A robust mouse model of HPIV-3 infection and efficacy of GS-441524 against virus-induced lung pathology

Lin, Yuxia; Khan, Mona; Weynand, Birgit; Laporte, Manon; Coenjaerts, Frank; Babusis, Darius; Bilello, John P.; Mombaerts, Peter; Jochmans, Dirk; Neyts, Johan

doi:10.1038/s41467-024-52071-5

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Published: 05 September 2024

A robust mouse model of HPIV-3 infection and efficacy of GS-441524 against virus-induced lung pathology

Nature Communications volume 15, Article number: 7765 (2024) Cite this article

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Abstract

Human parainfluenza virus type 3 (HPIV-3) can cause severe respiratory tract infections. There are no convenient small-animal infection models. Here, we show viral replication in the upper and lower airways of AG129 mice (double IFNα/β and IFNγ receptor knockout mice) upon intranasal inoculation. By multiplex fluorescence RNAscope and immunohistochemistry followed by confocal microscopy, we demonstrate viral tropism to ciliated cells and club cells of the bronchiolar epithelium. HPIV-3 causes a marked lung pathology. No virus transmission of the virus was observed by cohousing HPIV-3-infected AG129 mice with other mice. Oral treatment with GS-441524, the parent nucleoside of remdesivir, reduced infectious virus titers in the lung, with a relatively normal histology. Intranasal treatment also affords an antiviral effect. Thus, AG129 mice serve as a robust preclinical model for developing therapeutic and prophylactic strategies against HPIV-3. We suggest further investigation of GS-441524 and its prodrug forms to treat HPIV-3 infection in humans.

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Introduction

Human parainfluenza viruses (HPIVs, family Paramyxoviridae) are highly transmissible viruses that are responsible for common acute respiratory infections, ranging from colds and croup to bronchiolitis and pneumonia across all age groups, with the highest infection rate observed in young children^1,2. Of the four HPIV serotypes, HPIV-3 is the most virulent, primarily causing lower respiratory tract illnesses such as bronchiolitis and pneumonia, particularly in children under 5 years^3,4. HPIV-3 infection is an important cause of morbidity and mortality in immunocompromised individuals and the elderly⁵. There are no vaccines for the prevention, nor antiviral drugs for the treatment of HPIV infections. Akin to other paramyxoviruses, HPIVs are single-stranded, negative-sense RNA viruses and encode six essential proteins: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), hemagglutinin-neuraminidase (HN), and RNA-dependent-RNA polymerase (L).

BALB/c mice have been used in various HPIV-3 studies to assess immune responses to protein subunit vaccines^6,7. No robust HPIV-3 challenge models have been reported for immunocompetent mice, suggesting that they are not or not sufficiently susceptible to HPIV-3. Infection models for HPIV-3 have been described in hamsters, guinea pigs, cotton rats, ferrets, sheep, and non-human primates^5,8,9. Infection of hamsters results in low-level replication and mild bronchiolitis¹⁰; this model has been used occasionally. In cotton rats the virus replicates in the nasal turbinates and lungs; this model has been used in a few studies to assess the efficacy of antiviral drugs and vaccine candidates^11,12,13,14. In ferrets intranasal infection leads to replication in the lower airways and limited replication in the upper airways; no overt histological lesions were observed in the putative target cells¹⁵. A few older studies report hypersensitivity of the airways upon infection of guinea pigs^16,17,18. African green monkeys and newborn lambs also allow for viral replication, and broncho-interstitial pneumonia was noted in lambs^9,19. The non-human primate and newborn lamb models have been used for studies of vaccine candidates^9,20,21,22. Taken together, there are no robust HPIV-3 infection models in mice, hamsters or guinea pigs. The virus replicates well in the respiratory tract of cotton rats, ferrets, lambs, and African green monkeys, but these species are much less convenient as preclinical models than mice because of their larger body size, higher cost, limited availability, and more complex handling and husbandry. Moreover, these species are not inbred so the findings may be less reproducible due to differences in genetics.

Here, we set out to develop an inbred mouse HPIV-3 infection model for prophylactic and therapeutic modalities. The AG129 strain is a widely available inbred 129/Sv strain that carries a double knockout in receptor genes for IFNα/β and IFNγ²³ and supports the replication of several viruses^24,25,26. We show that AG129 mice support viral replication of HPIV-3 and develop bronchopneumonia and hyperplasia of pneumocytes upon intranasal inoculation. Using GS-441524, the parent nucleoside of remdesivir²⁷, we validated this model for antiviral studies.

Results

Replication potential of HPIV-3 in various mouse strains upon intranasal inoculation

