141207_1_additional_review_material_1101440_r467p7.pdf

nature portfolio

Double

-blind

peer review submissions:

write

DBPR

and your manuscript number

here

Corresponding

author(s):

oseph P.

asa

Last updated

by author(s): 2022-02-2

_________

Reporting

Summary

Nature

Portfolio

wishes

to improve

the reproducibility

of the work

that

we publish.

This

form

provides

structure

for consistency

and

transparency

in reporting.

For further

information

on Nature

Portfolio

policies,

see our

Editorial

Policies

and

the

Editorial

Policy Checklis

Statistics

For all statistical

analyses,

confirm

that

the following

items

are present

in the figure

legend,

table

legend,

main

text,

or Methods

section.

a Confirmed

D �

The

exact

sample

size

(n)

for each

experimental

group

condition,

given

as a discrete

number

and

unit

of measurement

D �

A statement

on whether

measurements

were

taken

from

distinct

samples

or whether

the same

sample

was

measured

repeatedly

D �

The

statistical

test(s)

used

AND

whether

they

are one-

or two-sided

Only common

tests

should

be described

solely

by name;

describe

complex

techniques

in the Methods

section.

� D

A description

of all covariates

tested

� D

A description

of any

assumptions

or corrections,

such

as tests

of normality

and

adjustment

for multiple

comparisons

D �

A full description

of the statistical

parameters

including

central

tendency

(e.g.

means)

or other

basic

estimates

(e.g.

regression

coefficient)

AND

variation

(e.g.

standard

deviation)

or associated

estimates

of uncertainty

(e.g.

confidence

intervals)

For null

hypothesis

testing,

the test

statistic

(e.g.

F, t, r)

with

confidence

intervals,

effect

sizes,

degrees

of freedom

and

value

noted

Give P values

as exact

values

whenever

suitable.

For Bayesian

analysis,

information

on the choice

of priors

and

Markov

chain

Monte

Carlo

settings

For hierarchical

and

complex

designs,

identification

of the appropriate

level

for tests

and

full reporting

of outcomes

Estimates

of effect

sizes

(e.g.

Cohen's

Pearson's

r),

indicating

how

they

were

calculated

Our web collection

on statistics for biologists

contains articles on many of the paints above.

Software and code

Policy

information

about

availability

of computer

code

Data

collection

Flow

cytometry

data

collected

by BD FACSDiva

software,

V8.0;

VRC07

concentrations

read

on a Singulex

Erenna

or SMCxPRO

(Singulex,

Alameda,

CA);

AAV8

capsid

Ab data

collected

on a Spectra

Max

microplate

spectrophotometer

using

proprietary

software

(Molecular

Devices,

Sunnyvale,

CA);

Tier 1 and Tier 2 VRC07

ADA

assay

data

collected

on MSD

600plate

reader

using

MSD

Workbench

software

(Rockville,

MD),

Tier 3 luminescence

dterminations,

Molecular

Devices

Paradigm

Multimode

Reader

wish

Software

Pro software

(San

Jose,

CA),

AAV8-VRC07

DNA

RT PCR data

was acquired

on an Applied

Biosystems

7900

HT (Waltham,MA).

lgGl

allotype

RT PCR data

was aquired

using

a CFX96

Touch

Real-Time

PCR detection

system

(CHAI,

Santa

Clara,

CA).

Pseudoviral

Neutralization

data

was acquired

on a Spectramax

L luminometer

(Molecular

Devices).

Data

analysis

Flow

cytometry

data

analysis,

FlowJo

software

(FlowJo,

Ashland,

OR),

Vl0.6.

Singulex

VRC07

determinations

analyzed

with

Sgx link or

SMCxPro(Singulex,

Alameda,

CA).

RT PCR data

for AAV8-VRC07

DNA

concentration

was analyzed

using

SDS 2.4.1

software

(Applied

Biosystems,

Foster

City,

CA).

Tier 3 VRC07

ADA

was analyzed

using

Labkey

Neutalization

Analysis

Tool

(LabKey,

San Diego,

CA)

For manuscripts

utilizing

custom

algorithms

or software

that are central

to the research

but not yet described

in published

literature,

software

must

be made

available

to editors

and

reviewers.

We strongly

encourage

code

deposition

in a community repository

(e.g. GitHub).

See the Nature Portfolio

guidelines

for submitting

code

& software

for further

information.

B27), mouse

anti-human

CD107a

BV650

(Biolegend

Catalog

#328638,

clone

H4A3)

and mouse

anti-human

CD3 H7APC

(BD

Bioscience

Cat # 641406,

clone

ASR) were used for flow cytometric analysis.

Mouse

anti-Human

lgG Fe unconjugated

secondary

antibody

used in the Singluex

VRC07

assay

was from ThermoFisher

(Cat # MH1015,

clone

HP6070).

For anti-AAV8

antibody

determinations

anti-human

lgG/A/M

was obtained

from two different

sources

(KPL Cat# #16-10-07

or more

recently

Jackson

lmmuno

Research

Cat# 309-065-064).

VRC07,

VRC 13 and 5C9 were produced

in house.

.....

Validation

All the antibodies

used in the study

were tested

for their reactivity

and specificity

prior to the use in the study,

but not further

validation

unless

noted.

Eukaryotic

cell lines

Policy

information

about

cell lines

Cell line source(s)

Authentication

Mycoplasma

contamination

Commonly

misidentified

lines

(See ICLAC

TZM-bl

cells were obtained

from the National

Institutes

of Health

AIDS Reagent

Repository

These

cells were not authenticated

Cell lines used for Tier 3 ADA responses

were tested

for mycoplasma,

and cells used for Ab neutralization

were not tested

for

mycoplasma.

No commonly

misidentified

lines were used.

Human research participants

Policy

information

about

studies

involving

human

research

participants

Population

characteristics

Recruitment

Ethics

oversight

Eight HIV-infected

individuals,

2 women

and 6 men, all on effective

ARV therapy

with vial loads

of <50, and CD4 counts

>300 between

the ages of 30-65

were enrolled in VRC603.

Exclusionary

criteria included

prior receipt

of monoclonal

anti

bodies

or gene therpy products,

active drug or alcohol

abuse,

anti-AAv8

antibody

levels of >1:90, active liver disease,

poorly

controlled

hypertension,

weight >115 kg, a history

of severe allergic response

within the preceding

2 years, pregnancy

and

any chronic

or clinically

significant

medical condition that in the opinion of the investi

gator would jeopardize

the safety or

rights

of the participant.

Volunteers

were recruited

from the greater

Washington,

DC, area by I RB-approved

written

and electronic

media.

This

included

fliers and advertisements

on the NIH campus

and Clinical

Center,

referrals

from the Office

of Patient

Recruitment

the NIH, contacting

participants

from previous

VRC trials,

fliers at local colleges

and universities,

HIV medical

provider

offices,

coffee

shops,

restaurants,

bars and grocery

stores,

gay pride

events,