key: cord-0694761-kvf2ty5m
authors: Silva-Cayetano, Alyssa; Foster, William S.; Innocentin, Silvia; Belij-Rammerstorfer, Sandra; Spencer, Alexandra J.; Burton, Oliver T.; Fra-Bidó, Sigrid; Le Lee, Jia; Thakur, Nazia; Conceicao, Carina; Wright, Daniel; Barett, Jordan; Evans-Bailey, Nicola; Noble, Carly; Bailey, Dalan; Liston, Adrian; Gilbert, Sarah C.; Lambe, Teresa; Linterman, Michelle A.
title: A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice
date: 2020-12-16
journal: Med (N Y)
DOI: 10.1016/j.medj.2020.12.006
sha: 52a51810a0bcb06e194b6fe89a94dd1ab27aeef9
doc_id: 694761
cord_uid: kvf2ty5m

Background The spread of SARS-CoV-2 has caused a global pandemic that has affected almost every aspect of human life. The development of an effective COVID-19 vaccine could limit the morbidity and mortality caused by infection, and may enable the relaxation of social distancing measures. Age is one of the most significant risk factors for poor health outcomes after SARS-CoV-2 infection, therefore it is desirable that any new vaccine candidates elicit a robust immune response in older adults. Methods Here, we use in-depth immunophenotyping to characterize the innate and adaptive immune response induced upon intramuscular administration of the adenoviral vectored ChAdOx1 nCoV-19 (AZD-1222) COVID-19 vaccine candidate in mice. Findings A single vaccination generates spike-specific Th1 cells, Th1-like Foxp3+ regulatory T cells, polyfunctional spike-specific CD8+ T cells and granzyme B producing CD8 effectors. Spike-specific IgG and IgM are generated from both the early extrafollicular antibody response and the T follicular helper cell-supported germinal centre reaction, which is associated with the production of virus neutralising antibodies. A single dose of this vaccine generated a similar type of immune response in aged mice, but of a reduced magnitude than in younger mice. We report that a second dose enhances the immune response to this vaccine in aged mice. Conclusions This study shows that ChAdOx1 nCoV-19 induces both cellular and humoral immunity in adult and aged mice, and suggests a prime-boost strategy is a rational approach to enhance immunogenicity in older persons. Funding BBSRC, Lister institute of Preventative Medicine, EPSRC VaxHub and Innovate UK

The current Coronavirus Disease 2019 (COVID-19) pandemic is caused by the zoonotic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) 1, 2 . The pandemic has affected almost every aspect of human life, and will continue to do so until effective vaccines or therapeutics are developed. SARS-CoV-2 infection is initiated when the trimeric 'spike' glycoprotein on the virion surface binds angiotensin-converting enzyme 2, allowing viral entry and initiating viral replication 3 . After an asymptomatic incubation period, the infection can cause highly heterogenous clinical outcomes, from negligible or mild symptoms to critical disease resulting in death 4 . One of the main risk factors for severe disease and death is age 4, 5, 6 . Therefore, development of a successful COVID-19 vaccine should aim to be effective in older adults 7, 8 . However, age-related changes in the immune system mean that older individuals often do not generate protective immunity after vaccination 9, 10, 11, 12 .

The effectiveness of COVID-19 vaccine candidates in older adults will ultimately be determined in clinical trials. Yet, pre-clinical studies in aged animals can be used to test alternative vaccine strategies or dosing regimens, and can be used to inform clinical strategy. Despite ageing occurring on different time scales in mice and people, many of the cellular and molecular changes that occur are conserved between the species 13, 14 . The response to vaccination is no exception 15, 16 ; after vaccination, both aged mice (>20 months old) and older humans have reduced vaccinespecific antibody formation, an impaired type I interferon response and fewer T follicular helper cells 17, 18, 19, 20 . This impaired immune response to vaccination in older individuals has been linked with reduced protection against subsequent infection 21, 22, 23, 24 . Importantly, interventions that enhance the immunogenicity of vaccines in aged mice are also effective in humans 17, 23, 25, 26 .

ChAdOx1 nCoV-19 is a chimpanzee adeno (ChAd)-vectored vaccine that encodes the full-length spike protein of SARS-CoV-2. Vaccination with ChAdOx1 nCoV-19 elicits spike-specific T cells that produce IFNγ and anti-spike antibodies in mice, pigs, macaques and people 27, 28, 29 . Here, we demonstrate that a single dose of ChAdOx1 nCoV-19 elicits a B and T cell response in 3-monthold adult mice, with formation of plasma cells, germinal centres and T follicular helper cells contributing to anti-spike antibody production. The development of humoral immunity is complemented by the formation of polyfunctional SARS-CoV-2 spike-specific Th1 cells and CD8 + T cells. In aged 22-month-old mice a similar cellular and humoral response was observed upon vaccination, but the formation of germinal centres and spike-specific CD8 + T cells that secrete granzyme B was impaired. Administration of a second dose enhanced the germinal centre response, spike-specific IgG and virus neutralizing antibody titre, and no deficit in spike-specific granzyme B producing CD8 + T cells was observed in aged mice. Together, this indicates that the immunogenicity of ChAdOx1 nCoV-19 can be enhanced in older individuals through the use of a prime-boost vaccination strategy.

To map where antigen drains upon immunisation, 3-month-old adult mice were immunised in the right quadriceps muscle with 20nm fluorescent nanoparticles that freely drain into lymphoid organs 30 . Twenty-four hours after injection, confocal microscopy revealed fluorescent nanoparticles in the medial iliac lymph node (iLN) and the spleen of all immunised mice (Fig. S1A) . These data suggest the iLN and spleen are sites of antigen drainage.