In preliminary experiments, we explored whether intranasal inoculation of five inbred mouse strains results in measurable viral replication: BALB/c, SCID, C57BL/6, Ifnar−/−, and AG129. Mice (n = 4 per strain) were intranasally inoculated with 1.5 × 10⁶ 50% tissue culture infective doses (TCID₅₀) of HPIV-3. Mice were killed at days 1, 3, 7, or 10 post-infection (p.i.), and viral loads in the lung were determined (Fig. S1a). Infectious virus titers in the lung of all BALB/c, SCID, C57BL/6, and Ifnar−/− mice remained below 2 log₁₀ TCID₅₀/mg lung sample at any time point (Fig. S1b, data not shown for BALB/c and SCID mice). In contrast, in AG129 mice, median infectious virus titers at day 1 p.i. were 2.9 log₁₀ TCID₅₀/mg lung sample (Fig. S1b). There was a minor gain in body weight of C57BL/6, Ifnar−/−, and AG129 mice (Fig. S1c). Histological examination with hematoxylin-eosin (H&E) staining of C57BL/6 mice revealed minimal lung pathology. In Ifnar−/− mice, peribronchial inflammation and perivascular inflammation were detected in the lung from day 7 p.i. and the severity increased until day 10 p.i., the end of the experiment. In AG129 mice, the first pathological changes, with very limited focal areas of peribronchial inflammation, were observed as early as day 1 p.i., and these increased over time to bronchopneumonia and hyperplasia of pneumocytes, with median cumulative lung pathology scores of 4.0 at day 7 p.i. (Fig. S1d). Based on these results, we decided to further explore AG129 mice as a model for HPIV-3 infection.

Kinetics of HPIV-3 replication in AG129 mice

Next, we infected AG129 mice intranasally with HPIV-3 and collected lung samples and nasal-pharyngeal-laryngeal-tracheal washes (NPLT-washes) at 0.5 h, 6 h, 1 day, 2 days, 3 days, 5 days, and 7 days p.i. to quantify infectious virus titers in the lung and NPLT-washes (Fig. 1a). Infectious virus titers in the lung peaked at day 1 p.i. (median value of 3.2 log₁₀ TCID₅₀/mg lung sample), followed by a gradual decrease from day 2 p.i. (median values of 2.5, 2.2, and 1.1 log₁₀ TCID₅₀/mg tissue) to undetectable levels at day 7 p.i. (Fig. 1b). Infectious virus titers in NPLT-washes persisted from day 1 to day 5 p.i. (~3.7 log₁₀ TCID₅₀/mL) and sharply declined to undetectable levels at day 7 p.i. (Fig. 1c). Infected mice did not present with overt changes in alertness or physical activity, except for a minor weight change at days 5 and 7 p.i. (Fig. 1d). Histological examination revealed focal areas of peribronchial inflammation in the lung at day 3 p.i. Pathological changes developed prominently from day 5 p.i., a time point at which viral replication was declining. At day 7 p.i., the lung parenchyma was severely affected, with substantial perivascular and peribronchial inflammation and hyperplasia of pneumocytes across the entire area (Fig. S2). Median histopathology scores at days 5 and 7 p.i. were 4.3 and 4.0, respectively (Fig. 1e). Taken together, we demonstrated that AG129 mice support sufficient viral replication and develop lung pathology upon intranasal inoculation of HPIV-3.

**Fig. 1: Kinetics of HPIV-3 replication in intranasally inoculated AG129 mice.**

Tropism of HPIV-3 to ciliated cells and club cells of the bronchiolar epithelium

HPIV-3 is a single-stranded negative-sense RNA virus. To determine the target cell types—the cellular tropism—in AG129 mice, we performed confocal imaging of sections stained in multiplex with the RNAscope technology, which visualizes a single RNA molecule as a dot or “punctum,” plural “puncta”²⁸, combined with immunohistochemistry (IHC), which visualizes an antigen as an immunoreactive (IR) signal. The precision of confocal imaging supports the robust characterization of a cell type or an individual cell as a target cell type or infected cell, respectively. We adopted the same workflow as we developed previously for postmortem samples of COVID-19 patients to visualize the patterns of infection with SARS-CoV-2, a single-stranded positive-sense RNA virus^{29,30,31,32,33}.

At the level of the lung, the respiratory tract consists mostly of bronchioles terminating in alveoli (Fig. 2). Most cells in the bronchiolar epithelium contain Krt8-IR signal. Major cell types in the bronchiolar epithelium are ciliated cells and club cells. An RNAscope probe for the gene Foxj1, encoding a transcription factor that is required for ciliogenesis, identifies ciliated cells (Fig. 2a). An RNAscope probe for the gene Scgb1a1, encoding an antimicrobial secretory protein that is also referred to as CC10, uteroglobin, or blastokinin, identifies club cells (Fig. 2a). The alveolar wall contains type I pneumocytes, which can be identified with an RNAscope probe for Ager, encoding a receptor for advanced glycosylation end products. Type II pneumocytes can be identified with an RNAscope probe for Sftpc, encoding surfactant associated protein C (Fig. 2a, j–l).

**Fig. 2: Tropism of HPIV-3 to ciliated cells and club cells of the bronchiolar epithelium of AG129 mice.**

As expected, sections through lung samples of uninfected mice did not yield RNAscope puncta for the viral hemagglutinin-neuraminidase gene HPIV-3-HN (Fig. 2b) nor for the viral nucleocapsid gene HPIV-3-NP (Fig. 2c). At day 1 p.i. (Fig. 2d, e) and day 3 p.i. (Fig. 2g–i), numerous ciliated cells and club cells harbored densely packed HPIV-3-HN and HPIV-3-NP puncta, colocalizing in the same cells. By contrast, at day 10 p.i., a small fraction of cells in the bronchiolar epithelium harbored HPIV-3-NP puncta (Fig. 2f). In the alveoli at day 5 p.i., neither cells harboring Ager puncta (Fig. 2j, k) nor cells harboring Sftpc puncta (Fig. 2l) harbored HPIV-3-NP puncta. Roundish cells were densely packed with HPIV-3-NP puncta and may represent intra-alveolar histiocytes (Fig. 2j–l).

