mRNA-encoded HIV-1 Env trimer ferritin nanoparticles induce ...

Article

mRNA-encoded HIV-1 Env
trimer ferritinnanoparticles induce monoclonal antibodies thatneutralize heterologous HIV-1 isolates in mice
Graphical abstract

Highlights

d mRNA-expressed HIV-1 Envs are well folded with optimal

stabilizing mutations

d mRNA-expressed stabilized Envs show preferential bnAb

binding

d mRNA-LNP elicit autologous tier 2 neutralizing antibodies

with key bnAb mutations

d Inducedmonoclonal antibodies with keymutations neutralize

heterologous viruses

Mu et al., 2022, Cell Reports 38, 110514March 15, 2022 ª 2022 The Authors.https://doi.org/10.1016/j.celrep.2022.110514

Authors

Zekun Mu, Kevin Wiehe,

Kevin O. Saunders, ..., Norbert Pardi,

Drew Weissman, Barton F. Haynes

[email protected] (B.F.H.),[email protected] (Z.M.),[email protected] (D.W.)

In brief

mRNA vaccines are highly effective

against COVID-19. Mu et al. demonstrate

the use of mRNA to express HIV-1 Env

trimers scaffolded on ferritin

nanoparticles. mRNA vaccination in mice

induced autologous tier 2 neutralizing

antibodies and key functional mutations.

Isolated monoclonal antibodies

neutralized heterologous HIV-1 isolates.

ll

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mRNA-encoded HIV-1 Env trimer ferritinnanoparticles induce monoclonal antibodiesthat neutralize heterologous HIV-1 isolates in miceZekunMu,1,2,* KevinWiehe,2,3 Kevin O. Saunders,1,2,4,5 Rory Henderson,2,3 DerekW. Cain,2 Robert Parks,2 DianaMartik,2

Katayoun Mansouri,2 Robert J. Edwards,2,3 Amanda Newman,2 Xiaozhi Lu,2 Shi-Mao Xia,2 Amanda Eaton,2

Mattia Bonsignori,2,3,10 David Montefiori,2,4 Qifeng Han,2,11 Sravani Venkatayogi,2 Tyler Evangelous,2 Yunfei Wang,2

Wes Rountree,2 Bette Korber,6 Kshitij Wagh,6 Ying Tam,7 Christopher Barbosa,7 S. Munir Alam,2 Wilton B. Williams,1,2,4

Ming Tian,8 Frederick W. Alt,9 Norbert Pardi,9 Drew Weissman,9,* and Barton F. Haynes1,2,3,12,*1Department of Immunology, Duke University School of Medicine, Durham, NC 27710, USA2Duke Human Vaccine Institute, Duke University School of Medicine, Durham, NC 27710, USA3Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA4Department of Surgery, Duke University School of Medicine, Durham, NC 27710, USA5Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA6Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM 87545, USA7Acuitas, Inc, Vancouver, Canada8Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics,Harvard Medical School, Boston, MA 02115, USA9Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA10Present address: Translational Immunobiology Unit, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, MD 20892, USA11Present address: Novo Nordisk Research Center China, Novo Nordisk A/S, Beijing 102206, China12Lead contact

*Correspondence: [email protected] (B.F.H.), [email protected] (Z.M.), [email protected] (D.W.)https://doi.org/10.1016/j.celrep.2022.110514

SUMMARY

The success of nucleoside-modified mRNAs in lipid nanoparticles (mRNA-LNP) as COVID-19 vaccines her-alded a new era of vaccine development. For HIV-1, multivalent envelope (Env) trimer protein nanoparticlesare superior immunogens compared with trimers alone for priming of broadly neutralizing antibody (bnAb) Bcell lineages. The successful expression of complex multivalent nanoparticle immunogens with mRNAs hasnot been demonstrated. Here, we show that mRNAs can encode antigenic Env trimers on ferritin nanopar-ticles that initiate bnAb precursor B cell expansion and induce serum autologous tier 2 neutralizing activityin bnAb precursor VH + VL knock-in mice. Next-generation sequencing demonstrates acquisition of criticalmutations, and monoclonal antibodies that neutralize heterologous HIV-1 isolates are isolated. Thus,mRNA-LNP can encode complex immunogens and may be of use in design of germline-targeting andsequential boosting immunogens for HIV-1 vaccine development.

INTRODUCTION

Nucleoside-modified mRNA COVID-19 vaccines encoding

SARS-CoV-2 trimeric spike protein have demonstrated the

robust nature of mRNA vaccines (Baden et al., 2020; Polack et

al., 2020; Sahin et al., 2020). In addition to success with clini-

cally-approved COVID-19 spike trimer vaccines, pre-clinical

success has been demonstrated with nucleoside-modified

mRNA encapsulated in lipid nanoparticle (mRNA-LNP) expres-

sion of Zika prM-E (Pardi et al., 2017), influenza hemagglutinin

(Pardi et al., 2018a, 2018b), and HIV-1 envelope (Env) in gp120

monomers or gp140 trimers (; Pardi et al., 2018a; Saunders

et al., 2021). However, recent studies have shown that protein

trimer nanoparticle (NP) multimers may be advantageous as im-

This is an open access article under the CC BY-N

munogens (Abbott et al., 2018; Havenar-Daughton et al., 2018;

Kato et al., 2020; Saunders et al., 2019; Tokatlian et al., 2019).

HIV-1 broadly neutralizing antibodies (bnAbs) may be disfa-

vored by the immune system due to their characteristics of

long heavy-chain complementarity-determining region 3

(HCDR3) loops and polyreactivity or autoreactivity that predis-

pose bnAbs to immune tolerance control (Havenar-Daughton

et al., 2018; Haynes et al., 2005, 2012, 2016, 2019; Huang

et al., 2020; Moody et al., 2016; Saunders et al., 2019;

Steichen et al., 2019; Zhang et al., 2016). Thus, the biology of

HIV-1 bnAbs has necessitated a strategy whereby the unmu-

tated common ancestor (UCA) or germline (GL) precursor of

bnAb B cell lineage is targeted with priming immunogens to

expand the bnAb precursor pool (Haynes et al., 2012, 2019;

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B

E

C D

Figure 1. Antigenicity of modified mRNA-encoded CH848 10.17DT gp160 with stabilizing mutations

(A) Stabilizing mutations tested in this study mapped onto structure of CH848 10.17DT SOSIP trimer (PDB ID: 6UM5). One protomer is shown in rainbow color,

and the other two protomers are shown in gray. Amino acid mutations are listed in boxes. Black fonts outside of boxes are mutations in v4.1, and blue fonts are

mutations in v5.2.8, in addition to v4.1 mutations. Residue 561C in v5.2.8 or redesigned HR1 region in UFOmutation is not shown due to lack of HR1 region in this

structure.

(B) Antigenicity of mRNA-expressed CH848 10.17DT, F14, and 113C-429GCG transmembrane gp160s measured by binding of bnAb PGT125 and DH270 UCA.

Data are shown as means ± standard error of the mean (SEM) of Phycoerythrin + (PE +) cell percentage among live cells from three independent experiments.

(C) eCD4-Ig binding reactivity to mRNA-expressed CH848 10.17DT, F14, and 113C-429GCG gp160s. Data shown are averages of mean fluorescent intensity

(MFI) from three independent experiments. Error bars, means ± SEM.

(legend continued on next page)

2 Cell Reports 38, 110514, March 15, 2022

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Jardine et al., 2013; McGuire et al., 2013). Env immunogens de-

signed to select for key antibody mutations, administered in a

specific order, are postulated to be able to guide antibody affinity

maturation toward bnAb breadth and potency (Bonsignori et al.,

2016, 2017; Havenar-Daughton et al., 2018; Haynes et al., 2012,

2016, 2019; Huang et al., 2020; Saunders et al., 2019; Steichen

et al., 2019; Zhang et al., 2016). However, guiding bnAb develop-

ment is difficult, because HIV-1 bnAbs are enriched in improb-

able functional somatic mutations that are required for neutrali-

zation potency and breadth (Bonsignori et al., 2017; Wiehe

et al., 2018). Rare somatic mutations are due to lack of targeting

by the somatic mutation enzyme activation-induced cytidine

deaminase (AID). To promote bnAb development, Envs will

need to engage B cell receptors that have accumulated func-

tional improbable mutations, thereby selecting intermediate

bnAb B cell lineage members to proliferate and evolve further

(Bonsignori et al., 2017; Haynes et al., 2012; Wiehe et al.,

2018). Whereas bnAbs arise in �50% of HIV-1-infected individ-

uals (Hraber et al., 2014), to date, potent and durable bnAbs have

not been induced in humans by vaccination.

HIV-1 Env is metastable and can adopt open and closed con-

formations (Tran et al., 2012; Ward and Wilson, 2017). The Env

open conformation exposes non-neutralizing antibody (nnAb)

epitopes that can create competition for Env antigen between

nnAb and bnAb precursors (Havenar-Daughton et al., 2017;

Lee et al., 2021; McGuire et al., 2014). To address the problem

of Env trimer opening and the exposure of non-neutralizing epi-

topes,multiple strategies have been designed to stabilize Env tri-

mers in native-like conformations (de Taeye et al., 2015; Gue-

naga et al., 2015; Henderson et al., 2020; Kong et al., 2016).

However, whether stabilizing mutations for mRNA expression

of complex multimers will result in the desired antigenicity and

immunogenicity of trimer multimer NPs is not known.

We have previously demonstrated that a HIV-1 Env trimer,

CH848, with two glycosylation sites in the first variable region

(V1), eliminated (CH848 N133D N138T, CH848 10.17DT), conju-

gated to a ferritin nanoparticle that was superior to the Env trimer

alone as an immunogen, and was capable of initiating a V3-

glycan bnAb lineage with key improbable mutations in bnAb

UCA heavy- and light-chain variable regions (VH + VL) knock-in

(KI) mice (Saunders et al., 2019).

Here, we determined stabilizing mutations in the CH848

10.17DT immunogen for formulation as mRNA-LNP. We demon-

strated thatmodifiedmRNAEnv expression resulted in Env bind-

ing to bnAbs in the forms of transmembrane gp160s, soluble

gp140 SOSIP trimers, or SOSIP trimer-ferritin NPs encoded by

mRNA (trimer-ferritin NPs, hereafter ‘‘NPs’’). Moreover, we

demonstrated that immunization of bnAb UCA VH + VL KI mice

with mRNA-LNP encoding CH848 10.17DT gp160s or NPs initi-

ated a V3-glycan bnAb B cell lineage, selected for bnAb lineage

B cells with B cell receptors (BCRs) bearing functional improb-

(D) Binding reactivity of nnAbs 17b and 19b to mRNA-expressed CH848 10.17D

eCD4-Ig. Data shown are means ± SEM of PE + cell percentage among live cell

(E) Binding of Env base antibodies to mRNA-encoded CH848 10.17DT, F14, and

negative control, and 2G12 was used as positive control to show gp160 expressio

See also Figures S1 and S2 and Table S1.

able mutations and induced high serum titers of tier 2 V3-glycan

bnAb N332-dependent autologous neutralizing antibodies.

RESULTS

Antigenicity of modified mRNA-encoded CH84810.17DT gp160s with stabilizing mutationsWe studied ten stabilization designs in CH848 10.17DT Env for

expression as mRNAs (de Taeye et al., 2015; Guenaga et al.,

2015; Henderson et al., 2020; Kong et al., 2016; Tran et al.,

2012; Ward and Wilson, 2017; Zhang et al., 2018) (Figure 1A

and Table S1; see STAR methods).We first designed mRNAs

with stabilizing mutations encoding CH848 10.17DT Envs as

transmembrane gp160s (Table S1). All mRNA constructs ex-

pressed and showed robust V3-glycan bnAb binding (Figure S1).

In particular, CH848 10.17DT F14, 113C-429GCG, and 113C-

431GCG gp160s exhibited binding reactivity to mature V3-

glycan bnAb PGT125 and the DH270 UCA equal to that of

10.17DT gp160 without stabilizing mutations (Figure 1B).

In contrast, DS, Vt8, and F14/Vt8 mutations decreased

DH270 UCA binding to 10.17DT gp160s (Figure S1B).

To evaluate expression of CD4-induced (CD4i) Env epitopes,

we examined the susceptibility of each Env gp160 to CD4 trig-

gering in transfected 293-F cells. Engineered CD4-Ig (eCD4-Ig)

(Fellinger et al., 2019) bound to CH848 10.17DT gp160 lacking

stabilizing mutations in a dose-dependent manner, but binding

to 10.17DT F14 and 113C-429GCG gp160s was minimal (Fig-

ure 1C). Next, we assessed whether mutations could stabilize

10.17DT gp160s in prefusion conformations and prevent V3

loop or C-C chemokine receptor type 5 (CCR5) co-receptor

binding site exposure. mRNA-transfected 293-F cells were

either untreated or treated with 20 mg/mL of sCD4, eCD4-Ig, or

CD4-IgG2 (Allaway et al., 1995), and Env conformation was

determined by binding of CCR5 co-receptor binding site nnAb,

17b or distal V3 loop nnAb, 19b. In the absence of CD4 treat-

ment, Env gp160s lacked binding to 17b or 19b. After treatment

with sCD4, eCD4-Ig, or CD4-IgG2, 10.17DT gp160 without sta-

bilizing mutations exhibited increased binding to both nnAbs

17b and 19b. In contrast, stabilizing the Env gp160s with F14

or 113C-429GCG mutations completely prevented CD4-

induced exposure of 17b and 19b epitopes (Figures 1D and

S1C). F14 and 113C-429GCG mutations are outside of the 17b

and 19b binding site; thus, these mutations themselves do not

interfere with 17b or 19b binding (Henderson et al., 2020; Zhang

et al., 2018). In addition, if F14 mutations themselves impair 17b

or 19b binding, then similar effects would be observed for any

Env strains. Thus, we performed surface plasmon resonance

(SPR) analysis of 17b and 19b binding to 10.17DT F14 and

CH505 M5 G458Y F14 SOSIP proteins. Some increase to 19b

binding was observed with 10.17DT F14 SOSIP after sCD4 treat-

ment. In contrast, robust 19b binding to CH505 M5 G458Y F14

T, F14, and 113C-429GCG gp160s with or without treatment with sCD4 and

s from three independent experiments.

113C-429GCG gp160s. Anti-Influenza hemagglutinin mAb CH65 was used as

n. Histograms shown are representative from three independent experiments.

Cell Reports 38, 110514, March 15, 2022 3

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SOSIP was observed with or without sCD4 (Figure S1E), demon-

strating that F14 mutations do not intrinsically impair 17b or 19b

binding. Additionally, anti-gp41 nnAb 7B2 against the immuno-

dominant epitope of gp41 (Pincus et al., 2003) showed low bind-

ing to mRNA-expressed 10.17DT Env gp160s, confirming mini-

mal exposure of this gp41 epitope (Figure S1C).

Next, we determined whether expressing Env trimers as trans-

membrane gp160s resulted in trimer base exposure (Turner

et al., 2021). Binding of three Env base antibodies, DH1029,

DH1312, and RM19R IgG were tested (Cottrell et al., 2020; Mar-

tin et al., 2020). Here, we report the negative-stain electron mi-

croscopy (NSEM) structures of DH1029 and DH1312 (Figure S2).

We did not observe binding of neither DH1029 nor DH1312 anti-

bodies and observed only minimal binding of RM19R IgG to

mRNA-expressed CH848 10.17DT, 10.17DT F14, or 113C-

429GCG gp160s (Figures 1E and S1D). Thus, 10.17DT F14 and

113C-429GCG gp160s were stabilized such that they preferen-

tially bound to bnAbs versus nnAbs, had minimal non-neutral-

izing epitope exposure after CD4 triggering, and limited expo-

sure of the trimer base.

CH848 10.17DT gp160 mRNA-LNP elicited autologoustier 2 neutralizing antibodies in vivo

On the basis of stability and antigenicity, we selected CH848

10.17DT F14 and 113C-429GCG gp160s to test their immunoge-

nicity in heterozygous V3-glycan bnAb DH270 UCA heavy- and

light-chain (VH+/–, VL

+/–) knock-in (DH270 UCA KI) mice (Saunders

et al., 2019). mRNAs encoding 10.17DT F14 or 113C-429GCG

gp160s were encapsulated in LNP for immunization (Figure 2A).

