customized mesh nebulizer to deliver the HRC4 lipopeptide aerosol deep into 153 the respiratory tract, to block virus infection. The particle size measurement of aerosol generated 154 following the nebulization of either HRC4 peptide or saline solution (0.9% NaCl) using a prototype 155 mesh nebulizer with a 3 µm pore sieve and a prototype face mask (Fig. 2a) was assessed by laser 156 diffraction (Fig. 2b). The nebulizer devices delivered particles with an average size of 4 µm, in 157 terms of the Volume Mean Diameter (VMD), of both peptide and saline solution, at a flow rate of 158 0.32 -0.46 ml/min (Fig. 2b, detailed in Table S1). Approximately 58% of particles were smaller 159 than 5µm, which is the aerosol size that reaches the airways 27 , where MeV infection initiates. 160 The inhibitory effects of HRC lipopeptide in vitro were evaluated before and after 161 nebulization to address the possibility that nebulization itself could inducing aggregation or 162 degradation of peptide with resultant loss of activity 26,27 ( Fig. 2c and d). Nebulization of HRC4 163 did not cause any loss of activity. Cytotoxicity of the nebulized HRC4 before and after nebulization 164 was evaluated in vitro using Vero-E6 cells (Fig. 2d). No measurable cytotoxicity was observed at 165 doses ranging from 0.5 nM to 4 µM, indicating that the HRC4 therapeutic index is higher than 500 166  (Table S1). After 172 nebulization, 40% of the total aerosolized product reached the respiratory tract, with 11.4% 173 distributed into the lungs (Fig. 3a). The deposition of the HRC4 peptide in the respiratory tract was 174 further analyzed using anti-HRC4 antibodies for the immunofluorescent detection of the peptides 175 in lungs of macaques, sampled either immediately after nebulization (15 min) or 16 h and 24 h 176 later ( Fig. 3b and S2). As expected from the scintigraphy imaging, analysis of all three lung regions 177 revealed the presence of HRC4 within the alveoli surface area, suggesting peptide distribution 178 throughout the lungs following the nebulization. 179 We further analyzed whether peptide could reach the blood circulation following the 180 nebulization. HRC4 was found in low concentration (below 1nM) in the serum of nebulized 181 animals, up to 96 h after a third nebulization (Fig. 3c), while it was absent in the urine. Low entry 182 of the peptide into the circulation did not lead to the active immunization of animals, as HRC4 183 specific antibodies were not found in the serum 28 days after nebulization (Fig. 3d). In addition, 184 no adverse effects (pyrexia, allergic reaction) were observed during the 28 days after peptide 185 nebulization. Histological analysis of lungs collected from nebulized animals did not reveal any 186 abnormalities (Fig. S3). 187 Biochemical parameters in the plasma and cellular composition of the blood were 188 evaluated immediately before treatment and 1, 2, 3, 6, and 28 days after nebulization of either 189 saline or HRC4 peptide in non-infected and MeV-infected NHP, to search for early and late toxic 190 effects of the aerosol delivery (Fig. 4) the infected animal P2, as observed in previous reports 17,47 , with total white blood cell and 280 lymphocytes counts decreased on day 6 following MeV infection in saline-treated animals, but 281 remained stable in HRC4-treated macaques ( Fig. 4B and S5). Leukopenia lasted longer in saline-282 treated macaques (days 6-16 p.i.) compared to animal P2 (days 9-13 p.i.). In infected saline-treated 283 animals, leukopenia was associated with lymphopenia, which was not observed in the fully 284 protected macaques P1 and P3 (Fig. S5). 285 Flow cytometry studies revealed a transient decrease in B cells (CD20 + ) between day 9-13 286 p.i. in mock-treated MeV-infected animals (Fig. S6). The proportion of CD3 + CD4 + and CD3 + CD8 + 287 T cells remained unchanged (Fig. S6) despite a decrease in absolute lymphocyte number (Fig. S6). 288 Evaluation of MeV-infected cell phenotype showed only a few CD14 + monocytes positive for GFP 289 between day 6 and 13 p.i, with CD4 + T lymphocytes and CD20 + B lymphocytes constituting the 290 main targets of the virus (Fig. 7a). Of the total cells infected, 40-60% were T cells and 20% were 291 B cells, with a peak of infection day 6 (C3) or day 9 (C1, C2 and P2) p.i.. The magnitude of 292 infection of CD3 + CD8 + T cells and CD14 + monocytes was lower (Fig. 7a) and the majority of 293 infected cells among PBMCs were CD4 + lymphocytes (Fig. 7b). Interestingly, peptide-treated 294 animal P2, who likely was infected later by transmission from its non-treated cage-mate, had a 295 very low percentage of all infected cell populations, ranging between 5-30 times lower than saline-296 treated animals, suggesting an anti-viral effect of HRC4 nebulization followed by secondary 297 infection from the co-housed actively infected C3 macaque (Fig. 7a). 298 299