Taken together, confocal imaging of sections subjected to multiplex fluorescence staining with the ultrasensitive RNAscope platform for RNA in situ hybridization combined with IHC enabled us to assign HPIV-3 tropism in AG129 mice to ciliated cells and club cells of the bronchiolar epithelium. We did not uncover evidence for tropism to type I or type II pneumocytes in the alveolar wall, but the absence of evidence does not equal the evidence of absence.

Prophylactic oral treatment of GS-441524 reduces viral loads and lung pathology

We reasoned that, if the infectious viruses detected in the lung reflected active replication, it should be possible to suppress viral replication by treatment with an inhibitor of HPIV-3 replication. We opted for GS-441524, the parent nucleoside of remdesivir (Veklury, Gilead Sciences), a drug that has been approved for the treatment of COVID-19³⁴. GS-441524 is intracellularly phosphorylated to the 5′-triphospate that inhibits the viral RNA-dependent RNA polymerase²⁷. GS-441524 and remdesivir inhibit the in vitro replication of parainfluenza viruses and other paramyxoviruses³⁵. AG129 mice were orally treated twice daily (BID) with vehicle or GS-441524 (50 mg/kg), starting 1 day before intranasal inoculation of HPIV-3. Treatment was continued for four consecutive days (Fig. 3a). As we had observed high viral titers in untreated mice at day 3 p.i. (Fig. 1), this timepoint was set as the endpoint for the treatment experiment. There was no weight loss or any clinical sign of adverse effects in either group (Fig. S3a). GS-441524 treatment led to a reduction of 3.1 log₁₀ TCID₅₀/mg lung sample (p < 0.0001) of infectious virus titer and a reduction of 1.3 log₁₀ TCID₅₀ equivalent/mg lung sample (p < 0.0001) of HPIV-3 RNA in the lung compared to the vehicle-treated group (Fig. 3b, c). No or only very limited infectious virus titers were detected in the GS-441524-treated animals (Fig. 3b). In contrast, no difference in infectious virus titers and HPIV-3 RNA in NPLT-washes was observed between the GS-441524-treated and the vehicle-treated groups (Fig. S3b, c).

**Fig. 3: Prophylactic oral treatment with GS-441524 of AG129 mice intranasally inoculated with HPIV3 reduces viral loads and lung pathology.**

Considering the kinetics of HPIV-3 replication and the late development of lung histopathology (day 5 p.i.), we conducted another experiment with the same setup but whereby lung samples were collected at day 6 p.i. to assess the impact of antiviral treatment on lung pathology (Fig. 3d). There was no weight loss or any clinical sign of adverse effects in either group at day 6 p.i. (Fig. 3e). The lungs of uninfected mice appeared normal. The lungs of the vehicle-treated group displayed inflammation, primarily in the centrilobular regions, accompanied by bronchopneumonia, perivascular inflammation, and peribronchial inflammation. In contrast, in GS-441524-treated mice only very focal and limited perivascular inflammation was noted within the nearly normal lung parenchyma (Fig. 3f). The median lung histopathology score in the vehicle-treated animals was 4.5 versus 1.5 in the GS-441524-treated mice (p = 0.0002) (Fig. 3g). Taken together, we demonstrated that oral treatment of GS-441524 significantly reduces viral loads and pathology in the lung of HPIV-3-infected AG129 mice.

A viral RNA species reflecting ongoing viral replication is drastically reduced in the target cell types of the bronchiolar epithelium by treatment with GS-441524

Our data on infectious virus titers of lung samples indicated that viral loads in the target cell types are affected by GS-441524 but do not provide information on the sites of inhibition of viral replication. We therefore performed confocal imaging of sections subjected to multiplex fluorescence staining with RNAscope and IHC (Fig. 4). Antigenome RNA species are not present in virions of this single-stranded, negative-sense RNA virus, but are transiently produced during the viral life cycle such that their presence reflects ongoing viral replication within target cells. We custom-designed an RNAscope HPIV-3-NP-sense probe, which we here refer to as the HPIV-3-NP-antigenome probe as a more intuitive reference to the virological significance of the puncta revealed by this probe.

**Fig. 4: Visualizing *HPIV-3* RNA species in vehicle-treated vs. GS-441524-treated AG129 mice.**

As expected, a section through a lung sample of an uninfected mouse did not yield RNAscope puncta for the HPIV-3-NP-antigenome probe (Fig. 4a). In vehicle-treated mice (Fig. 4b–f), a major fraction of cells harboring Foxj1 puncta (ciliated cells) or Scgb1a1 puncta (club cells) also harbored HPIV-3-NP-antigenome or HPIV-3-HN puncta. Interestingly, HPIV-3-NP-antigenome puncta resided perinuclear (around the nucleus), whereas HPIV-3-HN puncta diffusely filled the cells (Fig. 4b). We previously reported a similar perinuclear location of SARS-CoV-2-sense puncta, representing antigenome RNA^30,31. In sharp contrast, there were few (Fig. 4g, h) if any (Fig. 4i) HPIV-3-NP-antigenome or HPIV-3-HN puncta in sections through lung samples of GS-441524-treated mice. Taken together, by visualizing RNA species that reflect ongoing viral replication within target cells, we demonstrated that GS-441524 effectively reduces viral replication within the target cells of HPIV-3 in the bronchiolar epithelium—ciliated cells and club cells.

