Dam–Infant Rhesus Macaque Pairs to Dissect Age-Dependent Responses to SARS-CoV-2 Infection

The epidemiological evidence has consistently demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in young children are mostly mild with relatively low hospitalization rates (1, 2). This results in significantly decreased fatality rates of young (0- to 9-y-old) compared with old (>70-y-old) populations (3–9). Despite the increase in pediatric hospitalizations during emergence of the Delta variant, the proportion of children with severe disease after the Delta variant became predominant were similar to those earlier in the pandemic (10). This suggests that the increase in pediatric hospitalizations during this time is related to other factors, and that children are still at significantly lower risk to severe COVID-19 compared with adults.

Differences in local immune responses in the respiratory tract that include antiviral and proinflammatory mediators likely play a role in the age-dependent pathogenesis of SARS-CoV-2 (11). Indeed, children have increased expression of relevant pattern recognition receptors, including MDA5 (IFIH1) and RIG-I (DDX58), in the upper airways when compared with adults (12), suggesting that recognition of SARS-CoV-2 entry into respiratory cells is enhanced in children. This is relevant for recruitment of fast-acting innate immune cells such as neutrophils, which were shown to be higher in children during the acute phase of SARS-CoV-2 infection compared with adults (13). IFN responses are also key determinants of COVID-19 severity, and potent production levels of type III and, to a lesser extent, type I IFNs in the upper respiratory tract are associated with decreased viral load, milder COVID-19, and younger age (14). However, there remains a large gap in our understanding of how age-dependent factors mediate disease severity and recovery postinfection.

Nonhuman primate (NHP) models are valuable resources to address these questions related to SARS-CoV-2 pathobiology and to explore vaccine and drug-based interventions against COVID-19 (15, 16). We used a maternal–infant SARS-CoV-2 infection model to elucidate the age-dependent effects on viral pathogenesis. By using dam–infant pairs and inoculating both dams and infants at the same time, we were able to simultaneously analyze immune responses to SARS-CoV-2 infection in two different age groups while limiting genetic variation. We show that SARS-CoV-2 infection in dam–infant rhesus macaque pairs results in innate and adaptive immune differences, including SARS-CoV-2 neutralizing Ab (nAb) response kinetics and innate immune and profibrotic gene expression in the conducting airways. Our data highlight age-dependent differential immune and lung responses to SARS-CoV-2 infection and describe a model that can be used to investigate SARS-CoV-2 pathogenesis between infants and adults.

The four Indian origin rhesus macaques (Macaca mulatta) used in this study were housed at the California National Primate Research Center in accordance with the recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care International standards and with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). The Institutional Animal Use and Care Committee approved these experiments (study protocol no. 21702). All animals were challenged through combined intratracheal (2.0 ml for dams, 1 ml for infants) and intranasal (0.25 ml per nostril) inoculation with an infectious dose of 2.5 × 10⁶ PFU for the dams and 1.5 × 10⁶ PFU for the infants of SARS-CoV-2 (2019-nCoV/USA-WA1/2020). The stock was obtained from BEI Resources (NR-52281). The stock underwent deep sequencing to confirm homology with the WA1/2020 isolate. Virus was stored at −80°C prior to use, thawed rapidly at 37°C, and placed immediately on wet ice. Nasal swabs, pharyngeal swabs, bronchoalveolar lavage (BAL), blood, breast milk, and rectal swab samples were collected 1 wk before infection and every 2–3 d postchallenge. Nasopharyngeal, oropharyngeal, and buccal secretions were collected with FLOQSwabs (COPAN Diagnostics), placed in a vial with DNA/RNA Shield solution (Zymo Research), and stored at −70°C until further processing.