Humoral immune response in animals combatting MeV infection 300
MeV infection induces life-long immunity to reinfection, characterized by the generation of a 301 MeV-specific lymphocyte response 15 . We evaluated peripheral blood B cell phenotype and serum 302 antibody responses in MeV-infected macaques using flow cytometry, to track the presence of 303 unswitched (CD20 + CD27 + CD38 + IgD + ) memory B cells, secreting only IgM, and class-switched 304 (CD20 + CD27 + CD38 + IgD -) memory B cells, known to secrete IgG, IgA or IgE (Fig. 8a). Both B 305 cell populations increased from day 3-6 p.i. and day 9 p.i, respectively, in mock-treated animals 306 C2 and C3, although the response of C1 was much lower. The secondarily infected HRC4-treated 307 animal P2 displayed a similar but delayed increase in both class-unswitched and -switched B cell 308 populations. Notably, both P1 and P3 HRC4-treated animals were fully protected against MeV, 309 and B cell populations remained stable without any noticeable increase. 310 MeV-specificity of the B cell response was further confirmed by serological analysis (Fig.  311 8b and 8c). All saline-treated animals seroconverted after MeV infection, with a high MeV 312 antibody titer on day 28 p.i. The secondarily infected animal P2 had a slightly lower total antibody 313 titer (Fig. 8b). All seropositive animals secreted neutralizing antibodies with SN50 values ranging 314 between 546 and 3465 (Fig. 8c). The absence of seroconversion of HRC4-treated animals P1 and 315 P3 correlated with the lack of viral replication and the distinct composition of lymphoid blood 316 compartment in those animals, underlining the efficient and robust protection provided by the 317 nebulized HRC4 lipopeptides. 318 319 320

DISCUSSION 321
Airborne infection is transmitted through small aerosolized particles suspended in the air and is 322 responsible for spreading many important infectious diseases of humans and animals. In this study, 323 we pioneered a nebulization approach to inhibit highly contagious MeV infection in the NHP 324 model with fusion inhibitory peptides. As measles continues to present a significant health problem 325 worldwide 2 , there is a need for prevention modalities in addition to vaccination for those who 326 either cannot be vaccinated or do not respond appropriately to vaccination. In the current study, 327 we adopted an approach based on immunovirological and technological research, to develop a 328 drug and device that can be adapted to treat human patients. Fusion inhibitory HRC4 peptide 329 provided complete protection to MeV challenge after delivery by nebulization. This needle-free 330 therapy may find acceptance among people when compared to other routes of administration 48-50 . 331 The production of 4 µm aerosolized particles by the device used in this study supports its use for  (Fig. 2a). Deposition of aerosol was extrapolated based on the 99m Tc-DTPA signal 524 measured at the end of the nebulization using a gamma camera (Orbiter 75 Ecam, Siemens 525 healthcare, Erlangen, Germany) 68 . The nebulizer charge was measured by counting the 526 radioactivity in the syringe (that contained 99m Tc-DTPA) before and after loading the nebulizers. 