Mice immunized with 10.17DT F14 or 113C-429GCG gp160

mRNA-LNP developed serum-binding IgGs to 10.17DT trimer

and gp120 monomer (Figures 2B, S3A, and S3B). Three

10.17DT F14- and four 113C-429GCG gp160 mRNA-LNP-vacci-

nated mice had IgGs binding to the linear CH848 V3 peptide

epitope, indicating low levels of V3 loop exposure in vivo (Figures

2B and S3C). We observed non-detectable levels of binding to

gp41 in both groups of mice, suggesting that the gp41 on stabi-

lized 10.17DT gp160 Env was not immunogenic in vivo (Fig-

Figure 2. Immunogenicity of CH848 10.17DT gp160 mRNA-LNP in mice

(A) Immunization schema in DH270 UCA KI mice with CH848 10.17DT F14 and 1

(B)Week 5 serum IgG binding to CH848 10.17DT SOSIP trimer, 10.17D11 gp120,

under the curve (logAUC). Each dot represents an individual mouse (n = 6 each

difference was observed between two groups (p > 0.05).

(C) Measurement of serum IgG that block DH1029 binding to CH848 10.17DT SO

mice were used to show the induction of base-binding antibodies after SOSIP tri

blocking.

(D) Week 5 serum neutralizing antibody titers measured in TZM-bl reporting cells w

leukemia virus (MuLV) was used as negative control. Neutralization titers are repo

No significant statistical difference was observed between the two groups (p > 0

(E) Improbable G57R andR98Tmutation frequencies in DH270 VH KI gene after CH

10.17DT DS Sortase ferritin NP protein immunization and empty LNP immunizat

(F) CH848 10.17DT gp160 mRNA-LNP vaccination induced GC B cell responses

and 10.17DT-specific memory B cells among total memory B cells in splenoc

vaccinated DH270 UCA KI mice are shown. Each dot represents an individual m

(G) CH848 10.17DT gp160 mRNA-LNP vaccination induced GC Tfh cell respons

splenocytes after 10.17DT F14 or 113C-429GCGgp160mRNA-LNP vaccination in

individual mouse. Horizontal bar, mean. No significant statistical difference was o

Exact Wilcoxon Mann-Whitney U test.

See also Figures S3, S4, and S10.

ure S3D). As confirmation of no relevant exposure of the Env

base on 10.17DT F14 or 113C-429GCG gp160s, no DH1029

serum blocking activity was detected (Figure 2C). Both types of

stabilized Env gp160 mRNA-LNP immunization induced serum

binding IgGs to 10.17DT, 10.17DT F14 and 113C-429GCG

gp160s on the surface of transfected 293-F cells (Figure S3F).

Next, we asked whether CH848 10.17DT F14 and 113C-

429GCG gp160 mRNA-LNP immunization in DH270 UCA KI

mice elicited serumneutralizing antibodies. Neutralizing antibody

titers 1 week after the third immunization (week 5) were assessed

with a panel of seven pseudotyped HIV-1 strains sensitive to

either DH270 UCA or DH270 intermediate antibody 4 (DH270

IA4) (Bonsignori et al., 2017; Saunders et al., 2019) (Figures 2D

and S3G). CH848 10.17DT F14 and 113C-429GCG gp160

mRNA-LNP elicited autologous tier 2 neutralizing antibodies

against CH848 10.17DT pseudovirus, with ID50 geometric mean

titers (GMT) of 8103 and 8812, respectively. Neutralization was

dependent on V3-glycan at position N332 in all 10.17DT F14

and four out of six 10.17DT 113C-429GCG-vaccinated mice,

demonstrating the elicitation of bnAb V3-glycan autologous

neutralizing antibodies. Lower titers of neutralizing antibodies

against the V1-glycans restored CH848 10.17 virus were

observed. Strain-specific holes in glycan shield can elicit glycan

hole-targeted autologous tier 2 neutralizing antibodies (Bradley

et al., 2016; Crooks et al., 2015; McCoy et al., 2016). Computa-

tional prediction (Wagh et al., 2018) identified potential glycan

holes at positions 230 and 289 on CH848 10.17 Env (Figure S3E).

We constructed a glycan holes filled CH848 10.17DTmutant with

amino acid mutations D230N H289N, as well as P291S. Compa-

rable neutralization titers were observed against CH848 10.17DT

D230N H289N P291S virus, indicating that most neutralizing an-

tibodies elicitedby vaccinationwere not targeted to glycanholes.

CH848 10.17DT gp160 mRNA-LNP selected for keyDH270 bnAb mutations and elicited germinal centerresponsesNext, splenocytes from 1 week after the third immunization

(week 5) were subjected to next-generation sequencing (NGS)

13C-429GCG gp160 mRNA-LNP.

and V3 loop peptidemeasured by ELISA. Data shown are log-transformed area

group). Error bar, mean ± standard deviation (SD). No significant statistical

SIP trimer. Sera from a group of 10.17DT DS SOSIP trimer protein immunized

mer immunization. Dotted horizontal line indicates background cut-off at 20%

ith a panel of autologous and heterologous tier 2 HIV-1 pseudoviruses. Murine

rted as ID50. Each dot signifies an individual mouse. Horizontal bar, ID50 GMT.

.05).

848 10.17DT gp160mRNA-LNP immunization. Frequencieswere compared to

ion. Each dot represents an individual mouse. Horizontal bar, median.

in spleens. Frequencies of 10.17DT-specific GC B cells among total GC B cells

ytes of CH848 10.17DT F14 or 10.17DT 113C-429GCG gp160 mRNA-LNP

ouse. Horizontal bar: mean. **P < 0.01.

es in spleen. Frequencies of total Tfh and GC Tfh cells among CD4+ T cells in

DH270UCAKImicewere assessed by flow cytometry. Each dot represents an

bserved between the two groups (p > 0.05). Significance was determined using


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analysis. Both CH848 10.17DT F14 and 113C-429GCG gp160

mRNA-LNP selected the critical improbable G57R mutation in

the DH270 UCA VH KI gene that is necessary for the V3-glycan

bnAb B cell lineage to acquire heterologous neutralization

breadth (Bonsignori et al., 2017; Wiehe et al., 2018), with the me-

dians of mutation frequency at 5.4% and 3.4%, respectively.

Induced antibodies also acquired a second key improbable

VH R98T mutation. Frequencies of the improbable VH G57R

and the R98T mutations were comparable to those in a group

of DH270 UCA KI mice immunized with sortase-ligated

10.17DT DS Env ferritin NP protein (Figure 2E; p > 0.05, exact

Wilcoxon Mann-Whitney U test). Thus, in DH270 UCA KI mice,

10.17DT gp160 mRNA-LNP were immunogenic, induced potent

N332-dependent autologous tier 2 neutralizing antibodies, and

selected DH270 antibodies that acquired improbable mutations

required for acquisition of heterologous HIV-1 neutralization.

Splenocytes from week 5 were phenotyped for germinal cen-

ter (GC) responses by flow cytometry, using fluorophore-labeled

10.17DT SOSIP trimer tetramers to detect 10.17DT-specific B

cells (Figure S4). Both 10.17DT F14 and 113C-429GCG gp160

mRNA-LNP elicited 10.17DT-specific GC B cells and memory

B cells (Figure 2F). The average frequencies of 10.17DT-specific

GC B cells among total GC B cell population was 1.42% in the

10.17DT F14 gp160 mRNA-LNP group and 0.95% in the

10.17DT 113C-429GCG mRNA-LNP group. The 10.17DT F14

gp160 mRNA-LNP vaccinated group had higher frequencies of

10.17DT-specific memory B cells among total memory B cells

compared with the 10.17DT 113C-429GCG gp160 mRNA-LNP

vaccinated group (mean at 13.14% versus 3.88%, p < 0.01,

Exact Wilcoxon Mann-Whitney U test). Additionally, 10.17DT

gp160 mRNA-LNP elicited spleen Tfh cell and GC Tfh cell re-

sponses in both groups (Figures 2G and S4).

Antigenicity of modified mRNA-encoded CH84810.17DT SOSIP trimers with stabilizing mutationsThe antigenicity of mRNA-expressed Galanthus nivalis lectin

(GNL)-purified 10.17DT SOSIP trimers was measured by

enzyme-linked immunosorbent assay (ELISA) using a panel of

bnAbs and nnAbs (Figures 1A and 3A; Table S1). Each stabilized

construct encoded by mRNA efficiently bound to V3-glycan

bnAbs 2G12, PGT125, PGT128, and DH270 lineage Abs

DH270 UCA, DH270 IA4, and DH270.1. In particular, mRNA-en-

coded 10.17DT DS SOSIP trimers displayed greater binding

reactivity to bnAbs compared with other stabilizing mutations.

Consistent with our observations with 10.17DT gp160s, the Vt8

and F14/Vt8 mutations decreased DH270 UCA binding to

10.17DT SOSIP trimers. 10.17DT Vt8 and F14/Vt8 SOSIP trimers

also displayed lower binding to trimer-specific bnAb PGT151

compared with 10.17DT v4.1, DS, and F14 SOSIP trimers, sug-

gesting less native-like conformation of Envs. Little to non-

detectable binding to bnAbs was observed with v5.2.8 and

UFO mutations combined (v5.2.8 + UFO). All stabilized con-

structs tested presented low to non-detectable levels of binding

to most nnAbs, except for 10.17DT SOSIPv5.2.8, which dis-

played about binding to nnAbs 19b and F105 compared with

other stabilized Envs tested.

SPR analysis showed that sCD4 treatment of mRNA-ex-

pressed non-stabilized 10.17DT SOSIPv4.1 trimers increased


binding of nnAb 17b. In contrast, 10.17DT DS, F14, and F14/

Vt8 showed low to non-detectable binding to 17b with or without

sCD4 treatment. Although 10.17DT Vt8, v5.2.8, and v5.2.8 +

UFO trimers exhibited increased binding to 17b after sCD4 treat-

ment, the binding was at a lower response level compared

with 10.17DT SOSIPv4.1. Similar trends were observed for 19b

binding (Figure 3B).

We next used size exclusion ultra-performance liquid chroma-

tography (SE-UPLC) to define the folding of mRNA-encoded

CH848 10.17DT SOSIP trimers. The analytical SE-UPLC profile

of bnAb PGT151-purified 10.17DT DS SOSIP trimer protein indi-

cated that a well-folded 10.17DT SOSIP trimer was separated

and eluted from the column, as shown in Figure 4A. GNL-purified

mRNA-expressed 10.17 DT v4.1 and DS SOSIP trimer samples

showed a dominant peak of trimer that was 62% and 65% of the

total peak, respectively (Figures 4B and 4C). NSEM analysis of

10.17DTDS trimer confirmed the expression ofwell-foldedSOSIP

trimers from mRNA-transfected 293-F supernatant (Figure 4D).

Antigenicity of CH848 10.17DT trimer-ferritin NPs withstabilizing mutations encoded by modified mRNAsCH848 10.17DT NPs were designed by gene fusion of the

10.17DT SOSIP trimer gene with Helicobacter pylori (H. pylori)

ferritin gene (FtnA; GenBank: NP_223316) and were tested for

expression, stability, and antigenicity (Figure 5A). Moreover, we

asked whether we could further improve the antigenicity and

immunogenicity of 10.17DT Env by filling the glycan holes at posi-

tions 230and289andby introducing169Kmutation,which is crit-

ical to Env interaction with V2-glycan bnAbs (Doria-Rose et al.,

2012; Lee et al., 2017; McLellan et al., 2011). This 10.17DT Env

trimer (CH848 10.17DTD230NH289NP291SE169K)was termed

‘‘enhanced CH848 10.17DT’’ (CH848 10.17DTe). Since the

linker sequence connecting ferritin and Env protein would affect

the expression and assembly of NPs, we tested 10.17DTe

DS NPs with two different linkers, the sequences of which

were GGGSGGGGSGLSK (termed ‘‘2xGS linker’’) and

GGGSGGGGSGGGGSGLSK (termed ‘‘3xGS linker’’). We also

designed another NP construct using the H. pylori ferritin with a

N19Qmutation,which removedapotential N-linkedglycosylation

site at position 19 and added a glycine and a serine to theC termi-

nus of the ferritin protein (‘‘VRC ferritin’’) (Kanekiyo et al., 2013).

All CH848 10.17DT and 10.17DTe NPs exhibited effective

binding to V3-glycan bnAbs tested (Figure 5B). 10.17DTe NPs

displayed robust binding to V2-glycan bnAbs PGT145, PG9,

and VRC26.25, while V3-glycan bnAb and DH270 lineage anti-

body bindings were comparable to 10.17DT with E169, demon-

strating that 10.17DTe Envs had improved antigenicity by filling

glycan holes and restoring V2-glycan bnAb binding without im-

pairing V3-glycan targeted antibody binding. In contrast,

10.17DT and 10.17DTeNPs showed low to non-detectable bind-

ing to nnAbs (Figure 5B). Specifically, none of the NPs showed

binding to 17b, and the binding to 19b was low, except for

10.17DT 113C-429GCG NPs, indicative of an exposed distal

V3 loop.

SPR analysis following sCD4 treatment showed no increased

binding of nnAb 17b to CH848 10.17DT and 10.17DTe NPs and

low levels of binding of nnAb 19b (Figure 5C). Thus, 10.17DTNPs

could be expressed with mRNAs and bound to bnAbs efficiently

A

B

Figure 3. Antigenicity of modified mRNA-encoded CH848 10.17DT SOSIP trimers with stabilizing mutations

(A) BnAb/bnAb precursor and nnAb binding reactivity to mRNA-expressed CH848 10.17DT SOSIP trimers with various stabilizing mutations measured by ELISA.

Data shown are means of logAUC from three independent experiments.

(B) SPR sensorgrams of nnAb 17b or 19b binding to mRNA-expressed CH848 10.17DT SOSIP trimers with or without sCD4 treatment.

See also Table S1.


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A

D

B C Figure 4. ModifiedmRNA-expressedCH848

10.17DT DS SOSIP trimer is well folded

(A) Analytical SE-UPLC profile of CH848 10.17DT

SOSIP trimer protein standard purified by bnAb

PGT151.

(B and C) Analytical SE-UPLC profile of mRNA-

expressed GNL-purified (B) CH848 10.17DT

SOSIPv4.1 trimers and (C) DS SOSIP trimers.

Black curve indicates GNL-purified material from

mock transfection.

(D) Negative-stain electron microscopy (NSEM)

analysis of mRNA-expressedGNL-purified CH848

10.17DT DS SOSIP trimers. Shown on the left is a

representative NSEM micrograph of 10.17DT DS

SOSIP trimer; on the right are 2D classification of

well-folded trimers.

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with limited binding to nnAbs when optimized stabilizing muta-

tions were present.

Furthermore, mRNA-expressed 10.17DT DS SOSIP trimers

without ferritin bound to base-binding antibodies DH1029,

DH1312, and RM19R IgG. In contrast, DH1029 and DH1312

showed no binding to NPs, while RM19R IgG showed minimal

binding to 10.17DT NPs that were lower than binding to trimer

alone (Figure 5D).

We assessed whether mRNA-expressed CH848 10.17DT pro-

tein indeed self-assembled into NPs by NSEM. We purified

mRNA-transfected 293-F supernatants of 10.17DTe DS VRC

and 10.17DTeDS 3xGS linker NPs by bnAb PGT145 and demon-

strated that both mRNAs produced stabilized, well-folded NPs

(Figures 5E and S5). In addition, 2D classification analyses iden-

tified free Env trimers, unknown trimeric proteins, proteosome

molecules with seven-fold symmetry (Adams, 2003), species in

polygon shapes, and small unidentifiable particles (Figure S5).

Polygon-shaped particles have been observed in HIV-1 Env pro-

tein preparation by others (He et al., 2016) and represent

secreted host galectin-3-binding proteins (Gal-3BP) that

assemble into ring-like polymers (Muller et al., 1999; Sasaki

et al., 1998). Thus, many of these 2D classes were host proteins

co-purified with Env proteins. If we considered unknown trimeric

proteins as mRNA derived in non-native forms, then well-folded

NPs consisted of a minimum of 53.3% and 49.7% of total Env

protein particles in 10.17DTe DS 3xGS linker and 10.17DTe DS

VRC samples, respectively. If only folded NPs and SOSIP trimers

are considered mRNA derived and unknown trimeric proteins

host derived, the percentage of well-folded NPs among identifi-


able NPs and free trimers were 100% for

the 10.17DTe DS 3xGS linker NP sample

and 64.5% for the 10.17DTe DS VRC NP

sample (Figures S5D and S5H).