BAL was performed using a 20F rubber feeding tube with instillation of 20 ml of sterile physiologic saline followed by aspiration with a syringe. BAL samples were spun in the laboratory. The BAL cell pellet, together with 0.5 ml of supernatant, was then mixed with 1.5 ml of TRIzol LS (Thermo Fisher Scientific) and cryopreserved at −70°C. Additional aliquots of BAL supernatant were also immediately cryopreserved. Blood and milk were collected and processed as previously described (17–19).

Radiographs were obtained with a HF100+ ultralight imaging unit (MinXray, Northbrook, IL) at 50 kVp, 40 mA, and 0.1 s. Ventrodorsal, dorsoventral, right lateral, and left lateral radiographs were obtained prior to inoculation and on days 1, 3, 5, 7, 10, and 14 postinoculation. Radiographs were scored for the presence of pulmonary infiltrates by a board-certified veterinary radiologist, who was blinded to the experimental group and time point, according to a standard scoring system (0, normal; 1, mild interstitial pulmonary infiltrates; 2, moderate pulmonary infiltrates, perhaps with partial cardiac border effacement and small areas of pulmonary consolidation; 3, severe interstitial infiltrates, large areas of pulmonary consolidation, alveolar patterns, and air bronchograms). Individual lobes were scored, and scores per animal per day were totaled. At the end of the study, animals were euthanized, and a full necropsy was performed for tissue collection, including trachea for cell isolation and fixed lung tissues for histopathology.

Upon necropsy, tracheobronchial tissues were collected. Airway epithelial cells were isolated by placing tracheas in Eagle’s MEM (Joklik modification) (Lonza) containing 0.1% type XIV protease (Sigma-Aldrich), 50 U/ml penicillin, 50 μg/ml each streptomycin and gentamicin, and 100 µg/ml Geneticin (G418, Invitrogen) overnight. Cells were gently aspirated off and stored in liquid nitrogen prior to RNA isolation.

SARS-CoV-2 infection was determined by quantitative PCR (qPCR) of SARS-CoV-2 genomic (orf1a) and subgenomic (N) RNA.

A QIAsymphony SP (Qiagen, Hilden, Germany) automated sample preparation platform along with a virus/pathogen DSP midi kit and the Complex800 protocol were used to extract viral RNA from 800 µl of respiratory sample. A reverse primer specific to the orf1a sequence of SARS-CoV-2 (5′-CGTGCCTACAGTACTCAGAATC-3′) was annealed to the extracted RNA and then reverse transcribed into cDNA using SuperScript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA) along with RNase OUT (Thermo Fisher Scientific, Waltham, MA). The resulting cDNA was then treated with RNase H (Thermo Fisher Scientific, Waltham, MA) and added to a custom 4× TaqMan gene expression master mix (Thermo Fisher Scientific, Waltham, MA) containing primers and a fluorescently labeled hydrolysis probe specific for the orf1a sequence of SARS-CoV-2 (forward primer, 5′-GTGCTCATGGATGGCTCTATTA-3′; reverse primer, 5′-CGTGCCTACAGTACTCAGAATC-3′, probe, 5′-/56-FAM/ACCTACCTT/ZEN/GAAGGTTCTGTTAGAGTG GT/3IABkFQ/-3′). All PCR setup steps were performed using QIAgility instruments (Qiagen, Hilden, Germany). The qPCR was then carried out on a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, Waltham, MA). SARS-CoV-2 genomic (orf1a) RNA copies per reaction were interpolated using quantification cycle data and a serial dilution of a highly characterized custom RNA transcript containing the SARS-CoV-2 orf1a sequence. Mean RNA copies per milliliter were then calculated by applying an assay dilution factor of 11.7. The limit of quantification for this assay is ∼31 RNA copies/ml (1.49 log₁₀) with 800 μl of sample.

