1
Durability of ChAdOx1 nCov-19 (AZD1222) vaccination in people living with 1
HIV - responses to SARS-CoV-2, variants of concern and circulating 2
coronaviruses 3
Ane Ogbe*1, Mathew Pace*1, Mustapha Bittaye*4, Timothy Tipoe1, Sandra Adele1, 4 Jasmini Alagaratnam6,7, Parvinder K Aley8, M. Azim Ansari1, Anna Bara9, Samantha 5 Broadhead13, Anthony Brown1, Helen Brown1, Federica Cappuccini4, Paola 6 Cinardo10, Wanwisa Dejnirattisai11, Katie J. Ewer4, Henry Fok10, Pedro M. Folegatti4, 7 Jamie Fowler4, Leila Godfrey4, Anna L. Goodman10, Bethany Jackson10, Daniel 8 Jenkin4, Mathew Jones1, Stephanie Longet11,12, Rebecca Makinson4, Natalie G. 9 Marchevsky8, Moncy Mathew10, Andrea Mazzella10, Yama F. Mujadidi8, Lucia 10 Parolini1, Claire Petersen 6,7, Emma Plested8, Katrina M. Pollock9, Thurkka 11 Rajeswaran10 Maheshi N. Ramasamy8, Sarah Rhead8, Hannah Robinson8, Nicola 12 Robinson1,3, Helen Sanders4, Sonia Serrano13, Helen Stockmann7, Tom Tipton11,12, 13 Anele Waters10, Panagiota Zacharopoulou1, Eleanor Barnes1,2,3,4, Susanna 14 Dunachie1,2,14,15, Philip Goulder1,2,5, Paul Klenerman1,2,3, Gavin R. Screaton11, Alan 15 Winston6,7, Adrian V. S. Hill4, Sarah C. Gilbert4, Miles Carroll11,12, Andrew J 16 Pollard*3,8, Sarah Fidler*6,7, Julie Fox10,13*, Teresa Lambe*4, John Frater*1,2,3 17 18 1Peter Medawar Building for Pathogen Research, Nuffield Dept of Clinical Medicine, 19 University of Oxford, Oxford, UK 20 2Oxford University Hospitals NHS Foundation Trust, Oxford, UK 21 3NIHR Oxford Biomedical Research Centre, Oxford, UK 22 4The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, 23 UK 24 5Department of Paediatrics, University of Oxford, Oxford, UK 25 6Department of Infectious Disease, Faculty of Medicine, Imperial College London, 26 London, UK 27 7Department of HIV Medicine, St Mary's Hospital, Imperial College Healthcare NHS 28 Trust, London, UK 29 8Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Oxford, UK. 30 9NIHR Imperial Clinical Research Facility and NIHR Imperial Biomedical Research 31 Centre, London, UK 32 10Department of Infection, Harrison Wing and NIHR Clinical Research Facility, Guys 33 and St Thomas' NHS Trust, London, UK 34 11Wellcome Centre for Human Genetics, Nuffield Department of Medicine, University 35 of Oxford, Oxford, UK 36 12Public Health England, Porton Down, UK. 37 13NIHR Guy’s and St Thomas’ Biomedical Research Centre 38 14Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, 39 University of Oxford, Oxford, UK 40 15Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, 41 Thailand 42 43 *Contributed equally 44 45 46 Correspondence to: 47
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2
John Frater, Peter Medawar Building for Pathogen Research, Nuffield Dept of 48 Clinical Medicine, University of Oxford, Oxford, UK, OX1 3SY; 49 [email protected] 50 Ane Ogbe, Peter Medawar Building for Pathogen Research, Nuffield Dept of Clinical 51 Medicine, University of Oxford, Oxford, UK, OX1 3SY; [email protected] 52 53
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Author Contributions 54
JFr, Ao, SF, JFo, TL. AJP, SCG, AVSH, AA, KJE were involved in conceptualisation, 55
data curation, funding acquisition, supervision, methodology, writing and reviewing the 56
manuscript. AO, MP, SA, EA, MA, AA, AB, HB, MJ, LP, NR, TT, PZ were involved with 57
sample methodology and preparation, assay performance, data curation (CTV 58
assays), data analysis and writing and reviewing the manuscript. EB, SD, PG, PK were 59
involved in supervision, data curation, investigation, methodology and writing and 60
reviewing the manuscript. SL, ToT, MC oversaw MSD and ACE-inhibition assays; JA, 61
AB, CP, KMP, HS, AW, SF managed study recruitment at Imperial; PC, AM, TR, BJ, 62
MM, SB, HK, ALG, SS, AW, JFo managed study recruitment at Guy’s; WD, GRS were 63
involved in data curation (neutralisation), formal analysis, methodology and writing and 64
reviewing the manuscript. AB, JA, CP, KP, HS, AW, SF were involved in data curation, 65
methodology, project administration, supervision, investigation and writing and 66
reviewing the manuscript. PA was involved in project administration. MB, FC, PF, 67
JFow, DJ, RM, TL were involved in data curation, formal analysis, investigation, 68
methodology and writing and reviewing the manuscript. SB, HF, ALG, SS, AW, JFox 69
were involved in data curation, methodology, project administration, supervision, 70
investigation and writing and reviewing the manuscript. NGM, YM, EP, MR, SR, HR, 71
MV, AJP were involved in data curation, formal analysis, investigation, methodology 72
and writing and reviewing the manuscript. All authors critically reviewed and approved 73
the final version. 74
75
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Abstract 76
Duration of protection from SARS-CoV-2 infection in people with HIV (PWH) following 77
vaccination is unclear. In a sub-study of the phase 2/3 the COV002 trial 78
(NCT04400838), 54 HIV positive male participants on antiretroviral therapy 79
(undetectable viral loads, CD4+ T cells >350 cells/ul) received two doses of ChAdOx1 80
nCoV-19 (AZD1222) 4-6 weeks apart and were followed for 6 months. Responses to 81
vaccination were determined by serology (IgG ELISA and MesoScale Discovery 82
(MSD)), neutralisation, ACE-2 inhibition, gamma interferon ELISpot, activation-83
induced marker (AIM) assay and T cell proliferation. We show that 6 months after 84
vaccination the majority of measurable immune responses were greater than pre-85
vaccination baseline, but with evidence of a decline in both humoral and cell mediated 86
immunity. There was, however, no significant difference compared to a cohort of HIV-87
uninfected individuals vaccinated with the same regimen. Responses to the variants 88
of concern were detectable, although were lower than wild type. Pre-existing cross-89
reactive T cell responses to SARS-CoV-2 spike were associated with greater post-90
vaccine immunity and correlated with prior exposure to beta coronaviruses. These 91
data support the on-going policy to vaccinate PWH against SARS-CoV-2, and 92
underpin the need for long-term monitoring of responses after vaccination. 93
94
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Introduction 95
The global COVID-19 pandemic has led to over 200 million cases and 4.2 million 96
deaths (1). Vaccines which have been licensed against SARS-CoV-2 include the 97
AstraZeneca ChAdOx1 nCov-19 (AZD1222) adenoviral vectored vaccine, of which 98
over 1 billion doses have been made available worldwide. People living with HIV 99
(PWH) represent a high-risk group for adverse clinical outcomes from viral infections 100
such as influenza and COVID-19, with some evidence for higher hospitalisation and 101
mortality rates (2-6). This can in part be attributed to a state of immune cell depletion 102
and chronic immunopathology including immune activation and exhaustion which is 103
only partially restored by antiretroviral therapy (ART)(7, 8). Studies on influenza and 104
tetanus toxin vaccination in PWH have shown that antibody levels post-vaccination 105
were dependent on CD4 T cell count and activated T follicular helper (Tfh) cell 106
frequencies, which can vary widely in PWH (9, 10), resulting in broader concerns over 107
reduced responses to vaccines (11) and specific vaccination guidelines for PWH (12). 108
Some studies also report that vaccination of PWH may induce immune activation and 109
reactivate the HIV reservoir (13, 14). 110
111
ChAdOx1 nCov-19 containing SARS-CoV-2 full length spike has been shown to 112
induce potent humoral and cellular immune response in vaccine recipients (15-18). 113
We recently reported the safety and immunogenicity of the ChAdOx1 nCoV-19 114
vaccine in PWH up to 2 months post initial vaccination (16) and the durability of T and 115
B cell responses following natural infection with SARS-CoV-2 (19). There are, 116
however, few studies evaluating the durability of immunity following vaccination 117
against COVID-19 (20, 21). A recent open label phase I trial showed durable SARS-118
CoV-2 T and B cell immune response up to 6 months following vaccination in adults 119
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without HIV using a low dose of mRNA vaccine mRNA-1273 (21), with similar results 120
in another study using standard mRNA-1273 dosing (22). There have been no studies 121
to date reporting the durability of immune responses in PWH. 122
123
Since the rollout of COVID-19 vaccines, divergent mutations in the viral sequence in 124
the original SARS-CoV-2 strain have given rise to the alpha (B.1.1.7), beta (B.1.351), 125
gamma (P.1), and more recently delta (B.1.617.2) variants of concern (VOCs). 126
Infections with VOCs have become dominant in several countries (23). Studies of 127
symptomatic disease in fully vaccinated individuals report variable effectiveness 128
(ChAdOx1 nCoV-19 – alpha (74%), delta (67%) and BNT162b2 – alpha (93.7%), delta 129
(88%)), but with evidence for sustained protection from severe disease (24-26). 130
Nonetheless, breakthrough infections have been recorded and a significant proportion 131
of the world’s population remains unvaccinated (27). Understanding the ability of 132
immune responses generated in PWH to recognise VOCs is key to informing 133
vaccination strategies, especially in vulnerable populations. 134
135
Pre-existing cross-reactive T and B cell responses in individuals naïve to SARS-CoV-136
2 infection and vaccination to the circulating common cold coronaviruses (CCC) 137
HKU1, OC43, 299E and NL63 have been identified (28-34), however the impact of 138
this cross-reactivity is unclear. While some reports point to a beneficial role in 139
mitigating disease severity and the induction of neutralising antibodies in both 140
vaccination and natural infection (33, 35, 36), others report no biological function (37, 141
38) or a potential pathological role (39). 142
143
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In this open-label, non-randomised sub-study of male participants with HIV on ART 144
(CD4+ T cell count >350 cells/ul) receiving ChAdOx1 nCoV-19, we investigate the 145
immunological landscape six months after vaccination. We evaluate the durability of 146
the cellular and humoral immune response to SARS-CoV-2 and VOCs and assess the 147