Intranasal treatment of GS-441524 reduces viral loads and lung pathology

Since oral treatment of GS-441524 did not appear to reduce viral loads in NPLT-washes, we next explored whether intranasal treatment of GS-441524 would be able to do so. AG129 mice were intranasally treated once daily with GS-441524 (40 µL/mouse of a 30 mg/mL solution), starting 1 day before intranasal inoculation of HPIV-3. Mice were killed at day 3 p.i. and lung samples and NPLT-washes were collected (Fig. 5a). GS-441524 treatment reduced infectious virus titers by 2.3 log₁₀ TCID₅₀/mg lung sample (p = 0.0048) and HPIV-3 RNA by 1.1 log₁₀ TCID₅₀ equivalent/mg lung sample (p = 0.0071) compared to the vehicle-treated group (Fig. 5b, c). Interestingly, intranasal treatment with GS-441524 reduced infectious viral titers by 1.4 log₁₀ TCID₅₀/mL (p < 0.0001) and HPIV-3 RNA by 1.1 log₁₀ TCID₅₀ equivalent/mL (p < 0.0001) in NPLT-washes compared to the vehicle-treated group (Fig. 5d, e).

**Fig. 5: Intranasal treatment with GS-441524 of intranasally inoculated AG129 mice reduces viral loads in the lung and NPLT-washes and lung pathology.**

To assess the effect of intranasal treatment on lung pathology, lungs were collected at day 6 p.i. (Fig. 5f) using the same experimental setup. There is no significant difference in weight change between the two groups (Fig. 5g) and no clinical signs of adverse effects. In vehicle-treated mice, the lung exhibited perivascular inflammation, peribronchial inflammation, and hyperplasia of pneumocytes. In contrast, GS-441524 treatment significantly reduced lung pathology, with only a small area of bronchopneumonia, perivascular inflammation, and some hyperplasia of pneumocytes in the centrilobular regions (Fig. 5h). The median lung pathology score in vehicle-treated animals was 3.0 in GS-441524-treated mice, compared to 5.0 in vehicle-treated mice (p = 0.0070) (Fig. 5i).

A single intranasal prophylactic dose of GS-441524 inhibits HPIV-3 replication in the lung

Next, AG129 mice were treated prophylactically with a single intranasal dose of GS-441524 (40 µL/mouse of a 30 mg/mL solution) either at 6 h or 1 h before intranasal inoculation, and lungs were collected at day 3 p.i. (Fig. 6a). Prophylaxis with GS-441524 resulted in reductions of 0.8 log₁₀ TCID₅₀/mg lung sample (p = 0.0390) and 1.3 log₁₀ TCID₅₀/mg lung sample (p = 0.0390) in the −6 h and −1 h treatment groups, respectively (Fig. 6b). Consistently, this prophylaxis reduced HPIV-3 RNA by 1.1 log₁₀ TCID₅₀ equivalent/mg lung sample (p = 0.0392) and 1.8 log₁₀ TCID₅₀ equivalent/mg lung sample (p = 0.0060) in the −6 h and −1 h treatment groups, respectively (Fig. 6c). Thus, a single prophylactic treatment of GS-441524 resulted in inhibition of HPIV-3 replication in the lung.

**Fig. 6: A single intranasal prophylactic dose of GS-441524 in AG129 mice intranasally inoculated with HPIV-3 reduces viral loads in the lung.**

HPIV-3-infected mice do not transmit the virus to sentinels

Finally, we explored whether HPIV-3 can be transmitted from experimentally infected AG129 mice to sentinel mice. Six AG129 mice were intranasally inoculated with HPIV-3 and at day 1 p.i., each index mouse was co-housed in a cage with a sentinel mouse for 2 days. The index mice were killed at day 3 p.i., and the sentinel mice were killed the following day (Fig. S4a). There was no significant weight loss in either group (Fig. S4b). No virus was detected in the lung of all sentinels, and minute amounts in the NPLT-washes of one sentinel (Fig. S4c). Taken together, HPIV-3-infected AG129 mice do not readily transmit the virus to other mice in this experimental context.

Discussion

HPIV-3 infection is an important cause of lower respiratory tract illnesses in vulnerable populations, including children under 5 years, the elderly, and immunocompromised individuals. There are no vaccines nor antiviral drugs for the prevention and treatment of infections with HPIV-3 or other HPIVs. We here report a robust small-animal model for HPIV-3 infection that may serve as a preclinical model for antiviral studies.

We explored the replication capacity of HPIV-3 in the airways of mice following intranasal inoculation. Among the five inbred strains of mice we studied, AG129 mice were clearly the most susceptible. We ascribe their susceptibility to the failure of activation of the interferon pathways, which play a crucial role in the host antiviral response against viruses³⁶. AG129 mice lack a functional IFN α/β/γ immune response. HPIV-3 infection results in the production of type I and type II interferons³⁷, which inhibit HPIV-3 replication^38,39. Conversely, the rapid clearance of HPIV-3 replication in mice such as C57BL/6 and BALB/c mice can likely be ascribed to an efficient interferon response. Interestingly a patient, exhibiting a complete deficiency of IFNγ responsiveness due to mutations in the IFNγ receptor gene, was reported to experience repeated viral infections including HPIV-3 within a short interval of 5 months and suffered from severe pneumonia complicated by respiratory failure requiring mechanical ventilation⁴⁰.