CH848 10.17DT SOSIP trimer-ferritin NP mRNA-LNP inducedautologous tier 2 neutralizingantibodiesTo assess the immunogenicity of mRNA-

LNP encoding CH848 10.17DT NPs, we

immunized DH270 UCA KI mice (Figure 6A). All 10.17DT NP

mRNA-LNP elicited serum IgGs bound to 10.17DT trimer and

CH848 D11 gp120 proteins (Figures 6B, S6A, and S6B). None

of the NPs consistantly induced undesired V3 loop peptide or

gp41-binding antibodies, suggesting that NPs had stabilization

of the V3 loop in vivo (Figures 6B, S6C, and S6D). We detected

binding activity toH. pylori ferritin but did not observe any immu-

nized mouse serum cross-reactivity with human ferritin (Figures

S6E and S6F). Since a mouse antibody recognizing human

ferritin has been reported (Bayat et al., 2013), the lack of cross-

creativity to human ferritin was not due to limited mouse anti-

body repertoire but indeed suggested antibodies that react

with H. pylori ferritin do not cross-react to human ferritin. We

determined if immunized mouse serum contained antibodies

that could block the trimer base-binding antibody DH1029. No

blocking of DH1029 binding was observed in 10.17DT NP

mRNA-LNP vaccinated mice, except for one mouse vaccinated

with 10.17DT 113C-429GCG NP mRNA-LNP. In contrast, sera

from a control group of 10.17DT DS SOSIP trimer protein vacci-

nated DH270 UCA KI mice showed DH1029 blocking activity af-

ter the second and third immunizations (Figure 6C). Thus, vacci-

nation with 10.17DT NP mRNA-LNP in DH270 UCA KI mice did

not elicit trimer base-targeted antibodies, whereas 10.17DT DS

SOSIP trimer protein did.

Next, we assessed tier 2 serum neutralizing antibody titers af-

ter three immunizations against a panel of HIV-1 strains in the

TZM-bl neutralization assay. All CH848 10.17DT NP mRNA-

LNP elicited neutralizing antibodies against autologous tier 2 vi-

rus CH848 10.17DT in an N332-dependent manner (Figures 6D

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and S6G). Comparable neutralizing titers against glycan holes-

filled CH848 10.17DT 230N 289N 291S virus were observed,

indicating that the antibody responses were not directed at

glycan holes but rather were targeted to the V3-glycan bnAb

site. 10.17DTe DS VRCNPmRNA-LNP vaccinatedmice showed

higher neutralizing titers to CH848 10.17DT 230N 289N 291S vi-

ruses compared with 10.17DT DS (p < 0.05, Exact Wilcoxon

Mann-Whitney U test) and 10.17DT 113C-429GCG (p < 0.01,

Exact Wilcoxon Mann-Whitney U test) NP mRNA-LNP vacci-

nated groups. Thus, 10.17DT NPs encoded as mRNA-LNP effi-

ciently elicited tier 2 autologous neutralizing antibodies that tar-

geted the N332-dependent V3 glycan bnAb site.

CH848 10.17DT SOSIP trimer-ferritin NP mRNA-LNPelicited germinal center responses and selected for keyDH270 bnAb mutationsEach of the five CH848 10.17DT NPs selected the improbable VH

G57R mutation in DH270 UCA KI gene, with the highest median

of mutation frequency at 3.2% observed in 10.17DT 113C-

429GCG NP vaccinated mice. Similarly, the R98T mutation in

the DH270 UCA VH KI gene was also selected in all groups by

mRNA-LNP (Figure 6E). Thus, mRNA-LNP encoded 10.17DT

NP immunizations in DH270 UCA KI mice efficiently elicited

key improbable mutations.

Each of the five CH848 10.17DT NP mRNA-LNP induced

10.17DT-specific GC B cells and memory B cells in spleens (Fig-

ure 6F). Tfh cells and GC Tfh cells were also observed in all

10.17DT NP vaccinated mice (Figure 6G). No significant differ-

ence was observed among the five immunization groups (p <

0.05, Exact WilcoxonMann-Whitney U test). Interestingly, empty

LNP immunizations also elicited Tfh cell responses, albeit at a

much lower frequency, consistent with previous observations

that mRNA-LNP have adjuvant effects that favor Tfh cell and

GC responses (Alameh et al., 2021; Pardi et al., 2018a).

CH848 10.17DT trimer-ferritin NP mRNA-LNPimmunization induced heterologous neutralizing mAbsthat acquired improbable mutationsTo further assess antibody responses elicited by CH848

10.17DT NP mRNA-LNP, we injected DH270 UCA KI mice intra-

dermally (i.d.) or intramuscularly (i.m.) with 10.17DT DS NP

mRNA-LNP for six immunizations and sorted 10.17DT-specific

single memory B cells on 96-well plates and amplified immuno-

globulin (Ig) heavy- and light-chain variable regions by PCR (Fig-

ures 7A, 7B, and S7).We cloned a total of 397 Ig heavy- and light-

chain pairs, 228 (57%) pairs of which used DH270 KI genes

IGVH1-2 and IGVL2-23. Among these 228 DH270-like anti-

bodies, 173 (76%) antibodies have acquired at least one amino

Figure 5. Antigenicity of CH848 10.17DT trimer-ferritin NPs with stabili

(A) Design of CH848 10.17DT SOSIP trimer-ferritin fusion NPs.

(B) BnAb/bnAb precursor and nnAb binding reactivity to mRNA-expressed CH8

shown are means of logAUC from three independent experiments.

(C) SPR sensorgrams of nnAb 17b or 19b binding to mRNA-expressed CH848 1

(D) Binding reactivity of Env base antibodies DH1029, DH1312, and RM19R IgG t

measured by ELISA. Data shown are means of logAUC from at least two indepe

(E) Representative NSEM images and 2D classifications of mRNA-expressed, PG

See also Figures S2 and S5 and Table S1.


acid mutation (Figures 7C and S7B). We aligned all unique

VH1-2/VL2-23 Ig gene amino acid sequences with bnAb

DH270.6 and found that the Ig heavy-chain group accumulated

a total of 14 out of 19 (74%) DH270.6 probable mutations and

4 out of 8 (50%) DH270.6 improbable mutations (Figure S8).

Similarly, the Ig light-chain group accumulated 6 out of 9 (67%)

DH270.6 probable mutations and 4 out of 6 (67%) improbable

mutations (Figure S9). Specifically, 5 (2%) and 20 (9%) Ig heavy

chains acquired the DH270.6 bnAb G57R and the R98T improb-

able mutations, respectively, and 2 (1%) Ig light chains acquired

the DH270.6 bnAb L48Y improbable mutation. Two Ig heavy

chains had both G57R and R98T mutations (Figure 7D). Binding

reactivity of cloned antibodies was screened in ELISA (Table S3),

and antibodies with heterologous HIV-1 A.Q23 Env binding were

selected for further assessment. Among them, three mAbs

(DH270.mu84, DH270.mu85, and DH270.mu86) were identified

that showed strong binding to CH848 10.17DT, 10.17, and to

heterologous HIV-1 A.Q23 SOSIP trimers (Figure 7E). We as-

sessed neutralization of these three mAbs against a panel of

17 HIV-1 isolates that the first intermediate ancestor antibody

(DH270 IA4) in the DH270 lineage neutralizes (Figure 7F) (Saun-

ders et al., 2019). Each of the 3 mAbs neutralized autologous

tier 2 CH848 viruses and heterologous 92RW020 and 6101.1 vi-

ruses with titers comparable to DH270 IA4 (Saunders et al.,

2019). Antibody DH270.mu84 also neutralized each of the 13

other heterologous HIV-1 isolates tested. Antibodies

DH270.mu85 and DH270.mu86 neutralized 8 and 10 of 13 heter-

ologous isolates, respectively (Figure 7F). DH270.mu84 encoded

the VHG57R improbablemutation, DH270.mu86 encoded the VH

R98T improbable mutation, while DH270.mu85 had both the

G57R and R98T improbable mutations. Additionally,

DH270.mu86 acquired VL S27Y and VL S57N improbable muta-

tions (Figure 7G). One intriguing observation was that

DH270.mu84 makes a tyrosine mutation to the ‘‘invariant’’

cysteine that occurs before CDRL1 (C22Y). We hypothesized

that the C22Y mutation might enhance DH270.mu84 neutraliza-

tion potency or breadth due to increased antibody flexibility

(Klein et al., 2013). In summary, CH848 10.17DT DS NP

mRNA-LNP immunization induced heterologous tier 2 neutral-

izing DH270 antibodies with improbable mutations.

DISCUSSION

A major question addressed here is whether mRNA designs can

incorporate stabilizing mutations in trimers or NPs to optimize

immunogen expression and stability since the protein products

of mRNAs cannot be purified after mRNA-LNP injection

in vivo. In this study, we evaluated mutations that stabilize

zing mutations

48 10.17DT NPs with various stabilizing mutations measured by ELISA. Data

0.17DT NPs with or without sCD4 treatment.

o mRNA-expressed CH848 10.17DT DS SOSIP trimer or CH848 10.17DT NPs

ndent experiments.

T145-purified CH848 10.17DTe DS VRC and 10.17DTe DS 3xGS linker NPs.

A

B

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mRNA-encoded Envs expressed as transmembrane gp160s,

soluble SOSIP trimers, or single-gene mRNA trimer-ferritin

NPs. For mRNAs encoding the V3-glycan germline-targeting

CH848 10.17DT Env (Saunders et al., 2019), we showed that

F14 mutations optimally stabilized transmembrane gp160s, the

DS mutations best stabilized SOSIP trimers and NPs expressed

from mRNA.

mRNA-LNP encoding both transmembrane gp160s and solu-

ble NPs induced high titers of autologous bnAb-targeted tier 2

neutralizing antibodies with groups of mutations present in

bnAb intermediate antibodies in DH270 UCAKI mice (Bonsignori

et al., 2017; Saunders et al., 2019). Moreover, we observed

accumulation of mature bnAb DH270.6 mutations in vaccine-

inducedmAbs, althoughmutations are distributed across all iso-

lated mAbs. The next goal is to have mutations concatenated on

the same bnAb B cell clonal lineage members. To achieve this,

prolonged GC responses or enhanced recruitment of memory

B cells back into GCs will need to be induced by vaccination

to allow bnAb lineage B cell BCRs to acquire functional mutation

required for full bnAb neutralization breadth.

Therefore, we have promising mRNA-LNP vaccine candidates

for priming V3-glycan bnAb DH270 B cell lineage in two different

Env forms: as a transmembrane Env gp160 and as an NP. Trans-

membrane gp160 and NP forms encoded by mRNA-LNPs

showed similar immunogenicity in DH270 UCA KI mice. High-

avidity protein nanoparticles are concentrated in B cell follicles

(Martin et al., 2020; Tokatlian et al., 2019) and activate and recruit

low-affinity B cells (Kato et al., 2020). It is intriguing that mRNA-

LNP-delivered Env gp160s induced serum neutralization titers

and the accumulation of functional improbable bnAb mutations

that were comparable to NPs (Figure S10). The success of using

immunogens in transmembrane form has been demonstrated by

two COVID-19 mRNA-LNP vaccines (Baden et al., 2020; Polack

et al., 2020). However, the mechanisms of mRNA-delivered Env

transmembrane protein in eliciting B cell responses is less clear.

Some studies suggest that B cell recognition of membrane-

associated antigens plays a role of concentrating antigens for

B cell extraction, processing, and presentation (Carrasco and

Batista, 2006).

Several studies published recently explored the use of mRNAs

for HIV-1 vaccines in various forms, such as self-amplifying

mRNA (Aldon et al., 2021; Melo et al., 2019) or mRNA-encoded

virus-like particles (Zhang et al., 2021). These studies highlighted

the versatility of mRNA vaccine platform for HIV-1 vaccine devel-

Figure 6. Immunogenicity of CH848 10.17DT trimer-ferritin NP mRNA-L

(A) Immunization schema with CH848 10.17DT NP mRNA-LNP in DH270 UCA K

(B) Serum IgG binding to CH848 10.17DT SOSIP trimer, 10.17 D11 gp120, and V3

rest of groups). Horizontal bar, mean. Error bar, SD.

(C) Measurement of serum IgG that block DH1029 binding to CH848 10.17DT SO

previous studies were included as positive control. This positive control is the sam

(D) Serum neutralizing titers after CH848 10.17DT SOSIP NP mRNA-LNP immuni

Neutralization titers are reported as ID50. Each dot signifies an individual mouse.

(E) Improbable G57R and R98Tmutation frequencies in DH270 VH KI gene after CH

CH848 10.17DT Sortase ferritin NP protein immunizations and empty LNP immu

(F) Frequencies of CH848 10.17DT-specific GC B cells and memory B cells in sp

(G) Frequencies of total Tfh cells and GC Tfh cells in spleen of 10.17DT NP mRNA

Exact Wilcoxon Mann-Whitney U test, without any p value adjustment for multip

See also Figures S4, S6, and S10.


opment and stressed the need for careful studies to compare

and evaluate different mRNA technologies.

Eliciting bnAbs by vaccination has not been successful in hu-

mans. However, studies in HIV-1-infected individuals have

demonstrated that those who make bnAbs have higher levels

of T follicular helper (Tfh) cells (Locci et al., 2013; Moody et al.,

2016). Nucleoside-modified mRNA-LNP vaccines selectively

induce high levels of Tfh cells (Lederer et al., 2020; Pardi et al.,

2018a) and thus will be a key platform to test for bnAb lineage

initiation and selection of B cells with improbable functional mu-

tations that facilitate bnAb maturation. The CH848 10.17DTe DS

NP is currently in goodmanufacturing practice (GMP) production

to investigate the priming of such lineages in humans both as

mRNA-LNPs and as recombinant proteins.

In conclusion, we have demonstrated that single-chain

mRNAs can be designed to encode complex molecules and

that these immunogens are capable of selecting for difficult-to-

elicit improbable mutations critical for broad tier 2 virus neutral-

ization. The complex biology of HIV-1 bnAbs necessitates a vac-

cine strategy that utilizes a series of sequentially administered

Env immunogens that initially expand bnAb precursors and

then select for improbable mutations (Haynes et al., 2012,

2019; Saunders et al., 2019; Wiehe et al., 2018). Manufacturing

of complex nanoparticle protein immunogens in large scale is

faced with significant practical and funding challenges (Mu et

al., 2021). The use of mRNA-LNPs raises the possibility of mak-

ing such a complex immunization regimen both logistically

achievable and potentially cost effective.

Limitations of the studyThere are several limitations in this study. It is technically chal-

lenging to purify modified mRNA-encoded immunogens ex-

pressed in vivo and evaluate the stability directly. However, Pardi

et al. have compared the in vitro transfection of HEK293T cells

and dendritic cells with mRNA-LNPs and demonstrated mRNA

expression in each cell type to be at similar levels (Pardi et al.,

2015). Thus, the careful assessment of immune responses in an-

imal models for the induction of ‘‘off-target’’ antibody responses

targeting undesired epitopes on destabilized Env proteins is crit-

ical. Finally, the assessment of the immunogenicity of mRNA-

LNP in this study was performed in the DH270 UCA KI mouse

model. Although a valuable tool for testing HIV-1 vaccines, a

concern of this model is the relatively high frequency (�10%)

of DH270 UCA B cells among the naive B cell repertoire

NP in mice

I mice.

peptide. Each dot represents an individual mouse (n = 5 in Group 1; n = 6 in the

SIP trimer. 10.17DT DS SOSIP trimer protein vaccinated mice from one of our

e as that plotted in Figure 2C. Dotted horizontal line, 20% background cut-off.

zation measured in TZM-bl reporter cells. MuLV was used as negative control.

Horizontal bar, ID50 GMT.

848 10.17DTmRNA-LNP immunizations. Frequencies were compared to three

nizations. Horizontal bar, median.

lenocytes of 10.17DT NP mRNA-LNP vaccinated mice. Horizontal bar, mean.

