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iScience

Article

Attenuated humoral responses in HIV after SARS-

CoV-2 vaccination linked to B cell defects and

altered immune proﬁles

Emma Touizer,

Aljawharah

Alrubayyi,

Rosemarie Ford,

..., Emma Morris,

Dimitra Peppa,

Laura E. McCoy

d.peppa@ucl.ac.uk (D.P.)

l.mccoy@ucl.ac.uk (L.E.M.)

Highlights

PLWH have lower levels of

neutralizing anti bodies

(nAbs) after SARS-CoV-2

vaccination

Delayed pro duction o f

nAbs is associated with

greater memory B cell

(MBCs) disturbance

Additional doses increase

nAbs titers and breadth

despite persistent MBCs

perturbations

SARS-CoV-2 vaccina tion

elicits robust T cell

responses in PLWH

PLWH with spike-speciﬁc

TcellsbutnonAbshave

increased

CXCR3+CD127+C D8

cells

Touizer et al., iScience 26,

105862

January 20, 2023 ª 2023 The

Authors.

https://doi.org/10.1016/

j.isci.2022.105862

OPEN ACCESS

iScience

Article

Attenuated humoral responses in HIV

after SARS-CoV-2 vaccination linked to B

cell defects and altered immune proﬁles

Emma Touizer,

1,8

Aljawharah Alrubayyi,

1,2,8

Rosemarie Ford,

Noshin Hussain,

Pehue

n Pereyra Gerber,

Hiu-Long Shum,

Chloe Rees-Spear,

Luke Muir,

Ester Gea-Mallorquı

Jakub Kopycinski,

Dylan Jankovic,

Anna Jeffery-Smith,

Christopher L. Pinder,

Thomas A. Fox,

Ian Williams,

Claire Mullender,

Irfaan Maan,

4,5

Laura Waters,

Margaret Johnson,

1,6

Sara Madge,

Michael Youle,

1,6

Tristan J. Barber,

5,6

Fiona Burns,

5,6

Sabine Kinloch,

1,6

Sarah Rowland-Jones,

Richard Gilson,

4,5

Nicholas J. Matheson,

3,7

Emma Morris,

Dimitra Peppa,

1,4,6,

and Laura E. McCoy

1,8,9,

SUMMARY

We assessed a cohort of people living with human immunodeﬁciency virus

(PLWH) (n = 110) and HIV negative controls (n = 64) after 1, 2 or 3 SARS-CoV-2

vaccine doses. At all timepoints, PLWH had signiﬁcantly lower neutralizing anti-

bod y (nAb) titers than HIV-negative controls. We also observed a delayed devel-

opment of neutralizat ion in PLWH that was underpinned by a reduced frequency

of spike-speciﬁc memory B cells (MBCs). Improved n eutra lization breadth was

seen against the Omicron variant (BA .1) after the third vaccine dose in PLWH

but lower nAb responses persisted and were associated with global MBC

dysfunction. In contrast, SARS-CoV-2 vaccination induced robust T cell responses

that cross-recognized variants in PLWH. Strikingly, individuals with low or absent

neutralization had detectable functional T cell responses. These PLWH had

reduced numbers of circulating T follicular helper cells and an enriched population

of CXCR3

CD127

CD8

T cells after two doses of SARS-CoV-2 va ccination.

INTRODUCTION

People living with human immunodeﬁciency virus (HIV)[PLWH] appear to be at a higher risk of hospitaliza -

tion and worse clinical outcomes from COVID-19 disease, especially in the cont ext of cellu lar immunosup -

pression and unsuppressed HIV viral load.

Although combined antiretroviral therapy (cART) has

dramatically improved life expectancy in PLWH, t he persistence of immune dysfunction raises concerns

about the overall effectiveness and durabilit y of vaccine responses in this potentiall y more vulnerable pa-

tient group, in line with other immunocompromised groups

2,3

As a resul t, PLWH were included in either

priority group 4 (for clinically vulnerable PLWH, based on more advanced immunosuppression or co-mor-

bidities) or 6 ( all other PLWH)

in the UK for earlier COVID-19 vaccination than the general population. The

Joint Committee on Vaccin ation and Immunization (JCVI) advised to invite this patient g roup for an addi-

tional booster dose.

Previously, defects have been obser ved in serological vaccine responses i n PLWH on

cART. For example, after a full course of hepatitis B

or inﬂuenza vaccination

and long-term responses to

vaccination can b e shorter-lived in PLWH compared to the general population.

We and others h ave pre-

viously show n a failure to mount a ro bust antibody response follo wing COVID-19 vaccination in advanced

HIV infection with low CD4 T cell counts b elow 200 c ells/ mL.

8–12

Data on vaccine efﬁcacy and immunogenicity in PLWH remains limited (reviewed in

), and although there

are some conﬂicting results, meta-analyses

and recent studies

have shown reduced levels of sero con-

version and neutralization after a second dose of viral vector vaccine dose in PLWH receiving cART, with

lower CD4 T cell count/viremia and older age re sulti ng in a more impaired response and more rapid break-

through infection.

Assessment of vaccine efﬁcacy has been continually complicated by the ongoing

emergence of variants of concern (VOC), with the Alpha, Beta, Delta and Omicron variants being observed

to progressively evade antib odies

17,18

raised against the original Wuhan-Hu-1 strain in most vaccines. In

particular, data a fte r three v accine doses has been hard to assess because of the emerg ence o f Omicr on

Institute for Immunity and

Transplantation, Division of

Infection and Immunity,

University College London,

London, UK

Nufﬁeld Department of

Medicine, University of

Oxford, Oxford, UK

Cambridge Institute of

Therapeutic Immunology and

Infectious Disease,

Department of Medicine,

University of Cambridge,

Cambridge, UK

Mortimer Market Centre,

Department of HIV, Central

and North West London NHS

Trust, London, UK

Institute for Global Health,

University College London,

London, UK

The Ian Charleson Day

Centre, Royal Free Hospital

NHS Foundation Trust,

London, UK

NHS Blood and Transplant,

Cambridge, UK

These authors contributed

equally

Lead contact

*Correspondence:

d.peppa@ucl.ac.uk (D.P.),

l.mccoy@ucl.ac.uk (L.E.M.)

https://doi.org/10.1016/j.isci.

2022.105862

iScience 26, 105862, January 20, 2023 ª 2023 The Authors.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

OPEN ACCESS

concurrent with vaccination, and in PLWH data on humoral and cellular responses including against Om-

icron remains limited.

19–22

However, the data available thus far suggest that the third vaccine dose provides

a strong boost to antibody responses regardless of the CD4 T cell count, including in those who had

previously not serocon vert ed and induce s robust T cell r esponses.

19,21,22

Moreover, most studies on

SARS-CoV-2 vaccine responses in PLWH to date have mostly focused on e valuating humoral responses

and ge nerat ed limited data on function al T cell responses after 2 doses

or 3 doses.

19,20

Therefore, it re-

mains unclear what role HIV-associated immune dysfunction plays in serological and cellular outcome after

SARS-CoV-2 vaccination.

Inferior serological responses to vaccination in PLWH (on cART or unt reate d) are most commonl y linked to

HIV-induced immune destruction of CD4 T cells and imbalance of the CD4:CD8 T cell populations.

24,25

Despite effective cART, chronic i mmune activation in HIV can lead to exhaustion of the adaptive immune

system.

This can translate into impaired T cell responses, likely limiting T follicular helper (T

) cell help to

B cells, resulting in lower serologic al outputs. There i s also substantial e viden ce fo r dysfunction/exhaustion

in the B cell compartment during chronic infections that may limit antibody responses against the infecting

pathogen.

27,28

This B cell dysfunction persists to a variable degree after HIV viral suppression following

cART initiation,

29,30

but how these B cell defects impact serological responses to vaccination has not yet

been fully elucidated. Furthermore, there is substantial age-related decline in immune function leading

to senescenc e in both the T and B cell compartments, which may be accelerated in PLWH and c ould further

inﬂuence vaccine responses.

In this stu dy we h ave eval uated in a well-curat ed c ohort of PLWH on cART and HIV-negative controls

following three SARS-CoV-2 vaccine doses, the rel ation ship between humoral and functional T cel l re-

sponses. To achieve this goal, we have assessed how spike-speciﬁc memory B cell (MBC) responses, global

MBC proﬁles, CD4 and CD8 T cell phenotypes are linked with serological outcomes in PLWH to better un -

derstand which factors may modulat e immune responses to vacci nation.

RESULTS

Lower levels of seroconversion and neutralizing antibodies after SARS-CoV-2 immunization

in PLWH without a history of prior COVID-19 disease

Participants were recruited between January 2021 and April 2022 (n = 110 PL WH on cART and n = 64 HIV-

negative controls) as described in Table 1. Participants were sampled after 1, 2 or 3 doses of a SARS-CoV-2

vaccine and compared cross-sectionally. In addition, in 53 PLWH and 44 controls, responses were assessed

longitudinall y where sequential samples were available. Both study groups (HIV-negative and PLWH) w ere

divided according to their history of either previous SARS-CoV-2 infection (including infection prior or after

vaccination) or as SARS- CoV-2 naive. SARS-CoV-2 spike-speciﬁc IgG were tested for binding ag ain st t he S1

subunit of the SARS-CoV-2 spike protein in a semi-quantitative ELISA

32,33

to determine seropositivity.