Whereas intranasal inoculation of AG129 mice with HPIV-3 did not result in observable disease or discomfort, post-mortem examination revealed viral replication and major respiratory pathology. We found that HPIV-3 replicated well in the upper and lower respiratory tracts of AG129 mice, with a rapid and sustained peak occurring from days 1 to 3 p.i. in the lung and a peak from days 1 to 5 p.i. in the NPLT-washes. By day 7 p.i., the infectious viruses were cleared completely from the lung and NPLT-washes. Lung pathology became prominent when viral replication was declining or undetectable. Despite the productive replication of HPIV-3 in the respiratory tracts of AG129 mice, we did not observe mouse-to-mouse transmission of the virus, probably due in part to a lack of clinical signs such as coughing or sneezing with subsequent spread of the virus. In ferrets as well, no horizontal transmission of the virus was demonstrated in a direct contact transmission experiment¹⁵.

Infectious virus titers in the lung of AG129 mice appear comparable to those reported in cotton rats and are ~2 log₁₀ higher than viral titers in the lung of hamsters¹⁰, although a direct comparison is not possible. Viral loads in AG129 mice, however, are lower than those reported for patients^41,42,43. Since we collected NPLT-washes rather than nasal swabs, nasopharyngeal swabs, or tracheal aspirates, a direct comparison is difficult. Additionally, as humans are the natural host, it may not be unexpected that viral loads are higher in humans. Active viral replication in the lung was further confirmed by using RNAscope, using a probe targeting positive-sense RNA of the nucleocapsid gene. Confocal imaging of sections stained with RNAscope and IHC indicated that HPIV-3 replicates in ciliated cells and club cells of the bronchiolar epithelium. We did not observe obvious colocalization of the virus with type I or type II pneumocytes in the alveolar wall. In an in vitro model of human pseudostratified mucociliary airway epithelium, HPIV-3 was shown to mainly infect ciliated cells⁴⁴. HPIV-3 infection of a few type II pneumocytes was detected in lung organoids derived from human pluripotent stem cells⁴⁵. It remains to be demonstrated whether or not HPIV-3 has a tropism for pneumocytes in the human lung. In ferrets, the virus was proposed to target type I and type II pneumocytes¹⁵.

HPIV-3-infected AG129 mice developed peribronchial inflammation, perivascular inflammation, bronchopneumonia, and hyperplasia of pneumocytes. These features of bronchiolitis and pneumonia are also seen in HPIV-3-infected patients¹. HPIV-3-infected cotton rats present with bronchiolitis and interstitial pneumonia, but the pathological changes vary between the two cotton rat strains used¹¹. HPIV-3-infected ferrets present with viral infection of the lungs but with minimal histological abnormalities¹⁵. HPIV-3-infected hamsters develop only mild inflammation and mild interstitial pneumonia¹⁰. HPIV-3-infected guinea pigs display hyperreactivity of the trachea but there is little information on viral replication in their respiratory tract^16,17,18,46. HPIV-3-infected newborn lambs present with broncho-interstitial pneumonia characterized by bronchiolar hyperplasia and severe diffuse infiltration of macrophages in alveolar septae⁹.

To seek further evidence that HPIV-3 indeed replicates in the respiratory tracts of AG129 mice, we explored whether the antiviral agent GS-441524, the parent nucleoside of remdesivir, can reduce viral replication in the respiratory tracts. Intracellularly, GS-441524 is converted to its 5ʹ-triphosphate metabolite that inhibits the viral RNA-dependent RNA polymerase of coronaviruses and a number of other viruses/virus families including paramyxoviruses^{27,35,47,48,49,50}. Obeldesivir (GS-5245), an oral prodrug with enhanced GS-441524 bioavailability, is currently in phase 3 clinical trials^51,52,53. In the lung of HPIV-3-infected AG129 mice treated with oral doses of GS-441524, infectious virus titers were markedly reduced to even undetectable levels, and levels of HPIV-3 RNA were significantly reduced. Additionally, virtually no infected cells were detected in the respiratory tract by means of RNAscope. A single oral solution administration of 24 mg/kg GS-441524 (which is about half the efficacious dose of 50 mg/kg) given to uninfected mice, yielded a daily exposure (AUC_(0–24 h)) of 13,100 ng h/mL (Fig. S5). Assuming proportional increases in plasma exposures, the projected estimates following 50 mg/kg, twice-a-day (BID) administrations to mice would yield an AUC_(0–24 h) value of 56,500 ng h/mL. This was roughly twice the plasma GS-441524 exposures that were achieved in humans following oral administration of a 350 mg BID regimen of obeldesivir⁵⁴. These results support additional studies to establish the dose-dependent efficacy of oral obeldesivir treatment in this model.

Additionally, intranasal doses of GS-441524 decreased viral loads in the NPLT-washes as well as in the lung. Remarkably, even a single prophylactic intranasal dose at 6 or 1 h before infection yielded an antiviral effect. Together, these observations lend further support for our findings that HPIV-3 replicates well in the respiratory tract of AG129 mice. The finding that oral dosing (unlike intranasal dosing) did not reduce viral replication in the NPLT-washes may be explained by insufficient concentrations of the drug reaching the nasal epithelium.