potential role of cross-reactive CCC immune responses in the modulation of post-148
vaccine responses, presenting evidence for an interaction with the beta coronaviruses, 149
HKU1 and OC43. 150
151
Results 152
Participants 153
PWH (N=54; all male) were recruited as part of the ChAdOx1 nCoV-19 COV002 154
clinical trial (NCT04400838) in November 2020. Participants had undetectable VL 155
(<50 HIV RNA copies/ml) and a median CD4 count of 694 cells/µl (IQR 573.5 – 859.5) 156
at the time of recruitment. Most participants were of white ethnicity (81.5%). Other 157
reported ethnicities were Asian (3.7%), mixed (7.4%) and other (7.4%). 158
Demographically-matched HIV seronegative controls were provided from the 159
ChAdOx1 nCoV-19 COV002 clinical trial. All participants received ChAdOx1 nCoV-19 160
4-6 weeks apart and were followed for 6 months (Figure 1a, Table 1). 161
162
Persistent immune activation in PWH before and after vaccination 163
T cell immune activation and exhaustion were assayed at day 0 baseline, day 42 and 164
day 182 after first ChAdOx1 nCoV-19 vaccination (Figure 1b-g). There were 165
significantly higher frequencies of CD38+ HLA-DR+ expressing CD4+ and CD8+ cells 166
in PWH compared to HIV negative controls, consistent across all timepoints (Figure 167
1b and e, gating strategy in supplementary figure 1a). There was a transient 168
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increase in the frequency of CD38+ HLA-DR+ CD4 and CD8+ T cells 14 days after 169
vaccination in PWH which returned to pre-vaccination levels by 6 months 170
(Supplementary figure 1c, f). Expression of the immune checkpoint inhibitor PD-1 171
on CD4+ and CD8+ T cells was not significantly different between PWH and HIV 172
negative controls with no statistically significant changes after vaccination (Figure 1c 173
and f). The frequency of CD4+ and CD8+ PD-1 expressing cells fluctuated early after 174
vaccination in PWH but was restored to pre-vaccination levels at 6 months 175
(supplementary figure d, g, j and m). The frequency of functionally exhausted Tbet 176
(lo) Eomesodermin (Eomes) (hi) CD4+ and CD8+ T cells was higher in PWH 177
compared to HIV-negative individuals both before and after vaccination. (Figure 1d 178
and g, supplementary figure 1e, h, k and n). 179
180
Humoral immunity against ChAdOx1 nCoV-19 in PWH persists for 6 months. 181
We previously reported detectable antibody levels up to 56 days following ChAdOx1 182
nCoV-19 vaccination in PWH (16). To determine the further persistence of antibody 183
responses, total IgG for spike (S), receptor binding domain (RBD) and nucleocapsid 184
(N), as well as neutralising antibody levels were measured at days 0 and day 182. 185
Two independent ELISA technologies were used for binding IgG assays: a 186
standardised in-house total IgG against spike and Meso Scale Discovery (MSD) 187
binding assays measuring S, RBD and N antibody levels. Levels of anti-spike IgG 188
measured using the two assays were positively correlated (Supplementary figure 2a 189
and b, r=0.7, p<0.0001 and r = 0.9, p<0.0001 at Days 0 and 182, respectively; 190
Spearman). At day 182 post-vaccination, antibodies to S and RBD but not N were 191
significantly higher than at baseline (S: day 0 = 3/43 [6.9%], day 182 = 35/42 [83.3%]; 192
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9
RBD: day 0 = 0/43 [0%] and day 182 = 27/42 [64.2%]) (Figure 2a – c), consistent with 193
observed responses being driven by vaccination rather than infection. 194
195
Importantly, there was no difference in anti-spike antibody titres in HIV+ and HIV- 196
matched participants measured at 182 days after first vaccination, although with some 197
waning of responses in both groups after Day 56 (Figure 2d; Supplementary Figure 198
2d,e). Pre-vaccine baseline antibody titres correlated positively with early post-199
vaccination timepoints at day 14, and 28 but not 42, 56 and 182 (Supplementary 200
figure 2f, Supplementary table 1). 201
202 We next assessed the ability of antibodies from plasma collected 6 months after 203
vaccination to compete with SARS-CoV-2 for binding to ACE-2 using an ACE-2 204
inhibition assay and to neutralise SARS-CoV-2 using a live virus focus reduction 205
neutralisation assay (FRNT). FRNT was performed in a randomly selected subset of 206
the cohort for whom we have previously reported neutralisation antibody levels up to 207
day 56 (16). At day 182 post-ChAdOx1 nCoV-19 prime, antibodies capable of blocking 208
the SARS-CoV-2 ACE-2 interaction were present at significantly higher levels than at 209
pre-vaccination baseline (Figure 2e) and correlated strongly with anti-RBD antibodies 210
(Supplementary figure 2c). However, at the same time point antibody neutralisation 211
measured by FRNT live virus assay revealed titres below the assay detection limit in 212
nearly all participants (13/14; 92%) (Figure 2f). 213
214
Durable SARS-CoV-2 specific T cell responses are induced following ChAdOx1 215
nCoV-19 vaccination 216
Durability of vaccine-induced SARS-CoV-2-specific T cell immunity at 6 months was 217
assessed by IFNγ ELISpot and T cell proliferation assays. SARS-CoV-2 spike-specific 218
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ELISpot responses were maintained for 6 months in PWH following vaccination and 219
were equivalent to the HIV negative control group (Figure 3a and b; Supplementary 220
figure 4a). 221
222
For further resolution of the durability of T cell immunity, we used a T cell proliferation 223
assay which also allows distinction of different CD4 and CD8 T cell lineage responses. 224
The spike peptide pool was separated into S1 and S2. Gating strategy is shown in 225
Supplementary Figure 3a. The frequency of SARS-CoV-2 spike-specific proliferative 226
CD4+ and CD8+ T cell responses in PWH following vaccination were maintained at 227
levels significantly higher than at baseline for 6 months (Figure 3c - f). Longitudinal 228
responses to FECT controls remained unchanged while PHA responses were back to 229
baseline by day 182 (Supplementary figure 3b and c). There was no difference in 230
the magnitude of the vaccine-specific T cell proliferative responses between the HIV+ 231
and the HIV- cohorts (Supplementary Figure 4f - l). Although T cell responses in 232
PWH measured by IFNγ ELISpot peaked at day 14 and were then maintained to day 233
182, proliferative responses peaked later at day 42 and then contracted, such that day 234
182 responses were significantly lower than those measured at day 56 (Figure 3c-f). 235
These kinetics are similar to those observed with the anti-S antibody response 236
(Supplementary figure 2d and e). 237
238
Vaccine-reactive T cells are not differentially biased to a specific CD4+ subset. 239
Using CCR6 and CXCR3 expression to quantify Th1, Th2 and Th17 cells, we 240
interrogated the phenotype of circulating T cells following vaccination (gating strategy 241
in Supplementary Figure 1a). At 6 months post ChAdOx1 nCoV-19 vaccination, we 242
found redistributions in the phenotype of the CD4+ T cells in HIV+ volunteers with 243
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increases in Th1 (CXCR3+ CCR6-) and Th2 (CXCR3- CCR6-) (Figure 4a and b) but 244
not Th17 (CXCR3- CCR6+) or Tfh (CXCR5+ CD4+) (Figure 4c and d). None of these 245
populations correlated with anti-spike antibody levels 6 months after infection. 246
Although the hierarchy in cellular composition of the CD4+ T cell subsets was similar 247
in the HIV+ and HIV- cohorts, we found circulating frequencies of Th2 subsets to be 248
reduced while Th1 and Tfh subsets were significantly increased 6 months after 249
vaccination in PWH (Figure 4e, Supplementary Figure 5a – for HIV- control data). 250
251
The activation induced marker (AIM) assay was used to determine the phenotype of 252
vaccine-specific CD4+ T cells 6 months after ChAdOx1 nCoV-19 vaccination (gating 253
strategy in Supplementary figure 5b). Vaccine responses were compared with 254
concurrent HIV Gag and CMV responses (Figure 4f and g). Although AIM+ cells for 255
all antigens tested showed a Th17 bias (Supplementary Figure 5c), similar to HIV-256
Gag or CMVpp65-specific T cells there was no preferential skewing of the SARS-CoV-257
2-specific T responses to any CD4 T helper subset 6 months after vaccination (Figure 258
4h - k). 259
260
Responses to variants of concerns (VOCs) are preserved 6 months after 261
vaccination 262
Humoral and cellular immune responses to the major VOCs were measured 6 months 263
after vaccination. Inhibition of ACE-2 binding for alpha, beta and gamma variants was 264
increased compared to pre-vaccination baseline (Figure 5a), however there was 265
statistically significant reduction in ACE-2 inhibition for all three VOCs compared to 266
the original SARS-CoV-2 strain, which was more apparent in the beta and gamma 267
variants (Figure 5b). T cell proliferative responses to VOCs were comparable to the 268
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SARS-CoV-2 original strain, except for SARS-CoV-2 CD4 responses to S2 which were 269
moderately reduced across all VOCs tested (Figure 5c – f). HIV+ and HIV- 270
participants had similar magnitudes of T cell responses to S1 and S2 spike proteins of 271
all VOCs tested, with the exception of the CD8+ SARS-CoV-2 T cell proliferative 272
response targeting the S2 protein of the delta variant which showed a modest 273
reduction in HIV+ participants compared to HIV- controls (Figure 5g – j). 274
275
Modulation of ChAdOx1 nCoV-19 post vaccination responses by pre-existing 276
cross-reactive immunity 277
SARS-CoV-2 reactive T and B cells exist in unvaccinated COVID-19 naïve individuals 278
(Figure 3a – e, Supplementary figure 2d and e). To determine whether these pre-279
vaccine responses might reflect cross-reactivity to endemic circulating coronaviruses 280
of the alpha (NL63 and 299E) or beta (HKU1, OC43) genera, we also measured 281
responses to these viruses at baseline. 282
283
Based on the T cell proliferation assay, participants were divided according to those 284
with pre-vaccine baseline SARS-CoV-2 immune responses (‘baseline responders’, 285
(BR)) and those without pre-existing immunity (‘baseline non-responders’, (B-NR)). 286
Regardless of any pre-existing immunity, all donors mounted an immune response 287