Neutralizing antibodies (nAbs) were me asured against the ancestral vaccine-matched Wuhan Hu-1

SARS-CoV-2 (WT) strain by pseudovirus neutralization.

Approximately 90% of HIV-negative controls

and 80% of PLWH wi th no prior history of SARS-CoV-2 infection seroconv erted. However, alth ough over

82% of cont rols produced a neutr alizing response after o ne vacci ne d ose, only 29% of PLWH did so (Fi g-

ure 1A). As described,

prior history of SARS-CoV-2 infection was associated with a higher level of sero-

conversion and the development of nAbs in all individu als at every studied t imepoi nt regardless of HIV sta-

tus (Figure 1A).

Notably, PLWH had lower titers of nAbs than HIV-negative controls at all timepoints regardless of prior

SARS-CoV-2 infection (Figur es 1B and 1C). Overall, a similar trend was seen in binding responses

(Figure s S1A a nd S1B), and nAb titers correlated signiﬁcantly with both bindi ng titers for S1 IgG and

nAb titers obtained from a live virus neutralization assay (Figures S1C and S1D), as previously reported.

35,36

Given t hat this observational cohort incl udes a mixture o f SARS-CoV-2 vaccine type s, it was notable that

both binding and neutralizing titers remained signiﬁcantly lower in PLWH compared to controls when

only those who had received m RNA-based vaccines were co nsider ed (Figures S1E and S1F). A similar anal-

ysis for viral vector-based vaccines w as not feasible because of insufﬁcient numbers in the control group.

Both at the pre- and post-third vaccine dose timepoints, there were more SARS-CoV-2 naive PLWH that fail

to produce nAbs (Figures 1A, 1B, and S1A) compared to the control group. Owing to the cross-sectional

nature of the analysis, at the pre-third vaccine dose timepoint additional PLWH were recruited. However,

the observed differences persisted when PLWH were stratiﬁed for co-morbidities (Figure S1G).

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2 iScience 26, 105862, January 20, 2023

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Table 1. Co hort demographics

HIV- (n = 64) HIV+ (n = 110)

SARS-CoV-2-

(n = 27)

SARS-CoV-2+

(n = 37)

SARS-CoV-2-

(n = 65)

SARS-CoV-2+

(n = 45)

Clinical parameters

% female 76% 40% 8% 17%

% BAME 28% 24% 21% 44%

Median age (range) 33(21-65) 41(23-66) 53 (22-93) 49 (26-73)

cART – – 65 (100%) 45 (100%)

HIV viral load – – Undetectable (<50) Undetectable (<50)

Median CD4 count (range) – – 602 (22-1360) 560(200-1229)

Median CD4:CD8 ratio (range) – – 0.74 (0.13–3.05) 0.98 (0.37–2.55)

Co-morbidities

Diabetes, n – 1 5 3

Hypertension/CVD, n – 3 3 6

Renal disease, n – – 2 1

Liver disease, n – – 2 2

Respiratory disease, n – 1 3 1

Weakened immune system

inc. cancer/transplant, n

–– 8 1

Advanced HIV/HepB

co-infection, n

–– 4 –

Other – – Splenectomy

Sarcoidosis

Splenectomy

Timepoints

Post ﬁrst dose

N = 17 28 31 32

Median days post-previous

dose (range)

14 (12-74) 19 (12-60) 20 (12-102) 22 (13-82)

Vaccine (AZ |Moderna |Pﬁzer) 3 | 2 | 12 3 | 1 | 24 19 | 2 | 10 21 | 0 | 11

Post second dose

N = 18 25 30 24

Median days post-previous

dose (range)

39 (23-67) 26 (15-68) 20 (7-48) 21 (9-52)

Vaccine (AZ | Moderna | Pﬁzer) 3 | 3 | 12 2 | 1 | 23 17 | 1 | 12 12 | 0 | 12

Pre third dose

N = 21 26 39 16

Median days post-previous

dose (range)

129 (75-258) 129 (76-236) 125 (72-218) 119 (86-317)

Vaccine (AZ | Moderna | Pﬁzer) – – – –

Post third dose

N = 14 25 34 17

Median days post-previous

dose (range)

21 (13-43) 43 (18-129) 40 (9-149) 65 (7-140)

Vaccine (AZ |Moderna | Pﬁzer) 0 | 3 | 12 0 | 2 | 23 1 | 2 | 32 1 | 0 | 16

Cohort demographics, clinical characteristics and number of participants per timepoints for each group. All PLWH participants included in this study were on

cART. AZ = AZD1222; Moderna = mRNA-1273; Pﬁzer = BNT162b2.

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Figure 1. Weaker post vaccination antibody responses in SARS-CoV-2 naive PLWH

(A) Percentage of individuals with detectable neutralizing antibody response, non-neutralizing but binding response, or seronegative at each timepoint as

color-coded in the key. The headings above each graph show HIV status and previous SARS-CoV-2 exposure. N numbers for each group are indicated above

each column.

(B) WT pseudovirus neutralization reciprocal 50% inhibitory titers (ID50) in PLWH (blue) compared to HIV-negative controls (gray) stratiﬁed by vaccination

timepoint (on the x axis) for individuals without prior SARS-CoV-2 infectio n. The dotted line represents the lower limit of the assay (ID50 = 1:20). Where no

neutralization was detected, samples were assigned an ID50 of <1:20 a s this was the limit of assay detection. Each data point represents the mean of n = 2

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4 iScience 26, 105862, January 20, 2023

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Longitudinal samples fro m 53 PLWH and 44 controls we re then ev aluate d to assess binding ant ibody re-

sponses and nAb s over time after each vaccine dose. These i ncluded samples after the ﬁrs t dose and for

at least one additional timepoint, often including a baseline, post-second, pre-third and post-third sample

(Figure 1D). This analysis rev eal ed two clear t raje ctorie s of the de velopmen t of neut rali zation , ﬁrstly wh ere

nAbs were detected after a single vaccine dose,

deﬁned here as ‘‘standard neutra lizati on’’, and secondly

where neutral izatio n was no t ach ieved u ntil after the second dose or later, deﬁned as ‘‘delayed neutraliza-

tion’’. Most HIV- negati ve controls without prior SARS-CoV-2 infection show a standard neutralizat ion pro-

ﬁle, with only 3 individuals failing to mount a neutralizing response unti l after the second dose (Figure 1D),

and a similar effect was seenwithbindingresponses(Figures S1H–S1K). In contrast, two-thirds of SARS-

CoV-2 naive PLWH di d not make a detectable neutrali zing response until after the second dose and a sub-

stantial proportion of them lost detectable neutralizing activity before the third dose ( Figure s 1Aand1E).

However, both PLWH and HIV-negative controls with ahistoryofSARS-CoV-2infectionmadeastandard

neutralizing response (Figures 1F and 1G ). Therefore, having iden tiﬁed this delay ed neutralizati on pheno-

type in SARS-CoV-2 naive PLWH, we have evaluated its relationship with total CD4 T cell counts, which are

known to be important for SARS -CoV-2 vaccine respo nses in PLWH.

8–10,12

No signiﬁcant difference was

seen in median CD4 T cell count or CD4:CD8 T cell ratio between PLWH with standard or delayed neutral-

ization proﬁles (Figure s 1H and 1I); or correlate either with th e rapid development of neutralizatio n (Fig-

ure S1MandS1N).

Delayed neutralization is associated with lower frequency of spike-speciﬁc MBCs and a

perturbed MBC global phenotype

Spike is the SARS-CoV-2 glycopr otein and is the sole ant igen in most vaccines. It ha s been previousl y shown

that i nfection and vaccination produce spike-speciﬁc MBCs in proportio n to serological responses.

38–42

Given that the delay in neutralization observed more frequently in PLWH was not clearly associated with

peripheral CD4 T cell counts, we next assessed the relationship with global MBCs and spike-reactive

MBC frequency and phenotype, using a previously validated ﬂow cytometry panel, with memory B cells

deﬁned as CD19

CD20

CD38

lo/-

IgD- (Figure S2). This analysis was performed on available PBMC samples

after the ﬁrst vaccine dose, using SARS-CoV-2 naive baseline samples to determine the antigen-speciﬁc

gate (Figure 2A). We observed a signiﬁcantly lower frequency of spike- spe ciﬁc MBCs in SA RS-CoV -2 nai ve

participants after the ﬁrst dose as compared to those with a history of prior infection, regardless of HIV sta-

tus (Figure 2B). Moreover, a l ower freque ncy of spike-sp eciﬁc MBCs was observed in SARS-CoV-2 naive par-

ticipants who had a delayed neutralization response, although notably there were a small number of donors

in the standard neutralization group (Figure 2C).Inlinewiththis,thepercentageofspike-speciﬁcMBCs

showed a strong correlation with the nAb titer (Figure 2D) in agreement with previous ﬁndings during

SARS-CoV-2 convalescence .

Subsequent gating on CD21 and C D27 expression all owed the identi ﬁcation of four popul ations of class-

switched MBCs: CD21



CD27



atypical MBCs (also known as tissue-like memory); CD21



CD27

activated

MBCs; CD21

CD27

classical resting MBCs and C D21

CD27



switched naive (also known as intermediate

memory) MBCs (Figure 2E) as previously described.

