Safety, tolerability and viral kinetics during SARS-CoV-2 human challenge (2024)

SARS-CoV-2 human challenge causes rapid onset of upper respiratory tract infection with high peak viral loads.

Thirty-six healthy volunteers aged 18-29 years old were enrolled according to protocol-defined inclusion/exclusion criteria. Screening included assessments for known risk factors for severe COVID-19, including co-morbidities, low or high body mass index, abnormal safety blood tests, spirometry and chest radiography (Figure 1a & Protocol). The protocol had been given a favourable opinion by the UK Health Research Authority – Ad Hoc Specialist Ethics Committee (reference: 20/UK/2001 and 20/UK/0002). Written informed consent was obtained from all volunteers prior to screening and study enrolment. The study was overseen by a Trial Steering Committee (TSC) with advice from an independent Data and Safety Monitoring Committee (DSMB). The study was discussed with the Medicines and Healthcare products Regulatory Agency; since no medicinal product was being investigated, the study was deemed not a clinical trial according to UK regulations. As such, a EudraCT number was not assigned and the clinical study was registered with clinicaltrials.gov (identifier: NCT04865237). All participants were seronegative at screening by Quotient MosaiQ antibody microarray test and had no history of SARS-CoV-2 vaccination or infection. However, two participants seroconverted between screening and inoculation, resulting in 34 individuals in the per protocol analysis.

As this human challenge model was developed during the ongoing pandemic, with no directly comparable safety data and incomplete understanding of long-term effects following COVID-19, an adaptive protocol was designed with stepwise progression to ensure maximal risk mitigation during the early stages and progression only as data on the clinical features of human SARS-CoV-2 challenge was acquired. Following extensive screening, participants were admitted to individual negative pressure rooms in an in-patient quarantine unit, with 24-hour medical monitoring and access to higher level clinical support. At admission and before inoculation, volunteers were screened for coincidental respiratory infection using the Biofire FilmArray. Initial cohorts comprised 3 sentinel individuals followed by 7 additional participants. As per protocol, these first 10 challenged participants were assigned to receive pre-emptive remdesivir once two consecutive twelve-hourly nose or throat swabs showed quantifiable SARS-CoV-2 detection by PCR, with the aim of mitigating any unexpected risk of progression to more severe disease. Following review by the DSMB and TSC, pre-emptive remdesivir was deemed unnecessary and target recruitment of a further 30 individuals under the same conditions but without remdesivir was advised. A further sentinel cohort of 3 individuals was then challenged, with no pre-emptive remdesivir given. This was followed by 3 more groups of 7, 7 and 9 individuals, following exclusion of 4 volunteers shortly before virus inoculation due to detection of other respiratory viruses. Once pre-emptive remdesivir was no longer used, clinical severity criteria (i.e. persistent fever, persistent tachycardia, persistent severe cough, greater than mild CT imaging changes or SaO2 ≤94%) were defined for triggering of rescue treatment with monoclonal antibodies (Regeneron), but no such treatment was ultimately required. Participants were quarantined for at least 14 days post-inoculation and until they met virological discharge criteria (see Online Methods), with planned follow-up for 1 year to assess for prolonged symptoms, including smell disturbance and neurological dysfunction.

All participants were inoculated with 10 TCID₅₀ of SARS-CoV-2/human/GBR/484861/2020 (a D614G-containing pre-alpha wild-type virus; Genbank Accession number OM294022) by intranasal drops (Figure 1b). Eighteen participants (53% according to the per protocol analysis, [95% CI [35,70]) subsequently developed PCR-confirmed infection. This infection rate met the protocol-specified target of 50-70% and there was therefore no further dose escalation. Demographics between infected participants and those who remained uninfected were similar (Table 1).