527 Immediately after aerosol delivery, the animals were imaged using the gamma camera. The post-528 anterior static scintigraphy acquisition was performed for 120s. The amount of 99m Tc-DTPA 529 deposited into airways and stomach and remaining in the nebulizer was determined from the 530 digitalized images taking into account the tissue attenuation coefficients, previously determined 531 by perfusion scintigraphy (intravenous injection of 99m Tc-macroagregates of albumin). The organ 532 body outline was specified using a specific Region Of Interest (ROI), and the lungs were delineated 533 using the perfusion scan ROI. The aerosol dose delivered to different organs of NHPs is reported 534 as a percentage of the nominal dose placed in the nebulizer for that given experiment, taking into 535 account the decay of technetium for all measurements. and GAPDH primers if necessary (GAPDH FW: CACCCACTCCTCCACCTTTGAC, GAPDH 573 REV: GTCCACCACCCTGTTGCTGTAG). PCR amplification was recorded on a Step One plus 574 apparatus (Thermo). All samples were run in duplicates, and results were analyzed using the ABI 575 StepOne software v2.1 (Applied Biosystems). 576 577

Laser diffraction measurement 578
The aerodynamic performances of the aerosols generated by the prototype mesh nebulizer were 579 determined by laser diffraction using a Spraytec™ instrument (Malvern Instruments Ltd., 580 Malvern, UK) and the Spraytec inhalation cell (Malvern Instruments Ltd., Malvern, UK) 581 connected to an aspiration carried out by a vacuum pump set to 30-50L/min 69 . The prototype mesh 582 nebulizers (n=4) were loaded with 3 ml of either NaCl 0.9% or the HCR4 peptide (4 mg/ml, 583 dissolved as described above) and then connected to the inhalation cell. Nebulization duration was 584 notified at the end of the complete aerosolization of the loaded 3 ml. Diffraction data and volume 585 distribution were automatically registered by the Spraytec software. The volume mean diameter 586 VMD, in µm, the respirable fractions inferior to 5 µm (%< 5 µm) and inferior to 2 µm (%< 2 µm) 587 were calculated by the software. 588 The output of nebulizer was determined by the difference between the weight of the 589 nebulizer before and after nebulization and was expressed in percentage of the loaded volume. The 590 output rate of each nebulizer (in ml/min) was then determined as the ratio between the output and 591 the nebulization duration. At least, the residual volume corresponding to the volume of liquid 592 remaining in the reservoir at the end of the nebulization was also determined by weighting the 593 nebulizer before loading it and after nebulization. 594 595

Enzyme-linked immunosorbent assay (ELISA) 596
Determination of the HRC4 concentration in the serum and urine of macaques after the third 597 nebulization was determined by ELISA. Maxisorp 96 well plates (Nunc) were coated overnight 598 with purified rabbit anti-MeV-F HRC antibodies (Genescript) (5 µg/ml) in carbonate/bicarbonate 599 buffer pH 9.2 at +4°C. Plates were washed twice using PBS followed by incubation with 3% BSA 600 in PBS (blocking buffer) for 60 min. Then, the blocking buffer was replaced with 2 dilutions of 601 each sample in 3% PBS-BSA in duplicate and incubated for 90min at room temperature (RT). 602 Wells were washed 3 times using PBS, and the peptide was detected using an HRP-conjugated 603 rabbit custom-made anti-MeV F HRC antibody (1:1500) in blocking buffer for 2h at RT. Detection 604 of HRP activity was measured by using the TMB substrate (Thermo) and reading absorbance at 605 405 and 620nM on Multiskan FC reader (Thermo). The standard curves were established for each 606 peptide, using the same ELISA conditions as for the test samples and the detection limit was 607 determined to be 0.04nM. To assess the bioavailability of the HRC4 peptide on lungs, lung slices of paraffin embedded 633 organs of 5µm thickness were stained and imaged by confocal microscopy as described previously 634 24 . Briefly, after being blocked and permeabilized in 0.1% TritonX100, 5% BSA solution, slices 635 were sequentially incubated with a rabbit anti-HRC4 (Genscript) overnight at 4°C and with a 636 secondary goat anti rabbit alexa-555 (Thermo) and DAPI for 1H at room temperature. Slides were 637 imaged using a Zeiss LSM800 confocal microscope). 638  monocytes, CD4 + , CD8 + and CD20 + lymphocytes in MeV-infected cynomolgus monkeys by flow 957 cytometry, following the nebulization of either 0.9% NaCl (C) or HRC4 peptide (P). CD4 + T 958 lymphocytes were characterized as CD3 + CD8 -, and CD8 + T lymphocytes were characterized as 959 CD3 + /CD8 + ; B-lymphocytes were characterized as CD3 -/CD20 + cells.