Durability of ChAdOx1 nCov-19 (AZD1222) vaccination in ...

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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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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

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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

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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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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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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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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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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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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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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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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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27

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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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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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



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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



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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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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

✱✱✱✱ ✱✱✱✱✱✱✱


1

10

100

% w

ithin

CD

8+ T

cel

ls


✱✱✱✱ ✱✱✱✱✱✱✱✱

HIV+ HIV- HIV+ HIV- HIV+ HIV-1

10

100

% w

ithin

CD

4+ T

cel

ls



10

100

% w

ithin

CD

8+ T

cel

ls



1

10

100

% w

ithin

CD

4+ T

cel

ls


✱✱✱ ✱✱✱ ✱✱✱


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+



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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

)


✱✱✱✱

0 18210-1

100

101

102

103

104

105

106

SARS-CoV-2 N

AU

/ml (

log 10

)


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

****


0 28 56 1821

10

100

1000

6 month neuts


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


Anti-

spik

e Ig

G

ELIS

A U

nits

(EU

)

****

**** ***



https://doi.org/10.1101/2021.09.28.21264207


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


****

****

****

****

****

0 14 28 42 56 18205

10152020

40

60

80

% P

rolif

erat

ing

with

in C

D4+

CD4+ SARS-CoV-2 S1


****

****

****

****

***

✱✱✱

0 14 28 42 56 18205

10152020

40

60

80


% 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+


CD8+ SARS-CoV-2 S1

****

****

****

****

****

✱

0 14 28 42 56 18205

10152020

40

60

80


% 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


****

****

****

****

****

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


****

****

****

****

***

✱✱✱

0 14 28 42 56 1820.01

0.1

1

10

100


% 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+


CD8+ SARS-CoV-2 S1

****

****

****

****

****

✱

0 14 28 42 56 1820.001

0.01

0.1

1

10

100


% P

rolif

erat

ing

with

in C

D8+

CD8+ SARS-CoV-2 S2

****

****

****

****

***

✱✱

0 14 28 42 56 18210

100

1000

10000


SFU

/10^

6 P

BM

C

IFN-γ ELISpot

HIV+ Adults

HIV- Adults



https://doi.org/10.1101/2021.09.28.21264207


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


✱

0 42 1820

10

20

30

40

% w

ithin

CD

4+ T

cel

ls

Th1 HIV- by timepoints


0 42 1820

50

100

150

% w

ithin

CD

4+ T

cel

ls

Th2 HIV+ by timepoints


✱✱✱✱

✱✱✱

0 42 18260

70

80

90

100

% w

ithin

CD

4+ T

cel

ls



0 42 1820

10

20

30

40

% w

ithin

CD

4+ T

cel

ls

Th17 HIV+ by timepoints


✱✱✱✱

0 42 1820

5

10

15

20

25

% w

ithin

CD

4+ T

cel

ls



0 42 1820

10

20

30

40

% w

ithin

CD

4+ T

cel

ls

CXCR5+ HIV+ by timepoints


✱✱✱✱

0 42 1820

5

10

15

20

% w

ithin

CD

4+ T

cel

ls

CXCR5+ HIV- by timepoints


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

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 ✱✱✱✱

✱✱✱✱

CXCR5+ CD4+ (Tfh)J

CXCR3- CCR6+ (Th17)K

HIV+

HIV-



https://doi.org/10.1101/2021.09.28.21264207


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

-2 S1

VOC - Beta

S1

VOC - Gam

ma S1

VOC - Delt

a S1

0.01

0.1

1

10

100

% P

rolif

erat

ing

with

in C

D4+

SARS-CoV

-2 S2

VOC - Beta

S2

VOC - Gam

ma S2

VOC - Delt

a S1

0.01

0.1

1

10

100

% P

rolif

erat

ing

with

in C

D4+

✱

✱

✱

SARS-CoV

-2 S1

VOC - Beta

S1

VOC - Gam

ma S1

VOC - Delt

a S1

0.01

0.1

1

10

100

% P

rolif

erat

ing

with

in C

D8+

SARS-CoV

-2 S2

VOC - Beta

S2

VOC - Gam

ma S2

VOC - Delt

a S1

0.01

0.1

1

10

100

% P

rolif

erat

ing

with

in C

D8+

Figure 5: Responses to VOCs are preserved at 6 months post ChAdOx1 nCoV-19 vaccination in PWH

SARS-CoV

-2

VOC - Alph

a

VOC - Beta

VOC - Gam

ma0.01

0.1

1

10

100

unit/ml

ACE-2 Inhibition Assay

*

********

0 182 0 182 0 1820.01

0.1

1

10

100

unit/ml

ACE-2 Inhibition Assay

CoV-2 Alpha CoV-2 Beta CoV-2 Gamma

**** **** ****A B C D E F


0.01

0.1

1

10

100

% P

rolif

erat

ing

with

in C

D4+

VOC - Beta S1 VOC - Gamma S1 VOC - Delta S1


0.01

0.1

1

10

100

% P

rolif

erat

ing

with

in C

D4+



0.01

0.1

1

10

100

% P

rolif

erat

ing

with

in C

D8+



0.01

0.1

1

10

100

% P

rolif

erat

ing

with

in C

D8+


✱G H I J

ACE-2 Inhibition to VOC Proliferative T cell response to VOC

CD4+ SARS-CoV-2 S1 CD4+ SARS-CoV-2 S2 CD8+ SARS-CoV-2 S1 CD8+ SARS-CoV-2 S2

CD4+ S1 CD4+ S2 CD8+ S1 CD8+ S2



https://doi.org/10.1101/2021.09.28.21264207


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

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D4+

SARS-COV2 S1 BNR VS BR

**** *****

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D4+


**** * * * *****

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D8+

SARS-COV2 CD8 S2 BNR VS BR

**** * *****

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D8+


B-NR

BR

**** **** **** ******

A B

C D

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D4+


**** *****

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D4+


**** * * * *****

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D8+


**** * *****

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D8+


B-NR

BR

**** **** **** ******

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D4+


**** *****

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D4+


**** * * * *****

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D8+


**** * *****

D0 D14 D28 D42 D56 D1820.1

1

10

100


% P

rolif

erat

ing

with

in C

D8+


B-NR

BR

**** **** **** ******BN-R BR

BN-R BR

BN-R BR

BN-R BR

BN-R BR

BN-R BR

BN-R BR

BN-R BR

BN-R BR

BN-R BR

BN-R BR

0.001

0.01

0.1

1

10

100

% P

rolif

erat

ing

with

in C

D4+

✱✱

HKU1 CLADE 1

S1

HKU1 CLADE 1

S2

HKU1 CLADE 2

S1

HKU1 CLADE 2

S2 299E S1 299E S2 NL63 S1 NL63 S2 OC43 S1 OC43 S2

CCC Responders:

CCC Non - responders:

11

27

3

23

6

22

11

15 27

3

23

4

24

11

15

1

27

3

23

2

26

8

18

2

26

5

21

11

17

17

9

0

28

1

25

4

24

4

21

0.064

BN-R BR

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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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

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229E_APT69849

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229E_APT69869

NL63_AAS89767

OC43_AGT51660

G



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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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