A QIAsymphony SP (Qiagen, Hilden, Germany) automated sample preparation platform along with a virus/pathogen DSP midi kit and the Complex800 protocol were used to extract viral RNA from 800 µl of respiratory sample. The extracted RNA was then added to TaqMan fast virus one-step master mix (Thermo Fisher Scientific, Waltham, MA) containing primers and a fluorescently labeled hydrolysis probe specific for mRNA from the nucleocapsid gene of SARS-CoV-2 (forward primer, 5′-CGATCTCTTGTAGATCTGTTCTC-3′; reverse primer, 5′-GGTGAACCAAGACGCAGTAT-3′; probe 5′-/56-FAM/TAACCAGAA/ZEN/TGGAGAACGCAGT GGG/3IABkFQ/-3′). All PCR setup steps were performed using QIAgility instruments (Qiagen, Hilden, Germany). The qPCR was carried out on a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, Waltham, MA). SARS-CoV-2 subgenomic (N) RNA copies per reaction were interpolated using quantification cycle data and a serial dilution of a highly characterized custom RNA transcript containing the SARS-CoV-2 subgenomic nucleocapsid sequence. Mean RNA copies per milliliter were then calculated by applying an assay dilution factor of 5.625. The limit of quantification for this assay is ∼31 RNA copies/ml (1.49 log₁₀) with 800 μl of sample.

IgG binding to the stabilized SARS-CoV-2 spike (S) protein S-2P was measured in plasma using ELISA as previously described (20). Three hundred eighty-four–well plates were coated overnight with S protein (2 μg/ml) produced by the Protein Production Facility at the Duke Human Vaccine Institute. Plates were then blocked with assay diluent (PBS containing 4% whey, 15% normal goat serum, and 0.5% Tween 20). Ten serial 4-fold dilutions starting at 1:10 for plasma and undiluted for breast milk were added to the plates and incubated for 1 h, followed by detection with a HRP-conjugated mouse anti-monkey IgG (SouthernBiotech). The plates were developed by using a 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid peroxidase substrate system (Colonial Scientific), and absorbance was read at 450 nm with a SpectraMax microplate reader (Molecular Devices). Results were calculated as area under the curve (AUC) and EC₅₀ values. AUC values were calculated using the trapezoidal rule. EC₅₀ values were calculated by fitting a four-parameter logistic function using nonlinear regression. Pooled NHP convalescent serum to SARS-CoV-2 (BEI Resources, NR-52401) was used in all IgG assays to ensure interassay reproducibility, but standard curves were not developed given the lack of an SARS-CoV-2 rhesus macaque–specific IgG reagent of known concentration. IgA binding to S-2P was measured following a similar ELISA protocol but using as detection Ab anti-rhesus IgA 10F12-biotin (Nonhuman Primate Reagent Resource, catalog no. AB_2819304) followed by streptavidin-HRP (Pierce, catalog no. 21126).

SARS-CoV-2 Ags, including whole S (produced by the Protein Production Facility), S1 (Sino Biological, catalog no. 40591-V08H), S2 (Sino Biological, catalog no. 40590-V08B), receptor-binding domain (RBD; Sino Biological, catalog no. 40592-V08H), and N-terminal domain (NTD; Sino Biological, catalog no. 40591-V49H) were conjugated to MagPlex beads (Bio-Rad, Hercules, CA). The conjugated beads were incubated on filter plates (Millipore, Stafford, VA) for 30 min before plasma samples were added. Plasma samples were diluted in assay diluent (1% dry milk, 5% goat serum, and 0.05% Tween 20 in PBS [pH 7.4]) at a 1:1000-point dilution. Beads and diluted samples were incubated for 30 min with gentle rotation, and IgG binding was detected using a PE-conjugated mouse anti-monkey IgG (SouthernBiotech, Birmingham, AL) at 2 µg/ml. Plates were washed and acquired on a Bio-Plex 200 instrument (Bio-Rad, Hercules, CA), and IgG binding was reported as mean fluorescence intensity (MFI). To assess assay background, the MFIs of wells without samples (blank wells) were used, and nonspecific binding of the samples to unconjugated blank beads was evaluated.

