Biomarkers of protection against controlled human SARS-CoV-2 Delta variant breakthrough infection
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Background
Improved understanding of how variants cause breakthrough infection, despite pre-existing immunity, is needed to advance development of next-generation SARS-CoV-2 vaccines, including those that may provide cross-variant protection or block transmission. SARS-CoV-2 controlled human infection models (CHIMs) may therefore identify correlates of protection and accelerate the development of new interventions.
Methods
Healthy vaccinated adults aged 18-30 years were inoculated intranasally in a stepwise dose- escalation CHIM with doses from 1x10 2 TCID 50 to 1x10 6 TCID 50 of SARS-CoV-2 Delta variant. Within the 1x10 6 TCID 50 group, participants were selected for serum neutralising antibody titres (NT 50 ) ≤1:80. Post-inoculation, participants were quarantined for up to 14 days. Outpatient follow-up continued for 12 months. The primary aim was to elicit safe, well-tolerated Delta SARS-CoV-2 breakthrough infection at a rate of over 50%.
Findings
Forty-six participants were inoculated; 22 during dose-escalation with no resultant infections, and 24 at the highest dose, of whom 18 were screened for low serum neutralising antibodies. Sustained infection with mild-to-moderate symptoms occurred in 33% (6/18) of the sero- selected group, with highly variable viral loads, viral emissions and symptoms. Serum neutralising antibody, anti-N IgG and to a lesser extent, mucosal anti-S IgA and N-specific T cells most strongly predicted protection from virologically-defined infection. Higher neutralisation, serum and nasal anti-N IgG level, and N-specific T cell responses correlated with lower viral load, while baseline nasal anti-S IgA was associated with lower symptom scores. Transient infection was additionally observed in 6 participants and was associated with higher baseline N-specific T cell responses than those that developed sustained infection.
Interpretation
Susceptibility to SARS-CoV-2 breakthrough infection in those with hybrid immunity is strongly associated with low levels of pre-existing antibody, but the diversity of immune markers associated with protection implies that additional benefits may be conferred by multi-pronged immunity.
Funding
Wellcome Trust
Research in context
Evidence before this study
To identify other published SARS-CoV-2 controlled human infection models (CHIM), a search on PubMed was carried out on 4th March 2026. The search terms used were ((“controlled human infection”) OR (“human challenge”)) and ((SARS-CoV-2) OR (COVID-19)) and (“clinical trial”). Two clinical studies were identified.
The first study involved healthy adult 18-29 year olds, seronegative to SARS-CoV-2 inoculated with 1x101 TCID50 pre-Alpha (wild-type) SARS-CoV-2. Eighteen of 34 (53%) participants became infected. The model was safe and well-tolerated and there were no study-related serious adverse events. Mild to moderate symptoms were reported by 16 of 18 (89%) infected individuals while 2 had virtually no symptoms. 14 of 18 (78%) of participants reported smell disturbance measured by the University of Pennsylvania Smell Identification Test (UPSIT). Viral detection by qPCR became quantifiable in throat swabs from 40 hours (∼1.67 days) post- inoculation and nose swabs at 58 hours (∼2.4 days). Viral load peaked in the throat at 112 hours (∼4.2 days) post-inoculation and later at 148 hours (∼6.2 days) post-inoculation in the nose.
The second study involved healthy adult 18-30 year olds, seropositive to SARS-CoV-2 inoculated with escalating doses (1x10 1 -1x10 5 TCID 50 ) of the same pre-Alpha variant. Thirty- six participants were inoculated and no sustained infection meeting the protocol-defined definition of infection was induced. Five (14%) of 36 volunteers were considered to have transient (brief) infections, based on the kinetic of their PCR-positive swabs.
In the first study, functionally-complete protection was associated with early increases in innate and adaptive cell abundance in the nose post-inoculation, higher pre-existing chemokine levels (particularly CCL13) in the nasal lining fluid and cross-reactive T cell responses. Transient infections (PCR positivity outside of residual inoculum not meeting the protocol- defined criteria for infection) were present in both studies. In the second study, transient infection was associated with significantly lower baseline mucosal and systemic SARS-CoV- 2 antibody levels and significantly lower peripheral IFN-γ producing CD8 + T-cell against a SARS-CoV-2 peptide pool than uninfected participants.
Added value of this study
With near-universal seropositivity to SARS-CoV-2, understanding the factors that influence how variants cause breakthrough infection is critical. The optimisation of a model that can induce safe and tolerable infection in a large proportion of participants is necessary for SARS- CoV-2 CHIMs to be used as a tool for next-generation vaccine development.
Our study is the first SARS-CoV-2 CHIM of seropositive individuals to induce sustained, protocol defined infection, albeit with an infection rate of 33%. Despite the low number of infected individuals, we identified several potential correlates of protection against breakthrough Delta SARS-CoV-2 infection beyond serum neutralising antibodies.
Implications of all the available evidence
This study establishes the framework for SARS-CoV-2 CHIM conduct, using sero-selection to increase attack rate in the same way it is used for influenza human challenge studies, and a process for defining quantitative correlates of protection. Further work will optimise model parameters with Omicron subvariants to result in infection rates of ≥50% to support testing of novel interventions.