Global defects i n the balance of the se MBC subsets

have been identiﬁed previously in PLWH (reviewed in

), i nclud ing those on cART,

with increased

numbers of activated and atypical MBCs concurrent with a decrease in resting MBCs. This phenotype is

exempliﬁed in (Figure 2E) for a PLWH and an HIV-negative control. We have hypothesized that these

inherent defects may have an impact on the quality of serological responses after SARS-CoV-2 vaccination.

Figure 1. Continued

biological repeats, each measured in duplicates. N numbers match those in (A). Line represents median for each group. Statistical test: Mann Whitney

U-test (MWU).

(D) Longitu dina l ID

titers for HIV-negative controls without prior SARS-CoV-2 infection who provided at least two longitudinal samples, including a post

ﬁrst dose sample. Samples that were neutralizing after the ﬁrst dose are categorized as exhibiting a standard neutralizing response and colored gray,those

that only achieve neutralization after the second dose, exhibit a delayed neutralizing response and are color-coded in magenta. N numbers for each

category are indicated o n the graph.

(E) Shows the equivalent data for PLWH without prior SARS-CoV-2 infection.

(F) Shows the equivalent data for HIV-negative controls with prior SARS-CoV-2 infection.

(G) Shows the equivalent data for PLWH with prior SARS-CoV-2 infection.

(H) CD4 T cell counts and (I) CD4:CD8 T cell ratio for PLWH stratiﬁed by standard (gray) or delayed neutralization (magenta). N numbers are as per D-G. Line

represents median for each group. Statistical test: MWU. *p > 0.05; **p > 0.01; ***p > 0.001 and ****p > 0.0001. See also Figure S1.

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6 iScience 26, 105862, January 20, 2023

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Global phenotyping of the MBC response after the ﬁrst vaccine dose revealed that individuals with delayed

neutralization, consisting largely of PLWH, had signiﬁca ntly lower numbers of restin g MBCs (CD21

CD27

)

and greater numbers of both CD21



CD27

activated MBCs and CD21



CD27



atypical MBCs compared to

those with standard neutralization (Figure 2F). Moreover, lower frequencies of resting MBCs correlated

with lower nAb titers (Figure 2G), this a ssociation is driven by participants with standard n eutralization

and some individuals with delayed neu tral izatio n do have reasonable n umbers of resting MBCs. Similarly,

higher levels of at ypical MBCs signiﬁcantly correla te d with lower nAb tit ers when considering those with

standard neutralization, although the strength of this association was relatively weak (r = 0.4867) ( Fig-

ure 2H). Frequencies of more than 10% atypical MBCs were only observed in the delayed neutralization

group. Tog ether these ﬁn din gs suggest that the MBC subset pertu rbati ons seen in PLWH could account

for the low er serol ogical output.

Improved neutralization breadth after the thirdSARS-CoV-2doseinPLWHbutlowernAb

responses persist and are associat ed with global, but no t spike-speci ﬁc, MBC dysfunction

To assess the breadth of nAb responses across the cohort, samples from all timepoints were tested against

an Omicron pseudovirus (BA.1 strain), which represented the dominant circulating strain at the time of

the post third vaccine dose sampling. Owing to the substantial antigenic changes in the Omicron spike,

participants with no prior infection, over 50% of HIV-negative controls and more than 90% of PLWH were

not able to neutralize Omicron after the ﬁrst vaccine dose (Figure 3A). The second dose enabled most of

the control group to mount a neutralizing response whereas only a quarter of SARS-CoV-2 naive PLWH

had nAbs against Omicron. In the SARS-CoV-2 naive groups, the third dose enabled 100% of HIV-negative

controls to neutralize Omicron and increased the frequency of neutralization among PLWH to over 70%

(Figure 3A). As in the analysis of WT neutralization for individuals without prior SARS-CoV-2 infection,

median Omicron ID

titers were lower in SARS-CoV-2 naive PLWH compared to HIV-negative controls at all

timepoints (Figure 3B). In addition, there was no signiﬁcant difference when individuals with complex co-mor-

bidities were removed from the PLWH cohort at the third vaccine dose (Figure S3C) or whether they had pre-

viously been infected with SARS-CoV-2. These data suggest that the third vaccine dose was effective in both

boosting nAb titer and broadening the response to Omicron, especially in SARS-CoV-2 naive PLWH, thus

rendering their responses closer to those of SARS-CoV-2 naive HIV-negative controls (Figures 3B and 3C).

Next, we evaluate d cross-sectionally the B cell phenotype after th e third v acci ne dose. In contrast to the

ﬁrst vac cine dose, there was no signiﬁcant difference between the frequency of spike-speciﬁc MBCs

when individ uals were stratiﬁed by whether th ey had been previously infected with SARS -CoV- 2 or not (Fig-

ure 3D) regardless of HIV status. However, the frequency of spike-speciﬁc MBCs after the third dose corre-

lated with Omicron titers (Fi gure 3E).Thissuggeststhatafterthreevaccinedosestheseindividualshad

mounted a speciﬁc B cell response, and that the quantity of spike-speciﬁc B cells remained linked to the

improved neutralization pote ncy and breadth obser ved (Figure s 3A–3C). Given that all individuals as-

sessed after the third dose made a robust spike-speciﬁc MBC response, we wanted to evaluate further

Figure 2. Neutralization titer is a ssociat ed wit h the frequency of spike-speciﬁc MBCs after t he ﬁrst vaccine dose

(A) Spike-speciﬁc MBCs (CD19

CD20

CD38

lo/mid

IgD-excluding switched naive CD27



CD21

cell) according to dual positivity for spike-PE and spike-APC

to exclude non-speciﬁc binding in a representative naive pre-vaccine sample (left) or representativ e post-vaccine sample (right) after the ﬁrst vaccine dose.

(B) Percentage of spike-speciﬁc MBC after the ﬁrst vaccine dose stratiﬁed by prior SARS-CoV-2 infection. Line represents median for each group. Statistical

test: M-Whitney U test (MWU). Dotted lines represent lower limit of sensitivity of the assay (0.1% spike-speciﬁc MBCs, based on previous optimization).

(C) Percentage of spike-speciﬁc MBCs in SARS-CoV-2 naive donors after the ﬁrst vaccine dose, s tratiﬁed by delayed (magenta) or standard (gray )

neutralization proﬁle. Line represents median for each group. Statistical test: MWU. Dotted lines represent lower limit of sensitivity of the assay (0.1% spike-

speciﬁc MBCs).

(D) Correlation of the percent of spike-speciﬁc MBC with WT ID

titers stratiﬁed by PLWH (blue) and controls (gray) after the ﬁrst dose, statistical test:

Spearman’s rank correlation coefﬁcient.

(E) Distribution of MBCs (CD19

CD20

CD38

lo/mid

IgD-) subtypes according to CD27-BUV395 and CD21-BV711 in a representative HIV-negative donor

sample (left) or PLWH donor sample (right).

(F) Percentage of MBC subtypes (activated CD27

CD21



; resting CD27

CD21

; switched naive; switched naive CD27



CD21

and CD27



CD21



atypical)

after the ﬁrst vaccine do se stratiﬁed by delayed or standard neutralization proﬁle. Line represents median for ea ch group. Statistical test: MWU.

(G) Correlation of the percentage of resting CD27

CD21

MBCs with WT ID

titers stratiﬁed by delayed (magenta) or standard (gray) neutralization proﬁle

after the ﬁrst vaccine do se, statistical test: Spearman’s rank correlation coefﬁcient.

(H) Correlation of the percent of switched naive CD27



CD21

MBCs with WT ID

titers stratiﬁed by delayed (magenta) or standard (gray) neutralization

proﬁle after the ﬁrst vaccine dose, statistical test: Spearman’s rank correlation coefﬁcient. *p > 0.05; **p > 0.01; ***p > 0.001 and *** *p > 0 .0001.

See also Figure S6.

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14 iScience 26, 105862, January 20, 2023

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in the frequencies of the main T cell subsets in the two groups (Fig ures S6C and S6D). Among CD 4 T cell s,

there w as a reduction in ci rculat ing CXCR3

CXCR5

Tfollicularhelper(T

) subsets observed in HIV-pos-

itive nAb

/low

compared to nAb

high

donors (Figures 6A and 6B). The reduc ed abundance of CXCR3

CXCR5

in nAb

/low

HIV-positive subjects was further conﬁrmed by manual gating (Fi gures 6C, 6D,

and S3C). CXCR3

CXCR5

cells corre late d with SARS-CoV- 2 neutralizatio n l ev els in HIV-po sitiv e

SARS-CoV-2 naive individuals (r = 0.5294 p = 0.02388) (Figur e 6E), suggesting t hat reduced availabilit y of

cells could inﬂuence the magnitude of vaccin e-ind uced S ARS-C oV-2 antibody responses.