SARS-CoV-2 neutralization was assessed with S-pseudotyped viruses in 293T/ACE2 cells as a function of reductions in luciferase (Luc) reporter activity. 293T/ACE2 cells were provided by M. Farzan and H. Mu at Scripps Florida. Cells were maintained in DMEM containing 10% FBS, 25 mM HEPES, gentamicin (50 μg/ml), and puromycin (3 μg/ml). An expression plasmid encoding codon-optimized full-length S of the Wuhan-1 strain (VRC7480) was provided by B.S. Graham and K.S. Corbett at the Vaccine Research Center (NIH). The D614G amino acid change was introduced into VRC7480 by site-directed mutagenesis using the QuikChange lightning site-directed mutagenesis kit from Agilent Technologies (catalog no. 210518). The mutation was confirmed by full-length S gene sequencing. Pseudovirions were produced in HEK 293T/17 cells (American Type Culture Collection, catalog no. CRL-11268) by transfection using FuGENE 6 (Promega, catalog no. E2692) and a combination of S plasmid, lentiviral backbone plasmid (pCMV ΔR8.2), and firefly Luc reporter gene plasmid (pHR′ CMV Luc) (78) in a 1:17:17 ratio. Transfections were allowed to proceed for 16–20 h at 37°C. Medium was removed, monolayers were rinsed with growth medium, and 15 ml of fresh growth medium was added. Pseudovirus-containing culture medium was collected after an additional 2 d of incubation and was clarified of cells by low-speed centrifugation and 0.45-μm filtration and stored in aliquots at −80°C. Median tissue culture infectious dose assays were performed on thawed aliquots to determine the infectious dose for neutralization assays.

For neutralization, a pretitrated dose of pseudovirus was incubated with eight serial 5-fold dilutions of serum samples in duplicate in a total volume of 150 μl for 1 h at 37°C in 96-well flat-bottom poly-l-lysine–coated culture plates (Corning BioCoat). HEK 293T cells expressing ACE2 receptors were suspended using TrypLE select enzyme solution (Thermo Fisher Scientific) and immediately added to all wells (10,000 cells in 100 μl of growth medium per well). One set of eight control wells received cells and virus (virus control), and another set of eight wells received cells only (background control). After 66–72 h of incubation, medium was removed by gentle aspiration and 30 μl of Promega 1× lysis buffer was added to all wells. After a 10-min incubation at room temperature, 100 μl of Bright-Glo Luc reagent was added to all wells. After 1–2 min, 110 μl of the cell lysate was transferred to a black/white plate (PerkinElmer). Luminescence was measured using a PerkinElmer Life Sciences VICTOR2 luminometer. Neutralization titers are the serum dilution at which relative light units were reduced by either 50% or 80% compared with virus control wells after subtraction of background relative light units. Serum samples were heat-inactivated for 30 min at 56°C before an assay.

Whole-blood samples were stained fresh and acquired the same day. Fluorescence was measured using a BD Biosciences FACSymphony with FACSDiva version 8.0.1 software. Compensation, gating, and analysis were performed using FlowJo (version 10). Gating strategy for innate immune subsets in whole blood after gating on singlets was performed as previously described (21). Fluorochromes used included the following: CD3/CD20 allophycocyanin-Cy7, CD14 A700, CD8 BUV805, CD66 allophycocyanin, HLA-DR BV786, CD16 BV605, CD123 BV421, and CD11c PE-Cy7.