There are no drugs approved for the treatment of severe infections with any of the four HPIVs. The use of ribavirin, dosed either aerosolized or orally, with or without intravenous gamma globulin, has been reported in small and uncontrolled series and in case reports of immunocompromised patients with severe HPIV-3 infections. The therapeutic effect varies, from no effect to a decrease in mortality and improvement of symptoms^55,56. Additionally, in a few cases, the patients had to discontinue the treatment due to adverse effects, such as anemia⁵⁷. Thus, ribavirin is certainly not an ideal drug for the treatment of infections by HPIVs. Severe HPIV infections may develop in the elderly and patients with underlying chronic conditions and may even be fatal in immunocompromised patients, in particular in recipients of a hematopoietic stem cell transplant and in children with severe combined immunodeficiency disease syndrome^{40,55,56,58,59}. Since remdesivir has been approved for the treatment of SARS-CoV-2 infection and is safe and well tolerated, we propose that it (or obeldesivir) should be considered for the treatment of HPIV infections. Viral replication in immunocompromised patients may well be prolonged, such that antiviral efficacy can still be expected even when treatment is started relatively late. In immunocompetent patients at high or higher risk, such as those with underlying cardiac or lung disease, viral replication in the respiratory tract may be transient, (similar to our mouse model) such that early start of antiviral treatment might be of key importance.

In conclusion, we established a robust and convenient HPIV-3 infection model in AG129 mice that mimics, to some extent, the HPIV-3-induced bronchiolitis and (broncho)pneumonia that is observed in humans. This model will be instrumental for developing therapeutic and prophylactic modalities against this virus. We demonstrated in this model the therapeutic potential of GS-441524 and its prodrug forms for the treatment of HPIV-3.

Methods

Animals

C57BL/6 mice were purchased from Janvier Laboratories. Ifnar−/− mice (C57BL/6J mice with a knockout in the IFNα receptor 1 gene) and AG129 mice (129/Sv mice with knockouts in both IFNα/β and IFNγ receptor genes) were bred in-house. Ifnar−/− breeding pairs were a generous gift of Dr. Claude Libert, IRC/VIB, University of Ghent, Belgium. Breeding couples of AG129 mice were purchased from Marshall BioResources. The specific pathogen-free status of the mice is regularly checked at the KU Leuven animal facility of the Rega Institute. Inoculation was performed in 6–8 week-old female C57BL/6 and Ifnar−/− mice and in 8–12 week-old female and male AG129 mice. Mice were housed in individually ventilated isolator cages (IsoCage N Biocontainment System, Tecniplast) at a temperature of 21 °C, humidity of 55%, and subjected to 12:12 dark/light cycles. They had access to food and water ad libitum, and their cages were enriched with cotton and cardboard play tunnels for mice. Housing conditions and experimental procedures were approved by the Ethical Committee Dierproeven of KU Leuven (License P099/2022), following institutional guidelines approved by the Federation of European Laboratory Animal Science Associations. The animal work was conducted under Biosafety Level 2 conditions. There was no preference for the gender of the mice used in all experiments; the choice of sex depended on their availability. Mice were anesthetized by inhalation with isoflurane before infection and killed by administering 100 µL of intraperitoneal Dolethal before sample collection. Mice were monitored daily for any signs of disease.

Cells and virus

LLC-MK2 cells (rhesus monkey kidney, ATCC, CCL-7) were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum and 1.5% HEPES at 37 °C in a 5% CO₂ incubator. Endpoint titrations were performed using medium containing 2% fetal bovine serum instead of 10%.

HPIV-3, designated isolate 10-012854, was obtained from a nasopharyngeal wash collected from a patient in March 2010 at University Medical Center in Utrecht, The Netherlands. The isolate was propagated for 3 passages in LLC-MK2 cells to generate the virus stock used for inoculation, which has a titer of 3.8 × 10⁷ TCID₅₀/mL. Iketani et al. reported 9 mutations emerging in the HN gene of cultured isolates: L239F, P241L, R242K, H552Q, S554N, L555S, L555F, N556D, and T557I, and described that HPIV-3 isolation in cell lines resulted in a loss of viral entry properties required for fitness in humans⁶⁰. We did not observe any of these amino acid changes in the genome of the virus stock used for inoculation. The full genome of isolate 10-012854 is available in GenBank under accession number PP909812.

Compounds

GS-441524 was purchased from Excenen Pharmatech Co., Ltd. (China) and formulated as 15 mg/mL or 30 mg/mL stocks in 30% PEG₄₀₀ (Sigma) in PBS containing 1% DMSO.

HPIV-3 infection of mouse strains

Mice were anesthetized with isoflurane and inoculated intranasally with 40 µL of medium containing 1.5 × 10⁶ TCID₅₀ HPIV-3-isolate 10-012854 at day 0. Mice were monitored daily for weight changes and clinical signs. At indicated time points, mice were killed by intraperitoneal injection of 100 µL Dolethal. Nasal-pharyngeal-laryngeal-tracheal washes and lung samples were collected for quantification of infectious virus titers. A portion of the lung was fixed in 4% formaldehyde for histological analysis.

Treatment regimen

In oral treatment assays, AG129 mice were administered either vehicle (n = 12) or 50 mg/kg GS-441524 (n = 12) twice daily (BID) from day −1 to the endpoint. For intranasal treatment assays, AG129 mice were administered either vehicle (n = 11) or GS-441524 (40 µL/mouse of a 30 mg/mL solution) (n = 11) once daily (QD) from day −1 to the endpoint. At day 0, mice were anesthetized with isoflurane and intranasally inoculated with 40 µL of medium containing 1.5 × 10⁶ TCID₅₀ HPIV-3-isolate 10-012854. At day 3 p.i., mice were killed to collect nasal-pharyngeal-laryngeal-tracheal washes and lung samples for quantification of infectious virus titers and HPIV-3 RNA levels. At day 6 p.i., mice were killed to collect lung samples, and a portion of the lung was fixed in 4% formaldehyde for subsequent histological analysis.