J o u r n a l P r e -p r o o f 3 To characterise the early events of the immune response to ChAdOx1 nCoV-19 vaccination, we immunised adult C57BL/6 mice and assessed the induction of interferon signalling as well as the expansion of antigen presenting cell (APC) populations at day 1 and 2 post immunisation. At the site of immunisation, expression of the type I and 2 interferon inducible genes, Mx1 and Gbp2 respectively, was detected by day 1 in ChAdOx1 nCoV immunised mice compared to phosphate buffered saline (PBS) immunised controls (Fig. S1B) . In the draining iLN and spleen, type 2 conventional dendritic cells (cDC2s), Langerhans cells, macrophages and cDC1s were quantified by flow cytometry and all subsets were activated in response to ChAdOx1 nCoV ( Fig. S1C-E; gating strategy from 31 ). These data demonstrate that the ChAdOx1 nCoV-19 vaccine activates several APC populations required for the initiation of cell-mediated and humoral immunity 17, 32, 33 .

To assess the adaptive immune response to ChAdOx1 nCoV-19 in detail we used a highdimensional flow cytometry panel that contained antibodies that recognise multiple molecules used to define different lymphocyte subsets and their activation status (Table 1) . Mice were immunised with either ChAdOx1 nCoV-19 or PBS, and the immune response was assessed seven, fourteen and twenty-one days later. t-distributed Stochastic Neighbour Embedding (tSNE) and FlowSOM analysis grouped B cells into five clusters (Fig. 1A) . Cluster 1 corresponded to naïve B cells which were present in both ChAdOx1 nCoV-19 and PBS immunised mice, and four additional clusters which were over-represented in ChAdOx1 nCoV-19 immunised mice. The clusters represent activated B cells that express CD69 + (cluster 2) or CD86 + (cluster 5), a cluster of CD138 + IRF4 + antibody-secreting plasma cells (cluster 3) and Bcl6 + GL-7 + germinal centre B cells (cluster 4). Manual biaxial gating of these populations (Fig. S2A) showed that ChAdOx1 nCoV-19 immunisation induced a B cell response in the draining lymph node and spleen (Fig. 1B, C) , with early B cell activation and plasma cell formation occurring in the first week after immunisation, and the germinal centre response persisting over the three weeks assessed (Fig. 1B-G) . Formation of T follicular helper and T follicular regulatory cells accompanied the germinal centre response (Fig.  1H, I) . Antibodies binding the SARS-CoV-2 spike protein were induced by vaccination, and, as expected, the temporal induction of anti-spike IgM was faster than that of IgG, and serum IgA antibodies were not observed at high titre (Fig. 1J) . Of note, the method of quantification does not facilitate direct comparison between IgG and IgM titre. A mix of anti-spike IgG antibody subtypes was observed, with IgG 2 and IgG 1 subclasses persisting at later timepoints. At all timepoints, a predominantly Th1 dominant response (IgG 2 ) was measured (Fig. 1K) . The mean (and standard deviation) value for ratio of IgG 2 /IgG 1 on day seven was 4.8 (1.4), on day fourteen 2.3 (0.5), and on day twenty-one 2.8 (1.8) . This demonstrates that ChAdOx1 nCoV-19 immunisation stimulates B cell activation and differentiation, culminating in the production of anti-spike antibodies.

To investigate how ChAdOx1 nCoV-19 immunisation affects CD4 + T cells beyond the germinal centre-associated subsets, tSNE analysis followed by manual gating was performed ( Fig. 2A-B,  Fig. S2b, c) . tSNE/FlowSOM clustering showed that vaccination induced populations of T cells that express markers associated with T follicular helper cells (cluster 5), Th1 cells (cluster 2), Tbet expressing Th1-like Tregs (cluster 3) and CD69 + activated Treg cells (cluster 4) ( Fig. 2A) . The conventional CD4 + T cell response to ChAdOx1 nCoV-19 was characterised by early activation and proliferation, as well as the formation of CXCR3 + Th1 cells ( Fig. 2A-F) . The Foxp3 + regulatory T cell response was characterised by early expression of Ki67, indicative of proliferation, and the differentiation of CXCR3 + Th1-like cells (Fig. 2C, G) . To further characterise CD4 + T cell responses after vaccination, cells from the iLN were restimulated with PdBu/ionomycin and cytokine production was assessed. IL-2, TNFα, IL-10 and IFNγ were induced by cells isolated from animals that had been ChAdOx1 nCoV-19 vaccinated, with no difference observed in Th17-associated IL-J o u r n a l P r e -p r o o f 4 17 or the Th2-associated cytokines IL-4 and IL-5 (Fig. 2H, Fig. S3A-C) . The spike-specificity of this Th1-associated response in the draining lymph node was confirmed by restimulation of cells with peptide pools from SARS-CoV-2 spike protein (Fig. 2I) . In the spleen, spike-specific IL-2, TNFα, and IFNγ producing CD4 + T cells were observed, with triple producers persisting 21-days after immunisation (Fig. 2J, Fig. S3D ), consistent with previous work 27 . Therefore, ChAdOx1 nCoV-19 vaccination induces an early formation of Th1 cells and Th1-like Treg cells accompanied by the induction of spike-specific Th1-skewed cytokine-secreting cells.

