HIV envelope antibodies and TLR7 agonist partially prevent viral rebound in chronically SHIV-infected monkeys.

A key challenge for the development of a cure to HIV-1 infection is the persistent viral reservoir established during early infection. Previous studies using Toll-like receptor 7 (TLR7) agonists and broadly neutralizing antibodies (bNAbs) have shown delay or prevention of viral rebound following antiretroviral therapy (ART) discontinuation in simian-human immunodeficiency virus (SHIV)-infected rhesus macaques. In these prior studies, ART was initiated early during acute infection, which limited the size and diversity of the viral reservoir. Here we evaluated in SHIV-infected rhesus macaques that did not initiate ART until 1 year into chronic infection whether the TLR7 agonist vesatolimod in combination with the bNAb PGT121, formatted either as a human IgG1, an effector enhanced IgG1, or an anti-CD3 bispecific antibody, would delay or prevent viral rebound following ART discontinuation. We found that all 3 antibody formats in combination with vesatolimod were able to prevent viral rebound following ART discontinuation in a subset of animals. These data indicate that a TLR7 agonist combined with antibodies may be a promising strategy to achieve long-term ART-free HIV remission in humans.

Evaluation of Broadly Neutralizing Antibody Sensitivity by Genotyping and Phenotyping for Qualifying Participants to HIV Clinical Trials.

BACKGROUND: HIV envelope (env) diversity represents a significant challenge for the use of broadly neutralizing antibodies (bNAbs) in HIV treatment and cure studies. Screening for viral sensitivity to bNAbs to select eligible trial participants will be important to improve clinical efficacy; however, no universal approach has been established. METHODS: Pre-antiretroviral therapy plasma virus from participants in the Zurich Primary HIV Infection (ZPHI) study was genotyped and phenotyped for sensitivity to the bNAbs elipovimab (EVM, formerly GS-9722) and 3BNC117. The genotyping and phenotyping assessments were performed following the Clinical Laboratory Improvement Amendments of 1988 guidelines as required for entry into clinical trials. The genotypic-based prediction of bNAb sensitivity was based on HIV env amino acid signatures identified from a genotypic-phenotypic correlation algorithm using a subtype B database. RESULTS: Genotyping the plasma virus and applying env sensitivity signatures, ZPHI study participants with viral sensitivity to EVM and 3BNC117 were identified. ZPHI study participants with virus sensitive to EVM and 3BNC117 were also identified by phenotyping the plasma virus. Comparison of the genotypic and phenotypic sensitivity assessments showed strong agreement between the 2 methodologies. CONCLUSIONS: The genotypic assessment was found to be as predictive as the direct measurement of bNAb sensitivity by phenotyping and may, therefore, be preferred because of more rapid turnaround time and assay simplicity. A significant number of the participants were predicted to have virus sensitive to EVM and 3BNC117 and could, thus, be potential participants for clinical trials involving these bNAbs.

An advanced BLT-humanized mouse model for extended HIV-1 cure studies.

OBJECTIVE: Although bone marrow, liver, thymus (BLT)-humanized mice provide a robust model for HIV-1 infection and enable evaluation of cure strategies dependent on endogenous immune responses, most mice develop graft versus host disease (GVHD), limiting their utility for extended HIV cure studies. This study aimed to: evaluate the GVHD-resistant C57 black 6 (C57BL/6) recombination activating gene 2 (Rag2)γcCD47 triple knockout (TKO)-BLT mouse as a model to establish HIV-1 latency. Determine whether TKO-BLT mice could be maintained on antiretroviral therapy (ART) for extended periods of time. Assess the rapidity of viral rebound following therapy interruption. DESIGN: TKO-BLT mice were HIV-1 infected, treated with various ART regimens over extended periods of time and assayed for viral rebound following therapy interruption. METHODS: Daily subcutaneous injection and oral ART-mediated suppression of HIV-1 infection was tested at various doses in TKO-BLT mice. Mice were monitored for suppression of viremia and cellular HIV-1 RNA and DNA prior to and following therapy interruption. RESULTS: Mice remained healthy for 45 weeks posthumanization and could be treated with ART for up to 18 weeks. Viremia was suppressed to less than 200 copies/ml in the majority of mice with significant reductions in cellular HIV-1 RNA and DNA. Treatment interruption resulted in rapid viral recrudescence. CONCLUSION: HIV-1 latency can be maintained in TKO-BLT mice over extended periods on ART and rapid viral rebound occurs following therapy removal. The additional 15-18 weeks of healthy longevity compared with other BLT models provides sufficient time to examine the decay kinetics of the latent reservoir as well as observe delays in recrudescence in HIV-1 cure studies.

