Characterization and development of adeno-associated virus (AAV) vectors for expressing HIV-1 entry inhibitors Open Access

Leguizamo, Isai (Spring 2026)

Permanent URL: https://etd.library.emory.edu/concern/etds/4f16c4451?locale=en
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Abstract

The delivery of adeno-associated virus (AAV) vectors expressing broadly neutralizing antibodies

(bNAbs) represents a promising strategy for long-term prevention and treatment of HIV-1

infection. Unlike passive infusion of bNAbs, using AAV vectors allows for sustained bNAb

expression following a single administered dose. However, numerous studies have demonstrated

that durable expression of AAV-delivered bNAbs is significantly limited by host immune

responses, particularly through the development of host anti-drug antibodies. Using a novel

approach, we evaluated immune modulation through co-delivery of an AAV9 expressing surface

program death-ligand 1 (PD-L1) to provide an immunological shield to transduced cells. We

demonstrated that co-administration of AAV9.PD-L1 with an AAV9 expressing bNAb 10-1074

in rhesus macaques reduced the emergence of ADA and greatly improved the durability of serum

10-1074 concentration well above the therapeutic threshold (50ug/mL) throughout the duration of

the study. Macaques with sustained 10-1074 serum concentration were conferred protection

against ten repeated intrarectal SHIVAD8-EO challenges. In contrast, animals that received

AAV9.10-1074 alone, exhibited higher ADA responses, more rapid declines in circulating

antibody concentrations, and exhibited greater inflammation at injection sites. Building further

upon on these findings, we systematically evaluated six different transgene cassettes expressing

10-1074 with different promoter combinations (CMV, CMV/R, CBA, CASI, CB7, EF1α) in vitro,

with immune-deficient mice, and in rhesus macaques. We demonstrated that both promoter and

species selection, lead to differences in 10-1074 serum concentrations. The CB7 promoter yielded

the highest antibody concentrations in mice while the CMV/R promoter demonstrated superior

performance in macaques. Loss of 10-1074 concentration in macaques led to higher ADA

responses and antigen-specific IFN-γ T cell responses were focused on the 10-1074 heavy-chain

variable region. Circulating 10-1074 in macaques with measurable concentrations retained their

neutralizing activity against diverse HIV-1 isolates, confirming functional integrity of AAV-

expressed 10-1074. Lastly, we demonstrated that incorporation of the woodchuck hepatitis virus

post-transcriptional regulatory element (WPRE) greatly enhanced 10-1074 expression in both

mice and macaques. Together, these studies demonstrate that both immune modulation and

expression elements are critical for achieving sustained AAV-mediated antibody expression.

Table of Contents

Chapter One: Introduction 1

a. HIV Origins and Epidemiology

i. Origins of HIV

ii. History of the Epidemic

iii. Past and Current Impacts of the Epidemic

b. HIV Biology and Structure

i. HIV Groups and Clades

ii. Morphology

iii. HIV Genome and Essential Proteins

iv. HIV Replication Cycle (a. Attachment b. Uncoating & Integration c. mRNA synthesis

& Transport d. Assembly, Budding, Release & Maturation)

v. Cellular Targets & Tropism

c. HIV Pathogenesis

i. Transmission dynamics

ii. Viral Dissemination

iii. Innate and Adaptive Immune Response

iv. Viral Persistence and Depletion

d. Animal Models for HIV Infection

i. Importance of Animal models for HIV research.

ii. Small and Non-human Primates (NHP) models

iii. Animal models for Latency and Reservoir Establishment

e. ART, Preventatives, and Potential Treatment Strategies for HIV

i. History and Evolution of ART

ii. Classes and Mechanisms of Action

iii. Adverse Effects, Drug Resistance, and Clinical Challenges

iv. HIV Prevention Strategies and Potential Novel Therapies

f. Broadly Neutralizing Antibodies (bNAbs) targeting HIV

i. Discovery of bNAbs

ii. Targeted env Epitopes

iii. Fc-mediated Effector Functions (a. Antibody-dependent Cytotoxicity b. Antibody

Dependent phagocytosis c. Antibody-Dependent Complement-Mediated Lysis)

iv. Clinical Translation of bNAbs (a. Single-bNAb Trials in PLWH b. Combination

bNAb Trials c. bNAbs with LRAs and Immune Modulators)

v. Limitations and Future Directions

g.Adeno-Associated Virus (AAV) for expression of HIV entry inhibitors

i. Discovery of AAV

ii. AAV Morphology and Genome

iii. Transduction Pathway of rAAV (a. Receptor Engagement and Attachment b. Internalization c. Endosomal Trafficking d. Cytoplasmic escape and nuclear import e. Genome Release and Transgene Expression)

iv. Cassette Elements for Modulating Transgene Expression and Target Specificity (a. promoters b. CMV enhancer c. Introns d. Polyadenylation Signal e. Post-transcriptional Regulatory Elements)

v. Pre-Clinical and Clinical Translation of AAV Gene Therapy for HIV

vi. Limitations of AAV and Strategies (a. Host Anti-drug Antibodies b. Pre-existing Immunity to AAV Capsids c. Cellular Immune Responses to AAV Capsids)

h. Summary

Chapter Two: Protective efficacy of AAV-expressed bNAb co-delivered with PD-L1 56

Chapter Three: Characterization of expression elements for an AAV-delivered antibody in nonhuman primates when co-delivered with PD-L1 91

Chapter Four: Discussion 135

References: 144

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