Tracheal cells (epithelial, immune, and mesenchymal) were lysed in 350 μl of TRIzol, and RNA was extracted by the phenol/chloroform method and collected over an RNeasy column (Qiagen, Germantown, MD). The RNA concentration and integrity were determined on the NanoDrop 1000 (Thermo Fisher Scientific). RNA (1000 ng) from each sample was used as a template for preparing Illumina-compatible libraries using the TruSeq RNA library prep kit version 2 (Illumina). Library sizes were checked using D5000 high-sensitivity tape on the Agilent 2200 TapeStation, and pooled library concentrations were determined by a Qubit 3.0 fluorometer (Thermo Fisher Scientific). A library input of 1.8 pM with 1% PhiX (Illumina) spike-in was sequenced using the NextSeq 500 instrument with the NextSeq 500/550 high-output version 2.5 kit (Illumina) and generated paired-end 76-bp reads. Samples from all four animals (two dams and two infants) were processed and analyzed together.

RNA sequencing (RNA-seq) data were processed using the Trim Galore toolkit (22), which employs Cutadapt (23) to trim low-quality bases and Illumina sequencing adapters from the 3′ end of the reads. Only reads that were 20 nt or longer after trimming were kept for further analysis. Reads were mapped to the Mmul 10.99 version of the Macaca mulatta genome and transcriptome (24) using the STAR RNA-seq alignment tool (25). Only reads that mapped to a single genomic location were kept for subsequent analysis. The great majority of remaining trimmed and mapped reads (99.2%) were >70 bp long and the median length was 76 bp. Gene counts were compiled using the HTSeq tool (26). Only genes that had at least 10 reads (raw counts) in any given library were used in subsequent analysis (27). Normalization and differential expression were carried out using the DESeq2 (28) Bioconductor (29) package with the R statistical programming environment (30). To normalize for sequencing depth and RNA composition across different library sizes, DESeq2 corrects internally for these factors using the median of ratios method (28). The false discovery rate was calculated to control for multiple hypothesis testing. Gene set enrichment analysis was performed to identify Gene Ontology (GO) terms and pathways associated with altered gene expression for each of the comparisons performed. Heatmaps include only significantly upregulated or downregulated genes where both infants or both dams are higher/lower than each other to exclude genes where one animal dominated the response, potentially skewing the results.

The lungs were harvested and each lobe was separated. All lobes were cannulated with 18G blunt needles. All lobes were slowly infused with neutral buffered formalin at 30-cm fluid pressure. Once fully inflated (∼30 min) the main bronchus was tied off and the lungs were placed in individual jars of formalin and fixed for 72 h. Then they were sliced from the hilus toward the periphery into slabs ∼5 mm thick. Each slab was placed into a cassette recording its position in the stack and with further division of the slab into smaller pieces when required to fit into the cassette. Tissues were then held in 70% ethanol until processing and paraffin embedding, followed by sectioning at 5 μm and generation of H&E- and Masson trichrome–stained slides. Slides from every other slab of the right and left caudal lobes were examined independently by two American College of Veterinary Pathologists board-certified pathologists in a blinded manner.

Substance P (Thermo Fisher Scientific) and protein gene product 9.5 (MilliporeSigma) Abs were applied to 5-µm paraffin sections. The EnVision system (Agilent Technologies) was used as the detection system with 3-amino-9-ethylcarbazole (Agilent Technologies) as a chromogen. Paraffin sections were treated in an Ag unmasking solution (Vector Laboratories) at 100°C for 20 min before incubation with a primary Ab. The slides were counterstained with Gill’s hematoxylin (StatLab). Primary Abs were replaced by rabbit isotype control (Thermo Fisher Scientific) and run with each staining series as the negative controls.

All work described in this study was performed with approved standard operating procedures for SARS-CoV-2 in a biosafety level 3 facility conforming to requirements recommended in the Microbiological and Biomedical Laboratories, by the U.S. Department of Health and Human Service, the U.S. Public Health Service, the U.S. Centers for Disease Control and Prevention, and by the NIH.

The RNA-seq data presented in this article have been submitted to BioProject under accession number PRJNA904310 (https://www.ncbi.nlm.nih.gov/bioproject/904310).