We have next examined the CD8 T cell compartment in the two groups. Notably, a prominent cluster delin-

eated by the expression of CXCR3

CD127

CD38

CCR7

CD45RA

was signi ﬁcantl y enriched in PLWH

SARS-CoV-2- nAb

/low

(Figures 6F, 6G, and S6B). The high er abundance of CXCR3

CD127

CD38

CCR7

CD45RA

cells in PLWH SARS-CoV-2- n Ab

/low

was further conﬁrmed by manual gating

(p = 0.04) (Figures 6H, 6I, and S6C). Correlation an alysis of these populations showe d a positive association

between their frequencies and SARS-CoV-2-speciﬁc T cell res ponses following two vaccine doses i n PLWH

with nAb

/low

(Figure 6J), supporting the notion that these subsets could contribute to the observed induc-

tion of T cell responses in PLWH who lacked or generated low nAb responses. Overall, our analysis of the

global T cell proﬁle of individuals with low/absent nAbs but detectable functional T cell responses revealed

that reduced availability o f T

cells could contribute to the serological defect observed in conjunction with

the previously highlighted imbalance in MBCs. Mor eover , we have i dentiﬁ ed a subset of CD8 T cells that is

overrepresented in PLWH wi th low/absent nAbs and may enable stronger functional T ce ll responses, sup-

ported by rec ent ﬁndings showing that CXCR3

CD8 T cells are polyfunctional and associated with survival

in critical SARS-CoV-2 patients, and have been observed in other immunosuppressed groups.

53,54

DISCUSSION

Accumulating evidence suggests th at a br oad and we ll- coordinat ed immune response is required for

protection against se vere COVID -19 dise ase. The emergence of VOCs with inc reased abi lity t o evade

nAbs has reinforced the need for a more comprehensive assessment of adaptive immunity after vaccina-

tion, especially in more vulnerable g roups including some PLWH. Our data indicate that PLWH who are

well controlled on cART, elicited poorer humoral responses, in terms of magnitude and neutralizing ability

compared to HIV-negative donors following ﬁrst, second and third doses of SARS-CoV-2 vaccine. This was

related to global B cell but not anti gen- speciﬁ c B cell dysfunction. This suggests that t he overall distur-

bance in memory B cell homeost asis during HI V can limit the amount or quality of serum antibody pro-

duced indirectly, potentially by decreasing the total number of B cells available to participate in the anti-

gen-speciﬁc response. In contrast, the observation that antigen-speciﬁc B cells in these individuals are not

overtly dysfunctional suggests that lower serum titers are not the result of antigen-speciﬁc B cells failing to

respond fully, as has been suggested in some chronic diseases.

In contrast, T cell responses were com-

parable in the two groups and detectable, even in a sm all group of PLWH with very poor serological re-

sponses, suggesting a potentially important non-redundant immunological role for functional T cells.

Overall, our data reinforce the beneﬁcial effect of an additional vaccine dose in boosting adaptive immune

responses,

especially against circulating VOCs in this patient group.

Weaker humoral responses were observed in PLWH compared to HIV-negative controls after each dose of

vaccine when matched by prior SARS-CoV-2 status. Although the third dose largely narrowed the gap be-

tween PLWH and controls, and enabled Omic ron neutrali zation , 13% of SARS-CoV-2 naive PLWH st ill had

no nAbs after 3 vaccin e doses. This hig hlight s t he importance of r epeated vaccination in PLWH and sug-

gests additional doses/targeted vaccines could be merited , e specially given 28% of SARS-CoV-2 naive

PLWH failed to neutr alize Omicron after the third vaccine dose. Owing to known defects in germinal center

reactions in chronic HIV infection (as reviewed in

29,30,55

recall responses following vaccination in PLWH are

likely impaired resulti ng in lower tit ers and narrowe r neutrali zation breadt h. As such, addition al vaccina-

tions to stimulate additional afﬁnity maturation and diversiﬁcation of the response are likely needed to

achieve similar outcomes to those seen in HIV-negativ e individuals. In support of this, previousl y, it has

been shown that PLWH can be neﬁt fr om an ad ditional vaccine dose and accelerated sc hedule s durin g hep-

atitis B immunization.

Moreover, in other immunocompromised groups, repeated vaccination with a third

vaccine dose resulted in similarly improved levels of seroconversion and breadth against VOCs.

57,58

Previous studies among similar cohorts of PLWH with undetectable HIV viral loads have produced mixed

results, as pr evio usly reviewed.

SARS-CoV-2 viral vector vaccines have shown similar magnitude and

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durability of antibody responses to HIV-negative controls

23,59

but reduced levels of seroconversion and

neutralization have been reported after two doses in PLWH in a more recent study.

Furthermore, viral vec-

tor vaccines, lower CD4 T cell count/viremia and old age have been linked to lower serological responses

and breakthrough infection.

In terms of mRNA vaccines, both non-signiﬁcant

60,61

and signiﬁcant de-

creases in humoral responseshavebeenreportedinPLWH.

62–65

These differ ences may be be cause of

the size of cohorts examined and the range of immune reconstit ution in these PLWH. In contrast to previ ous

work,

8–10,12

we have found no association between the CD4 T cell count and serological outcome, which

could be beca use of insu fﬁcient power in this stu dy to de tect differences. The recent study by

demon-

strated a stronger humoral response after the third dose of vaccin e in PLWH, regardless of their CD4

T cell count, which is consistent with our ﬁndings,

Thus, the l ow er level of nAbs observed here in

PLWH could be in part owing to potential differences in boosting of memory responses to enable breadth

against Omicron after three vaccine doses.

Serological data correlated signiﬁcantly with frequency of spike-speciﬁc MBCs. The B cell phenotyping

conﬁrmed the charac teri stic and persistent defects seen in global MBCs in the setting of HIV (reviewed

). Speciﬁ cally, we have observed low er frequencies of resting MBCs and higher freq uenci es of atypical

and activated MBCs. This dysregulated MBC phenotype was also associated with a delay in developing

nAbs after the ﬁrst dose regardless of HIV status. Further evaluation in a group of individuals after the third

vaccine dose led to the interesting observation that although the global MBC landscape is still disrupted

with lower levels of resting MBCs and higher levels of atypical and switched naive MBCs in PLWH, this is not

reﬂected in the antigen -speciﬁc MBCs. Spike-speciﬁc MBCs present in P LWH had a similar memory B cel l

phenotype as HIV-ne gative con tro ls, albeit fe wer rest ing MBCs. H owever , higher l evels of gl obal atyp ical

MBCs, also observed in PLWH with lower neutralization at the third vaccine dose, suggest that the excess

atypical MBCs may be effectively exhausted, as has been described.

Therefore, SARS-CoV-2 serum anti-

body responses may be lower not because spike-speciﬁc responses are enriched within atypical MBCs

and therefore unable to progress to an antibody secreting phenotype (as has been postu lated for

HI V/HBV

27,67

), but r ather because of global MBC disturbance. Thus, we propose that this reduced nAb

to vaccination in PLWH may not be because of an alteration in the p henotype of antigen-speciﬁc cells

but rat her limited numbers of MBCs availa ble to partici pate in the antigen-speciﬁc response vi a the canon-

ical pathway.

In cont rast to serologi cal responses, SARS -CoV-2 vaccination elicited comparable T cell responses be-

tween PLWH and HIV-negat ive controls at all sampling points, and these responses were largel y preserved

against circulating VOCs, including Omicron, following three vaccine doses. These ﬁn dings are in line with

the recent observations showing a robust T cell response to SARS-CoV-2 after third dose in PLWH,

including to known VOCs, with either homologou s or hete rolo gous combin ation s of S ARS-CoV-2 vac-

cines.

19,20

Of interest, in a recent study despite the detection of si gniﬁcant T cell responses post a third

dose mRNA vacc ine in PLWH wh o had completed an mRNA primary c ourse, these resp onses were re-

ported to be impaired compared to the general population.

These results contra st our ﬁndi ngs and cou ld

be attributed to the diffe rent st udy design/ vaccine pl atforms a nd quanti ﬁcatio n of T cell r esponses using

whole blood assay stimulation and IFN-g quantiﬁcation via EL ISA.

Similarly, to the scenario seen in a nti -

body responses, pr ior SARS-Co V-2 infection also resulted in higher T ce ll responses to vaccination.

34,46,47

Of interest, detectable T cell responses were noted in a proportion of SARS-CoV-2 naive indi vidual s at

baseline,

23,45

which could repr esent p re-e xi sting cr oss-re activ e T cel l c ells due to past infecti on wi th ot her

coronaviruses.

An association between CD4 T cell counts and the magnitude of T cell responses was

observed in SARS-CoV -2 naive PLWH fo llowi ng vacci natio n, highl ight ing the relev ance of immun e cell

reconstitution in producing effective immunity to vaccination, especially in people who lack memory re-

sponses elicited by natural infection. In this cohort, PLWH were well-controlled on cART and had undetect-

able HIV viral loads. Both PLWH with, and without, prior SARS-CoV-2 exposure had similar median CD4

T cell counts (602 and 560 cells/mL, respectively) despite different serological outcomes. However, the

full impact of HIV-related immunosuppression, i n addition to other factors, includin g age, sex and pres-

ence of co-morbidities, in dampening effective and long-lived memory responses needs to be addressed

in future larger prospective studies. It is possible that different vaccine schedules, i.e., homologous versus

heterologous vaccination, could also account for the obse rved heterogen eity in cellu lar immune re-

sponses. A h eterol ogous viral vector ed/mRNA vaccinat ion has been described to lead to increased reac-

togenicity, combining the advantages from both vaccine classes.

Owing to li mited numbers, it has not

been possible t o addr ess the impact of differe nt vaccine platforms in our cohort. However, previ ous

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16 iScience 26, 105862, January 20, 2023

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work has shown that the adenovirus-based platforms induce a higher T cell response

70–72

whereas mRNA

vaccine generates a stronger antibody response.