ChAdOx1 nCoV-19 induces a CD8 + T cell response tSNE analysis of CD8 + T cells from the iLNs of ChAdOx1 nCoV-19 immunised and PBS control mice revealed distinct clustering seven days after immunisation. There were five CD8 + T cell clusters that were more abundant in ChAdOx1 nCoV-19 immunised mice, than in PBS immunised animals. Cluster 2 contains markers consistent with a CXCR5 + follicular CD8 cell population, cluster 3 is characterised by CXCR3 expression a marker of tissue homing, clusters 4 and 5 expresses CD69 and CD62L consistent with CD8 cells at an early stage of activation and cluster 6 expresses high levels of CXCR4 a chemokine receptor that facilitates bone marrow localisation of CD8 + T cells (Fig. 3A) . Together, this highlights that a diverse array of CD8 + T cell subsets are induced by ChAdOx1 nCoV-19. In order to understand how the response evolves over time, manual gating of different CD8 + T cell populations (Fig. S2D) , including those identified in the tSNE analysis, was done on samples taken seven, fourteen and twenty-one days after immunisation. In the iLN and spleen, the CD8 + T cell response to ChAdOx1 nCoV-19 was characterised by an increase in Ki67 expression, the upregulation of the activation markers CD69, CXCR3 and PD-1, as well as the formation of CD44 + CD62Lantigen-experienced T cells (Fig. 3B-F) . To characterise the overall production of granzyme B and cytokines by CD8 + T cells seven days after vaccination, cells from the iLN were restimulated with PdBu/ionomycin. There was a significant production of granzyme B, IL-2, TNFα and IFNγ in ChAdOx1 nCOV-19 vaccinated mice compared to PBS immunised mice (Fig. 3G) . The spike specificity of cytokine producing cells in the draining lymph node was confirmed by restimulation with SARS-CoV-2 peptide pools (Fig. 3H) . Restimulation of splenocytes with SARS-CoV-2 spike protein peptide pools from SARS-CoV-2 showed that spike-specific cytokine producing CD8 + T cells form in response to immunisation (Fig.  3I, Fig. S4A, B) . Spike-specific granzyme B producing CD8 + T cells formed early, and tended not to co-produce cytokines (Fig. 3I) . Both single and multiple cytokine producing spike-specific cells formed at all time points, with TNFα and IFNγ being the dominant cytokines expressed (Fig. 3I,  Fig. S4C ). With this dosing regimen, this data demonstrates that ChAdOx1 nCoV-19 stimulates a spike-specific CD8 + T cell response that peaks around the first week after vaccination.

To assess the CD8 + T cell response to ChAdOx1 nCoV-19 immunisation in the context of ageing, we immunised 3-month-old and 22-month-old mice and enumerated the CD8 + T cell types altered by vaccination (in Fig. 3 ) nine days after immunisation (Fig. 4A) . In the draining iLN, CD8 + T cells from aged mice expressed markers of activation and proliferation in response to ChAdOx1 nCoV-19. Unlike in younger adult mice, the frequency of CXCR3 + cells or antigen experienced cells did not increase in aged mice, compared to the PBS vaccinated group (Fig. 4B-D) . At this early timepoint, the frequency of central memory T cells was not altered in either younger adult or aged mice by ChAdOx1 nCoV-19 vaccination (Fig. 4E) . In the spleen, fewer Ki67 + CD8 + T cells were observed in aged mice after ChAdOx1 nCoV-19 vaccination, compared to younger adult mice (Fig.  4F) . The formation of spike-specific CD8 + T cells was assessed by restimulating splenocytes with SARS-CoV-2 spike protein peptide pools. Aged mice had a near absence of spike-specific granzyme B producing CD8 + T cells, but production of IFNγ and TNFα was not significantly J o u r n a l P r e -p r o o f impaired compared to younger mice (Fig. 4G) . Despite a trend to lower cytokine production by CD8 + T cells in aged mice, the proportion of polyfunctional spike-specific CD8 + T cells was not significantly diminished in aged mice after ChAdOx1 nCoV-19 vaccination (Fig. 4H) . This demonstrates that a single dose of ChAdOx1 nCoV-19 induces an altered CD8 + T cell response in aged mice characterised primarily by a failure to form spike-specific granzyme B-producing effector cells.

To determine whether a second dose could improve this response, we administered a booster dose of ChAdOx1 nCoV-19 one month after prime immunisation (Fig. 4I) . Nine days after boost, an increase in Ki67 + CD8 + T cells was not observed in the draining iLN (Fig. 4J) , possibly due to the kinetics of the secondary response being faster than the primary. A significant increase in CXCR3 + CD8 + T cells and antigen experienced cells was observed in aged mice after boost, with no change in the proportion of central memory cells in either age group (Fig. 4K-M) . Assessment of spike-specific T cells showed that the booster dose of ChAdOx1 nCoV-19 rescued the production of granzyme B producing CD8 + T cells in aged mice (Fig. 4N) . IFNγ production and cytokine polyfunctionality were similar to that following prime immunisation ( Fig. 4O, P) . This demonstrates that ChAdOx1 nCoV-19 is immunogenic in aged mice, and a booster dose can correct the age-dependent defect in the formation of spike-specific granzyme B-producing CD8 + T cells.

Nine days after primary immunisation of aged mice (Fig. 5A) , an increase in Ki67 + CD4 + T cells and CXCR3-expressing Th1 cells was observed in the draining lymph node of ChAdOx1 nCoV-19 immunised mice compared to PBS immunised control mice (Fig. 5B, C) . This was accompanied by an increase in the frequency of Th1-like Tregs in both adult and aged mice (Fig. 5D ). An increased frequency of these cell types was likewise observed in the spleen in response to ChAdOx1 nCoV-19 immunisation in both adult and aged mice ( Fig. 5E-G) . It is notable that, by these measurements, the response in aged mice is comparable to that in younger adults. The spikespecific CD4 + T cell response was assessed by restimulating splenocytes with SARS-CoV-2 spike protein peptide pools. As in young mice (Fig. 2) , the response in aged mice to ChAdOx1 nCoV-19 was Th1 dominated, however there were fewer spike-specific cytokine producing cells in aged mice nine days after a single immunisation (p<0.001, Fig. 5H , I).

A booster dose of ChAdOx1 nCoV-19 administered one month after prime (Fig. 5J ) stimulated Ki67 expression and the formation of CXCR3 + CD44 + Th1 cells, but not CXCR3 + Th1-like Treg cells in the draining lymph node of aged mice (Fig. 5K-M) . In the spleen, the booster dose did not enhance the frequency of Ki67 + CD4 + T cells or the formation of CXCR3 + conventional or regulatory T cells in adult or aged mice ( Fig. 5N-P) . In contrast to the response to primary immunisation, the number of spike-specific cytokine producing cells was comparable in adult and aged mice after booster immunisation (Fig. 5Q, R) . Together, this indicates that the CD4 + T cell response to ChAdOx1 nCoV-19 immunisation is largely intact in aged mice, with a slight deficiency in antigen-specific cytokine production that can be enhanced by a booster immunisation.