To investigate age-dependent differences in pathogenesis of SARS-CoV-2, two dams and their 6 mo-old infants were inoculated intranasally and intratracheally with SARS-CoV-2 (WA strain). Nasal and pharyngeal swabs, BAL, blood, breast milk, and rectal swabs were collected over time up to day 14 postchallenge (Fig. 1A).

To compare viral replication kinetics between the adult dams and infant macaques after SARS-CoV-2 infection, we performed both genomic (Orf1a gene) and subgenomic (N gene) PCR on nasal (dams only), pharyngeal, buccal, and rectal swabs. Nasal swabs were not collected from infants due to the restricted size of infant nostrils. As shown in (Fig. 1B, we did not detect significant differences in viral loads between adults and infants in pharyngeal or BAL supernatant or BAL pellet samples. Low levels of viral RNA were detected in one of the two infants in buccal and rectal swabs (Fig. 1B), whereas no viral RNA was detected in breast milk.

We evaluated clinical symptoms and radiographic changes overtime. Mild symptoms, including occasional sneezing, were reported. The radiographs were scored for the presence of pulmonary infiltrates, according to a standard scoring system (0–3 per lobe). Individual lobes were scored and scores per animal per day were totaled. Only the adult macaques had scores on multiple (≥2) days, and the highest scores observed (score of 5) were from one of the adult macaques (Supplemental Fig. 1). Overall, in both age groups the lesions in the lung were minimal and largely resolved, consisting of occasional foci of mild inflammation on H&E-stained sections. Trichrome-stained sections did not show an overt increase in fibrosis in either group (Supplemental Fig. 2). There was no significant evidence of interstitial pneumonia in any animal, only an occasional focal increase in interstitial cellularity and in some areas an increase in alveolar macrophages (Supplemental Fig. 2), as previously reported (31).

We evaluated innate immune cell subsets in the peripheral blood and observed no major differences in the proportion of cells between infants and dams after SARS-CoV-2 infection (Fig. 2A). Interestingly, however, circulating neutrophils (lineage⁻, CD66⁺) were higher in adults, and total HLA-DR⁺ (lineage⁻, CD66⁻) cells, comprised of monocytes and dendritic cell subsets, were higher in infants prior to inoculation with SARS-CoV-2 (Fig. 2A). For plasma cytokine responses, we observed a >2-fold increase in IL-6 (all four animals) and IP10 (CXCL10) (two dams, one infant) as reported previously (21), and the anti-inflammatory mediator IL-1RA (1 dam, 2 infants) within 1 d after SARS-CoV-2 infection (Fig. 2B). One infant demonstrated increased production of IL-17 and GM-CSF at several time points postinfection (Fig. 2B). Interestingly, IL-8 was upregulated >7-fold in three of the four animals at day 14 postinfection. However, we did not identify any specific cytokine signature that distinguished dams from infants.

We next measured anti-S binding and nAbs in serum and breast milk samples. With the limitation that the small group sizes preclude statistical significance, we found that whereas dams had higher serum S-binding IgG Ab titers than infants (Fig. 3A), infants demonstrated faster kinetics of nAb development. Infants had higher nAb titers at day 7 postinfection but similar nAb titers at day 14 postinfection compared with dams (Fig. 3B). S-binding IgG, but not IgA, was detected in breast milk of infected dams at day 14 postinfection (Fig. 3C). nAbs in breast milk were detected in one dam at day 14 postinfection (Fig. 3D). A multiplexed Ab binding assay did not detect differences in RBD, S1, S2, full S, and NTD-specific Abs between SARS-CoV-2–infected dam and infant macaques at day 14 (Fig. 3E).