following vaccination, however BR consistently showed higher magnitude CD4+ 288
(Figure 6a and b) and CD8+ T cell (Supplementary Figure 6a and b) responses to 289
SARS-CoV-2 S1 and S2 at most post-vaccination timepoints. Baseline SARS-CoV-2 290
CD4+ S2 (and to a lesser extent S1) T cell proliferation was positively correlated with 291
subsequent post-vaccine proliferative responses targeting the same regions 292
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(Supplementary Figure 7b and c, supplementary table 2a - d), which is of potential 293
interest as S2 is associated with regions of homology to other coronaviruses. 294
T cell and IgG responses to the endemic CCCs (HKU1 (clade 1 and 2), OC43, 299E 295
and NL63) in HIV-infected participants remained mostly unchanged by vaccination 296
with ChAdOx1 nCov-19 indicating that vaccination did not boost these responses 297
(Figure 7c, Supplementary Figure 8 and 9) however, IgG responses to SARS-CoV-298
1 and MERS-CoV in PWH were higher at 6 months (Figure 7a,b). 299
300
Focusing on baseline pre-existing responses – and dividing the cohort of PWH into 301
the SARS-CoV-2 BR and B-NR groups as before - participants with baseline 302
proliferative T cell responses to SARS-CoV-2 spike, also had T cell responses 303
targeting the S2 spike regions of CCCs, especially for the beta coronaviruses HKU1 304
and OC43 and alpha coronavirus 299E (Figure 6c and d, Supplementary Figure 6c 305
and d, Supplementary table 3). This was supported by humoral responses taken at 306
the same pre-vaccination timepoint, which showed strong correlations between 307
SARS-Cov-2 spike IgG levels and those of SARS-CoV-1, MERS-CoV-1 and HKU1 308
(Figure 7d-f; Supplementary Figure 9, supplementary table 4). Phylogenetic 309
analysis of spike sequences shows OC43 and HKU1 are the mostly closely related 310
CCCs to SARS-CoV-2 (Figure 7g). These data suggest that prior exposure to beta 311
coronaviruses and responses to the S2 homologous region may potentially be 312
associated with larger and more persistent T cell responses following SARS-CoV-2 313
vaccination. 314
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Discussion 315
Long-lasting immune responses against SARS-CoV-2 will be necessary to confer 316
protection from severe COVID-19. Although clinical management and effective 317
antiretroviral therapy (ART) have improved long-term outcomes for PWH – especially 318
in resource-rich countries – HIV-induced immunopathology evidenced by immune 319
activation and exhaustion does not recover to the levels found in HIV uninfected 320
subjects (40), raising concerns whether effective immune responses will persist after 321
vaccination. We show here for the first time in PWH that vaccine-induced immunity to 322
SARS-CoV-2 persists for at least 6 months by most assays, but with evidence that 323
responses are starting to wane. There were no significant differences in responses by 324
PWH and HIV- controls, extending the data from short-term responses reported 325
previously (16, 41). 326
327
We confirm the persistent immune activation - and, to a lesser degree, functional 328
exhaustion - in T cells in PWH on ART, but show that this does not impact the robust 329
humoral and cellular immune responses to ChAdOx nCoV-19 that persist for 6 months. 330
Reports on reactivation of the HIV reservoir and increased immune activation after 331
vaccination in PWH are conflicting (13, 14, 42), and although we found a transient 332
increase in the frequencies of T cells co-expressing CD38 and HLA-DR, this was 333
restored to baseline by six months. Further studies will be needed to determine any 334
impact on the HIV reservoir. 335
336
Vaccine design and regimen can skew the quality of the T cell response by the 337
preferential induction of one CD4+ T helper subset over another (43-48). ChAdOx-1 338
nCoV-19 responses show a qualitative skew towards the Th1 phenotype, with 339
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increased IFNγ, IL-2 and TNF-producing T cells shortly after vaccination (18). Other 340
studies in convalescent cohorts have linked a CCR6+ Th-17 cTfh phenotype with 341
reduced disease severity (49). Similar to others (44, 50), we found antigen-specific 342
CD4+T cells following vaccination were mostly a CCR6+ CXCR3- Th-17 phenotype. 343
We did not find SARS-CoV-2 spike-specific CD4+ T cells biased towards any 344
chemokine-expressing sub-population 6 months after vaccination, possibly reflecting 345
the longer duration between vaccination and analysis than in other studies. 346
347
Understanding durability of both humoral and cellular immunity to SARS-CoV-2 – both 348
likely key components of an effective response (49, 51, 52) - is key to understanding 349
long-term protection. When we assessed the longevity of the humoral and cellular 350
immunity in PWH 6 months after ChAdOx1 nCoV-19 vaccination, we found that 351
vaccine-mediated antibodies to spike or RBD remained elevated above baseline and 352
no different to HIV- controls. Similarly, T cell responses to spike were maintained at 353
magnitudes above baseline and demonstrated similar kinetics to HIV- participants. 354
Antibody function measured by ACE-2 binding inhibition was sustained at levels above 355
pre-vaccination, however live neutralisation assays did not detect antibodies in the 356
majority of the participants assayed at 6 months. Both assays identified the same 357
participants as low (n = 13) and high (n = 1) responders, and the ACE-2 binding 358
inhibition and SARS-CoV-2 RBD titres showed a strong positive correlation. We 359
speculate that although differences in positive responses between the two functional 360
assays could be as a result of function (neutralisation) versus antigenicity (ACE-2 361
binding inhibition), it could also in part, be due to assay sensitivity and differing 362
dynamic ranges between assays. 363
364
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16
SARS-CoV-2 convalescent plasma has been shown to have effective FC-mediated 365
antibody functions such as antibody dependent cellular phagocytosis (ADCP), 366
antibody dependent cellular cytotoxicity (ADCC) and complement dependent 367
cytotoxicity (CDC) (53-55), which are more durable than neutralisation (54). Non-368
neutralising functions were not evaluated in this study, and therefore we cannot 369
exclude that these are preserved in this cohort of PWH. Total spike IgG antibody and 370
T cell proliferative responses in PWH were significantly lower at 6 months after 371
vaccination compared to day 56. These results suggest detectable but waning T and 372
B cell responses at 6 months. Similar findings were reported for the mRNA-1273 373
COVID-19 vaccine, and found to be age-dependent, pointing to immune aging as a 374
contributing factor (20, 21). This comprehensive analysis of humoral and cellular 375
immunity is consistent with studies of COVID-19 in healthy adults and PWH showing 376
durable immune responses up to 7 months post infection (19, 52, 56, 57). Further 377
follow up at 12 months and beyond will be important to determine the longer-term 378
persistence of responses, especially when considering the value of booster doses. 379
380
The emergence of VOCs poses a potential roadblock to ending the pandemic. We 381
found humoral immunity to VOCs at 6 months to be at titres lower than those targeting 382
the original wild type (WT) SARS-CoV-2 strain, albeit still significantly higher than pre-383
vaccination levels. The magnitude of the T cell responses to VOCs were similar to 384
those targeting the WT SARS-CoV-2 strain for most VOCs tested apart from the CD4+ 385
S2 responses. For most of the VOCs, T cell responses in PWH did not differ from HIV- 386
controls. Similar observations regarding humoral immunity have been made with the 387
mRNA vaccine BNT162b2 although as most of these studies were done within 2 388
months of vaccination, information on durability of the response is lacking (58-60). 389
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17
One study assessing T cell responses between 21 – 28 days after full BNT162b2 390
vaccination found no differences between WT and VOC CD4 responses (58). This 391
study utilised a pool of spike peptide pools not parsed into its S1 and S2 regions, and 392
only a limited panel of VOCs were analysed. Importantly, emerging data from real 393
world effectiveness studies suggest that vaccination protects against death and 394
severe disease, even following infection with VOCs (24, 26) 395
396
Cross-reactivity from previous CCC infection may impact the measured SARS-CoV-2 397
immune response after vaccination and natural infection (32, 33, 61). We identified 398
measurable pre-vaccine antibody titres for SARS-CoV-2 S, RBD and N proteins in 399
PWH. Pre-vaccination SARS-CoV-2 S antibody levels strongly correlated with those 400
of contemporaneous beta coronaviruses SARS-CoV-1, MERS-CoV and HKU1 (of 401
which only the latter is likely to have been experienced by these UK study participants), 402
supporting the hypothesis that these titres result from previous infection with a similar 403
coronavirus and some cross-reactivity across coronaviruses. Supporting the antibody 404
data, the presence of cross-reactive T cells pre-vaccination (based on proliferative 405
potential following antigen challenge) was associated with higher magnitude post-406
vaccination T cell responses. 407
408
There is much debate over the significance of cross-reactive responses. Studies have 409
reported reduced disease severity in patients with CCC humoral responses and 410
regions of high homology to CCC capable of trans-priming SARS-CoV-2 T and B cell 411
responses (32, 33, 36). Pre-existing immunity was also shown to boost post vaccine 412
responses in low dose mRNA-1273 vaccine (21) although an explanatory mechanism 413
was not reported. Further investigations in large studies would be needed to fully 414
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18
elucidate the impact of baseline pre-existing immunity in post-vaccination response, 415
but we find clear evidence of higher magnitude immune responses in those with cross-416
reactivity. Although our data suggest that responses to CCC may help augment 417
subsequent vaccine responses against SARS-CoV-2, we have no evidence that on 418