The majority of clinically available vaccines are thought to provide protection by eliciting humoral immunity. Therefore, it was important to quantify the B cell response to ChAdOx1 nCoV-19 vaccination in the context of ageing. Early antibody production after vaccination arises from antibody-secreting cells generated in the extrafollicular plasma cell response, which is fast, but typically short-lived 34 . A comparable early plasma cell response was detected in the iLN of younger adult and aged mice after immunisation (Fig. 6A, B) , although there was an increase in the J o u r n a l P r e -p r o o f 6 proportion of IgM + plasma cells in aged mice (Fig. 6C ). An intact plasma cell response was coupled with an increase in anti-spike antibodies nine days after immunization. These were of only slightly lower titre in aged mice, and of similar IgG subclass distribution to younger animals, indicative of a predominantly Th1 dominated response ( Fig. 6D-F) .

Long-lived antibody-secreting cells typically arise from the germinal centre response 35 . The percentage, but not total number, of germinal centre B cells was reduced in aged mice compared to younger adult mice after ChAdOx1 nCoV-19 vaccination (Fig. 6G, H) . Like the plasma cell response, there were more IgM + germinal centre B cells in aged mice (Fig. 6I ). An increase in T follicular helper cells, but not T follicular regulatory cells, accompanied the lymph node germinal centre response in adult and aged mice (Fig. 6J, K) . In the spleen, germinal centres were easily visualised by microscopy in adult mice nine days after ChAdOx1 nCoV-19 vaccination, but were conspicuously absent in aged mice (Fig. 6L, and Fig. S5 ). Quantification of splenic germinal centres by flow cytometry confirmed impaired germinal centre formation in aged mice (Fig. 6N, O) . This was accompanied by fewer proliferating non-germinal centre B cells and T follicular helper cells in aged mice (Fig. 6P, Q) . As in the draining lymph node, splenic T follicular regulatory cells were not induced by ChAdOx1 nCoV-19 vaccination at this time point (Fig. 6R) . The impact of an impaired germinal centre response on spike-specific antibodies was observed 28 days after immunisation, with aged mice having lower titres of anti-spike IgM and IgG (Fig. 6S, T) , but a similar profile of IgG subclasses (Fig. 6U) . Together, these data indicate that whilst a single of dose of ChAdOx1 nCoV-19 can induce comparable extrafollicular plasma cell responses between younger adult and aged mice, the germinal centre response is compromised with age.

To test whether a prime-boost strategy can enhance the B cell response in aged mice, a primeboost approach was taken (Fig. 7A) . Nine days after boost, there were Ki67 + non-germinal centre B cells, plasma cells and germinal centre B cells in the draining lymph nodes of aged mice ( Fig.  7B-H) . Notably, the magnitude of the germinal centre response was larger in aged mice than in younger adult mice after boost ( Fig. 7F-H) and this was associated with increased T follicular helper and T follicular regulatory cell numbers (Fig. 7I, J) . A germinal centre response was not observed in the spleen of either adult or aged mice nine days after booster immunisation (Fig. 7K) . This demonstrates that a second dose of ChAdOx1 nCoV-19 can enhance the B cell response in aged mice. This improvement in the B cell response corresponded to an increase in anti-spike IgG, without skewing IgG subclasses, antibodies in every aged mouse that was given a booster immunisation ( Fig. 7L-O) . The post boost ratio of IgG 2 /IgG 1 was 3.7 (3.0) in younger adult and 2.6 (1.6) in aged mice (mean and standard deviation). The functional effect of the humoral immunity after both prime and boost immunisations was measured by SARS-CoV-2 pseudotyped virus microneutralisation assay. Nine days after prime immunisation, SARS-CoV-2 neutralising antibodies were at a lower titre in aged mice than measured in adult mice (Fig. 7P) . Nine days after boost, neutralising antibodies were detectable in all aged mice and had been boosted eightfold compared to post-prime, although the titre was lower than in younger adult mice (Fig. 7P) . The SARS-CoV-2 neutralising antibody titre positively correlated with anti-spike IgG titre in aged mice, indicating that the main limitation in age-dependent humoral immunity is quantity, rather than function (Fig. 7R) . This demonstrates that a booster dose of ChAdOx1 nCoV-19 can improve antispike humoral immunity in older mice.

J o u r n a l P r e -p r o o f 7 The development of an effective anti-SARS-CoV-2 vaccine represents an opportunity to limit the health, social and economic consequences of the current pandemic. Since older adults are more likely to have severe health outcomes after infection, a vaccination strategy that provides protection for this group is particularly desirable. Here, we provide the most detailed immunophenotyping analysis to date of how the murine immune system following ChAdOx1 nCoV-19 vaccination. This vaccine stimulates both cellular and humoral immunity. Vaccination of younger adult mice triggers formation of spike-specific polyfunctional CD8 + and Th1 cells that persist for at least 21 days after immunization. The first surge of antibodies come from short-lived plasma cells, that produce low levels of neutralizing antibodies. Germinal centres and T follicular helper cells are established later in the response, which contribute to longer term antibody titres. One dose of this vaccine stimulates an immune response in aged mice, but it is of reduced magnitude compared to that seen in younger adult animals. Importantly, the data presented here show that a second homologous immunisation was able to enhance the B cell, helper T cell and CD8 + T cell response in aged mice. In people, the same prime-boost approach had an acceptable safety profile and enhanced humoral immunity 29 , indicating that this is a rational vaccination approach for use in the older members of our communities -arguably the sector of society most in need of an effective vaccine to prevent COVID-19.