The hierarchical and principal component analyses and heatmap (Supplemental Fig. 3) of the differentially expressed genes of tracheal cells (epithelial, immune, and mesenchymal) demonstrated altered transcriptomic expression of infant and dam rhesus macaques on day 14 after SARS-CoV-2 infection, where mothers and offspring clustered with each other. To identify GO and hallmark gene sets associated with altered expression, gene set enrichment analysis was performed (1). The hallmark pathway analysis revealed significantly decreased trachea cell genes associated with the IFN-α and IFN-γ hallmark pathways in adult compared with infant macaques (Fig. 4A). Specifically, IFI6, XAF1, DDX60, OAS2, HERC6, MX2, IFI44L, IFIT1, SAMD9L, PARP14, and IFIT12 are decreased in both adults compared with their infants (Fig. 4B).

Alternatively, in the GO pathway analysis we identified an overwhelming signature of decreased cilia structure and function in adult compared with infant tracheas (Fig. 5). In fact, all GO pathways that were significantly enriched among downregulated genes (family-wise error rate adjusted p value of ≤0.05) were related to cilia structure and motility (Supplemental Table I). Considering that SARS-CoV-2 infection induces a dedifferentiation of multiciliated cells (32), these results suggest a prolonged impairment of cilia functions in adult compared with infant rhesus macaques 14 d after SARS-CoV-2 infection.

In both the GO and hallmark pathway analyses, there was significant enrichment among upregulated genes (family-wise error rate adjusted p value of ≤0.05) for pathways associated with wound repair and fibrosis in adult compared with infant macaques at day 14 postinfection. Of the 39 GO pathways that are significantly enriched among upregulated genes in the adult macaques (Supplemental Table I), the overwhelming majority were associated with extracellular matrix (ECM) organization and ECM metabolism. Similarly, the hallmark pathway analysis revealed multiple pathways associated with wound repair and fibrosis in adult macaques, including epithelial mesenchymal transition, angiogenesis, coagulation, hypoxia, apoptosis, and TGF-β signaling. Of the most differentially expressed genes of the upregulated pathways in adults compared with infants, all of them were associated with the ECM (Fig. 6A). Additionally, we stained trachea tissue for protein gene product 9.5 (PGP 9.5; also known as ubiquitin C-terminal hydrolase-L1 [UCH-L1]) (Fig. 6C–F) and substance P (Fig. 6G–J), proteins involved in wound healing (33, 34), and they were higher in infant compared with adult tracheas.

In this study, we evaluated age-dependent differences in SARS-CoV-2 pathogenesis by inoculating dam–infant rhesus macaque pairs with SARS-CoV-2. In line with previous studies in NHPs (15, 16, 35–38), we demonstrate that both infant and dam rhesus macaques became productively infected and exhibited no to mild clinical symptoms. Lung radiograph scores showed mild pulmonary infiltrates in both dams and infants; however, only in dams were pulmonary infiltrates observed for multiple days (≥2). Histologic examination of pulmonary infiltrates was extremely minimal and largely resolving when examined at day 14 postinfection. This is consistent with previous experiments (21).

Although viral loads in pharyngeal swabs and BAL samples did not differ between dam and infant macaques, differences in Ab response kinetics between the dams and infants were observed. For example, although there were higher anti-S IgG binding Ab titers in adult macaques at days 10 and 14 postinfection, infants exhibited higher nAb titers at day 7. These data suggest faster kinetics of SARS-CoV-2 nAb responses in infant macaques. In pediatric cohorts infected with SARS-CoV-2, results have been variable, and levels of nAbs have been reported as lower, higher, or not different compared with infected adults (39–43). However, age-dependent differences in nAb development have been observed for other pathogens. For example, in HIV-1–infected children, circulating broadly nAbs arise earlier in infection and have higher potency and breadth compared with adults (44–49). Future work using this model should include measurement of T cell subsets in both blood and lymphoid tissue as well as determination of breadth against multiple SARS-CoV-2 variants.