-LNP vaccinated mice. Horizontal bar, mean. Significance was determined by

le comparison. *p < 0.05, **p < 0.01.

A

B

E

G

F

C D

Figure 7. CH848 10.17DT DS trimer-ferritin NP mRNA-LNP vaccination elicited antibodies that acquired improbable mutations and

neutralization breadth

(A) Immunization schema with CH848 10.17DT DS NP mRNA-LNP in DH270 UCA KI mice.

(B) Representative gate for CH848 10.17DT Env-specific single memory B cell sorting from DH270 UCA KI mice splenocytes.

(C) Summary of Ig gene recovery by PCR from single-cell sortedmemory B cells. Ig gene pairs that usedDH270 IGVH1-2 and IGVL2-23 geneswere considered as

DH270-like Abs. Ig gene pairs that used only one of DH270 heavy chain or light chain, or endogenous mouse Ig genes are categorized as ‘‘Other Ab’’. Right: the

number and percentage of the 228 DH270-like Abs that had acquired at least one amino acid change in heavy or light chain. VH1-2/VL2-23 Abs without full length,

clean VDJ/VJ sequences are categorized as ‘‘Undetermined’’.

(D) Summary of improbable mutations in DH270-like Abs.

(E) Binding reactivity of mAbs DH270.mu84, DH270.mu85, and DH270.mu86 to a panel of HIV-1 Env SOSIP trimer proteins measured by ELISA.

(legend continued on next page)


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(Saunders et al., 2019). Future studies in outbred animal models,

such as rhesusmacaques, will be important to further assess the

immunogenicity of mRNA-LNP vaccines.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d RESOURCE AVAILABILITY

B Lead contact

B Materials availability

B Data and code availability

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Cell line

B Animals and immunizations

d METHOD DETAILS

B Design of CH848 10.17DT Envs with stabilizing muta-

tions

B Nucleoside-modified mRNA production

B Nucleoside-modified mRNA-LNP production

B Nucleoside-modified mRNA transfection in 293-F cell

line

B Evaluation of expression and folding of modified

mRNA-expressed CH848 10.17DT gp160s, SOSIP tri-

mers, and trimer-ferritin NPs

B Mouse serological analysis by ELISA

B HIV-1 pseudovirus neutralization assay

B Next-generation sequencing (NGS)

B Antibody sequence analysis

B Flow cytometric phenotyping of GC responses

B Isolation of CH848 10.17DT-specific neutralizing

monoclonal antibodies (mAbs)

d QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

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

celrep.2022.110514.

ACKNOWLEDGMENTS

We thank Holly Zoeller for assistance with SE-UPLC assays; Cindy Bowman,

Grace Stevens, and Austin Harner for help with animal studies; Victoria Gee-

Lai and Maggie Barr for help with ELISA assays, and Matthew Gardner at

Emory University and Michael Farzan at Scripps Research Institute Florida

for providing eCD4-Ig. We acknowledge Andrew Ward and Christopher Cot-

trell at Scripps Research Institute and Marit van Gils at Academisch Medisch

Centrum Universiteit van Amsterdam for kindly sharing with us RM19R IgG

plasmid. We also thank Cynthia Nagel for project management. This project

was supported by NIH, NIAID, Division of AIDS Integrated Preclinical and Clin-

ical AIDS Vaccine Development Grant AI135902 and by NIAID, Division of

AIDS Consortia for HIV/AIDS Vaccine Development (CHAVD) Grant

UM1AI144371.

(F) DH270.mu84, DH270.mu85, and DH270.mu86 neutralization activity against a

50% of virus replication (IC50) in TZM-bl assay. MuLV was used as negative con

(G) Improbable mutations in mAbs DH270.mu84, DH270.mu85, and DH270.mu8

quences. Mutations are highlighted, and improbable mutations are shown in red

See also Figures S7–S9 and Table S3.


AUTHOR CONTRIBUTIONS

Conceptualization, Z.M., K.O.S., D.W., and B.F.H.; Methodology, Z.M., R.P.,

K.O.S., and B.F.H.; Animal models, F.A., M.T.; Software, S.V. and K.J.W.;

Investigation, Z.M., K.J.W., K.O.S., R.H., D.W.C., R.P., D.M., A.E., B.K.,

K.W., K.M., R.J.E., A.N., X.L., S.X., M.B., D.M., Q.H., S.V., T.E., M.A.,

W.B.W., N.P., D.W., and B.F.H.; Statistical analysis, Y.W. and W.R.; Writing,

Z.M., K.O.S., K.J.W., and B.F.H.; Funding Acquisition, B.F.H.; Resources,

Y.T., C.B., N.P., D.W., and B.F.H.; Supervision, B.F.H.

DECLARATION OF INTERESTS

B.F.H., K.O.S., and K.W. have patent applications on some of the concepts

and immunogens discussed in this paper.

Received: August 7, 2021

Revised: January 9, 2022

Accepted: February 17, 2022

Published: March 15, 2022

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Goat F(ab’)2 Anti-Human IgG - (Fab’)2 (PE) Abcam Cat#ab98606; RRID:AB_10672217

FITC Rat Anti-Mouse IgG1, Clone A85-1 BD Biosciences Cat#553443; RRID:AB_394862

FITC Rat Anti-IgG2a/IgG2b, Clone R2-40 BD Biosciences Cat#553399; RRID:AB_394837

FITC Rat Anti-Mouse IgG3, Clone R40-82 BD Biosciences Cat#553403; RRID:AB_394840

PerCP/Cyanine5.5 anti-mouse

CD21/CD35 (CR2/CR1) antibody

Biolegend Cat#123416; RRID:AB_1595490

PE anti-MU/HU GL7 Antigen

(T/B Cell Act. Marker) antibody


PE-CF594 Rat Anti-Mouse CD93

(Early B Lineage) Clone AA4.1

BD Biosciences Cat#563805; RRID:AB_2738431

PE/Cyanine5 anti-mouse TER-

119/Erythroid Cells antibody


PE-Cy7 Rat Anti-IgM, Clone R6-60.2 BD Biosciences Cat#552867; RRID:AB_394500

APC-R700 Rat Anti-Mouse CD19 Clone 1D3 BD Biosciences Cat#565473; RRID:AB_2739253

BV510 Rat Anti-Mouse IgD Clone 11-26C.1 BD Biosciences Cat#563110; RRID:AB_2737003

BV605 Hamster Anti-Mouse CD95

Clone Jo2


BV650 Rat Anti-Mouse CD45R/B220

Clone RA3-6B2


BV711 Rat Anti-Mouse CD138 Clone 281-2 BD Biosciences Cat#563193; RRID:AB_2631190

BV786 Rat Anti-Mouse CD23 Clone B3B4 BD Biosciences Cat#563988; RRID:AB_2738526

FITC Rat Anti-Mouse CD4 Clone RM4-5 BD Biosciences Cat#553047; RRID:AB_394583

PE Rat anti-Mouse CD25 Clone 7D4 BD Biosciences Cat#558642; RRID:AB_1645250

PE-CF594 Hamster Anti-Mouse CD279 (PD-1)

Clone J43


PE-Cy7 Rat Anti-Mouse CD62L Clone MEL-14 BD Biosciences Cat#560516; RRID:AB_1645257

Biotin Rat Anti-Mouse CD185 (CXCR5)

Clone 2G8


APC-R700 Rat Anti-Mouse CD8a Clone 53-6.7 BD Biosciences Cat#564983; RRID:AB_2739032

BV421 Rat Anti-Mouse CD127 Clone SB/199 BD Biosciences Cat#566300; RRID:AB_2739672

BV510 Hamster Anti-Mouse CD3e

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BV570 anti-mouse/human CD11b

Antibody, Clone M1/70


BV605 Rat Anti-Mouse CD90.2 Clone 53-2.1 BD Biosciences Cat#563008; RRID:AB_2665477

Brilliant Violet 650TM anti-mouse

NK-1.1 Antibody, Clone PK136


BV711 Rat Anti-Mouse CD44

Clone IM7


Goat polyclonal Secondary Antibody

to Rabbit IgG - H&L (HRP)

Abcam Cat# ab97080; RRID:AB_10679808

Mouse Anti-Monkey IgG-HRP SouthernBiotech Cat# 4700-05; RRID:AB_2796069

High Sensitivity Streptavidin-HRP Thermo Fisher Scientific N/A

Chemicals, peptides, and recombinant proteins

Galanthus Nivalis Lectin (GNL), Agarose bound Vector Laboratories Cat#AL-1243

Streptavidin, AF647 conjugate Thermo Fisher Scientific Cat#S21374; RRID:AB_2336066

(Continued on next page)

e1 Cell Reports 38, 110514, March 15, 2022

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

m1-pseudouridine-50-triphosphate TriLink Cat#N-1081

CleanCap TriLink Cat#N-7413

HIV Env recombinant protein This paper N/A

Critical commercial assays

TransIT-mRNA Transfection Kit Mirus Cat#MIR2250

LIVE/DEAD Fixable Aqua Dead Cell Stain Kit Thermo Fisher Scientific Cat#L34957

SureBlue Reserve TMB 1-Component

Microwell Peroxidase Substrate

Seracare Cat#5120-0083

RNeasy Mini Kit Qiagen Cat#74104

SMARTer Mouse BCR IgG H/K/L Profiling Kit Takara Cat#634422

AMPure XP Beckman Coulter Cat#A63881

MiSeq Reagent Kit v3 (600 cycle) Illumina Cat#MS-102-3003

LIVE/DEADTM Fixable Near-IR Dead Cell Stain Kit Thermo Fisher Scientific Cat#L34975

Experimental models: Cell lines

Freestyle 293-F cell Thermo Fisher Scientific Cat#R79007

Human cell line TZM-bl NIH ARRRP Cat#8129

Experimental models: Organisms/strains

Heterozygous (VH+/-, VL+/-) DH270 UCA knock-in mouse Saunders et al., 2019 N/A

Recombinant DNA

See Table S2 for plasmids used for in vitro

transcription of modified mRNAs

This paper N/A

Software and algorithms

FACSDIVA BD Biosciences N/A

FlowJo version 10 BD Biosciences N/A

Prism version 9 GraphPad Software N/A

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RESOURCE AVAILABILITY

Lead contactFurther information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Barton F.

Haynes ([email protected]).

Materials availabilityNew plasmids generated in this paper will be shared by the lead contact upon request.

Data and code availabilityd Original data reported in this paper will be shared by the lead contact upon request.

d This paper did not generate original code.

d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell lineFreestyle 293-F cell line (Thermo Fisher Scientific, Cat# R79007) was purchased from Thermo Fisher and cultured in Freestyle 293

ExpressionMedium (Thermo Fisher Scientific, Cat# 12338-026). Cells weremaintained in 8%CO2 at 37�Cat a density between 0.33

106/mL to 33 106/mL. Mycoplasma test was performed when a new stock vial was thawed at Duke University Cell Culture Facility.

Animals and immunizationsThe DH270 UCA KI mice has been previously described (Saunders et al., 2019). For CH848 10.17DT gp160 mRNA-LNP immuniza-

tions, 8-12 weeks old mice were used. 12 DH270 UCA KI mice were split into two groups (N = 6 each group), each group had at least

one female mice. Mice were immunized intramuscularly (i.m.) with 20 mg of mRNA-LNP encoding CH848 10.17DT F14 gp160 and

Cell Reports 38, 110514, March 15, 2022 e2


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CH848 10.17DT 113C-429GCG gp160 every two weeks for three times. For CH848 10.17DT DS NP immunizations, 6-12 weeks old

mice were used, and each group had at least two female mice. Immunizations were performed similarly, except that the second and

the third immunizations were only one week apart. Control group mice were injected with 20 mg of empty LNP. Bleeding was per-

formed one week after each immunization. Necropsy was performed one week after the third immunization and blood, spleen,

and lymph nodes were collected. All mice were cared for in a facility accredited by the Association for Assessment and Accreditation

of Laboratory Animal Care International (AAALAC). All study protocol and all veterinarian procedures were approved by the Duke Uni-

versity Institutional Animal Care and Use Committee (IACUC).

METHOD DETAILS

Design of CH848 10.17DT Envs with stabilizing mutationsTen stabilization designs were tested in nucleoside-modified mRNA-encoded CH848 10.17DT Envs as transmembrane gp160s, sol-

uble SOSIP trimers, or trimer-ferritin NPs (Figures 1A and Table S1). The DS mutations introduce a disulfide bond in the closed Env

trimer and prevents CD4-triggered exposure of the CCR5 co-receptor binding site and the V3 loop (Kwon et al., 2015). The F14 mu-

tations are designed based on a structure of BG505 SOSIP trimer complexedwith BMS-626529, a small molecule that blocks soluble

CD4 (sCD4)-induced Env rearrangements (Pancera et al., 2017) and stabilize the SOSIP trimer by decoupling the allosteric confor-

mational changes triggered by CD4 binding (Henderson et al., 2020). Vt8 mutations stabilize the V3 loop in the prefusion, V1/V2-

coupled state (Henderson et al., 2020). The 113C-429GCG and 113C-431GCGmutations link the Env gp120 subunit inner and outer

domains through a neo-disulfide bond, resulting in prefusion stabilized Env trimer with impaired CD4 binding (Zhang et al., 2018). For

soluble gp140 trimer stabilization, we also tested SOSIPv4.1, v5.2.8 and uncleaved prefusion-optimized (UFO) mutations. Mutations

in v4.1 introduce hydrophobic amino acids to disfavor solvent exposure of the V3 loop and modify gp41 in the SOSIP.664 trimer,

which improve trimer formation and thermostability and decrease V3 loop exposure (de Taeye et al., 2015). The UFO design replaces

the bend between alpha helices in HR1 with a computationally designed linker and aims to minimize the metastability of HIV-1 gp140

trimer (Kong et al., 2016). Mutations in the v5.2.8 design are designed based upon v4.1 and combine an additional disulfide bond and

eight trimer-derived mutations that stabilize BG505 SOSIP trimers (Guenaga et al., 2015). Both v4.1 and v5.2.8 include an improved

hexa-arginine furin cleavage site R6 (Binley et al., 2002).

Nucleoside-modified mRNA productionModified mRNAs were produced by in vitro transcription using T7 RNA polymerase (Megascript, Ambion) on linearized plasmids en-

coding codon-optimized CH848 10.17DT gp160s, CH848 10.17DT SOSIP trimers, or CH848 10.17DT trimer-ferritin NPs. All the HIV-

1 modified mRNA constructs used in this study and their corresponding plasmids were listed in Table S2. One-methylpseudouridine

(m1J)-50-triphosphate (TriLink, Cat# N-1081), instead of UTP was used to produce nucleoside-modified mRNAs. Modified mRNAs

contain 101 nucleotide-long polyadenylation tails for optimized expression. Modified CH848 10.17DT SOSIPv4.1 trimer and CH848

10.17DT SOSIPv5.2.8 trimer mRNAs were capped using ScriptCap m7G capping system and ScriptCap 20-O-methyl-transferase kit

(ScriptCap, CellScript) (Pardi et al., 2013). Capping of all other in vitro transcribed mRNAs was performed co-transcriptionally using

the trinucleotide cap1 analog, CleanCap (TriLink, Cat# N-7413). All mRNAs were purified by cellulose purification, as described

(Baiersdorfer et al., 2019). All mRNAs were analyzed by agarose gel electrophoresis and were stored frozen at �20�C.

Nucleoside-modified mRNA-LNP productionNucleoside-modified mRNAs were encapsulated in LNP for mouse immunizations as previously described (Jayaraman et al., 2012;

Maier et al., 2013). Modified mRNAs in aqueous phase were rapidly mixed with a solution of lipids dissolved in ethanol. LNP formu-

lation contains ionizable cationic lipid (proprietary to Acuitas)/phosphatidylcholine/cholesterol/PEG-lipid. The cationic lipid and LNP

composition are described in US patent US10,221,127.

Nucleoside-modified mRNA transfection in 293-F cell line293-F cells were diluted to 0.7 3 106 cells/mL 24 h before transfection. On the next day, cells were diluted again to 13 106/mL and

seeded into tissue culture plates for transfection. 3 mg of mRNAs expressing gp160s were transfected into 6 mL of cells. For soluble

SOSIP trimers, 30 mL of cells were transfected with 12 mg of SOSIP-expressing mRNAs and 3 mg of Furin mRNAs. Transfection vol-

ume were doubled to 60 mL for trimer-ferritin NPs. TransIT-mRNA Transfection Kit (Mirus Cat# MIR2250) was used for mRNA trans-

fection following the manufacturer’s instructions. Transfected cells were cultured at 37�C with 8% CO2 and shaking at 120 rpm for

48 h (for gp160) or 72 h (for SOSIP trimers and trimer-ferritin NPs) before harvest.