Sample collection

Lung samples were collected and homogenized using bead disruption (Precellys) in 400 µL medium, followed by centrifugation (10,000 × g, 5 min) to pellet cell debris. The supernatant was then processed for endpoint virus titration. Nasal-pharyngeal-laryngeal-tracheal washes were collected with the following procedure. The skin surrounding the trachea of freshly killed mice was removed to expose the muscle surrounding the trachea. A small incision was made in the muscle to further expose the trachea. A small incision was made in the trachea and a catheter was inserted. Then, 400 µL of PBS was injected into the catheter using a syringe, and washes were collected from the nostrils. This washing procedure was repeated twice. The washes were then processed for endpoint virus titration.

Endpoint virus titration assay

The endpoint virus titration was conducted on LLC-MK2 cells in 96-well plates. Cytopathic effects induced by HPIV-3 were observed and scored under the microscope. Viral titers were calculated using the Reed and Muench method and expressed as the TCID₅₀ per milligram of lung sample or per milliliter of NPLT-wash.

RT-qPCR

RT-qPCR was conducted on a LIGHTcycler96 platform (Roche) using the iTaq universal probes one-step RT-qPCR KIT (Bio-Rad, Cat#1725141). The primers targeting the HN gene of HPIV-3 encoding hemagglutinin-neuraminidase were forward primer 5′-TGATGAAAGATCAGATTATGCAT-3′ and reverse primer 5′-CCAGGACACCCAGTTGTG-3′. The probe used was 5′-56-FAM/TGGACCAGG/ZEN/GATATACTACAAAGGCAAAATAATATTTCTC/3IABkFQ/-3′. The primers and probe were purchased from Integrated DNA Technologies, Inc. Equivalent standard serial dilutions of the HPIV-3 virus stock were employed to quantify viral RNA levels per milligram of lung sample or per milliliter of NPLT-wash.

Histology

Lung samples were fixed overnight in 4% formaldehyde and embedded in paraffin. Tissue sections (5 μm) were stained with H&E and scored blindly for lung damage by an expert pathologist. The 5 parameters, congestion, intra-alveolar hemorrhages, intra-alveolar edema, apoptotic bodies in the bronchus wall, and perivascular edema, were scored 0 or 1 for absent or present. The 3 parameters, perivascular inflammation, peribronchial inflammation, and vasculitis were scored on a scale of 0 to 3, depending on the number of inflammatory cells. Bronchopneumonia was scored on a scale of 0–3 depending on the extent of the pathology: 1 is ≤40%, 2 is between 41% and 60%, and 3 is ≥61% of the affected lung area⁶¹. A cumulative score was calculated by summing the scores of each parameter. The higher the number, the worse the pathology. The max score is 17. The epithelium in the images of Figs. 2 and 4 is referred to as “bronchiolar epithelium”, as the sections were made through parts of the lungs that are located peripherally.

RNAscope probes

Probes custom-designed by Advanced Cell Diagnostics against the gene encoding HPIV-3 nucleocapsid protein are: V-V-HPIV3-NP-C1 (Cat#1268201-C1), V-V-HPIV3-NP-C2 (Cat#1268201-C2), and reverse complement version V-V-HPIV3-NP-sense-C3 (Cat#1268848-C3). These custom-designed probes are commercially available. Catalog probes purchased from Advanced Cell Diagnostics are: V-HPIV3-HN-C1 (Cat#1223331-C1), Mm-Foxj1-C2 (Cat#317091-C2), Mm-Foxj1-C3 (Cat#317091-C3), Mm-Scgb1a1-C2 (Cat#420351-C2), Mm-Scgb1a1-C3 (Cat#420351-C3), Mm-Sftpc-C4 (Cat#314101-C4), Mm-Sftpb-No-XHs-C2 (Cat#539421-C2), and Mm-Ager-O1 (Cat#550791-C1).

RNAscope in situ hybridization

The RNAscope fluorescence manual assay was performed on lung FFPE sections at 5 µm thickness. Multiplex hybridization was detected with the Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics, Cat# 323110) according to the manufacturer’s protocol and as previously described^30,31. Briefly, slides were baked at 60 °C for 1 h, followed by deparaffinization in Xylene (Leica Biosystems, Cat# 3803665EG) and 100% ethanol wash. Sections were incubated in hydrogen peroxide for 15 min at room temperature. Tissue pretreatment was performed by steaming in target retrieval reagent (Advanced Cell Diagnostics, Cat#322000) for 30 min followed by incubation in Protease Plus (Advanced Cell Diagnostics, Cat#322330) at 40 °C for 30 min. RNAscope probes were hybridized at 40 °C for 2 h. Signal amplification steps were performed, followed by the development of specific HRP channels with different fluorophores assigned to each channel. The C4 channel probe was developed with the RNAscope 4-Plex Ancillary Kit (Advanced Cell Diagnostics, Cat#323120). Fluorophore dyes used were Opal 520 (Akoya Biosciences, Cat#FP1487001KT), Opal 570 (Akoya Biosciences, Cat#FP1488001KT), and Opal 690 (Akoya Biosciences, Cat#FP1497001KT). DAPI (Thermo Fisher Scientific, Cat#D1306) served as a nuclear stain. Slides were mounted in Mount Solid antifade (Abberior, Cat#MM-2011-2X15ML). Confocal images were taken on a Zeiss LSM 800.