Although there were not major differences in serum cytokines between adult and infant SARS-CoV-2–infected macaques, we observed increases in relevant proinflammatory cytokines. For example, IL-6 and IP10 (CXCL10) were increased in three of the four animals within 1 d after SARS-CoV-2, mirroring what was previously reported in rhesus macaques and other animal models of SARS-CoV-2 infection (37, 50, 51). Following SARS-CoV-2–infected macaques for >14 d would be worthwhile in observing late-onset proinflammatory signatures in older and infant animals. Additionally, measuring cytokines in tissues would give additional insight into age-dependent local responses to infection.

Increases in IFN-α and IFN-γ transcriptomic signatures were observed in infant macaques compared with dams. There were multiple IFN-stimulated genes (ISGs) that were upregulated in both infants compared with their dams, nearly all of which were previously shown to be significantly upregulated after SARS-CoV-2 infection, including IRF3, TLR3, DHX58, IFIH1, and DDX58 (12). Considering that trachea RNA-seq data were collected at day 14 postinoculation in our study, when viral loads were not detectable anymore, it is possible that levels of some ISGs are upregulated in infants at baseline compared with adults. Indeed, this has been demonstrated in multiple pediatric cohorts where ISGs (52) and cytokines and chemokine genes (12) were increased in SARS-CoV-2–uninfected children compared with adults, and an antiviral type I IFN gene signature was induced after SARS-CoV-2 infection in neonatal rhesus macaques (53). These data suggest the IFN response in children is preactivated in epithelial cells of the upper airways and stronger in immune cell subsets compared with adults.

There was an overwhelming signature of decreased cilia structure and function-related genes in adult compared with infant macaques. It has been previously demonstrated that SARS-CoV-2 infection leads to dedifferentiation of multiciliated cells in vitro and in vivo (32). Additionally, other groups have observed delayed repair responses after pulmonary injury in adults compared with neonates. For example, hypoxia- or LPS-induced lung injury resulted in less lung inflammation and apoptosis in neonatal compared with adult mice, which was dependent on increased NF-κβ activation (54–56). These data and our results support the hypothesis that the impacts of SARS-CoV-2 infection in the airways, including inflammation, apoptosis, and barrier permeability (that implicates cilia structure and function), are lessened in neonates and young children compared with adults.

We observed that multiple genes and pathways associated with ECM composition and metabolism, coagulation, angiogenesis, and hypoxia are increased in the trachea of dams compared with infants. These profibrotic transcriptomic signatures suggest greater activation of the cellular repair process in response to injury and apoptosis in the upper airway in adults compared with infants. Indeed, the deposition of multiple components of the lung ECM, including hyaluronan and fibrinogen (57–59) as well as profibrotic macrophage accumulation (59), has been associated with severe COVID-19 in adults. Alterations in ECM can be not only a consequence of lung fibrosis but also a driver of its progression (60), and acute respiratory distress syndrome in adults is more often associated with permanent alveolar simplification and fibrosis and higher morbidity and mortality outcomes compared with infants or children (61, 62). An increase in ECM and collagen genes in the adult macaques may be indicative of a dysregulation of injury repair.

Our study has limitations. First, we only infected two dam–infant macaque pairs, and more animal numbers are needed to determine statistical differences. Additionally, a mock-inoculated control group is necessary to decipher whether the differences observed are due to SARS-CoV-2 infection alone, the age-dependent maturation of tissues and the immune response, and/or experimental procedures. Finally, the time of euthanasia was not focused on evaluating acute inflammatory responses in tissues, as at 2 wk, virus replication is mainly gone and tissue responses reflect repair in this animal model, and we evaluated trachea instead of lung responses. However, our study is valuable in that it agrees with currently published data in SARS-CoV-2–infected pediatric and adult cohorts and furthers our understanding of why younger populations are less susceptible to severe COVID-19 compared with adults. Additionally, this model will allow further delineation of molecular mechanisms of age-dependent SARS-CoV-2 pathogenesis and assess efficacy of medical countermeasures in dam–infant pairs.

We thank the Duke Human Vaccine Institute Viral Genetics Analysis Core for support with RNA-seq.

The authors have no financial conflicts of interest.