their own they are potent enough to impact susceptibility to COVID-19. 419
420
In summary, we present a comprehensive immunological assessment of ChAdOx1 421
nCoV-19 in PWH 6 months after vaccination. We show that despite persistent immune 422
activation in PWH, PWH on ART and HIV-uninfected participants make equivalent T 423
and B cell responses following vaccination. However, both responses showed signs 424
of decline after 6 months. It is unknown what level of immunity is required to prevent 425
hospitalisation and mortality, but real-world data suggest vaccination is successful in 426
preventing severe disease and death even in the presence of transmissible and 427
virulent VOC (24, 26). A booster dose may become necessary in the future to maintain 428
long-term immunological memory to SARS-CoV-2 and the VOCs, especially for 429
susceptible cohorts and we must continue to carefully monitor this going forward. 430
Finally, we demonstrate that pre-existing SARS-CoV-2 cross-reactive immune 431
responses to the beta coronaviruses HKU1 and to a lesser extent OC43 are 432
associated with higher magnitude T cell responses following vaccination in PWH. 433
Together these data continue to reinforce the policy of ensuring all PWH are offered 434
vaccination against SARS-CoV-2. 435
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19
Methods 436
Study design and cohort 437
The cohort studied in this analysis has been described previously (16). Briefly, the 438
study comprised people living with HIV in an open-label non-randomised group within 439
the larger multicentre phase 2/3 COV002 trial. The participants in this single-arm group 440
comprised individuals with HIV who were stable on ART under routine follow-up at two 441
London UK National Health Service (NHS) clinics and received ChAdOx1 nCoV-19 442
vaccination according to the schedule of attendance. Recruitment was done in HIV 443
clinics at two centres in the UK (Imperial College NHS Trust and Guy’s and St Thomas’ 444
NHS Foundation Trust). Inclusion criteria were age 18–55 years, a diagnosis of HIV 445
infection, virological suppression on ART at enrolment (plasma HIV viral load <50 446
copies per mL), and a CD4 count of more than 350 cells/μL. The inclusion criteria for 447
the COV002 trial have been published in full elsewhere (15). Written informed consent 448
was obtained from all participants, and the trial was done in accordance with the 449
principles of the Declaration of Helsinki and Good Clinical Practice. Study approval in 450
the UK was done by the Medicines and Healthcare products Regulatory Agency 451
(reference 21584/0424/001-0001) and the South Central Berkshire Research Ethics 452
Committee (reference 20/SC/0145). Vaccine use was authorised by Genetically 453
Modified Organisms Safety Committees at each participating site. 454
The ChAdOx1 nCoV-19 vaccine was produced as previously described (17). 455
Participants received two standard intramuscular doses 4–6 weeks apart. For some 456
assays and where sample availability allowed, comparison was made with age- and 457
sex-matched participants who were HIV negative, aged 18–55 years, enrolled into the 458
main COV002 phase 2/3 randomised clinical trial, and randomly assigned (5:1) to 459
receive either ChAdOx1 nCoV-19 or MenACWY by intramuscular vaccination. The 460
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20
dose of vaccine administered was the same across both groups. Only participants 461
receiving the ChAdOx1 nCoV-19 vaccine were used for comparison. Full details of the 462
COV002 HIV-negative cohort have been published previously (15). 463
A screening visit where a full medical history, examination of all participants and blood 464
tests to exclude biochemical or haematological abnormalities (full blood count; kidney 465
and liver function tests) was done prior to enrolment. Participants with a history of 466
laboratory-confirmed SARS-CoV-2 infection by anti-N protein IgG immunoassay 467
(Abbott Architect, Abbott Park, IL, USA) at screening were excluded. For this study, 468
visits on day 0 (vaccine prime) and 182 were the main study timepoints used for 469
immunological analysis however for some assays other study visits - 14, 28 (vaccine 470
boost), 42, and 56 - are presented where available. As some participants did not attend 471
for their day 182 visit (n=6), there is a maximum of n=48 at this timepoint. 472
473
Mesoscale Discovery (MSD) binding assays 474
IgG responses to SARS-CoV-2, SARS-CoV-1, MERS-CoV and 475
seasonal coronaviruses were measured using a multiplexed MSD immunoassay. The 476
V-PLEX COVID-19 Coronavirus Panel 3 (IgG) Kit (cat. no. K15399U) from Meso Scale 477
Diagnostics, Rockville, MD USA. A MULTI-SPOT® 96-well, 10 spot plate was coated 478
with three SARS CoV-2 antigens (S, RBD, N), SARS-CoV-1 and MERS-CoV spike 479
trimers, as well as spike proteins from seasonal human coronaviruses, HCoV-OC43, 480
HCoV-HKU1, HCoV-229E and HCoV-NL63, and bovine serum albumin. Antigens 481
were spotted at 200−400 μg/mL (MSD® Coronavirus Plate 3). Multiplex MSD assays 482
were performed as per the instructions of the manufacturer. To measure IgG 483
antibodies, 96-well plates were blocked with MSD Blocker A for 30 minutes. Following 484
washing with washing buffer, samples diluted 1:1,000-10,000 in diluent buffer, or MSD 485
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standard or undiluted internal MSD controls, were added to the wells. After 2-hour 486
incubation and a washing step, detection antibody (MSD SULFO-TAG™ Anti-Human 487
IgG Antibody, 1/200) was added. Following washing, MSD GOLD™ Read Buffer B 488
was added and plates were read using a MESO® SECTOR S 600 Reader. The 489
standard curve was established by fitting the signals from the standard using a 4-490
parameter logistic model. Concentrations of samples were determined from the 491
electrochemiluminescence signals by back-fitting to the standard curve and multiplied 492
by the dilution factor. Concentrations are expressed in Arbitrary Units/ml (AU/ml). Cut-493
offs were determined for each SARS-CoV-2 antigen (S, RBD and N) based on the 494
concentrations measured in 103 pre-pandemic sera + 3 Standard Deviations. Cut-off 495
for S: 1160 AU/ml; cut-off for RBD: 1169 AU/ml; cut-off for N: 3874 AU/ml. 496
497
SARS CoV-2 spike IgG ELISA 498
Humoral responses at baseline and following vaccination were assessed using a 499
standardised total IgG ELISA against trimeric SARS CoV-2 spike protein as described 500
previously21. In brief, ELISA plates were coated with 2 μg/mL of full-length trimerised 501
SARS-CoV-2 spike glycoprotein and stored at 4°C overnight for at least 16 hours. After 502
coating, plates were washed 6 times with PBS/0.05%Tween and blocked with casein 503
for 1h at room temperature (RT). Thawed samples were treated with 10% Triton X-504
100 for 1h at RT and subsequently diluted in casein and plated in triplicate for 505
incubation for 2h at RT alongside two internal positive controls (controls 1 and 2) to 506
measure plate to plate variation. Control 1 was a dilution of convalescent plasma 507
sample and control 2 was a research reagent for anti-SARS-CoV-2 Ab (code 20/130 508
supplied by National Institute for Biological Standards and Control (NIBSC)). The 509
standard pool was used in a two-fold serial dilution to produce ten standard points that 510
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were assigned arbitrary ELISA units (EUs). Goat anti-human IgG (γ-chain specific) 511
conjugated to alkaline phosphatase was used as secondary antibody and plates were 512
developed by adding 4-nitrophenyl phosphate in diethanolamine substrate buffer. An 513
ELx808 microplate reader (BioTek Instruments) was used to provide optical density 514
measurement of the plates at 405mm. Standardised EUs were determined from a 515
single dilution of each sample against the standard curve which was plotted using the 516
4-Parameter logistic model (Gen5 v3.09, BioTek). Each assay plate consisted of 517
samples and controls plated in triplicate, with ten standard points in duplicate and four 518
blank wells. The assay LLOQ (representing the lowest IgG titres that can be reliably 519
and precisely quantified within a coefficient of variation of 25%) was determined 520
mathematically. This was based on the 4-PL function of the standard curve data from 521
250 independent experiments and represents the EU value corresponding to the 522
upper 95% confidence interval of the minimum asymptote of the 4-PL curve fit used 523
for modelling the assay standard curves. The value of 13 EU was calculated as the 524
assay LLOQ and this corresponds to an OD value of 0.2 for which the assay was 525
demonstrated to show linearity. 526
527
Focus reduction neutralization assay (FRNT) 528
Antibody neutralization was measured in a randomly selected subset of participants 529
using a Focus Reduction Neutralization Test (FRNT), as described previously (62) 530
where the reduction in the number of the infected foci is compared to a ‘no antibody’ 531
negative control well. Briefly, serially diluted Ab or plasma was mixed with SARS-CoV-532
2 strain Victoria and incubated for 1 hour at 37°C. The mixtures were then transferred 533
to 96-well, cell culture-treated, flat-bottom microplate containing confluent Vero cell 534
monolayers in duplicate and incubated for further 2 hours, followed by the addition of 535
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1.5% semi-solid carboxymethyl cellulose (CMC) overlay medium to each well to limit 536
virus diffusion. A focus forming assay was then performed by staining Vero cells with 537
human anti-NP mAb (mAb206) followed by peroxidase-conjugated goat anti-human 538
IgG (A0170; Sigma). Finally, the foci (infected cells) approximately 100 per well in the 539
absence of antibodies, were visualized by adding TrueBlue Peroxidase Substrate. 540
Virus-infected cell foci were counted on the classic AID ELISpot reader using AID 541
ELISpot software. The percentage of focus reduction was calculated and IC50 542
(reported as FRNT50) was determined using the probit program from the SPSS 543
package. 544
545
MSD ACE-2 inhibition assay 546
A multiplexed MSD immunoassay (MSD, Rockville, MD) was used to measure the 547
ability of human sera to inhibit ACE2 binding to SRAS-CoV-2 spike (B, B.1, 548