Strategies that are able to enhance humoral immunity in older individuals are key to vaccine efficacy, particularly as in some respiratory infections, a higher titre of antibodies are required to be protective 36, 37 . Antibody-secreting cells can form from two cellular pathways, but these are not equal in terms of quality or longevity. The extrafollicular response, produces an initial burst of antibodies early after antigenic challenge. This response is short-lived with no additional diversification of the B cell repertoire and thus its contribution to long-term immunity is minimal 34 . The germinal centre reaction, is a specialised microenvironment that produces memory B cells and long-lived antibody secreting plasma cells with somatically-mutated immunoglobulin genes 35, 38, 39 . The germinal centre is the only cellular source of long-lived plasma cells 40 , and the only known site where the B cell response can be altered in response to antigen. Nine days after ChAdOx1 nCoV-19 immunisation, the formation of plasma cells was comparable in young and aged mice, a time at which the majority of plasma cells derive from the extrafollicular response 40 . By contrast, the germinal centre response was diminished in aged mice, which is a well described deficit of the older immune system after vaccination 17, 41 . This indicates that poor germinal centre formation is the major barrier that must be overcome to improve humoral immunity in older individuals. A booster dose of ChAdOx1 nCoV-19 enhanced the magnitude of the germinal centre response in aged mice, to a greater extent than it did in younger animals. This may be because younger mice have produced higher titres of anti-vaccine vector antibodies, which limits the action of a second dose.

Another feature of the aged germinal centre reaction is reduced selection of high affinity B cells, resulting in a diminished quality of the response in ageing 19 . Whether the further diversification and affinity-based selection of the B cell receptor is important for protective humoral immunity to SARS-CoV-2 has yet to be established. However, antibodies isolated from spike-specific B cells from convalescent COVID-19 patients can be both potently neutralising and have low levels of somatic hypermutation 42, 43, 44 , indicating that extensive somatic hypermutation is not required for anti-SARS-CoV-2 neutralising antibody formation. Therefore, the main barrier to inducing protective humoral immunity to SARS-CoV-2 in older people may be an issue of enhancing the magnitude, rather than quality, of the germinal centre response. Our study shows this can be achieved in aged mice by giving a second dose of the vaccine.

J o u r n a l P r e -p r o o f 8 A rapid recall of memory CD8 + T cells in response to viral infections complements the humoral response by promoting efficient pathogen clearance and this becomes particularly important in scenarios where the protective ability or magnitude of neutralising antibodies is compromised 45 . A single dose of ChAdOx1 nCoV-19 vaccine induces a CD8 + T cell response in both adult and aged mice. There was, however, a profound defect in the generation of spike-reactive CD8 + T cells that produced granzyme B, a key cytotoxic effector molecule produced by effector CD8 + T cells. This failure to generate granzyme B + CD8 + T cells is a common feature in ageing, previously observed in aged mice after West Nile Virus infection 46 and in aged monkeys infected with SARS-CoV 47 . A similar trend for fewer granzyme B + CD8 + cells in older people was observed after influenza vaccination 48 . Consistent with reduced expression of granzyme B in T cells from older adults, T cells taken from older adults have an impaired ability to kill influenza infected cells ex vivo 49 . In a small study of older people, induction of granzyme B after influenza vaccination was a correlate of protection in older adults 37 . Here we demonstrate that a second dose of ChAdOx1 nCoV-19 was able to correct the defective granzyme B + CD8 + T cell response in aged mice, indicating that two doses of this vaccine is a better approach to enhance the cellular immune response in older bodies.

ChAdOx1 nCoV-19 is currently being trialed in older adults as part of a phase III trial, which will ultimately determine whether it is an effective vaccine for this demographic. Immunisation of people over 55 years of age with adenovirus type-5(Ad5)-vectored COVID-19 vaccine resulted in lower antibody titres in the older age group, suggesting that adenoviral vectored vaccine strategies may require more than one dose in older people 50 . However, as pre-existing immunity to Ad5, a naturally occurring human tropic adenovirus, can inhibit the response to this vaccine, it is possible that the reduced immunogenicity of this vaccine is due to an increased prevalence of antibodies to the vaccine vector in this age group. By contrast, the presence of anti-ChAdOx1 antibodies in the general population is low 51 . The work presented here demonstrates that one dose of this vaccine is immunogenic in aged mice, but this response can be significantly improved with a second booster dose. Given that a second dose of ChAdOx1 nCoV-19 is immunogenic with expected reactogenicity profile in humans 29 , this may be a viable strategy to enhance immunogenicity and possibly efficacy in older people.

This study used aged mice as a pre-clinical model to test the immunogenicity of ChAdOx1 nCoV-19 in older bodies. One limitation of this study was that SARS-CoV-2 infections were not performed to test the protection conferred by vaccination. SARS-CoV-2 does not naturally infect mice, to circumvent this limitation, mouse adapted strains have been generated 52, 53 . In order to bind to the mouse angiotensin-converting enzyme 2, SARS-CoV-2 requires amino acid substitutions in the receptor binding domain of the spike protein. As such, challenge with mouse adapted SARS-CoV-2 frequently mismatches vaccine encoded antigens and may not reflect realworld scenarios. The data presented in this manuscript show that a second dose of ChAdOx1-nCoV increases anti-spike IgG titre and SARS-CoV-2 pseudovirus neutralising antibody titre in aged mice, both of which are correlates of protection against SARS-CoV-2 infection in species that are susceptible to the wild type virus 54, 55 .

Research Council (BBS/E/B/000C0427, BBS/E/B/000C0428 and the Campus Capability Core Grant to the Babraham Institute), the Lister institute of Preventative Medicine, the EPSRC VaxHub (EP/RO13756/1) and Innovate UK (biEBOV: 971615). Teresa Lambe and Sarah Gilbert are Jenner Investigators. Michelle Linterman is an EMBO Young Investigator and a Lister Institute Prize Fellow. Jia Le Lee is supported by a National Science Scholarship (PhD) by the Agency for Science, Technology and Research, Singapore. 