72–74

The use of heterologous boosting strategies has

been shown to expand the quantity and the breadth of T cell immunity and improve the serological

response in PLWH.

70,75–7 7

Therefore, we speculate tha t d iffe rent vaccine plat for ms and/ or heterologous

versus homogeneous combinations could lead to different magnitude T cell responses in our cohort. How-

ever, there are insufﬁcient data to recommend the best vaccine approach to in duce a more effective, resil-

ient, and durable r esponse in PLWH. Instead, optimization of vaccine schedules requires a randomized

controlled trial to directly compare the immunogenicity of different vaccine platforms to design the

most effective vaccination schedules.

Overall humo ral responses correlated with the magnitude of T cell responses and our ﬁndings corroborate

the importance of T

cells supporting effective B cell responses after vaccination. Notably, in a small sub-

group of patients (serological non- or low-level responders), there were detectable T cell responses charac-

terized by a CXCR3+CD127+ CD8 T phenotype. This phenotype was not clearly related to HIV parameters or

presence of co-mor bidit ies. These T cell populations have been linked with i ncrease d survival in people in-

fected with SARS- CoV-2 and a re co nsistent w ith observ ation s in patie nt groups who lack B cell responses.

Upregulation of CXCR3 in vaccine-induc ed T cells with potential to home to lung mucosa in tuberculosis

suggests that the se CD8 T cells described herein could play a role in the protection against severe respira-

tory diseases su ch as SARS-CoV-2. One possibility is that in the absence of functional antibody responses,

the increased abund ance of viral antigens could drive CXCR3+ CD8

T cell proliferation, as these cells have

been shown to have an enhanced proliferative capacity

and improved effector differentiation.

80,81

These

CXCR3+ CD8

T cells could confer a degree of protection by localization to infected tissue compart-

ments,

79,82

and provide si te-s peci ﬁc responses, which are know n to be important in protection against res-

piratory disease.

CD8

T cells havealsobeen shown to expand following vaccination in patients receiving B

cell depleting therapies

and to contribute to vaccine mediated protection against SARS-CoV-2 in rhesus

macaques.

However, studies in larger cohorts with breakthrough infections are necessary to clearly eval-

uate the contribution of these CD8

T cell popu lations in va ccine-medi ated protection.

Overall, our data support the beneﬁt of a third SARS-CoV-2 dose in inducing nAbs against Omicron in

PLWH, as it does in the general popu lation. Mo reove r, our stu dy provi des new insight s into the reasons

why some PLWH f ail to produce effectiv e humoral responses via an in-depth assessment of B cell re-

sponses. Speciﬁcally, w e ﬁnd that glob al B cell dysfuncti on is relat ed to lower ser ologi cal output in t erms

of both bindi ng and neutralizing responses. A ntibody responses take longer to de velop in individuals with

greater global B cell dysfunction, which is most commonly seen in PLWH. Although a third SARS-CoV-2 vac-

cine dose improves neutralization potency and breadth for many, lower titers and MBC disturbance are still

observed. Prospective longi tudi nal studies are now needed to assess whe ther global B cell disturb ance

ﬂuctuates in PLWH on cART ov er time, what treatments/co-mor biditie s inﬂuence thi s and what level of B

cell dysfunction results in inferior clinical outcomes long-term with regards to infectious diseases, particu-

larly where vaccination has taken place. In parallel, CD8

T cell proﬁles and anti-viral T cell activity should be

monitored in such studies to u nderst and whether these cells do provid e the proposed immunological

compensation for defects in humoral immunity.

Limitations of the study

Our study has several limitations. These include a cross-sectional analysis, which precludes the establishment

of causal relationships. Our cohort is heterogeneous, with differences in sex, age and levels of immunosuppres-

sion that may contribute to the variability in the magnitude of responses. Moreover, the current analysis

provides an overview of responses after up to three vaccine doses, and therefore further work is required to

assess the durability and resilience of these responses against subvariants and additional vaccine doses. In

addition, we could not assess the impact of breakthrough infections on humoral and cellular immune response,

although some individuals were infected with SARS-CoV-2 after vaccination as noted above, because the

numbers of re-infections were too low across any given timepoint for meaningful analysis.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following:

d KE Y RE SOURCES TABLE

d RE SOURCE AVAILABILITY

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iScience 26, 105862, January 20, 2023 17

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Article

B Lead contact

B Materials availability

B Data and code availability

d EX PERIMENTAL MODEL AND SUBJ ECT DETAILS

B Ethics statement

B Patien t r ecruit ment and sampling

d ME THOD DETAILS

B PBMC isolation

B Semi-quantitative S1 ELISA

B Total IgG ELISA

B IgG puriﬁcation

B Pseudovi rus production

B Pseudovi rus neutralization

B Live neutralization

B Production of bi otinyl ated prote in

B B cell phenotypic ﬂow cy tometri c an alysis

B T cell phenotypic ﬂow cytometric analysis

B High-dimensional data analysis

B Ex vivo IFN-g ELISpot assay

B Overlapping pepti de pool s

d QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2022.105862.

ACKNOWLEDGMENTS

We are grateful to all the clinic staff and participants at the Mortimer Market Center and Ian Charleson Day

Center, in particular Rebecca Matthews, Aran Dhillon, Connor McAlpine, Paulina Prymas, Marzia Fiorino,

Thomas Fernandez, Jonathan Edwards and Hemat Nargis. The authors would like to thank James E Voss

of the Scripps Research Institute for the gift of Hela ACE2 expressing cells and Peter Cherepanov of the

Francis Cri ck I nstitut e for recombinant S1 antigen.

This study was supported by UKRI MRC grant (MR/W020556/1) to LE M, DP and EM and an NIH award

(R01AI55182) to (DP). LEM receives funding from the European Research Council (ERC) under the European

Union’s Horizon 2020 research and innovation program (Grant Agreement No. 757601) and i s supported by

an MRC Career Development Award (MR/R008698/1). NJM is supported by an MRC grant (MR/T032413/1),

an NHSBT grant (WPA15-02), the Wellcome Trust ISSF (204845/Z/16/Z), Addenbrooke’s Charitable Trust,

Cambridge University Hospitals (900239) and the NIHR Cambridge BRC. E T is supported by an MRC stu-

dentship (MR/N013867/1), AA by a Saudi Ministry of Education studentship (FG-350441) and TF by a Well-

come Trust Clinical PhD Fellowship (216358/Z/19/Z). NJM is supportedby an MRC g rant (MR/T032413/1),

an NHSBT grant (WPA15-02), the Addenbrooke’s Charitable Trust, Cambridge University Hospitals

(900239) and the NIHR Cambridge BRC.

AUTHOR CONTRIBUTIONS

Conceptualizati on: L.E.M. an d D.P. Invest igation: E.T., A.A., R.F., N.H. , P.P.G. , H-L.S., C.R -S., L.M. , E.G-M. ,

J.K., D.J., A.J-S., C.L. Preexisting and TAF Analysis: E.T., A.A., P.P.G., N.J.M., D.P., and L.E.M. Patient

Recruitment: D.P., I.W. , C.M., I.M., L.W., M.J., S.M., M. Y., T.J.B., F.B., R.G., a nd S.K. Resources: L.E.M.,

D.P., E.M., and N.J.M. Writing – Original Draft: E.T., A.A., D.P., and L.E.M. Writing – Review and Editing:

E.T., A.A., R.F., N.H., P.P.G., H-L.S., C.R-S., L.M., E.G-M., J.K., D.J., A.J-S., C.L.P., T.A.F., I.W., C.M., I.M.,

L.W., M.J., S.M., M.Y., T.J.B., F.B., S.K., S.R-J., R.G., N.J.M., E.M., D.P., and L.E.M. Supervision: L.E.M.,

D.P., E.M., N.J.M., and S.R-J. Funding A cquis ition: L.E.M., D.P., E.M., and N.J. M.

DECLARATION OF INTERESTS

The authors declare no c ompeting interests.

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18 iScience 26, 105862, January 20, 2023

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INCLUSION AND DIVERSITY

One or more of the authors of this paper self- iden tiﬁes as an underreprese nted et hnic m inorit y in science.

One or more of the authors of this paper se lf-ident iﬁed as a member of the LGBTQ + community.