Immunohistochemistry (IHC)

IHC for codetection of RNA and protein was performed for Cytokeratin 8 immunoreactivity after the final HRP blocker step. Slides were blocked in 10% donkey serum (Sigma-Aldrich, Cat#S30-100ML) in 0.1% Triton in PBS at room temperature for 1 h. Primary antibody Krt8, clone TROMA-1 (Sigma-Aldrich, Cat#MABT329) raised in rat, was diluted at 1:200 in 2% donkey serum in 0.1% Triton/PBS and incubated at 4 °C overnight. Slides were then washed in 0.1% Triton for three times for 5 min in PBS, followed by incubation with Alexa Fluor Plus 647 donkey anti-rat (Thermo Fisher Scientific, Cat#A48272) at 1:500 in 2% normal donkey serum in 0.1% Triton/PBS at room temperature for 1 h.

Transmission

Index AG129 mice aged 8–12 weeks (n = 6) were anesthetized with isoflurane and intranasally inoculated with 40 µL of medium containing 1.5 × 10⁶ TCID₅₀ HPIV-3-isolate 10-012854 at day 0. On the morning of day 1 p.i., each index AG129 mouse was co-housed with an uninfected contact (sentinel) AG129 mouse (n = 6) in a cage. Co-housing continued for 2 days, when the index mice were killed, and the sentinel mice were killed the following day. Lung samples were collected.

Pharmacokinetic study

Mouse pharmacokinetic studies were performed at Covance Laboratories (LabCorp Drug Development) as described previously⁶². Briefly, female BALB/c (n = 8) were administered GS-441524 (same formulation as used previously²⁷) at 24 mg/kg by oral gavage, and plasma concentrations (ng/mL) were quantitated at select timepoints over 24 h.

Statistics

GraphPad Prism (GraphPad Software, Inc.) was used to perform statistical analysis. Statistical significance was determined using the non-parametric Kruskal–Wallis test with Dunn’s multiple comparisons test for multiple comparisons or the Mann–Whitney U test for pairwise comparisons. p values of <0.05 were considered significant. Exact p values are provided except for p values smaller than 0.0001, which are presented as p < 0.0001. All measurements were taken from distinct samples.

Ethics

Housing conditions and experimental procedures were with the approval and under the guidelines of the Ethical Committee Dierproeven of KU Leuven (License P099/2022).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data generated and analyzed in this paper are included in this article. The deep sequencing data of the full genome of the isolate used in this study are available in GenBank under accession number PP909812. Additional imaging data related to the RNAscope analysis are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank Stijn Hendrickx, Lindsey Bervoets, and Carolien De Keyzer for their excellent technical assistance. We thank the staff at the Rega animal facility of KU Leuven for technical assistance with the animal experiments. We also thank the staff at the Department of Translation Cell and Tissue Research at KU Leuven for histology sample processing and staining. This project was funded by EU PANVIPREP (Project number 101137229) and EU DURABLE (Project number 101102733). Y.L. was supported by the China Scholarship Council (Grant no. 202006380126). Figures were generated using BioRender.

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Authors and Affiliations

KU Leuven Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Virology, Antiviral Drug & Vaccine Research Group, Leuven, Belgium
Yuxia Lin, Manon Laporte, Dirk Jochmans & Johan Neyts
Max Planck Research Unit for Neurogenetics, Frankfurt, Germany
Mona Khan & Peter Mombaerts
KU Leuven Department of Imaging and Pathology, Division of Translational Cell and Tissue Research, Leuven, Belgium
Birgit Weynand
Department of Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
Frank Coenjaerts
Gilead Sciences Incorporated, Foster City, CA, USA
Darius Babusis & John P. Bilello
VirusBank Platform, KU Leuven, Leuven, Belgium
Johan Neyts

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Contributions

J.N. acquired funding, designed the project, and wrote the manuscript. D.J. designed the project and assisted with the data analysis and manuscript writing. Y.L. designed and performed experiments, analyzed the data, made figures, and wrote the manuscript. P.M. designed the RNAscope and immunochemistry experiments and wrote the manuscript. M.K. designed and performed RNAscope and immunochemistry experiments, analyzed the data, made figures, and assisted with manuscript writing. B.W. performed the lung scoring and assisted with data analysis. F.C. isolated the HPIV-3 clinical isolate. D.B. and J.B. performed the pharmacokinetic study, analyzed the data, made the graph, and assisted with the manuscript writing. M.L. assisted with project design, data analysis, and manuscript writing. All authors have read and agreed with the content of the manuscript.

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Correspondence to Johan Neyts.

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D.B. and J.B. are employees of Gilead Sciences which owns GS-441524. None of the other authors have any competing interests.

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Lin, Y., Khan, M., Weynand, B. et al. A robust mouse model of HPIV-3 infection and efficacy of GS-441524 against virus-induced lung pathology. Nat Commun 15, 7765 (2024). https://doi.org/10.1038/s41467-024-52071-5

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Received: 18 April 2024
Accepted: 22 August 2024
Published: 05 September 2024
DOI: https://doi.org/10.1038/s41467-024-52071-5