B.1.1.7, B.1.351 or P.1). A MULTI-SPOT® 96-well, 10 Spot Plate (Plate 7) was 549
coated with eight SARS-CoV-2 spike and RBD antigens (B, B.1, B.1.1.7, B.1.351 550
or P.1). Multiplex MSD Assays were performed as per manufacturer’s instructions. 551
To measure ACE2 inhibition, 96-well plates were blocked with MSD blocker for 30 552
minutes. Plates were then washed in MSD washing buffer, and samples were 553
diluted 1:10 and 1:100 in diluent buffer. Importantly, an ACE2 calibration curve 554
which consists of a monoclonal antibody with equivalent activity against spike 555
variants was used to interpolate results as arbitrary units. Furthermore, internal 556
controls and the WHO international standard were added to each plate. After 1-557
hour incubation recombinant human ACE2-SULFO-TAG™ was added to all wells. 558
After a further 1-hour plates were washed and MSD GOLD™ Read Buffer B was 559
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added, plates were then immediately read using a MESO® SECTOR S 600 560
Reader. 561
562
Isolation of peripheral blood mononuclear cells (PBMC) from whole blood 563
PBMCs were isolated by density gradient centrifugation using Lymphoprep (Stem Cell 564
Technologies, Cambridge, UK). Buffy coats containing PBMCs were collected and 565
washed twice with pre-warmed R10 medium: Roswell Park Memorial Institute (also 566
known as RPMI) 1640 medium (Sigma Aldrich, St Louis, MO, USA) supplemented 567
with 10% heat-inactivated fetal calf serum (FCS; Sigma), 1 mM penicillin-streptomycin 568
solution (Sigma), and 2 mM L-glutamine solution (Sigma). After the second 569
centrifugation, cells were resuspended in R10 and counted using the Guava ViaCount 570
assay (Guava Technologies Hayward, CA, USA) on the Muse Cell Analyzer (Luminex 571
Cooperation). T-cell enzyme-linked immunospot assay (ELISpot) assays were done 572
on freshly isolated PBMCs, and CellTrace Violet (CTV; ThermoFisher Scientific, CA, 573
USA) T cell proliferation assay was done on cryopreserved samples. 574
575
Ex vivo IFNg ELISpot to enumerate antigen-specific T cells. 576
ELISpot assays were performed as described previously (17) using a validated 577
protocol with freshly isolated peripheral blood mononuclear cells (PBMCs) to 578
determine responses to the SARS-CoV-2 spike vaccine antigen at days 0 (before 579
vaccination), 14, 28 (boost), 42 and 56. Assays were performed using Multiscreen IP 580
ELISpot plates (Merck Millipore, Watford, UK) coated with 10 μg/mL human anti-IFNγ 581
antibody and developed using SA-ALP antibody conjugate kits (Mabtech, Stockholm, 582
Sweden) and BCIP NBT-plus chromogenic substrate (Moss Inc., Pasadena, MA, 583
USA). PBMC were separated from whole blood with lithium heparin by density 584
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25
centrifugation within four hours of venepuncture. Cells were incubated for 18–20 hours 585
in RPMI (Sigma) containing 1000 units/mL penicillin, 1 mg/mL streptomycin and 10% 586
heat-inactivated, sterile-filtered foetal calf serum, previously screened for low reactivity 587
(Labtech International, East Sussex, UK) with a final concentration of 10µg/ml of each 588
peptide. A total of 253 synthetic peptides (15mers overlapping by 10 amino acids) 589
spanning the entire vaccine insert, including the tPA leader sequence were used to 590
stimulate PBMC (ProImmune, Oxford UK). Peptides were pooled into 12 pools for the 591
SARS-CoV-2 spike protein containing 18 to 24 peptides, plus a single pool of 5 592
peptides for the tPA leader. Peptides were tested in triplicate, with 2.5 × 105 PBMC 593
added to each well of the ELISpot plate in a final volume of 100μL. Results are 594
expressed as spot forming cells (SFC) per million PBMCs, calculated by subtracting 595
the mean negative control response from the mean of each peptide pool response and 596
then summing the response for the 12 peptide pools spanning S1 and S2. 597
Staphylococcal enterotoxin B (0.02 μg/mL) and phytohaemagglutinin-L (10 μg/ mL) 598
were pooled and used as a positive control. Plates were counted using an AID 599
automated ELISpot counter (AID Diagnostika GmbH, algorithm C, Strassberg, 600
Germany) using identical settings for all plates, and counts were adjusted only to 601
remove artefacts. A lower limit of detection of 48 SFC/million PBMCs was determined 602
based on the minimum number of spots that could be detected. 603
604
T cell proliferation assay 605
T cell proliferation assay was done using cryopreserved PBMCs. Briefly, PBMCs were 606
thawed and washed twice with 1mL of PBS followed by labelling with CTV at a final 607
concentration of 2·5 μM for 10 min at room temperature. CTV, a DNA intercalating 608
dye, enables the measurement of the decrease in dye concentration following each 609
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26
cell division in proliferating cells in response to antigenic stimulation as described 610
previously (34). The labelling reaction was quenched with 4mL of fetal bovine serum 611
(FBS) at 4°C and cells were resuspended in RPMI medium supplemented with 10% 612
human blood group type AB serum (Sigma), 1 mM penicillin-streptomycin solution, 613
and 2 mM L-glutamine solution, and subsequently plated in a 96-well round bottom 614
plate at a plating density of 0·25 × 106 cells per well in duplicate wells (total of 615
0·5 × 106 cells per condition). Cells were stimulated with peptide pools (15nmers 616
overlapping by 11) spanning SARS-CoV-2 spike (S1 and S2), SARS-CoV-2 variants 617
of concern (beta, gamma and delta) and HCoVs (HKU-1 – 2 consensus clades, OC43, 618
NL63 and 299E) at a final concentration of 1 μg/mL per peptide. For antigenic control, 619
class 1 and 2 optimal peptides for FEC-T (flu, EBV, CMV, and tetanus) were pooled 620
at a final concentration of 1 μg/mL per peptide. Media, containing 0·1% dimethyl 621
sulfoxide (DMSO; Sigma) representing DMSO content in peptide pools, was used as 622
a negative control and 2 μg/mL phytohaemagglutinin L (Sigma) was used as positive 623
control. Cells were then incubated at 37°C, with 5% carbon dioxide and 95% humidity 624
for 7 days, with a change of media on day 4. At the end of the incubation period, cells 625
were stained using anti-human CD3, CD4, CD8, and a live cell discriminator 626
(Live/Dead near Infra-red, Life Technologies; ThermoFisher Scientific, CA, USA) as in 627
supplementary table 5. All samples were acquired using a BD Fortessa X20 (BD 628
Bioscience, San Jose, CA, USA) or MACSQuant x10 (Miltenyi Biotec, Bergisch 629
Gladbach, Germany). Responses above 1% were considered true positive based 630
mean of DMSO controls + 3x SD. Specificity of the assay has been previous reported 631
in (34). All datapoints presented represent a single participant and are presented as 632
background subtracted data. 633
634
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AIM Assay 635
Cryopreserved PBMCs from 25 HIV infected subjects were used for activation induced 636
marker (AIM) assay. Briefly, PBMCs were thawed in R10 (RPMI + 10% FCS, 1% 637
Pen/strep and 1% L-glutamine. Cells were washed, counted, and rested for 6 hours in 638
IMDM-10 (Iscove’s Modified Dulbecco’s Medium - Sigma, I3390 + 10% Human AB 639
serum, 1% Pen/strep and 1% L-glutamine) and 1ul/ml of benzonase nucleases 640
(70746-3, Merck). Following rest, cells were plated at 1-2 x106 cells/well in a 96 well 641
round bottom plate. Cells were then incubated for 24 hours at 37ºC and 5% CO2. After 642
stimulation cells were stained with the anti-human antibodies contained in 643
supplementary table 6. Stained cells were fixed in 4% PFA and acquired on a BD 644
LSR II flow cytometer. The data was analysed using FlowJo version 10 and Prism 645
version 9. Antigen-specific CD4+ and CD8+ T cells were gated using the gating 646
strategy described by Nielsen et al (44) and shown in supplementary figure 5 (for 647
CD4 T cells: CD25+ CD134(OX40)+ and CD25+ CD137+ and CD25+ CD69+; for 648
CD8+ T cells: CD25+ CD137+ and CD25+ CD69+). Chemokine receptors CCR6 and 649
CXCR3 were used as an unbiased way of analysing T cell skewness independent of 650
cytokine kinetics. 651
652
Ex vivo activation and exhaustion assays 653
Cryopreserved PBMCS were thawed in 30mls of RPMI media supplemented with 10% 654
FBS, 1% Pen-strep and 1% L-glutamine (R10). Cells were counted and rested for an 655
hour at a cell density of 2 x 106 per ml of R10 in the presence of benzonase 656
endonucleases (70746-3, Merck). Following rest, 2-3 million cells were used for each 657
of the panels. Cells were washed in staining buffer (420201, Biolegend). This was 658
followed by blocking FC receptors (422302, Biolegend) for 10 minutes at room 659
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28
temperature (RT) and live cell staining using L/D aqua (L34966, Life Technologies). All 660
cells were then washed in preparation for antibody staining. For ex vivo 661
immune activation panel, antibodies for assessing immune activation (as listed 662
in supplementary table 7) was used as a cocktail and added to the cell pellet. Cells 663
were subsequently incubated at 37°C for 15 minutes which was followed by 664
a wash and fixation in 4% paraformaldehyde (PFA) for 10 minutes at RT. PFA was 665
washed off and cells resuspended in PBS for acquisition on flow cytometer. For 666
immune exhaustion panel, antibodies for surface markers were prepared in a cocktail 667
which was added to cells and incubated for 15 minutes at 37°C (supplementary table 668
8). Cells were then prepared for intranuclear stain using FOXP3 669
fixation/permeabilisation kit (Life technologies). Briefly, 100ul of fixation buffer was 670
added to cells and incubated at RT for 30 minutes. This was followed by cellular 671
permeabilisation using the permeabilisation buffer contained in the aforementioned kit. 672
Antibody cocktails were prepared in permeabilisation buffer, added to cells and 673
allowed to incubate for 30 minutes at RT. Following staining, cells were washed and 674
resuspended in PBS for acquisition. All data was acquired on a BD LSR II flow 675
cytometer and fluorescent minus one (FMO) gates were used to set gates for markers 676
of interest. Gating strategies are as shown in supplementary figure 1a and b 677
678
Phylogenetic analysis 679