Table 1

Participant baseline physical and demographic characteristics, selected clinical features and adverse events.
Group			Total	Infected (sero- negative)	Uninfected (sero- negative)	Uninfected (sero- positive)
Group			(n=36)	(n=18)	(n=16)	(n=2)
Characteristic
	Age (years)
		Mean (SD)	21.8 (2.9)	22.2 (2.9)	20.8 (2.2)	26.5 (3.5)
		Min, Max	18, 29	18, 27	18, 25	25, 29
	Gender, n (%)
		Male	26 (72)	12 (67)	14 (88)
		Female	10 (28)	6 (33)	2 (12)	2 (100)
	Race, n (%)
		White or Caucasian	33 (92)	17 (94)	14 (88)	2 (100)
		Mixed Ethnicity	3 (8)	1 (6)	2 (12)
	BMI (kg/m²)
		Mean (Range, SD)	23.2 (19.6-29.7, 2.6)	22.8 (19.9-26.4, 2.2)	23.4 (19.6-29.7, 3.0)	25.2 (23.3-27.1, 2.7)
Symptoms
	Report of any symptoms on 2 consecutive days, n (%)		22 (61)	17 (94)	5 (31)
Fever
	>37.8⁰C, n (%)			7 (39)
C-Reactive Protein
	CRP > 5mg/L, n (%)			5 (28)
Antibody Titres at 28 days p.i.
	Neutralising antibody titre (median)			863.5 (IQR 403)	Undetectable*	167.5
	Spike-specific IgG titre (ELU/mL, median)			1549 (IQR 1865)	Undetectable*	178
Adverse events
	Any serious adverse event
	Clinically significant adverse events thought to be associated with viral infection that occurred or worsened during the observation period
	Smell disturbance
		During Quarantine		12
		Day 28		11	2**	1^#
		Day 90		4
		Day 180		5
	Low white cell count (<2.0 x10⁹/L)			1
	Low lymphocytes (<0.75 x10⁹/L)			9
	Low neutrophils (<1.0 x10⁹/L)			3
	Low neutrophils (<0.5 x10⁹/L)			1
	Epididymal discomfort			1
*One participant was naturally infected with SARS-CoV-2 between discharge from quarantine and day 28 post-inoculation (p.i.), so is excluded. Their neutralising antibody titre at day 28 was 472 and their spike-specific IgG was 536.6.
**One subject reported a runny nose that was hindering smell and quickly resolved, 1 subject. reported partial smell loss having had ‘natural’ COVID 2 weeks before. Neither subject had a significant change in UPSIT compared to baseline
# Subject reported smell disturbance only after performing the UPSIT, score was not significantly different to baseline

In the 18 infected individuals, viral shedding by qPCR became quantifiable in throat swabs from 40 hours (median, 95% CI [40,52]) (~1.67 days) post-inoculation, significantly earlier than in the nose (p=0.0225, where initial viral quantifiable detection occurred at 58 hours (95% CI [40,76]) (~2.4 days) post-inoculation (Figure 2a & 2b). This was initially closely paralleled by viable virus measured by focus forming assay (FFA), which was also quantifiably detected earlier in the throat than in the nose (p=0.0058, Figure 2b). Viral loads (VL) increased rapidly thereafter, with qPCR peaking in the throat at 112 hours (95% CI [76,160]) (~4.7 days) post-inoculation and later at 148 hours (95% CI [112,184]) (~6.2 days) post-inoculation in the nose (Figure 2a & 2c). However, at its peak, VL was significantly higher in nasal samples at 8.87 (95% CI [8.41,9.53]) log10 copies/mL and 3.9 (95% CI [3.34,4.42]) log10 FFU/mL than in the throat at 7.65 (95% CI [7.39,8.24]) log10 copies/mL and 2.92 (95% CI [2.68,3.56]) log10 FFU/mL (p<0.0001 for qRT-PCR and p=0.0024 for FFA, Figure 2d).

In both nose and throat, viral detection continued at high levels for several days and high cumulative VLs by area under the curve (AUC) were therefore seen, particularly in the nose (median 9.03, 95% CI [8.65,9.43] copies/mL by qPCR)(Figure 2e). In all infected participants, quantifiable virus by qPCR was still present at day 14 post-inoculation which necessitated prolonged quarantine of up to 5 extra days until qPCR Ct values had fallen to <33.5 in two consecutive nasal and throat swabs (as per protocol-defined discharge criteria). At these later timepoints, VLs by qRT-PCR were more erratic, with low level qPCR positivity remaining in 15/18 (83%) at discharge. At day 28 post-inoculation 6/18 (33%) remained qPCR positive in the nose and 2/18 (11%) in the throat but by day 90 all participants were qPCR negative. Of the participants not meeting infection criteria and deemed uninfected, low level non-consecutive viral detections were observed only by qPCR in the nose of 3 participants and throat of 6 participants (Extended Figure 1a & 1b).