Sarah Gilbert and Teresa Lambe are named on a patent application covering ChAdOx1 nCoV-19. The remaining authors declare no competing interests. The funders played no role in the conceptualisation, design, data collection, analysis, decision to publish, or preparation of the manuscript. tSNE/FlowSOM analyses of CD4 + T cells from 3-month-old (3mo) mice seven days after immunization with ChAdOx1 nCoV-19 or PBS, on heat map red indicates high expression, yellow low expression. Heatmaps of the manually gated CD4 + Foxp3 -(B) and Foxp3 + CD4 + (C) T cell populations indicated at seven, 14 and 21 days after immunisation in the iliac lymph node (right) and spleen (left), the gating strategy for these populations is shown in Fig S2. Here the frequency of each cell subset in each ChAdOx1 nCoV-19 immunised mouse has been expressed as the log2 fold change over the average frequency in PBS immunised mice (n=5). Bar charts showing the number of CD69 + CD62L + CD44 -CD4 + Foxp3 -(D), Ki67 + CD4 + Foxp3 -(E) CXCR3 + non-Tfh cells (F) and CXCR3 + Th1-like Treg cells (G) CD4 + cells in the iliac lymph node of ChAdOx1 nCoV-19 or PBS immunised mice, at the indicated timepoints post immunisation. H. Analysis of cytokine production six hours after PdBu/ionomycin stimulation of iliac lymph node cells from 3-month-old mice seven days after immunization with ChAdOx1 nCoV-19 or PBS. Stacked bar plots show the number of CD4 + Foxp3cells singly or co-producing IFNγ, IL-2 or TNFα in the iLN seven days after immunization (I) or spleen at the days post immunisation (J), six hours after restimulation with SARS-CoV-2 peptide pools, each bar segment represents the mean and the error bars the standard deviation. In D-G bar height in corresponds to the mean and each circle represents one biological replicate. P-values are calculated using a student's t-test with Holm-Sidak multiple testing correction. taken from 3-month-old or 22-month-old mice nine days after immunization with ChAdOx1 nCoV-19 or PBS. Bar height in B-G, J-O corresponds to the median and each circle represents one biological replicate. In H, P, each bar segment represents the mean and the error bars the standard deviation. The Shapiro-Wilk normality test was used to determine whether the data are consistent with a normal distribution, followed by either an ordinary one-way ANOVA test for data with a normal distribution or a Kruskal Wallis test for non-normally distributed data alongside a multiple comparisons test. Data are representative of two independent experiments (n=4-8 per group/experiment). Cartoon of prime-boost immunization strategy. Percentage of proliferating Ki67 + (K), CXCR3 + CD44 + CD4 T cells (L) and CXCR3 + CD44 + Foxp3 + Treg cells (M) in the draining iliac lymph node. Percentage of Ki67 + CD44 + (N), CXCR3 + CD44 + CD4 + Foxp3 -T cells (O) and CXCR3 + CD44 + Foxp3 + Treg cells (P) in the spleen of 3-month-old or 22-month-old mice nine days after immunization with ChAdOx1 nCoV-19 or PBS. Q, R. Number of CD4 + Foxp3 -T cells producing IFNγ, IL-2, IL-4, IL-5, IL-17 or TNFα six hours after restimulation with SARS-CoV-2 peptide pools, in (R) and the number of single and multiple cytokine producing CD4 + T cells are represented in stacked bar charts. Bar height in corresponds to the median and each circle represents one biological replicate. In I, R, each bar segment represents the mean and the error bars the standard deviation. The Shapiro-Wilk normality test was used to determine whether the data are consistent with a normal distribution, followed by either an ordinary one-way ANOVA test for data with a normal distribution or a Kruskal Wallis test for non-normally distributed data alongside a multiple comparisons test. Data are representative of two independent experiments (n=4-8 per group/experiment). 

IgG (F) anti-spike antibodies and IgG subclasses (U) 28 days after immunization. Bar height in corresponds to the median and each circle represents one biological replicate. The Shapiro-Wilk normality test was used to determine whether the data are consistent with a normal distribution, followed by either an ordinary one-way ANOVA test for data with a normal distribution or a Kruskal Wallis test for non-normally distributed data alongside a multiple comparisons test, for ELISA data analyses were done on log transformed values. Data are representative of two independent experiments (n=4-8 per group/experiment). and nine days after boost immunization. P-Q SARS-CoV-2 neutralising antibody titres in sera were determined by micro neutralisation test, expressed as reciprocal serum dilution to inhibit pseudotyped virus entry by 50% (IC 50 ). Samples below the lower limit of detection (LLoD) are shown as half of the LLoD. R. Linear regression of serum dilution to inhibit pseudotyped virus entry by 50% (IC 50 Log10) and serum anti-spike ELISA titre (Log10) in 22-month-old mice, nine days post boost. Bar height in corresponds to the median and each circle represents one biological replicate. The Shapiro-Wilk normality test was used to determine whether the data are consistent with a normal distribution, followed by either an ordinary one-way ANOVA test for data with a normal distribution or a Kruskal Wallis test for non-normally distributed data alongside a multiple comparisons test. For ELISA data analyses were done on log transformed values. In B-O, are shown from one of two independent experiments (n=4-8 per group/experiment), in P-R the data are pooled from two experiments.

Lead contact: Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Michelle Linterman (michelle.linterman@babraham.ac.uk) Materials Availability: This project did not generate new unique reagents Data and Code Availability: This project did not generate any new code or novel datasets EXPERIMENTAL MODEL AND SUBJECT DETAIL Mouse housing and husbandry C57BL/6Babr mice were bred, aged and maintained in the Babraham Institute Biological Support Unit. No primary pathogens or additional agents listed in the Federation of European Laboratory Animal Science Association recommendations 56 were detected during health monitoring surveys of the stock holding rooms. Ambient temperature was ~19-21°C and relative humidity 52%. Lighting was provided on a 12 hr light: 12 hr dark cycle including 15 min 'dawn' and 'dusk' periods of subdued lighting. After weaning, mice were transferred to individually ventilated cages with 1-5 mice per cage. Mice were fed Mouse Breeder and Grower CRM (P) VP diet (Special Diet Services) ad libitum and received seeds (e.g. sunflower, millet) at the time of cage-cleaning as part of their environmental enrichment. All mouse experimentation was approved by the Babraham Institute Animal Welfare and Ethical Review Body. Animal husbandry and experimentation complied with existing European Union and United Kingdom Home Office legislation and local standards (PPL: J o u r n a l P r e -p r o o f 13 P4D4AF812). Young mice were 10-12 weeks old, and aged mice 93-96 weeks old when experiments were started. Mice that had tumours, which can occur in aged mice, were excluded from the analysis.