Evaluation of expression and folding of modified mRNA-expressed CH848 10.17DT gp160s, SOSIP trimers, andtrimer-ferritin NPsThe expression and folding ofmodifiedmRNA-encodedCH848 10.17DT transmembrane gp160s, Soluble SOSIP trimers, and trimer-

ferritin NPs were defined as follows. For CH848 10.17DT transmembrane gp160s, flow cytometry was used to measure binding of

a panel of bnAbs and nnAbs. BnAb binding reactivity indicated successful expression of gp160 Envs on cell surface with desired

antigenicity. Binding of nnAbs 17b and 19b measured the ability of various stabilizing mutations to keep the gp160 Envs in prefusion


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conformation and to decrease the exposure of non-neutralizing epitopes CCR5 binding site and distal V3 loop. Additionally, 7B2

binding was used to measure the exposure of gp41.

For CH848 10.17DT soluble trimer, total Env formswere purified byGalanthus nivalis lectin (GNL) frommodifiedmRNA-transfected

293-F cell supernatant. A panel of nnAbs and bnAbs were used in ELISA to measure the expression of non-neutralizing and neutral-

izing epitopes. Binding of nnAbs 17b and 19b after CD4 triggering were measured by SPR. To assess the percent of trimeric Envs in

GNL-purified materials, size-exclusion ultra-performance liquid chromatography (SE-UPLC) analysis was performed with PGT151

affinity-purified CH848 10.17DT SOSIP trimer as a standard. Finally, Negative-stain Electron Microscopy (NSEM) analysis was per-

formed to confirm trimer formation in GNL-purified 293-F transfection supernatant.

Similar antigenicity measurement and SPR analysis was performed on GNL-purified 293-F cell supernatant transfected with modi-

fied mRNA expressing CH848 10.17DT trimer-ferritin NPs to evaluate the expression of well-folded Env trimers on the ferritin nano-

particle. Additionally, transfected 293-F supernatant were affinity purified with PGT145-conjugated beads, which exclude host

glycan proteins that may be purified by GNL. PGT145-purified materials were analyzed by NSEM to confirm the assembly of

CH848 10.17DT DS ferritin NPs.

Flow cytometry

Binding of bnAbs to CH848 10.17DT gp160s was performed by flow cytometry as previously described (Henderson et al., 2020; Sa-

unders et al., 2021). Briefly, modified mRNA-transfected 293-F cells were harvested 48 h after transfection and were washed once

with 1% BSA in PBS. Then, cells were incubated with 10 mg/mL of bnAbs in V-bottom 96-well plates for 30 min at 4�C. Cells were

then washed with 1% BSA in PBS and incubated with Goat F(ab’)2 Anti-Human IgG - (Fab’)2 (PE) (Abcam Cat# ab98606,

RRID:AB_10672217) for 30 min at 4�C in dark. Then, cells were washed once with PBS and dead cells were stained with LIVE/

DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen Cat# L34957, 1:1000 dilution in PBS) for 15 min at 4�C in dark, then washed twice

and re-suspended in 1%BSA in PBS. Flow cytometric data were acquired on a LSRII High-throughput system using FACSDIVA soft-

ware (BD Biosciences) and were analyzed with FlowJo software (FlowJo). The percentage of 293-F cells that were PE positive was

shown in the results.

Measurement of binding of nnAbs after CD4 treatment has been described previously (Henderson et al., 2020). Briefly, mRNA-

transfected 293-F cells were first incubated with 20 mg/mL of soluble CD4 (sCD4), eCD4-Ig or CD4-IgG2 for 10 min at 4�C. Cellswere washed once with 1% BSA in PBS and then incubated with 10 mg/mL of nnAbs 17b, 19b or 7B2 for 30 min at 4�C. Then, cellswere incubated with Goat F(ab’)2 Anti-Human IgG - (Fab’)2 (PE) and dead cells were stained with LIVE/DEAD Fixable Aqua Dead Cell

Stain Kit. Data acquisition and analysis were the same as described above.

Galanthus nivalis lectin purification of SOSIP trimers and trimer-ferritin NPs

293-F cells transfected with modified mRNAs expressing SOSIP trimers or trimer-ferritin NPs were harvested 72 h after transfection

and were centrifuged for 30 min at 3000 rpm to remove cells and debris. Supernatant were first filtered using a 0.22 mm vacuum filter

and were then concentrated by 50-fold using 10 kDaMWCO concentrators. Concentrated supernatant was incubated with 200 mL of

agarose boundGalanthus nivalis lectin (GNL) (Vector Laboratories Cat# AL-1243) with gentle rotation at 4�C overnight. The next day,

GNL agarose beads were washed with MES wash buffer (20 mM MES, 130 mM NaCl, 10 mM CaCl2 pH 7.0) for three times, and

SOSIP trimers or trimer-ferritin NPs were eluted by 500 mM Methyl alpha-D-mannopyranoside in MES wash buffer. The eluates

were then dialyzed to 10 mM Tris-HCl pH8 500 mM NaCl using 30kDa MWCO spin concentrators. GNL-purified SOSIP trimers or

trimer-ferritin NPs were snap-freezed and stored in �80�C.Enzyme-linked immunosorbent assay (ELISA)

Binding reactivity of modified mRNA-expressed CH848 10.17DT SOSIP trimers and trimer-ferritin NPs to bnAbs and nnAbs was

measured by enzyme-linked immunosorbent assay (ELISA). In brief, HIV-1 antibodies were coated onto 384-well assay plates in

0.1MSodium bicarbonate overnight at 4�C. GNL-purified SOSIP trimers or trimer-ferritin NPs with serial dilutions were then captured

on the plates. Next, poly-serum fromCH848 10.17DT-immunized rhesusmacaque was incubated for 1 h at room temperature. Then,

Mouse Anti-Monkey IgG-HRP (SouthernBiotech Cat# 4700-05, RRID:AB_2796069) was incubated for 1 h at room temperature and

plates were developed with SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate (Seracare Cat# 5120-0083) for

15 min and were stopped with 1% HCl solution. Absorbance at 450 nm were determined by SpectraMax Plus 384 microplate reader

(Molecular Devices) and log area-under-curve (log AUC) were calculated using Prism (Graphpad) and shown in figures.

The base binding antibody assay was performed similarly. Briefly, base binding antibody DH1029 was coated onto plates to cap-

ture samples. Then, a rabbit serum was incubated before detection with Goat polyclonal Secondary Antibody to Rabbit IgG - H&L

(HRP) (Abcam Cat# ab97080, RRID:AB_10679808). The plate development, data acquisition, and analysis were the same as

described above.

Size-exclusion ultra-performance liquid chromatography (SE-UPLC)

Size exclusion chromatography of modified mRNA-expressed GNL-purified CH848 10.17DT SOSIP trimers was performed using a

Waters Acquity H-Class Bio UPLC System with a Waters Acquity UPLC BEH SEC 450A, 2.5 mm, 4.63 150 mm column (Waters Cor-

poration). An isocratic elution with a mobile phase of 20 mM sodium phosphate 300 mM NaCl pH 7.4, and a flow rate of 0.2 mL/min,

was used for the analysis with a quaternary pump. Samples and protein standardsweremaintained at 5–8�C in the auto-sampler rack

prior to injection at a volume of 10 mL. Samples and protein standards with a concentration greater than 1.0 mg/mL were diluted to a

down to 1.0 mg/mL using Type 1 water. The column temperature was set to 30�C with detection at a wavelength of 214 nm using a

photodiode array detector.


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Negative-stain electron microscopy (NSEM)

Negative-stain electronmicroscopy (NESM) analysis of modifiedmRNA-expressed CH848 10.17DT SOSIP trimers and trimer-ferritin

NPs were performed as previously described (Saunders et al., 2017; Williams et al., 2021).

Surface plasmon resonance (SPR)

SPR analyses of modified mRNA-expressed SOSIP proteins incubated with and without sCD4 against distal V3 loop antibody 19b

and CCR5 binding site antibody 17b were obtained using the Biacore S200 instrument (Cytiva). Antibodies 19b and 17b were immo-

bilized onto a CM3 sensor chip to a level of 2000-4000RU. A negative control Influenza IgG1 antibody (CH65) was also immobilized

onto the sensor chip for reference subtraction. Modified mRNA-expressed GNL-purified CH848 10.17DT SOSIP trimers or trimer-

ferritin NPs were diluted down in HBS-N 1x running buffer to 0.5–2.0 mg and incubated with a 2-83 higher dose of soluble CD4

(4.4 mg) (Progenics Therapeutics). Proteins incubated with and without sCD4 were injected over the sensor chip surface using the

High performance injection type for 180s at 30 mL/min. The protein was then allowed to dissociate for 600s followed by sensor surface

regeneration of two 20 s injections of glycine pH 2.0 at a flow rate of 50 mL/min. Results were analyzed using the BIAevaluation Soft-

ware (Cytiva). Protein binding to the CH65 immobilized sensor surface as well as buffer binding were used for double reference sub-

traction to account for non-specific protein binding and signal drift.

Mouse serological analysis by ELISASerum IgG antigen binding assay

Serum IgG binding to HIV-1 antigens was measured by ELISA as previously described (Saunders et al., 2019).

DH1029 blocking assay

DH1029 blocking by vaccinated mouse sera was performed in ELISA. Briefly, 384-well assay plate were coated with 2 mg/mL

PGT145. Then, 0.125 mg/mL of CH848 10.17DT E169K SOSIP trimer were captured for 1 h at room temperature. Next, mouse

sera at 1:50 dilution or DH1029 mAb in serial dilution (2-fold dilution starting at 2 ug/mL) were incubated for 1 h. Next, biotinylated

DH1029 were added to the plate at 0.05 mg/mL for 1 h and binding were detected by High Sensitivity Streptavidin-HRP (Thermo

Fisher Scientific, Cat #21130). Plate development and data acquisition were the same as described above.

HIV-1 pseudovirus neutralization assayNeutralization assays were performed in TZM-bl reporter cells as described (Mascola et al., 2005). The analyses of possible glycan

holes onCH848 10.17DTEnvwas performed usingmethod described before (Wagh et al., 2018). Potential glycan holes were found at

amino acid positions 230 and 289. To restore glycosylation at these two sites, Asparagine (N) mutations at positions 230 and 289

(230N 289N) were made. In addition, a Serine (S) mutation at position 291 (291S) was made to complete the NXT/S glycosylation

sequence.

Next-generation sequencing (NGS)We performed next-generation sequencing (NGS) on mouse antibody heavy and light chain variable genes using an Illumina

sequencing platform. First, RNA was purified from splenocytes using a RNeasy Mini Kit (Qiagen, Cat# 74,104). Purified RNA was

quantified via Nanodrop (Thermo Fisher Scientific) and used to generate Illumina-ready heavy and light chain sequencing libraries

using the SMARTer Mouse BCR IgG H/K/L Profiling Kit (Takara, Cat# 634,422). Briefly, 1 mg of total purified RNA from splenocytes

was used for reverse transcription with Poly dT provided in the SMARTer Mouse BCR kit for cDNA synthesis. Heavy and light chain

geneswere then separately amplified using a 50 RACE approachwith reverse primers that anneal in themouse IgG constant region for

heavy chain genes and IgK for the light chain genes (SMARTer Mouse BCR IgG H/K/L Profiling Kit). The DH270 UCA KI mousemodel

has the light chain gene knocked into the kappa locus, therefore kappa primers provided in the SMARTer Mouse BCR kit were used

for light chain gene library preparation. 5 mL of cDNA was used for heavy and light chain gene amplification via two rounds of PCR;

PCR1 used 18 cycles and PCR2 used 12 cycles. During PCR2, Illumina adapters and indexeswere added. Illumina-ready sequencing

libraries were then purified and size-selected by AMPure XP (Beckman Coulter, Cat# A63881) using kit recommendations. The heavy

and light chain libraries per mouse were indexed separately, thus allowing us to deconvolute the mouse-specific sequences during

analysis. Libraries were quantified using QuBit Fluorometer (Thermo Fisher). Mice were pooled by groups for sequencing on the Il-

lumina MiSeq Reagent Kit v3 (600 cycle) (Illumina, Cat# MS-102-3003) using read lengths of 301/301 with 20% PhiX.

Antibody sequence analysisNGS data analysis and the analysis of improbable mutation frequencies was performed as described (Wiehe et al., 2018). Animals

with <1000 NGS reads likely reflect technical errors during NGS library preparation and thus were excluded from analysis.

Flow cytometric phenotyping of GC responsesFor immunophenotyping of murine B cells and Tfh cells, spleens from immunized mice one week after the third immunization were

processed into single-cell suspensions and treated with ACK lysis buffer to remove red blood cells. Splenocytes (2 3 106) were

suspended in 100 mL PBS/2% FBS. To detect antigen-specific B cells, fluorochrome-mAb conjugates and fluorochrome-conjugated

CH848 10.17DT Envs were prepared as a master mix at 23 concentration, then 100 mL of 23 master mix was added to an equal

volume of cells (Figure S4). Staining for T cell subsets was conducted in the same manner, with the additional step for detection


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of biotinylated mAb with Streptavidin–APC. Cells were incubated at 4�C for 20 minutes, then washed with PBS. Cells were resus-

pended in 100 mL PBS containing Near-IR Live/Dead (Thermo Fisher Scientific) at 1:1000, and incubated at room temperature for

20 min. Cells were washed in PBS/2% FBS, then re-suspended in PBS/2% formaldehyde. Cells were analyzed on a BD LSRII

(BD Biosciences). Data were analyzed using FlowJo v10 (FlowJo).

Isolation of CH848 10.17DT-specific neutralizing monoclonal antibodies (mAbs)Antibody cloning, screening, and mAbs expression

Immunoglobulin (Ig) gene were cloned from sorted single B cells as previously described (Liao et al., 2009). Briefly, complementary

DNA (cDNA) of Ig genes were amplified by reverse-transcription with SuperScript III First-Strand Synthesis System (Thermo Fisher

Scientific, Cat# 18080051) using random hexamer oligonucleotides as primers. Ig gene cDNA was then used as template in nested

PCR for heavy and light chain gene amplification using AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Cat #4398881).

Mouse Ig-specific primers and DH270 variable region-specific primers were used to amplify mouse endogenous Ig genes and

DH270 KI Ig genes. Agarose gel electrophoresis was used to identify positive PCR amplification and Ig genes were recovered by

Sanger sequencing. Following sequencing, contigs of PCR amplicon sequences were assembled, and Ig genes were inferred

with human Ig gene library andmouse Ig gene library in Cloanalyst. PCR reactionswith successful Ig sequence recoverywere purified

using AMPure XP kit (Beckman Coulter, Cat# A63881). Purified PCR product was used for overlapping PCR to generate a linear anti-

body expression cassette. The expression cassette was transiently transfected with into 293i cells with ExpiFectamine 293 Trans-

fection Kit (Thermo Fisher Scientific, Cat# A14525). The supernatant was harvested 72 h after transfection and screened in ELISA

binding assays with a panel of protein of interests. The genes of selected heavy chains were synthesized with human IgG1 backbone

(GenScript). Kappa and lambda chains were synthesized similarly. To express mAbs plasmids were prepared for transient transfec-

tion using the Plasmid Plus Mega Kit (Qiagen, Cat #12981). Heavy and light chain plasmids were co-transfected into 293i cells using

ExpiFectamine 293 Transfection Kit for antibody production.

QUANTIFICATION AND STATISTICAL ANALYSIS

Exact Wilcoxon Mann-Whitney U tests were performed without any adjustment for multiple comparisons. Significant results were

indicated in figures and figure legends as: * P < 0.05; ** P< 0.01.