In contrast, viable virus was detectable by FFA for a more limited duration: 156 hours (median, 95% CI [120,192]) (6.5 days) in the nose and in the throat for 150 hours (95% CI [132,180]) (6.25 days; Figure 2f). The average time post-inoculation to clearance of viable virus was 244 hours (95% CI [208, 256]) or 10.2 days from the nose and 208 hours (95% CI [172,244]) or 8.7 days from the throat (Figure 2g). VLs by qPCR and FFA were significantly correlated in both nose and throat (Extended Figure 3a & 3b). Although there was a striking degree of concordance between the shape and magnitude of individuals’ VL curves (Figure 2a) and between VLs in the nose and throat (Figure 2i), greater inter-individual variability was observed in timing of VL between nose and throat (Extended Figure 4). Despite relatively high levels of late qPCR detection, the latest that viable virus could be detected was day 12 post-inoculation in the nose and day 11 in the throat (Figure 2g). In contrast, swabs by qPCR that became undetectable in quarantine during the resolution phase first occurred at 352 hours (95% CI [340,364]) (~14.6 days) in the nose and 340 hours (95% CI [304,352]) (~14.7 days) in the throat although some later continued to fluctuate around the limits of quantification and detection (Figure 2h).

Of the first 10 participants prospectively assigned to receive pre-emptive remdesivir on PCR-confirmed infection, 6 became infected. No apparent differences were seen in VL by qPCR (Extended Figure 2a) or FFA (Extended Figure 2b) between remdesivir-treated and untreated infected individuals and cumulative virus (AUC) was similar (Extended Figure 2c). While there was an apparent trend towards lower mean nasal VL during the treatment period and delayed VL peak in the 6 remdesivir-treated individuals (Extended Figure 2d), this was not observed in the throat, primarily driven by one individual and was not statistically significant. With no significant differences between remdesivir-treated and untreated participants, infected individuals were therefore analysed together.

Thus, following SARS-CoV-2 human challenge, viral shedding begins within 2 days of exposure, rapidly reaching high levels with viable virus detectable up to 12 days post-inoculation, and significantly higher VL in the nose than the throat despite its later onset.

Serum neutralising antibodies are mounted rapidly following SARS-CoV-2 challenge infection

The rapid onset of infection was reflected in serum antibody responses. No increase in serum antibodies by microneutralisation or anti-spike protein IgG ELISA was observed in those deemed uninfected, even where isolated viral detections had occurred, except for one participant who acquired natural COVID-19 after discharge from quarantine (Figure 3a & 3b). In contrast, serum antibodies were generated in all infected participants with neutralising antibody titres of 425 (median, IQR 269) at 14 days post-inoculation and a further rise to 863.5 (IQR 403) at 28 days (Figure 3a). A slower rise was seen in spike protein-binding IgG measured by ELISA, with a median increase to 192.5 (IQR 393.1) ELU/mL at day 14 followed by an increment by day 28 to 1549 (IQR 1865) ELU/mL (Figure 3b). Of note, in the two participants who seroconverted between screening and inoculation, both neutralising and S protein binding antibodies were detectable at admission to the quarantine unit on day -2 pre-inoculation. Both individuals were excluded from the per protocol infection rate analysis but remained uninfected, with no change in their serum antibody levels post-inoculation.

SARS-CoV-2 human challenge infection causes mild disease with no evidence of serious safety signals

Following infection, symptoms by self-reported diary (Supplementary Table 1) became apparent from 2-4 days post-inoculation (Figure 4a) when symptoms started diverging from challenged but uninfected individuals, who reported both fewer and milder symptoms with no consistent pattern (Figure 4a and Extended Figure 1c). Symptom scores exhibited greater variability than VLs, with inconsistent onset and peak cumulative daily scores ranging from 0 to 29. Symptoms were most frequent in the upper respiratory tract and included nasal stuffiness, rhinitis, sneezing and sore throat (Figure 4b, 4c and Extended Figure 5). Systemic symptoms of headache, muscle/joint aches, malaise and feverishness were also recorded. There was no difference in symptoms between remdesivir-treated and untreated individuals (Extended Figure 6). All symptoms were mild-to-moderate, with peak symptoms (at 112 hours post-inoculation (95% CI [88,208]) aligning closely with peak VL in the nose, which was significantly later than peak VL in the throat by FFA (88 hours, 95% CI [76,112], p=0.0114) (Figure 4d, Extended Figure 4). However, despite the temporal association between nasal VL and symptoms, there was no correlation between the amount of viral shedding by qPCR or FFA and symptom score AUC (Figure 4e & 4f).

Seven participants (39% of infected) had temperatures of >37.8°C. Otherwise there were no notable disturbances in any clinical assessments, including daily spirometry and thoracic CT scans. No serious adverse events were reported and no criteria for commencing rescue therapy were met. A total of 18 adverse events deemed probably or possibly related to virus infection were largely due to transient and non-clinically significant leukopenia and neutropenia, and mild muco-cutaneous abnormalities during the quarantine period (Table 1 and Supplementary Table 2).