We used protein BLAST to download all human coronavirus S protein sequences from 680
NCBI database. We then randomly chose 3 sequences for each of the human 681
coronavirus species. HKU1 was consisted of two clades and we chose three isolates 682
for each clade (c1 and c2). We used MAFFT to align all chosen human corona viruses, 683
SARS-CoV, MERS-CoV and SARS-CoV-2 S protein sequences. We then calculated 684
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29
the pairwise distances between the sequences and built a neighbour joining tree using 685
MATLAB. 686
687
Statistical analysis 688
This study was not powered to a specific endpoint and the sample size was based on 689
practical recruitment considerations in line with other subgroups of the COV002 study. 690
We analysed all outcomes in all participants who received both doses of the 691
vaccination schedule and with available samples, unless otherwise specified. We log-692
transformed serological, FRNT50, and ELISpot data for analysis. FRNT50 titres less 693
than 20 were given the value 10 for statistical analysis. We present medians and IQRs 694
for immunological endpoints. We used non-parametric analysis (Spearman’s rho) for 695
correlations between two immunological endpoints. For comparison of two non-696
parametrically distributed unpaired variables, we used the Wilcoxon rank sum (Mann 697
Whitney U) test. For comparison of two non-parametrically distributed paired datasets, 698
we used the Wilcoxon matched pairs signed rank test. We used the χ2 test for 699
comparison of ELISpot responses. Missing data were not imputed. We did all analyses 700
using R (version 3.6.1 or later), and Prism 9 (GraphPad Software). The COV002 study 701
is registered with ClinicalTrials.gov, NCT04400838, and is ongoing. 702
703
Role of the funding source 704
The funders of the study had no role in the study design, data collection, data analysis, 705
data interpretation, or writing of the report. All authors had full access to all the data in 706
the study and had final responsibility for the decision to submit for publication. 707
708
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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 29, 2021. ; https://doi.org/10.1101/2021.09.28.21264207doi: medRxiv preprint
30
Acknowledgements 709
This Article reports independent research funded by UK Research and Innovation 710
(MC_PC_19055), Engineering and Physical Sciences Research Council 711
(EP/R013756/1), Coalition for Epidemic Preparedness Innovations, and NIHR. We 712
acknowledge support from Thames Valley and South Midland's NIHR Clinical 713
Research Network and the staff and resources of NIHR Southampton Clinical 714
Research Facility and the NIHR Oxford Health Biomedical Research Centre. PMF 715
received funding from the Coordenacao de Aperfeicoamento de Pessoal de Nivel 716
Superior, Brazil (finance code 001). ALF was supported by the Chinese Academy of 717
Medical Sciences Innovation Fund for Medical Science, China (grant number 2018-718
I2M-2–002). MAA is supported by the Wellcome Trust and Royal Society 719
(220171/Z/20/Z). KJE is an NIHR Biomedical Research Centre senior research fellow. 720
AJP and EB are NIHR senior investigators. M.C., S.L. and ToT. are funded by a U.S. 721
Food and Drug Administration Medical Countermeasures Initiative grant 722
75F40120C00085. The views expressed in this publication are those of the authors 723
and not necessarily those of the NIHR, FDA or the UK Department of Health and 724
Social Care. We thank the volunteers who participated in this study. 725
726
Declaration of interests 727
Oxford University has entered into a partnership with AstraZeneca for further 728
development of ChAdOx1 nCoV-19 (AZD1222). AstraZeneca reviewed the data from 729
the study and the final manuscript before submission, but the authors retained editorial 730
control. SCG is cofounder of Vaccitech (a collaborator in the early development of this 731
vaccine candidate) and named as an inventor on a patent covering use of ChAdOx1-732
vectored vaccines (PCT/GB2012/000467) and a patent application covering this 733
. CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 29, 2021. ; https://doi.org/10.1101/2021.09.28.21264207doi: medRxiv preprint
31
SARS-CoV-2 vaccine. TL is named as an inventor on a patent application covering 734
this SARS-CoV-2 vaccine and was consultant to Vaccitech. PMF is a consultant to 735
Vaccitech. AJP is Chair of the UK Department of Health and Social Care’s JCVI, but 736
does not participate in policy advice on coronavirus vaccines, and is a member of the 737
WHO Strategic Advisory Group of Experts (SAGE). AVSH is a cofounder of and 738
consultant to Vaccitech and is named as an inventor on a patent covering design and 739
use of ChAdOx1-vectored vaccines (PCT/GB2012/000467). 740
. CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 29, 2021. ; https://doi.org/10.1101/2021.09.28.21264207doi: medRxiv preprint
32
Tables and table legend 741 742 Table 1: Demographic information for HIV infected and HIV uninfected receiving 743 ChAdOx1 nCoV-19 744 745 746
HIV infected HIV uninfected
Assay All reported assays
ELISpot and ELISA
Activation/Exhaustion Panel, proliferation assay, and AIM assay
n 54 50 10 Sex Male 54 (100%) 26 (52%) 10 (100%) Female 0 (0%) 24 (48%) 0 (0%)
Age in years 42.5 (37.2 - 49.8)
38.5 (29.2 - 45.0)
Ethnicity White 44 (81%) 40 (80%) 6 (60%) Black 0 (0%) 1 (2%) 1 (10%) Asian 2 (4%) 8 (16%) 2 (20%) Mixed 4 (7%) 0 (0%) 0 (0%) Other 4 (7%) 1 (2%) 1 (10%) Antiretroviral therapy Y 54 (100%) NA
NA
N NA NA Plasma HIV VL <50 NA NA
CD4 count > 350 cells/ul 694.0 (573.5 - 859.5) NA NA
747
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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 29, 2021. ; https://doi.org/10.1101/2021.09.28.21264207doi: medRxiv preprint
33
Figures and Figure Legends 748 749
750 751 Figure 1: PWH show higher baseline immune activation and exhaustion. 752 A) Schematic showing vaccination schedule for ChAdOx1 nCoV-19 in PWH. Frequency of (B) CD38+ HLA-DR+, (C) PD1+ (D) 753 Tbet(lo) Eomes(hi) cells within CD4+ and (E) CD38+ HLA-DR+, (F) PD1+ (G) Tbet(lo) Eomes(hi) cells within CD8+ T cells. 754 Comparison of two groups by two-tailed Mann-Whitney U test. Where indicated * = <0.05, ** = <0.01, *** = < 0.001 and **** = <0.0001. 755
HIV+ HIV- HIV+ HIV- HIV+ HIV-0.1
1
10
100
% w
ithin
CD
4+ T
cel
ls
Day 0 Day 42 Day 182
✱✱✱✱ ✱✱✱✱✱✱✱
HIV+ HIV- HIV+ HIV- HIV+ HIV-0.1
1
10
100
% w
ithin
CD
8+ T
cel
ls
Day 0 Day 42 Day 182
✱✱✱✱ ✱✱✱✱✱✱✱✱
HIV+ HIV- HIV+ HIV- HIV+ HIV-1
10
100
% w
ithin
CD
4+ T
cel
ls
Day 0 Day 42 Day 182
HIV+ HIV- HIV+ HIV- HIV+ HIV-1
10
100
% w
ithin
CD
8+ T
cel
ls
Day 0 Day 42 Day 182
HIV+ HIV- HIV+ HIV- HIV+ HIV-0.1
1
10
100
% w
ithin
CD
4+ T
cel
ls
Day 0 Day 42 Day 182
✱✱✱ ✱✱✱ ✱✱✱
HIV+ HIV- HIV+ HIV- HIV+ HIV-1
10
100
% w
ithin
CD
8+ T
cel
lsDay 0 Day 42 Day 182
✱ ✱
Figure 1: PLWH show higher baseline immune activation and exhaustion
A
B C D
E F G
HIV+
HIV-
CD38+ HLA-DR+ within CD4+
CD38+ HLA-DR+ within CD8+
PD1+ within CD4+
PD1+ within CD8+
Tbet(lo) Eomes(hi) within CD4+
Tbet(lo) Eomes(hi) within CD8+
. CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 29, 2021. ; https://doi.org/10.1101/2021.09.28.21264207doi: medRxiv preprint
34
756 757 Figure 2: Antibody levels against SARS-CoV-2 6 months after ChAdOx1 nCoV-19 vaccination. 758 IgG levels for SARS-CoV-2 (A) Spike (B) RBD and (C) N protein measured at day 0 (baseline) and day 182 (6 months post-759 vaccination) using the MSD ELISA assays. (D) Comparison between antibody kinetics in HIV+ and HIV- across all available 760 timepoints. (E) ACE-2 inhibition assay at baseline and 6 months post-vaccination and (F) Live-virus focus reduction neutralisation 761 assay (FRNT) on n =15 HIV+ donors on day 0, 28, 56 and 182. Comparison of two timepoints within the same group was done by 762 Wilcoxon matched pair sign ranked test. Comparison of two groups was done by two-tailed Mann-Whitney U test. Where indicated * 763 = <0.05, ** = <0.01, *** = < 0.001 and **** = <0.000. Dotted lines in A – C indicate cut off points determined for each SARS-CoV-2 764 antigen (S, RBD and N) based on pre-pandemic sera + 3X SD. 765
0 182100
101
102
103
104
105
106
AU
/ml (
log 10
)
SARS-CoV-2 S
✱✱✱✱
Time since vaccination (days)
Figure 2: Antibody levels against SARS-CoV-2 6 months after ChAdOx1 nCoV-19 vaccination
A
0 182100
101
102
103
104
105
106
SARS-CoV-2 RBD
AU
/ml (
log 10
)
Time since vaccination (days)
✱✱✱✱
0 18210-1
100
101
102
103
104
105
106
SARS-CoV-2 N
AU
/ml (
log 10
)
Time since vaccination (days)
B C
ED F
SARS-CoV-2 ACE-2 inhibition assay
0 1820.01
0.1
1
10
100
unit/
ml
SARS-CoV-2 ACE-2 Inhibition Assay
****
Time since vaccination (days)
0 28 56 1821
10
100
1000
6 month neuts
Time since vaccination (days)
FRN
T50
(rec
ipro
cal)
plas
ma
dilu
tion
0 14 28 42 56 182
1
10
100
1000
10000
G12 vs G5d
HIV+ve
HIV-ve
Time since vaccination (days)
Anti-
spik
e Ig
G
ELIS
A U
nits
(EU
)
****
**** ***
. CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 29, 2021. ; https://doi.org/10.1101/2021.09.28.21264207doi: medRxiv preprint
35
766
767 768 Figure 3: T cell responses following ChAdOx1 nCoV-19 vaccination are durable in PWH. 769 (A) T cell response measured using peptides pools against SARS-CoV-2 S1 and S2 antigens by IFNγ ELISpot across all timepoints. 770 (B) comparative analysis of IFNγ T cell responses in HIV+ and HIV- volunteers. Proliferative T cell responses to (C) SARS-CoV-2 S1 771 and (D) SARS-CoV-2 S2 in CD4+ T cells across all available timepoints. Proliferative T cell responses to (E) SARS-CoV-2 S1 and 772 (F) SARS-CoV-2 S2 in CD8+ T cells across all available timepoints. Comparison of two timepoints within the same group was done 773 by Wilcoxon matched pair sign ranked test. Comparison of two groups was done by two-tailed Mann-Whitney U test. Where indicated 774 * = <0.05, ** = <0.01, *** = < 0.001 and **** = <0.000. Dotted lines in C - F indicate threshold for true positive based mean of DMSO 775 controls + 3x SD. 776
Figure 3: T cell responses following ChAdOx1 nCoV-19 vaccination are durable in PWH.
0 42 1820
5
10
15
20
Time since Vaccination (days)
% P
rolif
erat
ing
with
in C
D8+
CD8+ SARS-CoV-2 S2
HIV+ Adults
HIV- Adults
IFN! ELISpot
C D E F
A B
0 14 28 42 56 18210
100
1000
10000
SFU
/10^
6 PB
MC
IFNγ ELISPOT
Time since vaccination (days)