Cell Reports, Volume 38

Supplemental information

mRNA-encoded HIV-1 Env trimer ferritin

nanoparticles induce monoclonal antibodies

that neutralize heterologous HIV-1 isolates in mice

Zekun Mu, Kevin Wiehe, Kevin O. Saunders, Rory Henderson, Derek W. Cain, RobertParks, Diana Martik, Katayoun Mansouri, Robert J. Edwards, AmandaNewman, Xiaozhi Lu, Shi-Mao Xia, Amanda Eaton, Mattia Bonsignori, DavidMontefiori, Qifeng Han, Sravani Venkatayogi, Tyler Evangelous, Yunfei Wang, WesRountree, Bette Korber, Kshitij Wagh, Ying Tam, Christopher Barbosa, S. MunirAlam, Wilton B. Williams, Ming Tian, Frederick W. Alt, Norbert Pardi, DrewWeissman, and Barton F. Haynes

Table S1. Stabilizing mutations studied for modified mRNA-encoded CH848 10.17DT transmembrane gp160s, soluble SOSIP trimers, and trimer-ferritin nanoparticles. Related to Figure 1.

Env form Design Mutations Reference

Transmembrane DS 201C-433C Kwon et al., 2015

gp160s F14 68I, 204V, 208L, 255L Henderson et al., 2020

Vt8 203M, 300L, 302L, 320M, 422M Henderson et al., 2020

F14/Vt8 F14+Vt8 Henderson et al., 2020

113C-429GCG 113C-429C, 428G, 430G Zhang et al., 2018


Soluble SOSIP trimers v4.1

501C-605C, 559P, R6, ΔMPER, 535M, 543N/Q,

316W, 64K De Taeye et al., 2015

v5.2.8 V4.1, 66R, 73C-561C, L165, Q432, R429, K65, T106, E49,

D47, R500 Guenaga et al., 2015

DS 201C-433C Kwon et al., 2015

F14 68I, 204V, 208L, 255L Henderson et al., 2020

Vt8 203M, 300L, 302L, 320M, 422M Henderson et al., 2020

F14/Vt8 F14+Vt8 Henderson et al., 2020

UFO HR-1 re-designed, 501C-605C Kong et al., 2016

v5.2.8+UFO v5.2.8+UFO Guenaga et al., 2015; Kong et al., 2016

Multimeric trimer-ferritin NPs DS 201C-433C Kwon et al., 2015


Table S2. Plasmids used for in vitro transcription of each mRNA construct. Related to Figure 1.

mRNA Construct Plasmid

CH848 10.17DT gp160 ccap1-TEV-CH848-10.17-DTgp160-A101

CH848 10.17DT DS gp160 ccap1-TEV-CH848-10.17-DT-DS gp160-A101

CH848 10.17DT F14 gp160 ccap1-TEV-CH0848-10.17-DTgp160_F14-A101

CH848 10.17DT Vt8 gp160 ccap1-TEV-CH848-10.17-DTgp160-VT8-A101

CH848 10.17DT F14/Vt8 gp160 ccap1-TEV-CH0848-10.17-DTgp160_F14_VT8-A101

CH848 10.17DT 113C-429GCG gp160

ccap1-TEV-CH0848.d949.10.17gp160_N133DN138T_113C-429GCG-A101

CH848 10.17DT 113C-431GCG gp160

ccap1-TEV-CH0848.d949.10.17gp160_N133DN138T_113C-431GCG-A101

CH848 10.17DT SOSIPv4.1 trimer

cap1-TEV-CH848.3.D0949.10.17CHIM1.6R.SOSIP.664V4.1_N133DN138T-A101

CH848 10.17DT SOSIPv5.2.8 trimer

cap1-TEV-CH848.3.D0949.10.17CHIM.SOSIPV5.2.8_N133DN138T-A101

CH848 10.17DT F14 SOSIP trimer ccTEV-CH848-10.17-DT-F14 SOSIP-A101

CH848 10.17DT Vt8 SOSIP trimer ccTEV-CH848-10.17-DT-Vt8 SOSIP-A101

CH848 10.17DT F14/Vt8 SOSIP trimer ccTEV-CH848-10.17-DT-F14Vt8 SOSIP-A101

CH848 10.17DT 113C-429GCG SOSIP trimer

ccap1-TEV-CH848.3.10.17ch.SOSIP_DT_113C-429GCG-A101

CH848 10.17DT UFO-BG SOSIP trimer

ccap1-TEV-CH848.d949.10.17 N133D N138T SOSIP.UFO-BG-A101

CH848 10.17DT DS trimer-ferritin NP

ccap1-TEV-CH848.3.D0949.10.17chDS.SOSIP_N133DN138T-ferritin-A101

CH848 10.17DT 113C-429GCG trimer-ferritin NP

ccap1-TEV-CH848.10.17ch.SOSIP_DT_113C-429GCG-ferritin-A101

CH848 10.17DTe DS 3xGS linker trimer-ferritin NP

CH848.3.D0949.10.17_N113D_N138T_D230N_H289N_P291S_E169K_DS_SOSIP_LSK_ferritin_3xGSlinker

CH848 10.17DTe DS 2xGS linker trimer-ferritin NP

CH848.3.D0949.10.17_N113D_N138T_D230N_H289N_P291S_E169K_DS_SOSIP_LSK_ferritin_2xGSlinker

CH848 10.17DTe DS VRC trimer-ferritin NP

CH848.3.D0949.10.17_N133D_N138T_D230N_H289N_P291S_E169K_DS.SOSIP_VRCferritin

CH65 2G12 DH1029 DH1312 RM19R IgG10.17DT 74.90 3800.33 177.00 77.23 255.3310.17DT F14/Vt8 78.90 1346.00 152.33 69.63 217.0010.17DT DS 93.13 2569.33 165.00 83.50 270.3310.17DT Vt8 79.20 1417.00 161.33 74.73 351.0010.17DT F14 79.23 4204.00 173.00 76.17 281.6710.17DT 113C-429GCG 77.10 4200.33 156.67 76.50 255.0010.17DT 113C-431GCG 82.30 3572.67 156.33 73.90 273.67

D

CH65 17b 19b 7B2

No CD4 0.07 4.77 3.32 6.63+sCD4 0.07 43.93 26.77 8.29

+eCD4-Ig 0.07 72.77 69.83 26.67+CD4-IgG2 0.13 58.40 54.07 8.01

No CD4 0.36 0.68 13.68 21.53+sCD4 0.41 0.53 14.73 20.83

+eCD4-Ig 0.38 0.37 17.84 20.60+CD4-IgG2 0.39 0.53 22.74 19.60

No CD4 0.07 1.61 1.86 6.53+sCD4 0.04 1.95 2.06 6.17

+eCD4-Ig 0.05 3.86 4.19 5.44+CD4-IgG2 0.04 2.48 2.60 6.16

No CD4 0.70 0.86 2.41 11.52+sCD4 0.54 0.87 4.24 11.72

+eCD4-Ig 0.58 11.76 14.60 10.52+CD4-IgG2 0.60 11.78 13.76 11.29

No CD4 0.78 1.13 7.63 5.62+sCD4 0.24 0.40 7.11 6.46

+eCD4-Ig 0.24 0.53 9.56 5.70+CD4-IgG2 0.30 0.49 14.20 6.06

No CD4 0.07 1.88 2.26 7.65+sCD4 0.04 2.03 2.33 8.37

+eCD4-Ig 0.08 1.74 2.25 8.45+CD4-IgG2 0.06 2.11 2.23 10.10

No CD4 0.04 2.62 2.53 10.09+sCD4 0.07 2.67 2.50 9.59

+eCD4-Ig 0.09 2.52 2.40 6.32

+CD4-IgG2 0.14 2.43 2.49 10.02

10.17DT

10.17DT DS

10.17DT F14

10.17DT Vt8

10.17DT F14/Vt8

10.17DT 113C-429GCG

10.17DT 113C-431GCG

CH65 CH01 PG9 PGT125 DH270 UCA10.17DT 0.07 37.30 9.87 82.90 76.0710.17DT DS 0.12 25.33 15.61 64.97 39.6010.17DT F14 0.07 37.57 6.45 85.73 81.6310.17DT Vt8 0.09 13.31 1.65 86.13 34.7710.17DT F14/Vt8 0.09 8.48 1.15 82.50 35.4010.17DT 113C-429GCG 0.07 35.27 0.89 86.37 78.0310.17DT 113C-431GCG 0.04 18.33 0.90 85.87 77.07

A

B C

Singlets75.1

FSC-A

FSC-

HSingle Cells

91.3

SSC-A

SSC-

H

Live cells77.4

SSC-A

LIVE

/DEA

D-B

V510

PE+81.0

DH270 UCA-PE

SSC-

A

Figure S1. Antigenicity of modified mRNA-encoded CH848 10.17DT transmembrane gp160s. Related to Figure 1.(A) Gating strategy for flow cytometric analysis of antibody binding to modified mRNA-expressed CH848 10.17DT gp160s on 293-F cell surface. Figure shows representative gating of DH270 UCA binding to CH848 10.17DT gp160. (B) Binding of bnAbs to modified mRNA-expressed CH848 10.17DT transmembrane gp160s. Data shown were means of PE+ cell percentage among total live cells from three independent experiments.(C) Binding of nnAbs 17b, 19b, and 7B2 to CH848 10.17DT gp160 after CD4 triggering. Three forms of CD4: soluble CD4 (sCD4), eCD4-Ig, and CD4-IgG2 were used. Binding of Abs were detected by Goat anti-Human IgG F(ab’)2 secondary antibody labeled with PE. Data shown were means of PE+ cell percentage among total live cells from three independent experiment.(D) Binding of Env base antibodies to CH848 10.17DT gp160s. Data shown were averages of mean fluorescence intensity (MFI) from three independent experiment. Influenza hemagglutinin Ab CH65 and HIV bnAb 2G12 were used as negative control and positive control, respectively.(E) 17b and 19b binding to F14 stabilized CH848 10.17DT and CH505 M5 G458Y SOSIP trimers measured by SPR. The data demonstrated that F14 mutations do not intrinsically impair 17b or 19b binding.

E

A B C

300 Å

D90° 90°

30 Å

5,868particles

4,672particles

3,984particles

2,399particles

2,831particles

50 Å

300 Å

DH1029

E F G

H90° 90°

300 Å

30 Å

50 Å8,999

particles4,912

particles4,339

particles

300 Å

DH1312

I

J 90° 90°

30 Å

30 Å

DH1029 DH1312 RM19R

Figure S2. NSEM structures of SOSIP complex with DH1029 and DH1312, and comparison to RM19R. Related to Figures 1 and 5.(A) Raw micrograph of DH1029 Fab complexed with CH505M5chim.6R.SOSIP.664v4.1_G458Y/GnTI- Env trimer.(B) Representative 2D class averages of Fab-bound trimers.(C) 3D classification shows two classes with two Fabs bound (gray and yellow), one class with weaker density for one of the Fabs (cyan), and two junk particle classes (purple and pink). The number of particles is indicated below each class.(D) Final reconstruction from the combined particles from the first two classes in C.(E) Raw micrograph for DH1312 Fab complexed with CON-Schim.6R.DS.SOSIP.664_OPT_N130D_N135K_N138S_N141S/293F Env trimer.(F) Representative 2D class averages of Fab-bound trimers.(G) 3D classification shows one class with three Fabs bound (gray), one class with weaker density for one of the Fabs (yellow), and one junk particle classes (cyan). The number of particles is indicated below each class.(H) Final reconstruction from the combined particles from the first class in C.(I) Comparison of SOSIP complexes with DH1029, DH1312, and RM19R show side by side. For RM19R, the cryo-EM map (EMD 21227) has been low-pass filtered to 20 Å. (J) Superposition of DH1029, DH1312 and RM19R shown in three orthoganal views.

10.17DT F14 gp16010.17DT 113C-429GCG gp160

mRNA-LNP immunogens:

CH848 10.17DT trimer CH848 10.17DT Δ11 gp120 CH848 V3 peptide

gp41

A CB

D

10.17DT F14 gp160 mRNA-LNP 10.17DT 113C-429GCG gp160 mRNA-LNP

Mock10.17DT

10.17DT DS

10.17DT 113C-429GCG

101

102

103

104

Cell surface-expressed 10.17DT gp160 Envs

MFI

-1 5 -1 5 -1 5 -1 5

Mock10.17DT

10.17DT DS

10.17DT 113C-429GCG

101

102

103

104

Cell surface-expressed 10.17DT gp160 Envs

MFI

-1 5 -1 5 -1 5 -1 5Week

Week

F

G

MuLV 92RW020CH848.d949.10.17

6101CH848.d949.10.

17DT.N332TQ23

CH848.d949.10.17.DT

CH848.d949.10.17.DT.D230N.H289N.P291S

Immuniza�on

<200 <200 <200 <200 316 <200 3465 4016<200 <200 204 <200 37 <200 4771 6898<200 <200 <200 <200 <20 <200 5641 6797<200 <200 406 <200 56 <200 18342 24220<200 <200 584 <200 <20 <200 8550 11164<200 <200 745 <200 106 <200 19357 21662<200 <200 615 <200 29 <200 15519 13945<200 <200 424 <200 <20 <200 7191 7092<200 <200 480 <200 41 <200 6126 5534<200 <200 331 <200 <20 <200 5569 4540<200 <200 <200 <200 5767 <200 6130 7959<200 <200 <200 <200 23357 <200 20061 16434

ID50nt

<200160-999

1000-9999>10000

IC50 (1/dilu�on) in TZM-bl cells

CH848 10.17DT F14 gp160 mRNA-

LNP

CH848 10.17DT 113C-429GCG gp160 mRNA-

LNP

0 2 4 6

0

5

10

15

Study weeks

Log

AUC

0 2 4 6

0

5

10

15

Study weeks

Log

AUC

0 2 4 6

0

5

10

15

Study weeks

Log

AUC

0 2 4 6

0

5

10

15

Study weeks

Log

AUC

E

Figure S3. Serum antibody binding and neutralization activity after CH848 10.17DT gp160 mRNA-LNP immunizations in DH270 UCA KI mice. Related to Figure 2.(A-D) ELISA logAUCs of vaccinated mouse serum IgG binding to (A) CH848 10.17DT DS SOSIP trimer; (B) CH848 10.17 Δ11 gp120; (C) CH848 V3 loop peptide; (D) gp41 subunit.(E) Predicted possible glycan holes on CH848 10.17 Env. PNGL: potential N-linked glycosylation site.(F) Week 5 serum antibody binding to modified mRNA-expressed CH848 10.17DT, CH848 10.17DT DS, and CH848 10.17DT 113C-429GCG gp160s on 293-F cell surface. Each dot signifies an individual mouse (N = 6 each group). Data shown are MFI measured by flow cytometry.(G) Serum neutralizing titers after CH848 10.17DT gp160 mRNA-LNPs immunization. Serum neutralization titers one week after the third immunization in DH270 UCA KI mice were measured using TZM-bl reporter cells. Data shown were ID50s. Murine leukemia virus (MuLV) was used as negative control.

CH848DT-sp.GC B cells

CH848DT-sp.IgG+ Non-GC B cells

A

GC TFH

TFH

Antigen-specific B cell gating

Tfh cell gatingB

Figure S4. Flow cytometric gating strategies for antigen-specific B cells and Tfh cell populations. Related to Figures 2 and 6.(A) The gating strategy to identify CH848 10.17DT-specific germinal center B cells and memory B cells.(B) The gating strategy to identify T follicular helper (Tfh) and germinal center Tfh (GC Tfh) cells.

Figure S5. NSEM analysis of modified mRNA-expressed CH848 10.17DT DS trimer-ferritin NPs. Related to Figure 5.(A) Representative raw image of modified mRNA-expressed, bnAb PGT145-purified CH848 10.17DT DS 3xGS linker trimer-ferritin NPs.(B) Green circles indicate particles automatically picked by the software. From 60 images, a total of 12,365 particles were picked. (C) 2D class averages. Labels indicate tentative identification of particle types for trimer-ferritin nanoparticles (NP), apparent Env trimers (E), galectin-3 binding proteins (G), unknown trimeric proteins (T), and proteosomes (P). (D) Table indicating the number of classes, the number of particles in those classes, and the percentage of the total particles for each identified type.(E) Representative raw image of modified mRNA-expressed, bnAb PGT145-purified CH848 10.17DT DS VRC trimer-ferritin NPs.(F) Green circles indicate particles automatically picked by the software. From 60 images, a total of 12,365 particles were picked. (G) 2D class averages. Labels indicate tentative identification of particle types for trimer-ferritin nanoparticles (NP), apparent Env trimers (E), galectin-3 binding proteins (G), unknown trimeric proteins (T), and proteosomes (P). (H) Table indicating the number of classes, the number of particles in those classes, and the percentage of the total particles for each identified type.