Mice were immunised in the right quadriceps femoris muscle with 50µL of either 1x10 8 infectious units of ChAdOx1 nCoV-19 in phosphate buffered saline (PBS) alone, 50µL 0.02µm yellow-green fluorescent Carboxylate-Modified Microspheres (Invitrogen # F8787) in phosphate buffered saline (0.5% solids, final injected concentration). At the indicated timepoints post vaccination, blood, the right medial iliac lymph node, spleen and right quadriceps femoris muscle were taken for analysis.

For T and B cell flow cytometric stains a single cell suspension was prepared from the iliac lymph node and half the spleen was generated by pressing the tissues through a 70 µm mesh in 2% FBS in PBS. Cell numbers and viability were determined using a CASY TT Cell Counter (Roche). 2×10 6 cells were transferred to 96-well plates for antibody staining. Samples were blocked with 100µl of 2.4G2 Fc Block (made in house) for 20 min at 4°C. Cells were then stained with surface antibody mix for 2hrs at 4°C and then were fixed with the eBiosciences Foxp3/Transcription Factor Staining Buffer (#00-5323-00) for 30 min at 4°C. Cells were then washed with 1x Permeabilisation buffer (eBioscience #00-8333-56) twice and stained with intracellular antibody mix in 1x Permeabilisation buffer at 4°C overnight. For cytokine staining, splenic cells were stimulated with a pool of SARS-CoV-2 spike protein immunodominant domain peptides, (Miltenyi Biotec #130-126-700) at a 0.6 µM concentration (approx.1µg/ml), whilst lymph node cells were stimulated with 0.5µg/ml of Phorbol 12,13 dibutyrate (PdBu, Tocris Bioscience, #), 0.75µg/ml of Ionomycin calcium salt (Tocris Bioscience, #), both in warm complete RPMI (10% FCS, 1% Pen/Strep, 1% glutamine, 1% sodium pyruvate, 1% MEM NAA, 1% HEPES and 55µM-2-mercaptoethanol) for 4hrs at 37°C, 5%CO2. Cytokine secretion was then blocked with 22µg/ml of Brefeldin A (Tocris Bioscience, #) in warm complete RPMI for 2hrs at 37°C, 5%CO2. The cells were then stained with surface antibody mix for 20 minutes at 4°C and were subsequently fixed with 2% formaldehyde for 30min at room temperature. After two wash steps with 1x Permeabilisation buffer (eBioscience #00-8333-56), the cells were stained with intracellular antibody mix in 1x Permeabilisation buffer, supplemented with 20% 2.4G2 Fc Block at 4°C overnight. Following overnight staining, samples were washed twice with 1x Permeabilisation buffer and once with 2% FBS in PBS and acquired on a Cytek TM Aurora. Cells for single colour controls were prepared in the same manner as the fully stained samples. The antibodies used for surface and overnight staining are listed in the Key Resources Table. Manual gating of flow cytometry data was done using FlowJo v10 software (Tree Star). tSNE, FlowSOM and heatmap analysis were performed on iLN samples from day 7 postvaccination using R (version 4.0.2) using code that has previously been described 57 . The antibodies used for surface staining are listed in Table 1 . After fixation, all tissues were dehydrated in 30% sucrose (Sigma #S0389) overnight, embedded in Optimum Cutting Temperature (OCT) medium (VWR #25608-930) on dry ice and stored at −80 °C. The frozen tissues were cut into 10µm sections using a cryostat (Leica Biosystems) at −20°C and again stored at −80°C. For imaging yellow-green fluorescent carboxylate-codified microspheres, slides were first air-dried and then washed in PBS three times after which DAPI staining was performed (Invitrogen #D1306; diluted 1:10 000 in PBS) for 10 minutes at room temperature protected from light. Slides were then washed three times with PBS and coverslips were mounted on the slides using Hydromount mounting medium (National Diagnostics #HS-106). For antibody stains, the slides were first air-dried and then hydrated in 0.5% Tween 20 in PBS (PBS-T). The slides were blocked in PBS containing 2% BSA and 10% goat serum for 2hrs, washed three times with PBS-T and then permeabilised with PBS containing 2% Triton X (Sigma #X100) for 45min. Following three wash steps with PBS-T, the slides were stained with primary antibody mix, which included AF647-conjugated rat anti-mouse IgD (clone 11-26 c.2a, Biolegend; 1:200), FITC-conjugated rat anti-mouse Ki67 (clone SolA15, Invitrogen; 1:100), rat anti-mouse biotin-conjugated CD21/35 (clone 8D9, ThermoFisher Scientific; 1:400) and hamster anti-mouse CD3ε (clone 500A2, ThermoFisher Scientific; 1:200), in PBS-T containing 1% BSA at 4°C overnight. The next day, slides were washed three times with PBS-T and incubated with secondary antibody mix, which included AF568-conjugated goat anti-hamster IgG (Thermofisher, 1:1000) and BV421-conjugated Streptavidin (Biolegend, 1:1000), in PBS-T containing 2% goat serum for 2hr at room temperature. Slides were then washed three times with PBS-T, PBS and dH 2 O and coverslips were mounted using Hydromount mounting medium (National Diagnostics #HS-106). Images were acquired using a Zeiss 780 confocal microscope with 10x and 20x objectives and analysed using ImageJ.