Received: November 1, 2022

Revised: December 4, 20 22

Accepted: December 20, 2022

Published: J anua ry 20, 2023

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22 iScience 26, 105862, January 20, 2023

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Goat anti-human F(ab)’2 Jackson ImmunoResearch Cat# 109-006-006; RRID: AB_2337553

Alkaline phosphatase-conjugated

goat anti-human IgG

Jackson ImmunoResearch Cat#109-055-098; RRID: AB_2337608

FITC Mouse Anti-Human IgG BD Biosciences Cat # 560952; RRID:AB_10563419

BV786 Mouse Anti-Human CD19 BD Biosciences Cat # 740968; RRID:AB_2740593

BUV395 Mouse Anti-Human CD27 BD Biosciences Cat # 563815; RRID:AB_2744349

PE-Cy7 Mouse Anti-Human IgD BD Biosciences Cat # 561314; RRID:AB_10642457

APC/Cyanine7 anti-human IgM Antibody BioLegend Cat # 314520; RRID:AB_10900422

Alexa Fluor 700 Mouse Anti-Human CD20 BD Biosciences Cat # 560631; RRID:AB_1727447

BV711 Mouse Anti-Human CD21 BD Biosciences Cat # 563163; RRID:AB_2738040

PE-CF594 Mouse Anti-Human CD38 BD Biosciences Cat # 562288; RRID:AB_11153122

Brilliant Violet 510 anti-human CD3 Antibody BioLegend Cat # 317332; RRID:AB_2561943

Brilliant Violet 510 anti-human CD14 Antibody BioLegend Cat # 301842; RRID:AB_2561946

APC/Cy7 anti-human CD197 (CCR7) BioLegend Cat # 353212; RRID:AB_10916390

Brilliant Violet 650 anti-human

CD127 (IL-7Ra) Antibody

BioLegend Cat # 351325; RRID:AB_11125369

Brilliant Violet 650 anti-human CD3 Antibody BioLegend Cat # 317324; RRID:AB_2563352

Brilliant Violet 711 anti-human CD27 Antibody BioLegend Cat # 302833; RRID:AB_11219201

Brilliant Violet 785 anti-human CD38 Antibody BioLegend Cat # 303530; RRID:AB_2565893

Alexa Fluor 700 anti-human CD45RA Antibody BioLegend Cat # 304120; RRID:AB_493763

Brilliant Violet 421 anti-human

CD279 (PD-1) Antibody

BioLegend Cat # 329920; RRID:AB_10960742

PE/Dazzle 594 anti-human CD4 Antibody BioLegend Cat # 300548; RRID:AB_2563566

Brilliant Violet 711 anti-human CD8a Antibody BioLegend Cat # 301044; RRID:AB_2562906

Brilliant Violet 510 anti-human CD14 Antibody BioLegend Cat # 301842; RRID:AB_2561946

Brilliant Violet 510 anti-human CD19 Antibody BioLegend Cat # 302242; RRID:AB_2561668

BB515 Rat Anti-Human CXCR5 (CD185) BD Biosciences Cat # 564624; RRID:AB_2738871

BV605 Mouse Anti-Human CD56 BD Biosciences Cat # 562780; RRID:AB_2728700

PE-Cy7 Mouse Anti-Human CD25 BD Biosciences Cat # 335824; RRID:AB_2868687

PE-Cy5 Mouse Anti-Human CD183 BD Biosciences Cat # 551128; RRID:AB_394061

PerCP-eFluor 710 Anti-Human CD3 eBioscience Cat # 46-0037-42; RRID:AB_1834395

APC Anti-Human CD19 BioLegend Cat # 302212; RRID:AB_314242

Brilliant Violet 421 Streptavidin BioLegend Cat # 405226

anti-human IFN-gamma mAb 1-D1K, puriﬁed Mabtech Cat # 3420-3-1000; RRID:AB_907282

Anti-human IFN-g mAb (7-B6-1), biotin Mabtech Cat # 3420-6-250; RRID:AB_907273

PE-Streptavidin Agilent Cat # PJRS25-1

APC-Streptavidin Agilent Cat # PJ25S

Virus strains

SARS-CoV-2 (lineage B) isolate

SARS-CoV-2/human/Liverpool/REMRQ0001/2020

Ian Goodfellow (University of Cambridge)

isolated by Lance Turtle (University of

Liverpool) and David Matthews and

Andrew Davidson (University of Bristol)

N/A

(Continued on next page)

OPEN ACCESS

iScience 26, 105862, January 20, 2023 23

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Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

Biological samples

Donor blood samples Mortimer Market Centre and Ian

Charleson Day Centre,

RoyalFree Hospital

N/A

Chemicals, peptides, and recombinant proteins

PepTivator CMV pp65, human Miltenyi Biotec Cat # 130-093-438

PepTivator SARS-CoV-2 Prot_S Delta Miltenyi Biotec Cat # 130-128-763

PepTivator SARS-CoV-2 Prot_S Com Miltenyi Biotec Cat # 130-127-953

PepTivator SARS-C oV-2 Prot_S Omicron Miltenyi Biotec Cat # 130-129-928

Wuhan Hu-1 Alpha Biochem Shanghai Customized

Wuhan Hu-1 Beta Biochem Shanghai Customized

Alpha Mutation Pool Biochem Shanghai Customized

Beta Mutation Pool Biochem Shanghai Customized

BD Cytoﬁx/Cytoperm Fixation/

Permeabilization Solution Kit

BD Biosciences Cat # 554714; RRID:AB_2869008

Phytohemagglutinin-L (PHA-L) Sigma-aldrich Cat # 11-249-738-001

mmPACT AMEC Red Substrate, Peroxidase 2B Scientiﬁc Cat # SK-4285

Vectastain ELite ABC PK-6100 2B Scientiﬁc Cat # PK-6100

LIVE/DEAD Fixable Blue Dead Cell Stain Invitrogen Cat #L23105

BD CompBeads anti-mouse Ig, k BD Biosciences Cat # 552843; AB_10051478

PEI-Max Polysciences, Inc Cat # 23966

Biotin Sigma-Aldrich Cat #B4639

Imidazole Sigma-Aldrich Cat #I2399

Recombinant biotinyl ated spike protein This study and

N/A

Recombinant biotinyl ated RBD protein This study and

N/A

SARS-CoV-2 spike S1 re protein Peter Cherepanov Laboratory

(The Francis Crick Institute)

N/A

Critical commercial assays

Bright-Glo Luciferase kit Promega Cat #E2650

Experimental models: Cell lines

HEK-293T/17 AmericanType Culture Collection ATCC CRL-11268

FreeStyle 293F Thermoﬁscher Scientiﬁc Cat #R79007

HeLa-ACE2 James Voss Laboratory (The

Scripps Research Institute)

N/A

HEK-293T cells expressing Renilla luciferase

(Rluc) and SARS-CoV-2 Papain-like

protease-activatable circularly permuted

ﬁreﬂy luciferase (FFluc)

Nicholas Matheson Laboratory

(University of Cambridge)

N/A

Recombinant DNA

HIV-1 luciferase reporter vector (pCSLW) Seow et al.

N/A

HIV-1 packaging construct (p8.91) Zufferey et al.

SARS-CoV-2 spike WT (Wuhan hu-1) Katie Doores Laboratory

(King’s College London)

N/A

SARS-CoV-2 spike Omicron (BA.1/B.1.1.259.1) Katie Doores Laboratory

(King’s College London)

N/A

(Continued on next page)

OPEN ACCESS

24 iScience 26, 105862, January 20, 2023

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RESOURCE AVAILABILITY

Lead contact

More information and req uests for reage nts should be directed to and will be fulﬁlled by th e lead contact ,

Laura E McCoy (l.mc coy@ucl.ac.uk).

Materials availability

This study did not generate unique resource s or materials.

Data and code availability

Any additional information required to reanalyze the data reported in this study is available from the lead

contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Ethics statement

The protocols and study documents for the study were approved by the local Research Ethics Committee

(REC) Berkshire (REC 16/SC/0265) and South Central - Hampshire B (REC 19/SC/0423). All subjects enrolled

into the study provided written informed consent. The study complied with all relevant ethical regulations

for work with human participants and conformed to the Helsinki declaration principles and Good Clinical

Practice (GCP) guidelines.

Patient recruitment and sampling

There were 110 HIV + par ticipan ts who were virally supp ressed and on cART and 64 HIV-n egative heal thy

controls were recruited as part of either the Jenner II or the Vaccine in Clinical Infection (VCI) cohorts.

PBMCs and plasma (o r ser um) w ere col lected at the following t imepoi nts: base line, post-ﬁrst dose

(R12 day s following the ﬁrst dose), post-second dose (%70 days following the second dose), pre-third

dose (R70 days following the second dose) to evaluat e wani ng response,

41,90

and post-third dose

(>7 days following the third dose). Foll owing each dose days cut-off was chosen to allo w development

of a de-novo (or recall) immune respon se foll owing vacci nati on.

Participants received a mix of avail able

SARS-CoV-2 v accination (Pﬁzer-BioNTech’s BNT162b2; Moderna’s mRNA-1273 or Astra-Zeneca’s

AZD1222) according to Joint Committee on Vaccination and Immunization, UK, guidelines.

Not eve ry

participant was sampled at all timepoints. At each visit, participants were asked to report any history of

SARS-CoV-2 infection.

Between vaccinations, 7 previously SARS-CoV-2 naı

ve participants (3 HIV-, 4 HIV+) reported a SARS-CoV-2

infection, as such, any subsequent timepoints were moved into the ‘prior SARS-CoV-2 infection’ group for

analysis. Similarly, 4 participants with prior SARS-CoV-2 reported a further infection (3 HIV-, 1 HIV+). All par-

ticipants were recruite d at the Mor timer Market Cent re for Sexual Health and HIV Researc h and the Ian

Charleson Day Centre at the RoyalFree Hospital (London, UK) following written informed consent as

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

RBD-Avi-His tag Katie Doores Laboratory

(King’s College London)

N/A

SARS-CoV-2 Spike Avi-His tag Katie Doores Laboratory

(King’s College London)

N/A

BirA Addgene Cat # 20856

Software and algorithms

Flowjo 10.8.1 FlowJo, LLC https://www.ﬂowjo.com

GraphPad Prism 9.0.0 GraphPad https://www.graphpad.com

Cytobank Beckman Coulter https://premium.cytobank.or g

Other

MSCRN-IP DURA 0.45UM CLEAR 50/PK Millipore Cat # MAIPN45 50

OPEN ACCESS

iScience 26, 105862, January 20, 2023 25

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part of a study approved by the local ethics board committee. Additional information about demographic

and sampling can be found in Table S1.