Worst-casescenario

Best-casescenario

Worst-casescenario

Best-casescenario

10.17DT DS trimer-ferritin NP10.17DT 113C-429GCG trimer-ferritin NP10.17DT DS 3xGS linker trimer-ferritin NP

10.17DT DS 2xGS linker trimer-ferritin NP10.17DT DS VRC trimer-ferritin NPEmpty LNP

gp41

0 1 2 3 4 50

5

10

15

Study weeks

Log

AUC

CH848 V3 peptide

0 1 2 3 4 50

5

10

15

Study weeks

Log

AUC

CH848 10.17DT Δ11 gp120

0 1 2 3 4 50

5

10

15

Study weeks

Log

AUC

Human ferritin

Study weeks

0 1 2 3 4 50

5

10

15

Log

AUC

Helicobacter pylori Ferritin

0 1 2 3 4 50

5

10

15

Study weeks

Log

AUC

CH848 10.17DT trimer

0 1 2 3 4 50

5

10

15

Study weeks

Log

AUC

A CB

D FE

mRNA-LNP immunogens:

MuLV CH848.10.17 CH848.10.17.N332T CH848.10.17.DT CH848.10.17.DT.N332TCH848.10.17.DT.D230N.H

289N.P291S92RW020.2 6101.10

Immuniza�on

<160 <160 nt 4344 <160 5301 <160 170<160 <160 nt 7134 <160 5542 <160 <160<160 <160 nt 6034 <160 6018 172 285<160 <160 nt 4682 <160 3480 <160 185<160 194.5 <160 7362 <160 7803 <160 <160<160 <160 nt 4187 <160 5321 <160 197<160 <160 nt 1966 <160 1629 <160 <160<160 532 <160 15206 <160 20932 <160 177<160 <160 nt 4071 <160 5360 <160 168<160 532 <160 4396 <160 6340 <160 172<160 <160 <160 1519 <160 1936 <160 318<160 <160 nt 12110 <160 14835 <160 186<160 <160 nt 4549 <160 6242 <160 192<160 <160 nt 12339 <160 23328 <160 190<160 <160 nt 6308 <160 7913 <160 <160<160 <160 nt 3590 <160 5466 <160 219<160 <160 nt 1501 <160 1187 <160 <160<160 324.5 <160 10127 <160 16398 <160 <160<160 <160 nt 5041 <160 6892 <160 <160<160 <160 nt 6573 <160 12384 <160 185<160 <160 nt 4749 <160 5580 <160 163<160 <160 nt 5448 <160 9133 <160 225<160 <160 nt 9814 <160 15247 <160 160<160 415.5 <160 13412 <160 24893 <160 360<160 325 <160 14835 <160 18650 <160 492<160 467 <160 20052 <160 36649 <160 193<160 <160 nt 3117 <160 7575 <160 172<160 <160 nt 4399 <160 8302 <160 261<160 <160 nt <160 <160 <160 <160 <160<160 <160 nt <160 <160 <160 <160 <160<160 <160 nt <160 <160 <160 <160 261<160 <160 nt <160 <160 <160 <160 432<160 <160 nt <160 <160 <160 <160 225<160 <160 nt <160 <160 <160 <160 <160

ID50nt

<160160-999

1000-9999>10000

CH848 10.17DT DS 2xGS linker LSK ferri�n NP

mRNA-LNP

CH848 10.17DT DS VRC ferri�n NP mRNA-LNP

Empty LNP

ID50 (dilu�on) in TZM-bl cells

CH848 10.17DT DS ferri�n NP

mRNA-LNP

CH848 10.17DT 113C-429GCG

ferri�n NP mRNA-LNP

CH848 10.17DT DS 3xGS linker LSK ferri�n NP

mRNA-LNP

G

Figure S6. Serum IgG binding activity after CH848 10.17DT trimer-ferritin NP mRNA-LNP immunizations in DH270 UCA dKI mice. Related to Figure 6.(A-F) ELISA logAUCs of vaccinated mouse serum IgG binding to (A) CH848 10.17DT DS SOSIP trimer; (B) CH848 10.17 Δ11 gp120; (C) CH848 V3 loop peptide; (D) gp41 subunit; (E) Helicobacter pylori ferritin, and (F) human ferritin.(G) Serum neutralizing titers after CH848 10.17DT trimer-ferritin NP mRNA-LNP immunizations. Serum neutralization titers one week after the third immunization in DH270 UCA KI mice were measured using TZM-bl reporter cells. Data shown were ID50s. Murine leukemia virus (MuLV) was used as negative control.

Total mutants Total mutants Total mutants48401 110 86 79 76 49 68 4548402 40 28 25 26 19 25 1848404 58 31 27 36 29 29 2348405 55 40 40 35 28 35 2848407 43 19 17 22 16 15 1348411 52 34 33 34 28 33 2948412 39 25 23 23 19 23 18

Total 397 263 244 252 188 228 174

VH1-2 # VL2-23 # VH1-2/VL2-23 #

1

2

Group Animal ID mAb #

A

B

Figure S7. Isolation of CH848 10.17DT-specific antibodies. Related to Figure 7.(A) Gating strategy used for single cell sort of CH848 10.17DT-specific memory B cells from vaccinated mice splenocytes. (B) Summary of single B cell sort and cloning of Ig genes by PCR. A total number of 617 B cells were sorted on 96-well plates. 397 pairs of heavy and light chains were successfully recovered by sequencing of PCR amplicon. Among these heavy and light chain pairs, 263 of heavy chains were DH270 VH1-2, 252 of light chains were DH170 VL2-23, and 228 pairs were both DH270 heavy chain VH1-2 and DH270 light chain VL2-23. 169 of 228 DH270-like antibodies have acquired at least one amino acid change. DH270.mu84 was isolated from animal 48402; DH270.mu85 was isolated from animal 48411; DH270.mu86 was isolated from animal 48412.

!"#$ %""&

Q V Q L V Q S G A E V K K P G A S V K V S C K A S G Y T F T G Y Y M H W V R Q A P G Q G L E W M G W I N P N S G G T N Y A Q K F Q G R V T M T R D T S I S T A Y M E L S R L R S D D T A V Y Y C A R G G W I S L Y Y D S S G Y P N F D Y W G Q G T L V T V S S

. . . . . . . . . Q M . N . . . . . . . . . A P . . . . . . D F . I . . L . . . . . . . . Q . . . . M . . Q T . R . . T . R N . . . . . . . . . . . . . G . . . . . . R S . T . . . . . I . . . T T . . . . . . . . . . . Y . . . . . H . . . . . . L . . . . 1 9 8

. . . . . . . . T . . . . . . . . . . . . . . . . . . . . . D . . L . . L . . . . . . D . . . . . . . . . . . . . S . . . . . . . . . F . . A . . . . . . . . . . . . . . . T . . . . . . F . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . R . . . . . L . D . . V . . . . . . . . . . . . . . . . . . S K T . . A . D . . N . . . . I I . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

. . . . . . . . . . . M . . . . A . . . . . M . . . . . L . D . . L . . . . . . . . . . . . . . . . . . . H R . . S D H . . . . . . . . . I . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . . . . T R . S . . . . . . . . . . . . . . . V . . . . . . . . . . T . . . . . . . . . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . . . . T R . S . . . . . . . . . . . . . . . V . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . R . . . M . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . H R . . A . D . . . . . . . . . . . . . . . . . . . H . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . M . . . . . . . . . . . . D . . . . . . . . . . . . . . . . . . . F . . . . . . . . F E . . . . D . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . 1 0

. . . . . . . . . . . . . . . . . . . . . . M . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . H R . . A . D . . M . . . . . . . . . . . . . N . V H . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . V . D . . I . . . . . . . . . . . . . . . . . . . . . . . S . S . . . . . . . . . . . . . . . . N . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N F . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . E . . . . D . . I . . . . . . . . . . . . . . . . . . . . R . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . . . . F F . . . . . . N . . . . . G . . . . . . . . . . . . . . . . . . . 2 0

M C T . . T . . . . . . N . . . . . . . . . . . . . . . . . D H . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . A . . . . . N . . . Y . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1

. . . . . . . . T . . . . . . . . . . . . . . . . . . . . . D . . I . . . . R . . . . . . . . . . . . . . . . . N R . F . E Q . . . . . . L . . . S . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . . S . I . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . . I . . . . . . . . . . . . . . . . . . . S . . . . . . . . N . E . . . . . . . . . . . R . . F . . . . . . . . . . . . . . . . . T . . . . . . . . . . . A . . . . . . . . . . . . . A . . . 3 1

. . . . . . . . T . . . . . . . . . . . . . . . . . . . . . D . . . . . . . . . . . . . . . . . . . . . . . . . S R . F . . Q . . . . . . L . . . S . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . S . . . . . . 1 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . H R . . A . S . . . . . . . . . . . . . . . . . . . . L . . N . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . K . . . R . F V E . . . . . . . . . . . . . . . . . . . D . K S . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . R . . . . . . . . . I . D . . L . . . . . . . . . . . . . . . . . . . . . . . . . D . . . I . . . . . . . . . . . . T . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . Q . . . . . . . D . . I . . . . . . . . . . . . . . . . . . . . T . . R . F E . . . . . . . . . . . . S . . . . . F . . . N . . I . . . . . . . . . T T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1

. . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . M . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . D . N S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0

. . . . . . . . . . . . . . . . . . . . . . Q . . . . . . . D . . I . . . . . V . . H . . . . . . . . . . . . . . R . F E . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . N . . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L K D . . I . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . S . . . . . . . N . . . . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

. . . . . . . . . . . Q . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . . R . . S . C . . . . E . . . . . . . . . . . N . . . . . . K . . T . . . A . . . . . . . . . . . . . . . . . . . . . . . . H . . . . . . . . . . . 4 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . . I . . . . . . . . . . . . . . . . . . . . . . . . . S V . . . . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . K . . . R . F V E . . . . . . . . . . . . . . . . . . . D . K S . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . L . . . . . . . . . . . . . . . . . . . . . . . K . S . . . . . D . . . . . . . . . L . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . R . . . . . . . . 1 0

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . . I . . . . . . . . . . . . . . . . F . S K T . . R . F . . . . . D . . . V . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0

. . . . . . . . . . . . . . . . . . R . . . . . . . . . L . D . . . . . . . . . . . . . . . . . . . . . . . T . . R . H V E N . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . . T . K . . . . D . . I . . . . . . . . . . P . . . . . . . . . . . . S . S . . . . . . . . . . . . . . . . N . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . . I . . . . . . . . . . . . . . . . F . . K . . . R . F . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . F . . S T . . R . . . . . . . . . . . . . . . . . . . . . N . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . . P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K S . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0

. . . . . . . . . . . . . . . . . . . . . . M . . . . S . N D . . I . . . . . . . . . . . . . . . . . . . T T . . R D F V . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . R M . . . . . 4 0

. . . . . . . . . . G . . . . . . . . . . . . . . . . . . . D . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . 1 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . I L . D . . . . . . . . . . . . . . . . . . . . . . . T . . R . F D . . . . . . . . . . . . . . . . . . . . . . N . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . . . . R . . S E . . . . . . . . . . . . . . . G . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . . . . . . . . . . . 4 1

. . . . . . . . . . . . . . . . . . T . . . . . . . . . . . D . . L . . . . . . . . . . . . . . . . . . S K T . . S . D . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . A . . D . . I . . . . . . . . . . . . . . . . . . . . T . S R . F . E . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

. . . . . . . . . . . R . . . . . M . . . . . . . . F . L . D . . I . . . . . . . . . . . . . . . . . . . . . . . S . N . . . . . . . . . . . . . . . . . . . S L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . Q . . . . . 2 0

. . . . . . . . . . . . . . . . . . R . . . . . . . . . . . D . . I . . I . . . . . . . F . . . . . L . . . T . A R . . V . . . . . . . . . . . . . . . . A . . . . . N . . G . . . . . . . . . . T . S . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . . L . . . . . . . . . . . . . . . . . . . . . . . R . F E D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . 1 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . T H . . . A . N . . . . . . . . . . . . . . . . . . . . . . . . . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0

. . . . . . . . . . . . R . . . . . . . . . . . . . F . . . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . 2 0

. . . . . . . . . . . . R . . . . . . . . . . . . . F . . . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . R . . . . . L . D . . I . . . Q . . . . . . . . . . . . . . . . T . . A . S . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . P . . . . . . . . . . . . S . N . . T . . . . . . . . . . . . F N . . . . . . . G . K . . . S . . . . . . . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . P . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . F . . . . . . . . . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . . . . . . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . L . . . . . . . . . . . . . . . . . . . . . . . S . . I E . . R . . F . V . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . S . . . . . . 1 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . . L . . . . . . . . . . . . . . . . . . . K T . . R . F . . . . . D . . . V . K . . . . . . V . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . . . . . R . S E E . . . . . . . . . . . . . V G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . . . . . F . L S D . . I . . I . . . . . . E . . . . . . . . . . . . . S . D . L R . . . . . . . . . . . . . N . . . . . . . . . T . . . . . . . F . . . . . . . . . . . . . . . . . . . . . . . . . . P . . . . . 3 0

. . . . . . . . . . . . . . . . . . . . . . R . . . . . . . D . . I . . . . . . . . . . . . . . . . . . . . . . . R . . I E . . . . . F . . . . . . . . . . . . . . . . . . . . . . . . . . . . T T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . . I . . . . . . . . . . . . . . . . . . . . T . S R . . . . . . . . . . . . . . . . . . . . T . . D . . G . N . . . . . . F . . . . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . 3 0

. . . . . . . . P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . K R . . A . N V . . . . D . . . L . . . . . . N . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

. . . . . . . . V . . . . . . . . . . . . . . . . . . . L . D . . . . . I . . . . . . . . . . . . . . . T . T . . R . . E . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . D . . I . . . . . . . . . . . . . . . . . . . . . . . R . . V E . . . . . . . . . . . . . V N . . . L . . . . . M . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . . I . . . . . . . . . . . . . . . . . . . . T . . R . F . E . . . D . . . . . K . . . . . . . . . . M R . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . 4 0

. . . . . . . . . A . . . . . . . . . . . . E . . . . . . . D . . I . . . . L . . . . . . . . . . . . . . . . . . . . F P . . . . D . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . 2 0

. . . . . . . . . . . . . . . . . M . . . . . . . . . . . . D . . L . . . . . . . . . . . . . . . . . . . . . . . A . F V . . . . . . . . . P . . . . . T . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

. . . . . . . . T . . . . . . . . . . . . . . . . . . . L . D . . . . . . . . . . . . . . . . . . . . . . . T . . R . . . E . . . . . . S . . . . . . . N . . . . . . . . . . . . . . . . F . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