****
****
****
****
****
0 14 28 42 56 18205
10152020
40
60
80
% P
rolif
erat
ing
with
in C
D4+
CD4+ SARS-CoV-2 S1
Time since vaccination (days)
****
****
****
****
***
✱✱✱
0 14 28 42 56 18205
10152020
40
60
80
Time since vaccination (days)
% P
rolif
erat
ing
with
in C
D4+
CD4+ SARS-CoV-2 S2
****
****
****
****
**
✱✱✱
0 14 28 42 56 18205
10152020
40
60
80
% P
rolif
erat
ing
with
in C
D8+
Time since vaccination (days)
CD8+ SARS-CoV-2 S1
****
****
****
****
****
✱
0 14 28 42 56 18205
10152020
40
60
80
Time since vaccination (days)
% P
rolif
erat
ing
with
in C
D8+
CD8+ SARS-CoV-2 S2
****
****
****
****
***
✱✱
T cell proliferation
0 14 28 42 56 18210
100
1000
10000
SFU
/10^
6 PB
MC
IFNγ ELISPOT
Time since vaccination (days)
****
****
****
****
****
0 14 28 42 56 1820.01
0.1
1
10
100
% P
rolif
erat
ing
with
in C
D4+
CD4+ SARS-CoV-2 S1
Time since vaccination (days)
****
****
****
****
***
✱✱✱
0 14 28 42 56 1820.01
0.1
1
10
100
Time since vaccination (days)
% P
rolif
erat
ing
with
in C
D4+CD4+ SARS-CoV-2 S2
****
****
****
****
**
✱✱✱
0 14 28 42 56 1820.001
0.01
0.1
1
10
100
% P
rolif
erat
ing
with
in C
D8+
Time since vaccination (days)
CD8+ SARS-CoV-2 S1
****
****
****
****
****
✱
0 14 28 42 56 1820.001
0.01
0.1
1
10
100
Time since vaccination (days)
% P
rolif
erat
ing
with
in C
D8+
CD8+ SARS-CoV-2 S2
****
****
****
****
***
✱✱
0 14 28 42 56 18210
100
1000
10000
Time since vaccination (days)
SFU
/10^
6 P
BM
C
IFN-γ ELISpot
HIV+ Adults
HIV- Adults
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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 29, 2021. ; https://doi.org/10.1101/2021.09.28.21264207doi: medRxiv preprint
36
777 778 Figure 4: SARS-CoV-2 specific T cells are not preferentially biased for any CD4+ T cell subsets. 779 Ex vivo frequencies of (A) CXCR3+ CCR6- (Th1), (B) CXCR3- CCR6- (Th2), (C) CXCR3- CCR6+ (Th17), and (D) CXCR5+ within 780 CD4+ T cells in HIV+ volunteers measured at day 0, 42 and 182 using ex vivo T cell phenotyping. (E) comparative analysis of 781 frequencies of ex vivo CD4+ T cell frequencies in HIV+ and HIV- volunteers at day 182 (6 months post vaccination). Measurement 782 of frequencies of antigen specific T cells including SARS-CoV-2 S1 and S2, HIV gag and CMVpp65 using activation induced marker 783 (AIM) assay in (F) CD4+ and (G) CD8+ T cells. For CD4 T cells, antigen specific cells were: CD25+ CD134(OX40)+ and CD25+ 784 CD137+ and CD25+ CD69+; for CD8+ T cells, antigen specific cells were: CD25+ CD137+ and CD25+ CD69+ Frequencies of (H) 785 CXCR3+ CCR6-(Th1), (I) CXCR3- CCR6- (Th2), (J) CXCR3- CCR6+ (Th17), and (K) CXCR5+ CD4+ T cells within antigen specific 786 (AIM+) T cells in HIV+ volunteers. Comparison of two timepoints within the same group was done by Wilcoxon matched pair sign 787 ranked test. Comparison of two groups was done by two-tailed Mann-Whitney U test. Where indicated * = <0.05, ** = <0.01, *** = < 788 0.001 and **** = <0.000. 789
S1 S2
HIV
GA
G
CM
V PP
65
0.001
0.01
0.1
1
10
Freq
. of C
D8+
Ag
Sp c
ells
(%)
✱✱✱✱
✱✱✱✱
✱✱✱✱
Figure 4: SARS-CoV-2 specific T cells are not preferentially biased for any CD4+ T cell subsets
F G
AIM+ CD4+ T cells AIM+ CD8+ T cells
A
Total Circulating CD4+ T cells
0 42 1820
10
20
30
40
% w
ithin
CD
4+ T
cel
lsTh1 HIV+ by timepoints
Time since vaccination (days)
✱
0 42 1820
10
20
30
40
% w
ithin
CD
4+ T
cel
ls
Th1 HIV- by timepoints
Time since vaccination (days)
0 42 1820
50
100
150
% w
ithin
CD
4+ T
cel
ls
Th2 HIV+ by timepoints
Time since vaccination (days)
✱✱✱✱
✱✱✱
0 42 18260
70
80
90
100
% w
ithin
CD
4+ T
cel
ls
Th2 HIV- by timepoints
Time since vaccination (days)
0 42 1820
10
20
30
40
% w
ithin
CD
4+ T
cel
ls
Th17 HIV+ by timepoints
Time since vaccination (days)
✱✱✱✱
0 42 1820
5
10
15
20
25
% w
ithin
CD
4+ T
cel
ls
Th17 HIV- by timepoints
Time since vaccination (days)
0 42 1820
10
20
30
40
% w
ithin
CD
4+ T
cel
ls
CXCR5+ HIV+ by timepoints
Time since vaccination (days)
✱✱✱✱
0 42 1820
5
10
15
20
% w
ithin
CD
4+ T
cel
ls
CXCR5+ HIV- by timepoints
Time since vaccination (days)
CXCR3+ CCR6- (Th1) CXCR3- CCR6- (Th2) CXCR3- CCR6+ (Th17) CXCR5+ CD4+ (Tfh)
HIV+ HIV- HIV+ HIV- HIV+ HIV- HIV+ HIV- HIV+ HIV- HIV+ HIV-0
5
10
1520406080
100
% w
ithin
CD
4+ T
cel
ls
✱✱✱✱
✱✱✱✱
CXCR3+ CCR6- (Th1)
CXCR3- CCR6- (Th2)
CXCR3- CCR6+ (Th17)
Th subsets within bulk CD4 T cells 6 months after vaccination
✱✱✱
✱✱✱
CXCR5+ (Tfh)
CXCR5+ PD1+ (Tfh)
CD25+ CD127lo (Treg)
B C D E
S1 S2
HIV
GA
G
CM
V PP
65
0.01
0.1
1
10
Freq
. of C
D4+
Ag
Sp c
ells
(%)
✱✱
✱✱✱✱
✱✱✱✱
Frequency within AIM+ CD4+ T cells
CXC
R3+
CC
R6-
(Th1
)
CXC
R3-
CC
R6-
(Th2
)
CXC
R3-
CC
R6+
(Th1
7)
0
20
40
60
80
100
% w
ithin
Ag
spec
ific
cells
S1 S2
HIV
GA
G
CM
V PP
65
0
10
20
30
40
Freq
. with
in A
g sp
ecifi
c ce
lls (%
) CXCR3+ CCR6- (Th1)
CXC
R3+
CC
R6-
(Th1
)
CXC
R3-
CC
R6-
(Th2
)
CXC
R3-
CC
R6+
(Th1
7)
0
20
40
60
80
100
% w
ithin
Ag
spec
ific
cells ✱✱✱✱
✱✱✱✱
S1 S2
HIV
GA
G
CM
V PP
65
0
20
40
60
80
100
Freq
. with
in A
g sp
ecifi
c ce
lls (%
) CXCR3- CCR6- (Th2)
CXC
R3+
CC
R6-
(Th1
)
CXC
R3-
CC
R6-
(Th2
)
CXC
R3-
CC
R6+
(Th1
7)
0
20
40
60
80
100
% w
ithin
Ag
spec
ific
cells ✱✱✱✱
✱✱✱✱
S1 S2
HIV
GA
G
CM
V PP
65
0
20
40
60
80
100
Freq
. with
in A
g sp
ecifi
c ce
lls (%
) CXCR3- CCR6+ (Th17)
CXC
R3+
CC
R6-
(Th1
)
CXC
R3-
CC
R6-
(Th2
)
CXC
R3-
CC
R6+
(Th1
7)
0
20
40
60
80
100
% w
ithin
Ag
spec
ific
cells ✱✱✱✱
✱✱✱✱
S1 S2
HIV
GA
G
CM
V PP
65
0
20
40
60
80
100
Freq
. with
in A
g sp
ecifi
c ce
lls (%
) CXCR5+ CD4+ (cTfh)
CXC
R3+
CC
R6-
(Th1
)
CXC
R3-
CC
R6-
(Th2
)
CXC
R3-
CC
R6+
(Th1
7)
0
20
40
60
80
100
% w
ithin
Ag
spec
ific
cells ✱✱✱✱
✱✱✱✱
H ICXCR3+ CCR6- (Th1) CXCR3- CCR6- (Th2)
CXC
R3+
CC
R6-
(Th1
)
CXC
R3-
CC
R6-
(Th2
)
CXC
R3-
CC
R6+
(Th1
7)
0
20
40
60
80
100
% w
ithin
Ag
spec
ific
cells
S1 S2
HIV
GA
G
CM
V PP
65
0
10
20
30
40
Freq
. with
in A
g sp
ecifi
c ce
lls (%
) CXCR3+ CCR6- (Th1)
CXC
R3+
CC
R6-
(Th1
)
CXC
R3-
CC
R6-
(Th2
)
CXC
R3-
CC
R6+
(Th1
7)
0
20
40
60
80
100
% w
ithin
Ag
spec
ific
cells ✱✱✱✱
✱✱✱✱
S1 S2
HIV
GA
G
CM
V PP
65
0
20
40
60
80
100
Freq
. with
in A
g sp
ecifi
c ce
lls (%
) CXCR3- CCR6- (Th2)
CXC
R3+
CC
R6-
(Th1
)
CXC
R3-
CC
R6-
(Th2
)
CXC
R3-
CC
R6+
(Th1
7)
0
20
40
60
80
100
% w
ithin
Ag
spec
ific
cells ✱✱✱✱
✱✱✱✱
S1 S2
HIV
GA
G
CM
V PP
65
0
20
40
60
80
100
Freq
. with
in A
g sp
ecifi
c ce
lls (%
) CXCR3- CCR6+ (Th17)
CXC
R3+
CC
R6-
(Th1
)
CXC
R3-
CC
R6-
(Th2
)
CXC
R3-
CC
R6+
(Th1
7)
0
20
40
60
80
100
% w
ithin
Ag
spec
ific
cells ✱✱✱✱
✱✱✱✱
S1 S2
HIV
GA
G
CM
V PP
65
0
20
40
60
80
100
Freq
. with
in A
g sp
ecifi
c ce
lls (%
) CXCR5+ CD4+ (cTfh)
CXC
R3+
CC
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)
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)
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40
60
80
100
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ific
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HIV-
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37
790 Figure 5: Responses to VOCs are preserved at 6 months post ChAdOx1 nCoV-19 vaccination in PWH. 791 (A) ACE-2 binding inhibition assay for alpha, beta and gamma VOCs measured at day 0 (baseline) and at day 182 (6 months post-792 vaccination) in HIV+ volunteers. (B) Comparison between ACE-2 binding inhibition of SARS-CoV-2 WT strain and alpha, beta and 793 gamma VOCs in HIV+ volunteers. Comparison between proliferative T cell responses to SARS-CoV-2 WT strain and beta, gamma 794 and delta VOCs in (C) CD4+ S1, (D) CD4+ S2, (E) CD8+ S1 and (F) CD8+ S2 in HIV+ volunteers. Comparative analysis of (G) CD4+ 795 S1, (H) CD4+ S2, (I) CD8+ S1, and (J) CD8+ S2 T cells responses to VOCs in HIV+ (solid circles) and HIV- (open circles). Comparison 796 of two timepoints within the same group was done by Wilcoxon matched pair sign ranked test. Comparison of two groups was done 797 by two-tailed Mann-Whitney U test. Where indicated * = <0.05, ** = <0.01, *** = < 0.001 and **** = <0.000. Dotted lines in C- J indicate 798 threshold for true positive based mean of DMSO controls + 3x SD. 799
SARS-CoV
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ing
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ing
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Figure 5: Responses to VOCs are preserved at 6 months post ChAdOx1 nCoV-19 vaccination in PWH
SARS-CoV
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VOC - Gam
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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 29, 2021. ; https://doi.org/10.1101/2021.09.28.21264207doi: medRxiv preprint
38
800
801 802 Figure 6: Pre-existing cross-reactive CD4+ T cell responses in PWH measured at baseline are associated with high 803 magnitude T cell responses post ChAdOx1 nCoV-19 vaccination. 804 Baseline CD4+ SARS-CoV-2 responses were split into baseline responders (BR, proliferation >1%, black circles and black lines) and 805 baseline non-responders (B-NR, Proliferation <1%, yellow circles and yellow lines) and CD4 T cell responses post vaccination were 806 analysed at all available timepoints for (A) SARS-CoV-2 S1 and (B) SARS-CoV-2 S2. T cells responses targeting (C) S1 and (D) S2 807 proteins in endemic CCCs are measured at baseline in BR and B-NR. Comparison of two timepoints within the same group was done 808 by Wilcoxon matched pair sign ranked test. Comparison of two groups was done by two-tailed multiple Mann-Whitney U test. CCC 809 responses among participants were compared using fisher’s exact test and listed in supplementary table 3. P values as indicated or 810 * = <0.05, ** = <0.01, *** = < 0.001 and **** = <0.000. Dotted lines in indicate threshold for true positive based mean of DMSO controls 811 + 3x SD 812
Figure 6: Pre-existing cross-reactive CD4+ T cell responses in PWH measured at baseline are associated with high magnitude T cell responses post ChAdOx1 nCoV-19 vaccination
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Time since vaccination (days)
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Time since vaccination (days)
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**** **** **** ******BN-R BR
BN-R BR
BN-R BR
BN-R BR
BN-R BR
BN-R BR
BN-R BR
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ing
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HKU1 CLADE 1
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27
4
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16
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20