METHOD DETAILS

PBMC isolation

Whole blood was colle cted i n hepari n-coated tube s. PBMC s were i solated from wh ole blood via de nsity -

gradient sedimentation. Whole blood was ﬁrst spun v ia centrifugation for 5 minat 800g. Plasma was then

collected, ali quo ted and store d at 80



C for further use. Remaining blood was diluted with RPMI (Gibco,

Paisley, UK), layered over an appropriate volume of Ficoll (Cytiva, Uppsala, Sweden) and then spun via

centrifugation for 20 minat 800g without brake. The PBMC layer was collected and washed with RPMI to

be spun via centrifugation f or 10 minat 400g. PBMCs were stained with t rypan blue and counted using

Automated Cell Counter (BioRad, Hercules, Cal iforn ia, USA). PBMCs were then cryo preserve d in a cry ovial

in cell recovery freezing medium containing 10% dimethyl sulfoxide (DMSO) (Honeywell, Seetze, Germany)

and 90% heat-inactivated fetal bovine serum (FBS) and stored at 80



C in a Mr. Frosty freezing container

overnight before being tr ansferr ed into liquid ni tro gen for further stor age. If pr esent , serum s eparator

tubes were spun at 400g for 5 min to collect serum and then stored at 80



Cforfurtheruse.

Semi-quantitative S1 ELISA

This assay was set up previously by our lab.

45,91

In a 96-half-well NUNC Maxisorp

plate (Nalgene, NUNC

International, Hereford, UK), three columns were coated overnight at 4



Cwith25mLofgoatanti-human

F(ab)

2 (Jackson ImmunoResearch, Ely, UK) (1:1000) in PBS, the o ther nine columns were coated with

25 mL of SARS-CoV-2 WT S1 protein (a kind gift from Peter Cherepanov (Ng et al., 2020), The Francis Crick

Institute) at 3 mg/mLinPBS.Thenextday,plateswerewashedwithPBS-T(0.05%TweeninPBS)andblocked

for 1 hour (h) at room temperature (RT) with assay buffer (5% milk powder PBS-T). Assay buffer was then

removed and 25 mL of patient plasma at dilutions from 1:501:10,000 in assay buffer added to the S1-

coated wells in duplicate. Serial dilutions of known concentrations of IgG were added to the F(ab)

IgG-coated we lls in triplicate to generate an internal stand ard c ur ve. Aft er 2 h of incubation at RT, plates

were washed with PBS-T and 25 mL alkaline phosphatase (AP)-conjugated goat anti-human IgG (Jackson

ImmunoResearch) at a 1:1000 dilution was add ed to each well and incubated for 1 hat RT. Plates were

then washed w ith PBS-T, and 25 mL of AP substrate (Sigma Aldrich, St Loui s, Missouri, USA) added. Optical

density (OD) was measured using a Multiskan

FC (Thermo Fisher-Scientiﬁc, Horsham, UK) plate reader at

405 nm and S1-speciﬁc IgG titers were interpolated from the IgG standard curve using 4PL regression

curve-ﬁtting on GraphPad Prism 9 (GraphPad, San Diego, California, UK).

Total IgG ELI SA

To measure total IgG levels in plasma, a 96-half-well NUNC Maxisorp plate (Nal gen e) was entirely coated

overnight at 4



Cwith25mLofgoatanti-humanF(ab)

2 (1:1000). As above, plates were washed in PBS-T and

blocked for 1 hat RT i n assay buffer. 25 mL of seri al dilutions of patient plasma (1:1 00 to 1:10

) were added in

duplicates to the plate alongside known concentrations of IgG in triplicates. As above, after 2 h of incuba-

tion at RT, p lates were w ashed with PB S-T and 25 mL AP-conjugated goat anti-human IgG was added and

then incubated for 1 hat R T. Pl ate s were washed wi th P BS-T , and 25 mL of AP substrate added. ODs were

measured using a Multiskan FC plat e read er at 405 nm and total IgG titers interpol ated f rom the IgG stan-

dard curve using 4 PL regression curve-ﬁtting on GraphPad Prism 9.

IgG puriﬁcation

As the PLWH participants in this study were on cART which can interfere with the lentivirus-based pseudo-

type neutralization assay IgG was puriﬁed from plasma using a Pierce 96-well pro tein G spin plate

(ThermoFischer Scientiﬁc). Plasma was incubated in wells containing protein G at RT for 30 min. The

captured IgG was then eluted with 0.1M Glycine (pH = 2-3) twice into 2M Tris (p H = 7.5-9) buffer. To remove

Tris/Glycine buffer from the puriﬁed IgG , the elua te was concen trated (Thermo Scientiﬁc Pierce Prote in

Concentrator PES, 50K MWCO, 0.5 mL) and washed thrice at 10000 rpm for 10 min before quantiﬁcation

by measuring absorbance of 280 nm on a NanoDrop

(ThermoFischer, Rockford, Illinois, UK). The entire

volume of puriﬁed IgG was then ﬁltered sterile using a 0.22 mm PDVF hydrophilic membrane Fil trE X ﬁlter

plate (Corn ing, Corning, NY, U SA) and stored at 4



Cforfurtheruse.

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26 iScience 26, 105862, January 20, 2023

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Pseudovirus production

In a T75 ﬂask, 3 3 10

HEK-293T cells were seeded in 10 mL of complete DMEM Dulbecco’s Modiﬁed Eagle’s

Medium (Gibco) suppl emente d with 10% FBS and 50 mg/mL penicillin-streptomycin. The next day, the

following transfection mix was prepared: 1 mL of Opti-MEM

(Gibco); 90 mL of PEI-max (1 mg/mL);

10 mg of p8.91 HIV-1 gag/pol packaging plasmid

; pCSLW HIV-1 luciferase reporte r vector p lasmid

and 5 mg of either S ARS-CoV-2 spike plasmid of interest, speciﬁcally WT (Wuhan-hu-1) or Omicron (BA.1/

B.1.1.529.1)

as indicated in the results section. The transfection mix was left to incub ate for 20 min before

being added to the cells and left to incubate at 37



C5%CO

for 72 h before being collected and ﬁltered

through a 0.45 mm ﬁlter (Millipore, Cork, Ireland) and either used directly in an assay or stored at 80



Pseudovirus neutralization

Neutralization assays were performed in 96-well plates by adding either duplicate serial dilutions of neat

plasma in complete Dulbecco’s Modiﬁed Eagle medium (Thermo F isher Scientiﬁc-UK) (DMEM) starting

at 1:20 dilution for HIV-negative samples or the appropriate amount of puriﬁed IgG for H IV + s amples

to give a starting dilution equivalent to 200 or 400 mg/mL of IgG as based on total IgG. These dilutions

were incub ated with the appropri ate amount of ﬁl tered pseudotyped vi rus for 1 h at 37



C5%CO

before

adding 10000/mL HeLa-ACE2 cells

(kind gift from James Voss, The Scripps R esearch I nstitut e, USA ) in

100 mL per well. After a 72 h incubation at 37



C5%CO

, the supernatant was removed, and cells lysed.

Bright-G lo

luciferase substrate (Promega, Madison, Wisconsin, USA) was added, and relative light

unit (RLU) values were read on a Glomax (Promega) or BioTek Synergy

H1 (Agilent) plate reader.

RLU readouts were used to calculate the reciprocal inhibitory dilution at which 50% of the virus activity is

neutralized by plasma (ID

) for each sampl e on GraphPad Prism 9.

Live neutralization

The SARS-CoV -2 v irus used in th is st udy was the wild-t ype (l ineage B) isolate S ARS- CoV-2/ human/Li ver-

pool/REMRQ0001/2020, a kind gift from Ian Goodfellow (University of Cambridge, UK), isolated by Lance

Turtle (University of Liverpool, UK) and David Matthews and Andrew Davidson (University of Bristol,

UK)

93,94

( Plasma was heat-inactivated at 56



C for 30 mins before use, and neutralizing antibody titers at

50% inhibition (NT

) measured as previously described.

87,95,96

In brief, luminescent HEK293T-ACE2-30F-

PLP2 repo rter cells (clone B 7) expressing SARS-CoV-2 Papain-like protease-activatable circularly permuted

ﬁreﬂy luciferase (FFl uc) were seeded in ﬂat-bottomed 96-well plates. The n ext day, S ARS-CoV- 2 viral stock

(MOI = 0.01) was pre-incubated with a 3-fold dilution series of each sample f or 2 hat 37



C, t hen added to

the cells. 16 h post-infection, cells were lysed in Bright-Glo

Luciferase Buffer (Promega) diluted 1:1 with

PBS and 1% NP-40, and F Fluc activ ity measured by luminometr y. Experimen ts were conduct ed in duplicate .

To obtain NT

, titration curves were plotted as FFluc vs log (serum dilution), then analyzed by non-linear

regression using the Sigmoi dal, 4PL, X is log(co ncentration) function in GraphPad P rism. NT

were quan-

titated when (1) at least 50% inhibition was observed at the lowest serum dilution tested (1:10, or 1:20 for

pre-diluted samples), and (2) a sigmoidal curve with a good ﬁt was generated. Samples with no detectable

neutralizing activity were assigned an arbitrary NT

equivalent to the lower limit of q uant iﬁc ation.