10 20 30 40 50 60 70 80 90 100 110 120

Mu484_Hu48405_PL008_VH1_H6 Mu484_Hu48405_PL008_VH1_H10Mu484_Hu48405_PL008_VH1_D11Mu484_Hu48402_PL003_VH1_H9 Mu484_Hu48402_PL003_VH1_H7 Mu484_Hu48402_PL003_VH1_H2 Mu484_Hu48402_PL003_VH1_G7 Mu484_Hu48402_PL003_VH1_G5 Mu484_Hu48402_PL003_VH1_F9 Mu484_Hu48402_PL003_VH1_F7 Mu484_Hu48402_PL003_VH1_F2 Mu484_Hu48402_PL003_VH1_E8 Mu484_Hu48402_PL003_VH1_E7 Mu484_Hu48402_PL003_VH1_E2 Mu484_Hu48402_PL003_VH1_D5 Mu484_Hu48402_PL003_VH1_B7 Mu484_Hu48402_PL003_VH1_B6 Mu484_Hu48402_PL003_VH1_B3 Mu484_Hu48412_PL006_VH1_H10Mu484_Hu48412_PL006_VH1_G5 Mu484_Hu48412_PL006_VH1_F2 Mu484_Hu48412_PL006_VH1_F10Mu484_Hu48412_PL006_VH1_E7 Mu484_Hu48412_PL006_VH1_E2 Mu484_Hu48412_PL006_VH1_E11Mu484_Hu48412_PL006_VH1_D7 Mu484_Hu48412_PL006_VH1_D2 Mu484_Hu48412_PL006_VH1_C8 Mu484_Hu48412_PL006_VH1_C2 Mu484_Hu48412_PL006_VH1_B8 Mu484_Hu48412_PL006_VH1_B7 Mu484_Hu48412_PL006_VH1_B3 Mu484_Hu48412_PL006_VH1_A8 Mu484_Hu48401_PL002_VH1_H6 Mu484_Hu48401_PL002_VH1_H11Mu484_Hu48401_PL002_VH1_G9 Mu484_Hu48401_PL002_VH1_G3 Mu484_Hu48401_PL002_VH1_G10Mu484_Hu48401_PL002_VH1_F4 Mu484_Hu48401_PL002_VH1_F3 Mu484_Hu48401_PL002_VH1_F2 Mu484_Hu48401_PL002_VH1_F11Mu484_Hu48401_PL002_VH1_E7 Mu484_Hu48401_PL002_VH1_E5 Mu484_Hu48401_PL002_VH1_E2 Mu484_Hu48401_PL002_VH1_D6 Mu484_Hu48401_PL002_VH1_D5 Mu484_Hu48401_PL002_VH1_D11Mu484_Hu48401_PL002_VH1_C9 Mu484_Hu48401_PL002_VH1_B9 Mu484_Hu48401_PL002_VH1_B3 Mu484_Hu48401_PL002_VH1_B2 Mu484_Hu48401_PL002_VH1_B10Mu484_Hu48401_PL002_VH1_A7 Mu484_Hu48401_PL002_VH1_A5 Mu484_Hu48401_PL002_VH1_A3 Mu484_Hu48401_PL002_VH1_A11Mu484_Hu48401_PL001_VH1_H8 Mu484_Hu48401_PL001_VH1_G6 Mu484_Hu48401_PL001_VH1_G10Mu484_Hu48401_PL001_VH1_E10Mu484_Hu48401_PL001_VH1_D9 Mu484_Hu48401_PL001_VH1_D5 Mu484_Hu48401_PL001_VH1_D3 Mu484_Hu48401_PL001_VH1_C9 Mu484_Hu48401_PL001_VH1_B9 Mu484_Hu48401_PL001_VH1_B5 Mu484_Hu48401_PL001_VH1_B10Mu484_Hu48401_PL001_VH1_A8 Mu484_Hu48401_PL001_VH1_A10Mu484_Hu48404_PL007_VH1_F9 Mu484_Hu48404_PL007_VH1_F8 Mu484_Hu48404_PL007_VH1_F7 Mu484_Hu48404_PL007_VH1_F6 Mu484_Hu48404_PL007_VH1_E7 Mu484_Hu48404_PL007_VH1_E6 Mu484_Hu48404_PL007_VH1_E3 Mu484_Hu48404_PL007_VH1_E10Mu484_Hu48404_PL007_VH1_D8 Mu484_Hu48404_PL007_VH1_D4 Mu484_Hu48404_PL007_VH1_D2 Mu484_Hu48404_PL007_VH1_C4 Mu484_Hu48404_PL007_VH1_C3 Mu484_Hu48404_PL007_VH1_C10Mu484_Hu48404_PL007_VH1_B8 Mu484_Hu48404_PL007_VH1_B7 Mu484_Hu48404_PL007_VH1_B2 Mu484_Hu48404_PL007_VH1_B10Mu484_Hu48404_PL007_VH1_A9 Mu484_Hu48404_PL007_VH1_A8 Mu484_Hu48404_PL007_VH1_A7 Mu484_Hu48404_PL007_VH1_A4 Mu484_Hu48404_PL007_VH1_A11Mu484_Hu48411_PL005_VH1_H4 Mu484_Hu48411_PL005_VH1_H11Mu484_Hu48411_PL005_VH1_G5 Mu484_Hu48411_PL005_VH1_G4 Mu484_Hu48411_PL005_VH1_G3 Mu484_Hu48411_PL005_VH1_G2 Mu484_Hu48411_PL005_VH1_G10Mu484_Hu48411_PL005_VH1_E9 Mu484_Hu48411_PL005_VH1_E6 Mu484_Hu48411_PL005_VH1_E3 Mu484_Hu48411_PL005_VH1_D6 Mu484_Hu48411_PL005_VH1_D3 Mu484_Hu48411_PL005_VH1_C9 Mu484_Hu48411_PL005_VH1_C7 Mu484_Hu48411_PL005_VH1_C3 Mu484_Hu48411_PL005_VH1_C11Mu484_Hu48411_PL005_VH1_B11Mu484_Hu48411_PL005_VH1_B10Mu484_Hu48411_PL005_VH1_A9 Mu484_Hu48407_PL001_VH1_H9 Mu484_Hu48407_PL001_VH1_H4 Mu484_Hu48407_PL001_VH1_F11Mu484_Hu48407_PL001_VH1_E8 Mu484_Hu48407_PL001_VH1_E6 Mu484_Hu48407_PL001_VH1_E4 Mu484_Hu48407_PL001_VH1_D2 Mu484_Hu48407_PL001_VH1_C9 Mu484_Hu48407_PL001_VH1_C7 Mu484_Hu48407_PL001_VH1_C4 DH270.6 DH270_UCA3_HC

%"#$ !"& !#$ %"& %#$ !& "'%#$ %& "#$ "'%&!"#$#%&'()*&+$+%,%#-

Figure S8. CH848 10.17DT trimer-ferritin NP mRNA-LNP immunization elicited antibodies with heavy chain somatic mutations shared with DH270.6. Related to Figure 7.Heavy chain amino acids of isolated VH1-2/VL2-23 DH270-like mAbs aligned with DH270 UCA and DH270.6. Dots represent amino acids that are in common with DH270 UCA. Mutations shared with DH270.6 are highlighted in colors according to mutation probability. The numbers to the right of each row are total numbers of probable mutations and improbable mutations observed, respectively.

Q S A L T Q P A S V S G S P G Q S I T I S C T G T S S D V G S Y N L V S W Y Q Q H P G K A P K L M I Y E V S K R P S G V S N R F S G S K S G N T A S L T I S G L Q A E D E A D Y Y C C S Y A G S S T V I F G G G T K L T V L . . . . . . . . . . . . . . . . . . . . . . . . . K Y . . . . H D . . . . . . . Y . . . V . . Y . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . R . . . . . . . . . . . F G . . A . . V C . . . . . V . . . 9 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . . . . . . . . K . . . . . . . . . . . . . N . . . . . . . T . . . . . . . . . . . . . . V . . . . . . . A . . . . . . . . . . . . . . . . V . . . . . S . . 1 0. . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . D . . . . . . . . . . . . . . . . . . . . .. . . . E . . . . . . . . . . . . . . . . . . . . . . . . . V . . . N . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . L . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . I . . . . R . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . T . . . . . . K . S . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . E . V . A . . . . . . . . . . . . . . . . . . . . . V . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . T . . . . . . K . S . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . S . . . . E . V . A . . . . . . . . . . . . . . . . . . . . .V . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . V . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . 2 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . L . . . . . . . . . S . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . N . . . . N . . . . . . . . . . . . . V . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . I . . . . . . . . . . . 2 0. . . . . . . . . . . . A . . . . . . . . Y S . S . . . . . N . . . I . . . . . . .. . . . . . . . . . . N . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . . . . . . . A . . . . . . . . . . . . . . . . . . . . . V . . . 1 0. . . . . . . . . . . . . . . . . . . . . Y . . . N . . . . N . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . V . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . G . . . . . . . . .. . . . . . . . . . . . . . . I 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . Y . . . . . N . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . V . . . 2 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . H . V . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . I 1 0. . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . Y . . . . . . H Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A T . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . .N . . . . . . . . . . . . . . . . . . . . . . T . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . Q . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . T . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . L . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . N . . I . N . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . H . . . . . . A . . . . . . . . . . . . . . V . . . . . . . . . . . . . . L . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . I . . . . . . . . . . . . R . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . .. . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . I . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . T . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . . . T . . . . . . . . . T . W . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . A . . . . . . . . . . . . I . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . T . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . I . N . . . . . . . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . F . . . . . . . Y . . . . . . . . . . . . N . . . . . . . . . . . . . . . D . . . . . . . . . . . . . . A . E . . . . . . . . . . . . . . . . . . R . . . . 2 0. . . . . .. . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . H . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . . . . V . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . A T . . F . . . . . . . . A . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . I L . . . . . . . . . . . . . . . .. . . S . . . . . . . . . . . . . . . . . . R . . . V . . . . . . . . . . . . . . V . . . . . . . . . . 1 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . I . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . Y . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . . . . . . . . . . I . . . . . . . . . . D . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . V . . . L . . . . . . . . . . . . D . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . A T . . F . . . . . . . . A . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . .. . L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . N L . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . A T . . F . . . . . . . . A . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D H . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . .. . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . D . . . . . . . . . . N . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . V . . . 3 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . A T . . F . . . . . . . . A . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . N L . . V . . . . . . . . N . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . 2 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . .. . . . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . E . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A G . . F . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . A T . . F . . . . . . . . A . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A .. . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . R . . C . . . . C F . . D . . . . . . . . . . . T D . . . . . . . . . . . . E . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y. . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . A . . . . . N . D . . . . . . . . . . . . . . . I . . . . N . W . . . . . . . . . . . . . . S . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . V . . . 3 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . W . . . . . . . . . . . . . . . . . .. . . . . . . . . . A . . . . . . . . . . . . . . . . . . R . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . S . . . . . A . . . . . . . . . . . . I . . . . . . . . V . . . 1 0. . . . . . . . . .. . . . . . . . . . . . . . S . . . . . N . . . . . . . . . . . . . V . . . . . . . . C N . A . . I Y . . . . . . N . . . A . F . . . . . . . . . . A . . . H . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . A . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . N . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . M . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . N . . . N . . . . . . . . . . . . . . . . . . . . . . T . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . V . . . 1 0. . . . . . . . . . . . . . . . P . . . . . . . . . . . I . T . . . . . . . . . . . . . . . R . . . . . . . . W . . . . . . . . . . . R . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . . . . . . . . . . . V . . . . . . . . . . . S . . A . . . . . . . . . D . . . . . . . . . . R . . . G . . . . . . . . . . . . . I . . . . . . . . . . . 2 1. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . L . . . . . . . . . .. . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . V . . I 1 0. . . . . . . . . . . . . . . . . . . . . . S . . . . . . . I . . . . . . . . R . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . D . . . . . . . . . . . . . . . . . . . . . . . A S . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . I . T . . . . . . . . . . . . . . . . . . . . . I T . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . A . N . . . . . . . . . . . . . . .. . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . N . . . . . . . . . . . . A . . . . D . . . . . . . . . . . . . . . . . . . . . . A . . . H . . . . . . . . . I . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . .. . . . . . . . . . . . . . . . . W . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . N . . . . T . . . . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . L . . . . . . V . . . 1 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . N . . . . . . . . . . . L . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . .. . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . V . . I 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . D Y N . A . R . . . C . . . . Q . . . M . . . . . . . . . G . . A V . . . S . Q . . . . . I . M . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . G . . F . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . D . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . .. . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . S . . . . . . . . . . . . F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . V . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . R . . . . . . . . . . . . N . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . V . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . F . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . D . . . . . . . . . . . . . . . . . . . . I . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . I . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . I . N . . . . . . . . . Y . . . . . Q . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A A . . . . . . . . . . . . . . . . . . . . . . . . . 2 0. . . . . . . . . . . . . . . . . . . . . F . . . . . . I . N . . . . . . . . . . . . . . . . . L . . . . N . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . T . . . . N . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . A . . . . . . . . . N . . V . . . . . . . . . . 2 0. .. . . . . . . . . . . . . . . . . . . . . . . . T . . . T . . . . . . . . . . . . . . . . Y . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . S . . . . . . . . V . . . 2 1. . . . . . . . . . . . . . . . . . . F . . . . . . . . . . N . . . . . . . R . . T . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . G . . . . . . . . . . . . . . . . . . . . R . . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . W . . . . . . . . . . . . . . . . . . . . . . . . . . Q . A . . . . . . . . . . . . . . M . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . N . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D S . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . D . . . . . . . . . . . . . . . . . L . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . A . . . . Y . . . . . . . I . L . . . . . .. . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . G G . . . N . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . L . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . N . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . S . . . . . . . . . .. . . . . . . . . . . N . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . G T . . . . . . . . . . . . . . . . . . . . V . . . 2 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V G . . . . . . . . . . . . . V . . . . . . . . . . 2 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . T . D . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 2 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . K . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . I . . . . . . . . . . . . . . . . . . . . A . E . . . . . . .. . . . I . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . . . G . . . F . . . . . . . . . . M . . . . . . . . . . 0 1. . . . . . . . . . . . . . . . . . . . . . . . . N Y . . . N . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . S . . . . . . . . . . . . 1 1. . . . . . . . . . . . . L . . . . . . . Y . . . . . . . A N . . . . . . . . . . . . . . . . . L . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . L . . . . . . V . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . S . . . . .N . . . . . . . . H . . . . . . . . . . . . . N . . A . . A . . . . . . . . . . . . . . . I . . . . . . . . A . . . . . . . . V . . . I . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . D . . . . . R . . . . E . . . F . . . . . N . . . . . . . . . . . . . . . . D . . . . . . . . . . . . . V . . . . . . . . . . . . I . . . . . . . . . . . . 2 0. . . . . . . . . . . . . . . . . . . . . Y . . . . . . I . . . . F . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . S . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . Y . . . D . . . . N . . . . . . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . V . . . . . . . . . . . . . . L . . . . . . V . . . 2 0. . G . . . . . . . . . . . . . . . . L . . . . . . . . . . N . A . . . . . . K . . . . . . . . . . . . . N . . A . . . . . . . . . . . . . . A . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . 1 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . L . N . . . . . . . . L . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . 0 0. . . . . . . . . . . . . . . . . . . . . . . . . . N . I . T . . . . . . . . H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . V . . . . . . . . G . . . I . F . . . . . . . . . . 0 1

10 20 30 40 50 60 70 80 90 100 110

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Figure S9. CH848 10.17DT trimer-ferritin NP mRNA-LNP immunization elicited antibodies with light chain somatic mutations shared with DH270.6. Related to Figure 7.Light chain amino acids of isolated VH1-2/VL2-23 DH270-like mAbs aligned with DH270 UCA and DH270.6. Dots represent amino acids that are in common with DH270 UCA. Mutations shared with DH270.6 are highlighted in colors according to mutation probability. The numbers to the right of each row are total number of probable mutations and improbable mutations, respectively.

mRNA-LNP gp

160s

mRNA-LNP N

Ps10-1

100

101

102

G57R

Mut

atio

n fre

quen

cy %

mRNA-LNP gp

160s

mRNA-LNP N

Ps10-1

100

101

102

R98T

Mut

atio

n fre

quen

cy %

mRNA-LNP gp

160s

mRNA-LNP N

Ps103

104

105

CH848 10.17DT

IC50

(1/d

ilutio

n) in

TZM

-bl c

ells

CH848 10.17DTD230N.H289N.P291S

mRNA-LNP gp

160s

mRNA-LNP N

Ps103

104

105

IC50

(1/d

ilutio

n) in

TZM

-bl c

ells

Figure S10. Comparing mRNA-LNP-encoded CH848 10.17DT gp160s and mRNA-LNP-encoded SOSIP trimer-ferritin NPs. Related to Figures 2 and 6.(A) Comparison of mutation frequencies of DH270 heavy chain improbable G57R and R98T mutations between CH848 10.17DT transmembrane gp160 (blue circle) and CH848 10.17DT SOSIP trimer-ferritin NP (red square) mRNA-LNP vaccinations in DH270 UCA KI mice. (B) Comparison of serum neutralization titers of autologous tier 2 CH848 10.17DT HIV-1 strains between CH848 10.17DT transmembrane gp160 (blue circles) and CH848 10.17DT SOSIP trimer-ferritin NP (red squares) mRNA-LNP vaccinations in DH270 UCA KI mice. Bar, geometric mean.Exact Wilcoxon Mann-Whitney U test.

A

B

p = 0.3678 p = 0.1096

p = 0.0843 p = 0.5899

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