Standardised ELISA was performed to detect SARS-CoV-2 FL-S protein -specific antibodies in sera. MaxiSorp plates (Nunc) were coated with 100 or 250 ng/well FL-S protein overnight at 4 °C for detection of IgG or IgM and IgA, respectively, prior to washing in PBS/Tween (0.05% v/v) and blocking with Blocker Casein in PBS (Thermo Fisher Scientific) for 1 h at room temperature (RT). Standard positive serum (pool of mouse serum with high endpoint titre against FL-S protein), individual mouse serum samples, negative and an internal control (diluted in casein) were incubated for 2 hours at room temperature for detection of specific IgG or 1h at 37 o C for detection of specific IgM or IgA. Following washing, bound antibodies were detected by addition of alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Sigma-Aldrich) for 1h at room temperature or addition of AP-conjugated goat anti-mouse IgM or IgA (Abcam and Sigma-Aldrich, respectively) and addition of p-Nitrophenyl Phosphate, Disodium Salt substrate (Sigma-Aldrich). An arbitrary number of ELISA units were assigned to the reference pool and optical density values of each dilution were fitted to a 4-parameter logistic curve using SOFTmax PRO software. ELISA units were calculated for each sample using the optical density values of the sample and the parameters of the standard curve. The IgG subclass ELISA were performed according to the protocol described for detection of specific IgM or IgA in the serum. In addition, all serum samples were diluted to 1 total IgG ELISA unit and then detected with anti-mouse IgG subclass-specific secondary antibodies (Southern Biotech or Abcam). The results of the IgG subclass ELISA are presented using optical density values instead of the ELISA units used for the total IgG ELISA. The ratio of IgG 2 /IgG 1 was calculated for each animal as sum of optical density values (IgG 2b + IgG 2c ) divided by the optical density value of IgG 1 and represented as mean values with standard deviation (SD).

Lentiviral-based SARS-CoV-2 pseudotyped viruses were generated in HEK293T cells incubated at 37 °C, 5% CO 2 as previously described 28 . Briefly, cells were seeded at a density of 7.5 x 10 5 in 6 well dishes, before being transfected with plasmids as follows: 500 ng of SARS-CoV-2 spike, 600 ng p8.91 (encoding for HIV-1 gag-pol), 600 ng CSFLW (lentivirus backbone expressing a firefly luciferase reporter gene), in Opti-MEM (Gibco) along with 10 µL PEI (1 µg/mL) transfection reagent. A 'no glycoprotein' control was also set up using the pcDNA3.1 vector instead of the SARS-CoV-2 S expressing plasmid. The following day, the transfection mix was replaced with 3 mL DMEM (Dulbecco's Modified Eagle's medium) with 10% fetal bovine serum (DMEM-10%) and incubated for 48 and 72 hours, after which supernatants containing pseudotyped SARS-CoV-2 (SARS-CoV-2 pps) were harvested, pooled and centrifuged at 1,300 x g for 10 minutes at 4 °C to remove cellular debris. Target HEK293T cells, previously transfected with 500 ng of a human Angiotensin-converting enzyme 2 (ACE2) expression plasmid (Addgene, Cambridge, MA, USA) were seeded at a density of 2 × 10 4 in 100 µL DMEM-10% in a white flat-bottomed 96-well plate one day prior to harvesting SARS-CoV-2 pps. The following day, SARS-CoV-2 pseudotyped viruses were titrated 10-fold on target cells, and the remainder stored at -80 °C. For micro neutralisation tests, mouse sera were diluted 1:20 in serum-free media and 50 µL was added to a 96-well plate in triplicate and titrated 2-fold. A fixed titred volume of SARS-CoV-2 pseudotyped viruses was added at a dilution equivalent to 10 5 signal luciferase units in 50 µL DMEM-10% and incubated with sera for 1 hour at 37 °C, 5% CO 2 (giving a final sera dilution of 1:40). Target cells expressing human ACE2 were then added at a density of 2 x 10 4 in 100 µL and incubated at 37 °C, 5% CO 2 for 72 hours. Firefly luciferase activity was then measured with BrightGlo luciferase reagent and a Glomax-Multi + Detection System (Promega, Southampton, UK). Pseudovirus neutralization titres were expressed as the reciprocal of the serum dilution that inhibited luciferase expression by 50% (IC 50 ).

All experiments were performed either twice or three times with 3-8 mice per group. Data was first tested for gaussian distribution using a Shapiro-Wilk test. Then data that was consistent with a normal distribution was analysed with either a student's t-test for comparing two data set, or oneway ANOVA test for data with multiple groups. If the data did not follow a normal distribution then a Mann-Whitney test was used for comparing two data sets and a Kruskal Wallis test for multiple comparisons. For ELISA data p-values were generated on log transformed data. All p-values shown are adjusted for multiple comparisons where multiple tests were performed on the same data. Analyses were performed within the Prism v8 software (GraphPad).

Nakaya Context and significance: Effective COVID-19 vaccines will play a central role in the exit strategy from the global pandemic. However, older persons often do not generate protective immunity upon vaccination due to age-dependent changes in their immune system. Because older people are more likely to have poor clinical outcomes after SARS-CoV-2 infection, vaccine strategies that elicit an optimal immune response in older bodies are urgently required. Researchers from the Babraham Institute in Cambridge, UK, performed pre-clinical testing of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice to assess how ageing influences the immune response to this vaccine. The results show that a "primeboost" vaccine regime enhances immunogenicity in aged mice, indicating that this approach is a rational strategy for vaccinating older persons. 

A Novel Coronavirus from Patients with Pneumonia in China

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SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese Center for Disease Control and Prevention

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Specific humoral immunity in the elderly: in vivo and in vitro response to vaccination

The mouse as a model organism in aging research: usefulness, pitfalls and possibilities

Mechanisms underlying T cell ageing

Age-related factors that affect B cell responses to vaccination in mice and humans

Influence of immune aging on vaccine responses

Rejuvenating conventional dendritic cells and T follicular helper cell formation after vaccination

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The adjuvant GLA-SE promotes human Tfh cell expansion and emergence of public TCRbeta clonotypes

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Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19

Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind

Nanoparticles target distinct dendritic cell populations according to their size

Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species

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T cell responses are better correlates of vaccine protection in the elderly

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Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy

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Ad26 vaccine protects against SARS-CoV-2 severe clinical disease in hamsters

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units

Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition

We are grateful to Dr. Martin Turner, Dr. Geoff Butcher, Marc Wiltshire, Paul Symonds and Prof. Michael Wakelam for their support. We thank Dr. Hayley Sharpe and Dr. Heidi Welch for technical assistance. This study was supported by funding from the Biotechnology and Biological Sciences