Production of bi otinylated protein

To produce biotinylated spike and receptor binding domain (RBD) protein, HEK-293F cells were seeded at

1 3 10

cells/mL in Freestyle 293 Expression Medium (Gibco). The next day, a transfection mix was

prepared (for 200 mL of cells) of 72 mg of spike-Avi-His tag or RBD-Avi-His tag plasmid and 18 mgofBirA

plasmid

36,88

into 11 mL of Opti-MEM

,alongside2mLofPEI-Max and 3 mL of 10 mM biotin, and left

to incubate at 37



C5%CO

in a shaking incu bator for 7 days beforeharvestingforpuriﬁcation. The

supernatant was puriﬁed using an imidazole (Sigma-Aldrich) buffer at a ﬁnal concentration o f 20 mM during

binding to the His GraviTrap (Cy tiva) column and 500 mM imidazole for el utio n. The e lu ted p ro tein was

then concentrated with a 100KD Amicon  Ultra concentrator (Millipore) and washed with PBS before

quantiﬁcation using a NanoDrop

. Biotinylated protein was then further puriﬁed through size exclusion

chromatography using an AKTA pure system with a Superdex 200 Increase 10/300 GL column

(Sigma-Aldrich) to select for fractions c ontaining trimeric spike or RBD protein.

B cell phenotypic ﬂow cytometric analysis

As previously described ,

1 mg of biotinylated spike with either streptavidin-conjugated allophycocyanin

(APC) (Agilent , Santa Clara, Cali fornia , USA) or phycoerythri n ( PE) (Ag ilen t) and 0.5 mg of biotinylated RBD

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with BV421 (BioLegend, San Diego, California, USA) were incubated for 30 minutes in the dark to generate

ﬂuorochrome-linked biotinylated tetramers. Previously cryopreserved aliquots of 5 3 10

or 10 3 10

cell

aliquots of PBMCs were quickly thawed in PBS, t hen stained with a panel of phenotyping antibodies and

biotinylated tetramers (see Table S2) or phenotyping antibodies only for FMO controls. PBMCs were

then washed with PBS and ﬁxed in Cytoﬁx /C ytoper m (BD, Franklin Lake s, New Jersey, USA) buffer.

Compensation controls we re prepar ed accordin g to manufacturer’s instructions using Anti-Mouse Ig, k

CompBeads (BD). Samples were acq uired on an LSRFortessa (BD) ﬂow cytometer. Data was analyzed

(see Figure S2 fo r gating s trategy) on FlowJo v10 (BD). For further analysis of the phenotype of spi ke-spe -

ciﬁc MBCs, analysis was limited to samples for which at least 50 cells were acquired in the CD19

CD20

CD38lo/-IgD-MBCs(excludingCD27



CD27

cells) spike-PE + spike-APC +gateaspreviouslydeﬁned.

T cell phenotypic ﬂow cytometric analysis

Puriﬁed cryopreserved PBMCs samples were thawed and rested for 2 hours at 37



CincompleteRPMIme-

dium (RPMI supplemented with penicillin-streptomycin, L-Glutamine, HEPES, non-essential amino acids,

2-Mercaptoethanol, and 10% FBS) (Gibco).

After 2-hour incubation, cel ls were washed and pl ated in a

96-round bottom plate at 0.5-1 3 10

per wel l and stained for chemokin e markers (CXCR3, CC R7 and

CXCR5) for 30 minutes at 37



C.Cellswerethenwashedandstainedwithsurfacemarkersat4



Cfor

20 min with different combinations of antibodies (see Table S 3) in the pre sence of ﬁxable live/dead stain

(Invitrogen, Eugene, Oregon, USA). After 20 min of incubation, cells were washed with PBS, and ﬁxed

with 4% paraformaldehyde for 15 minat RT. Samples were acqu ired on a LSRFortessa X-20 using

FACSDiva version 8.0 (BD) and subsequent data analysis was performed using FlowJo v10 (BD). The

gating str ategi es used for ﬂow cytometry experiment s ar e provided in Figures 6 and S6.

High-dimensional data analysis

Visualization of high-dimensi onal single-cell data (viSNE)

and FlowSOM

analyses were performed using

the Cytobank platform (https://www.cy tobank. org ). Concatenated ﬁl es w ere used to evaluat e overall CD4

and CD8 T cell landscape in different group s. Cell s were manually g ated for lymphocy tes, singl ets, CD14



CD19



live cells, CD3

and CD4

or CD8

and then subjected t o viSNE analysis. The viSNE clustering anal-

ysis was performed on 8 parameters (CCR7, CD45RA, CD127, PD-1, CD38, CXCR5, CXCR3, CD25). Equal

event sampling was selected across all samples. FlowSOM was then performed using the same markers

outlined previously for viSNE and with the following parameters: number of clusters 100, number of meta-

clusters 10; the size of clusters 15 pixels (Cytobank default).

Ex vivo IFN-g ELISpot as say

This assay was performed using cryopreserved PBMCs samples. Brieﬂy, 96-well ELISpot plates (S5EJ044I10;

Merck Millipore, Darmstadt, Germany) pre-wetted with 30 mL of 70% ethanol for 2 min before washi ng with

200 mL of sterile PBS. Anti-IFN-g coating antibody (10 mg/mL in PBS; clone 1-D1K; Mabtech, Nacka Strand,

Sweden) was then added and the p lat es incubated overnight at 4



C. Prior to addition of cells, ELISpot

plates were washe d wit h PBS and blocked with R10 (RPMI supplemented with penicill in- strept omycin,

L-glutamine, and 10% FBS) for a minimum of 2 hat 37



C. PBMCs samples were thawed and rested for 2

hours at 37



CinR10.Cellswerethenaddedat23 10

cells/well, in duplicat e, and stimulated with over-

lapping pepti de po ols at 2 mg/mL for 16–18 hat 37



C. Unstimulated cells were used as a negative control

while PHA (10 mg/mL, Sigma-Aldrich) stimulated cells were used as a positive control. Plates were then

washed with 0.05% Twee n/PBS (Sigma Aldrich) and incubated for 2 h with an IFN-g dete ctio n antibody

(1 mg/mL; clone mAb-7B6-1; Mabtech) fo llowed by 1 h incubation with AP-conjugate d streptavidin

(1:1000 in PBS, Mabtech). Plates were t hen washed and visualized using the VECTASTAIN Elite ABC-

HRP kit according to the manufacturer’s instructions (Mabtech). Antigen-speciﬁc T cell responses were

quantiﬁed by subtracting the number of spots in unstimulated cells from the peptide stimulated cells.

An additional threshold of > 5 SFU/10

PBMCs w as used. Partici pants who lacked T cell responses to the

positive stimuli (PHA) or where antig en-speciﬁc respon ses found to be lower than two standard deviations

of negative controls were excluded from t he results.

Overlapping peptide pools

For the detection of antigen-speciﬁc T cell responses, puriﬁed cryopreserved PBMCs were stimulated with

the following peptide pools: (1) Wild-type S ARS-CoV-2 spike ; SARS-CoV -2 spike PepTiv ato r protein

pools (Miltenyi B iotec, Gladbach, Germany) were used to test T cell responses against ful l spike prot eome.

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28 iScience 26, 105862, January 20, 2023

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(2) VOC spike-speciﬁc peptide pools; the WuhanHu-1 and variant pools containing peptides from the Wu-

han Hu-1, Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (BA.1/B.1.1.529.1) sequences (9, 19,

32 and 83 peptides, respecti vely) were used to deﬁne T c ell responses to mutat ed Spi ke sequences in

SARS-CoV-2 variants. Alpha and Bet a peptide pools were synt hesized by GL Bioche m Shanghai Ltd, China

and previously used in.

The corresponding controls to Alpha and Beta pools with Wuhan Hu-1amino acid

sequences were compar ed in parallel within the same don or. Delta and Omicron pools were obtained from

Miltenyi Biotec. (3) Non-SARS-CoV-2 antigens: Peptide pools of the pp65 prot ein of human cytomegalo-

virus (CMV) (Miltenyi Biotec), or HIV-1 Gag peptide pools (NIH AIDS Reagent Repository) were used as pos-

itive/neg ative controls.

QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical analysis were carried out in GraphPad Prism 9.0 (GraphPad). All tests were two- ta iled . Mann-

Whitney U-test (MWU) was used to compare unpaired, non-parametric datawhilst Wilcoxon matched-pairs

sign rank test (WMP) was used to compare paired, non-parametric data. Non-parametric Spearman test

was used for correlat ion analysis b etween two sets of data. Where app rop riate, median for groups is

shown. Statistical signiﬁcance in the ﬁgures is sho wn as p value >0.05 (*); >0.01 (**); >0.001 (***)

and >0.0001 (****).

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Do we know now whether viral vector-based vaccines exhibit this same trend in PLWH? Were there any commonalities (perhaps some sort of comorbidity) between these 4/5 SARS-CoV2 naive patients that produced no detectable nAb, even after 3 doses? I assume these are the same ~4 patients with co-morbidities in figure S1g? Is there any explanation as to why two patients had no humoral or cell-mediated responses after 2/3 doses of the vaccine? Is something that has been reported elsewhere?

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