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CD4+ SARS-CoV-2 S1 CD4+ SARS-CoV-2 S2
CD4+ CCC S1 CD4+ CCC S2
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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 29, 2021. ; https://doi.org/10.1101/2021.09.28.21264207doi: medRxiv preprint
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813 Figure 7: Cross reactive humoral immune responses among betaCoVs. 814 Antibody titres against (A) SARS-CoV, (B) MERS-CoV, and (C) HKU-1 spike proteins measured at day 0 (baseline) and day 182 (6 815 months post-vaccination) in HIV+ participants. Correlation between baseline antibody titres for SARS-CoV-2 and (D) SARS-CoV-1, 816 (E) MERS-CoV, and (F) HKU-1 spike protein at baseline. (G) Phylogenetic tree showing relationship between coronaviruses. 817 Correlation was performed via Spearman’s rank correlation coefficient and comparison of two timepoints within the same group was 818 done by Wilcoxon matched pair sign ranked test. Where indicated ns = not significant, * = <0.05, ** = <0.01, *** = < 0.001 and **** = 819 <0.0001. Dotted lines in A – B indicate cut off points determined for each SARS-CoV-2 antigen based on pre-pandemic sera + 3X 820 SD.821
Figure 7: Cross reactive humoral immune responses among betaCoVs
D E F
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10 100 1000 100001
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100
1000
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Day
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ERS-
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100000
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D0 D182100
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105
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log 1
0)Time since vaccination
(days)
✱✱✱✱
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103
104
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log 1
0)
Time since vaccination (days)
A B C
0.1
OC43_AIL49500
HKU1_c1_AGW27863
NL63_AGT51366
HKU1_c2_ABD75521
OC43_AIX10760
HKU1_c1_ABD96197
229E_APT69849
SARS-CoV
NL63_AUF73167
MERS-CoV
HKU1_c2_AYN64561
HKU1_c1_AGW27881
229E_AIW52755
SARS-CoV-2
HKU1_c2_ABD75633
229E_APT69869
NL63_AAS89767
OC43_AGT51660
G
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40
Supplementary figure legends 822 823 Supplementary figure 1: Flow cytometry gating strategy and kinetics of cells 824 expressing activation and exhaustion marker. 825 Gating strategy for (A) activation panel and (B) exhaustion panel. All gates were set 826 based on fluorescent minus one (FMO). Frequency of cells expressing (C) CD38+ 827 HLA DR+, (D) PD-1+, (E) Eomes(hi) Tbet(lo) within CD4+ T cells on HIV+ participants 828 and frequency of cells expressing (F) CD38+ HLA DR+, (G) PD-1+, (H) Eomes(hi) 829 Tbet(lo) within CD8+ T cells on HIV+ participants. Frequency of cells expressing (I) 830 CD38+ HLA DR+, (J) PD-1+, (K) Eomes(hi) Tbet(lo) within CD4+ T cells on HIV- 831 participants and frequency of cells expressing (L) CD38+ HLA DR+, (M) PD-1+, (N) 832 Eomes(hi) Tbet(lo) within CD8+ T cells on HIV- participants. Comparison of two 833 timepoints within the same group was done by Wilcoxon matched pair sign ranked 834 test. Where indicated * = <0.05, ** = <0.01, *** = < 0.001 and **** = <0.0001 835 836 837 Supplementary figure 2: Humoral immune responses against SARS-CoV-2 in 838 PWH. 839 (A) Correlations between antibody levels measured using MSD assay and in-house 840 total IgG ELISA at (A) day 0, (B) day 182 and (C) correlations between day 182 SARS-841 CoV-2 RBD levels and ACE-2 binding inhibition assay. (D) Antibody levels in HIV+ 842 participants measured across all timepoints presented as dot plots and, (E) before-843 after plots to show individual responses. (F) Correlation plot showing correlation matrix 844 between SARS-CoV-2 humoral immune response across all proteins, assays and 845 timepoints. Correlation was performed via Spearman’s rank correlation coefficient and 846 correlation matrix was created using corrplot package on R studio. Size and Colour of 847 the heatmap corresponds to the correlation coefficient. Comparison of two timepoints 848 within the same group was done by Wilcoxon matched pair sign ranked test. Where 849 indicated * = <0.05, ** = <0.01, *** = < 0.001 and **** = <0.0001. 850 851 852 Supplementary figure 3: Gating strategy and T cell response to control antigens 853 (FECT) and mitogen (PHA). 854 (A) Gating strategy for proliferation assay. All gates were set based on DMSO controls. 855 Background was subtracted and responses were assigned positive if they were >1% 856 after background subtraction. (B) PHA responses in CD4+ and CD8+ T cells at 857 longitudinal timepoints. (C) FECT responses in CD4+ and CD8+ T cells at longitudinal 858 timepoints. Comparison of two timepoints within the same group was done by 859 Wilcoxon matched pair sign ranked test. Where indicated * = <0.05, ** = <0.01, *** = 860 < 0.001 and **** = <0.000. Dotted lines in indicate threshold for true positive based 861 mean of DMSO controls + 3x SD. 862 863 864 865 Supplementary figure 4: Longitudinal T cell responses to SARS-CoV-2 in HIV+ 866 and HIV- subjects following ChAdOx1 nCoV-19 vaccination. 867 (A) IFNγ ELISpot responses in HIV- volunteers at day 0, 42 and 182. T cell proliferative 868 response to (B) SARS-CoV-2 S1, (C) SARS-CoV-2 S2 in CD4+ T cells in HIV- 869 volunteers and T cell proliferative response to (D) SARS-CoV-2 S1, (E) SARS-CoV-2 870 S2 in CD8+ T cells in HIV- volunteers. Comparison of T cell proliferative responses to 871
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(F) SARS-CoV-2 S1, (G) SARS-CoV-2 S2 in CD4+ T cells (H) SARS-CoV-2 S1, (I) 872 SARS-CoV-2 S2 in CD8+ T cells in HIV+ and HIV negative volunteers at day 0, 42 873 and 182. Comparison of two timepoints within the same group was done by Wilcoxon 874 matched pair sign ranked test. Comparison of two groups was done by two-tailed 875 multiple Mann-Whitney U test. Where indicated * = <0.05, ** = <0.01, *** = < 0.001 876 and **** = <0.000. Dotted lines in indicate threshold for true positive based mean of 877 DMSO controls + 3x SD. 878 879 880 Supplementary figure 5: Phenotype of total and SARS-CoV-2 S-specific 881 circulating CD4+ T cells 882 (A) Ex vivo frequencies of (A) CXCR3+ CCR6+ (Th1), CXCR3- CCR6- (Th2), CXCR3- 883 CCR6+ (Th17), and CXCR5+ within CD4+ T cells in HIV- volunteers measured at 884 various timepoints. (B) gating strategy for AIM assay. All cells expressing CD25+ 885 CD134 (OX40) and CD25+ CD137+ and CD25+ CD69+ were Boolean gated as 886 antigen specific cells. Gating was set based on DMSO control and all responses were 887 background subtracted. CXCR3 and CCR6 quadrant gate for antigen specific 888 population was set based on expression in bulk CD4 T cell population. (C) Frequency 889 of CD4 T cell subsets in SEB, SARS-CoV-2 S1, SARS-CoV-2 S2, HIV GAG and 890 CMVpp65. Comparison of two timepoints within the same group was done by 891 Wilcoxon matched pair sign ranked test. Comparison of two groups was done by two-892 tailed multiple Mann-Whitney U test. Where indicated * = <0.05, ** = <0.01, *** = < 893 0.001 and **** = <0.000. 894 895 896 Supplementary figure 6: Pre-existing cross-reactive immunity in PWH measured 897 at baseline are associated with high magnitude CD8+ T cell responses post 898 ChAdOx1 nCoV-19 vaccination 899 Baseline CD8+ SARS-CoV-2 responses were split into baseline responders (BR, 900 proliferation >1%, black circles and black lines) and baseline non-responders (B-NR, 901 Proliferation <1%, yellow circles and yellow lines) and CD8 T cell responses post 902 vaccination were analysed at all available timepoints for (A) SARS-CoV-2 S1 and (B) 903 SARS-CoV-2 S2. T cells responses targeting (C) S1 and (D) S2 proteins in endemic 904 CCCs are measured at baseline in BR and B-NR. Comparison of two timepoints within 905 the same group was done by Wilcoxon matched pair sign ranked test. Comparison of 906 two groups was done by two-tailed multiple Mann-Whitney U test. CCC responses 907 among participants were compared using fisher’s exact test and listed in 908 supplementary table 3. P values as indicated or * = <0.05, ** = <0.01, *** = < 0.001 909 and **** = <0.000. Dotted lines in indicate threshold for true positive based mean of 910 DMSO controls + 3x SD. 911 912 913 Supplementary figure 7: Relationship between baseline and post vaccination. 914 timepoints for CD4+ and CD8+ proliferative T cells. 915 Correlation plots showing correlation matrix for (A) CD4+ SARS-CoV-2 S1, (B) CD4+ 916 SARS-CoV-2 S2, (C) CD8+ SARS-CoV-2 S1, (D) CD8+ SARS-CoV-2 S2. Correlation 917 was performed via Spearman’s rank correlation coefficient and correlation matrix was 918 created using corrplot package on R studio. Size and Colour of the heatmap 919 corresponds to the correlation coefficient. Where indicated * = <0.05, ** = <0.01, *** = 920 < 0.001 and **** = <0.0001. 921
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Supplementary figure 8: Responses to CCC in PWH 922 T cell proliferative responses to CCCs HKU-1 clade 1 and 2, OC43, 299E, NL63 S1 923 and S2 in CD4+ and CD8+ T cells measured on day 0, 14, 28, 42, 56 and 182. 924 Comparison of two timepoints within the same group was done by Wilcoxon matched 925 pair sign ranked test. P values as indicated or * = <0.05, ** = <0.01, *** = < 0.001 and 926 **** = <0.000. Dotted lines in indicate threshold for true positive based mean of DMSO 927 controls + 3x SD. 928 929 930 Supplementary figure 9: Relationship between antibody responses for SARS-931 CoV-2 and CCCs spike in PWH 932 (A) Correlation plot showing correlation matrix between different circulating HCoVs. 933 (B) Antibody titres at day 0 and day 182 for OC43 Spike, 299E spike and NL63 spike 934 proteins in HIV+ participants. Correlation was performed via Spearman’s rank 935 correlation coefficient and correlation matrix was created using corrplot package on R 936 studio. Size and Colour of the heatmap corresponds to the correlation coefficient. 937 Comparison of two timepoints within the same group was done by Wilcoxon matched 938 pair sign ranked test. Where indicated * = <0.05, ** = <0.01, *** = < 0.001 and **** = 939 <0.0001. 940
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1123
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