SARS-CoV-2 human challenge infection commonly causes smell disturbance

To assess the degree and kinetics of smell disturbance, University of Pennsylvania Smell Identification Tests (UPSITs) were conducted. No smell disturbance was observed during quarantine in uninfected individuals (Extended Figure 1d). However, 12 infected participants (67%) reported some degree of smell disturbance. While other symptoms peaked with nasal VLs, the nadir of UPSIT scores was 6-7 days later (Figure 4a, Extended Figure 4). Complete smell loss (anosmia) occurred in 9 individuals (50%), but most experienced rapid improvement before day 28. Although at day 28 some smell disturbance was still reported by 11 participants (61%), by day 180 this number had fallen to 5. Of these, only one individual still had measurable smell impairment at 180 days post-inoculation, although this was improving both subjectively and objectively (UPSIT at baseline=31, day 11=9, day 28=11, day 90=17, day 180=23). Two of the remaining reported mild parosmia and two had mild reduction in smell subjectively (although UPSIT scores had normalised). Six individuals received smell training advice, including 2 who also received treatment with short courses of oral and intranasal steroids.

Anosmia is therefore a common feature of human SARS-CoV-2 challenge that generally onsets several days later than viral shedding and resolves quickly in most individuals. Together, these findings indicate that human SARS-CoV-2 challenge at this inoculum dose has low risk of causing severe symptoms in healthy young adults but leads to large amounts of nasopharyngeal virus even in the absence of respiratory or systemic disease.

Antigen testing by lateral flow assay is strongly associated with virus detection by quantitative culture

Lateral flow assay (LFA) rapid antigen tests are commonly used to identify potentially infectious people in the community but their usefulness in early infection is unknown. To test the performance of LFA over the entire course of infection, antigen testing was performed using the same morning nose and throat swab samples assessed for VL. None of the uninfected participants had a positive LFA test at any time, whereas all infected individuals had positive LFA for ≥2 days (Extended Figure 7). Despite earlier viral detection in the throat by other methods, median time to first detection by daily LFA tests was the same in nose and throat at 4 days (range 2-8) post-inoculation (Figure 5a). This was on average 24-48 hours after first qPCR positivity (Figure 5b) and within 24 hours of FFA (Figure 5c). Of note, in 9 of 18 infected individuals, viable virus became detectable by FFA one or more day before the first positive LFA. Towards the end of infection, the last LFA detection mainly occurred 24-72 hours after viable virus detection had ceased.

To assess the relationship between VL and probability of a positive LFA, logistic regression models were fitted using generalised estimating equations to control for repeated within-participant assessments. Log₁₀ VL was a significant predictor (P<2x10⁻⁵) of LFA positivity with an odds ratio of 5.01 (95% CI [2.93,8.57]) when predicting LFA from FFA in nose (Figure 5e). Area under the receiver operating characteristic curves (AUROC) were high at 0.96 for nasal qPCR, and 0.89 for throat qPCR (Extended Figure 8a) but lower for FFA, particularly in the throat (AUC 0.69). To test longitudinal performance as infection progressed, the sensitivity and specificity of LFA when compared with qPCR and FFA were calculated for each day post-exposure (Figure 5f). With both tests and anatomical sites, sensitivity of LFA was limited at the beginning and end of acute illness. However, from ~4 days post-inoculation, LFA demonstrated high sensitivity as a surrogate for qPCR or FFA-positivity. Overall, LFA was highly specific although some “false positives” were observed in relation to FFA (but not qPCR).

Where asymptomatic/pre-symptomatic LFA testing programmes exist, testing is usually recommended twice weekly. To model the differential impact of LFA testing frequency that incorporate viral dynamics throughout infection, the mean proportion of VL AUC that had yet to occur (and might be responsible for transmission if undiagnosed) by the time of a first positive LFA test with testing cadences of 1-7 days was modelled. For both FFA (Figure 5g) and qPCR (Extended Figure 8b), infection would be recognised at or before >90% of the VL AUC had occurred if testing was daily. As the period between tests increased, the proportion of VL AUC declined with twice-weekly testing capturing 70-80% of virus and weekly testing still exceeding 50% if nose and throat swabs were combined. Thus, LFA positivity is strongly associated with culturable virus and therefore contagiousness and can be highly effective as a trigger for interventions to interrupt transmission.