Distinct HIV-1 Neutralization Potency Profiles of Ibalizumab-Based Bispecific Antibodies.

BACKGROUND: Preexposure prophylaxis using antiretroviral agents has been shown to effectively prevent human immunodeficiency virus type 1 (HIV-1) acquisition in high-risk populations. However, the efficacy of these regimens is highly variable, which is thought to be largely due to the varying degrees of adherence to a daily intervention in the populations. Passive immunization using broadly neutralizing antibodies (bNAbs) against HIV-1, with their relatively long half-life and favorable safety profile, could provide an alternative to daily preexposure prophylaxis. However, most bNAbs have a limited breadth, only neutralizing 70%-90% of all HIV-1 strains. METHODS: To overcome the problem of limited antiviral breadth, we proposed that targeting human CD4 and HIV-1 envelope proteins simultaneously may improve virus-neutralization breadth and potency. Therefore, we constructed bispecific antibodies (biAbs) using single-chain variable fragments of anti-gp120 bNAbs fused to ibalizumab (iMab), a humanized monoclonal antibody that binds human CD4, the primary receptor for HIV-1. RESULTS: Some of our biAbs neutralized 100% of HIV-1 strains tested in vitro at clinically achievable concentrations. Distinct neutralization patterns were observed in this panel of biAbs. Those biAbs with specificity for the CD4-binding site on gp120 demonstrated 100% breadth, as well as slightly improved potency compared with iMab. In contrast, biAbs with specificity for the V1-V2 apex epitope or the V3-glycan epitope on gp120 demonstrated dramatically improved potency; some showed limited gain in neutralization breadth, whereas others (eg, PGT128-LM52 and 123-iMab) improved to 100% breadth. CONCLUSION: Our data suggest that this panel of iMab-based biAbs could be used to probe the parameters for potent HIV-1 neutralization. Moreover, a few of these biAbs warrant further studies and possibly clinical development.

V1/V2 Neutralizing Epitope is Conserved in Divergent Non-M Groups of HIV-1.

BACKGROUND: Highly potent broadly neutralizing monoclonal antibodies (bNAbs) have been obtained from individuals infected by HIV-1 group M variants. We analyzed the cross-group neutralization potency of these bNAbs toward non-M primary isolates (PI). MATERIAL AND METHODS: The sensitivity to neutralization was analyzed in a neutralization assay using TZM-bl cells. Twenty-three bNAbs were used, including reagents targeting the CD4-binding site, the N160 glycan-V1/V2 site, the N332 glycan-V3 site, the membrane proximal external region of gp41, and complex epitopes spanning both env subunits. Two bispecific antibodies that combine the inhibitory activity of an anti-CD4 with that of PG9 or PG16 bNAbs were included in the study (PG9-iMab and PG16-iMab). RESULTS: Cross-group neutralization was observed only with the bNAbs targeting the N160 glycan-V1/V2 site. Four group O PIs, 1 group N PI, and the group P PI were neutralized by PG9 and/or PG16 or PGT145 at low concentrations (0.04-9.39 µg/mL). None of the non-M PIs was neutralized by the bNAbs targeting other regions at the highest concentration tested, except 10E8 that neutralized weakly 2 group N PIs and 35O22 that neutralized 1 group O PI. The bispecific bNAbs neutralized very efficiently all the non-M PIs with IC50 below 1 µg/mL, except 2 group O strains. CONCLUSION: The N160 glycan-V1/V2 site is the most conserved neutralizing site within the 4 groups of HIV-1. This makes it an interesting target for the development of HIV vaccine immunogens. The corresponding bNAbs may be useful for immunotherapeutic strategies in patients infected by non-M variants.

Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells.

HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs.

Neutralization properties of simian immunodeficiency viruses infecting chimpanzees and gorillas.

UNLABELLED: Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Ig(mim2), CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4(+) T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection. IMPORTANCE: SIVcpz is widespread in wild-living chimpanzees and can cause AIDS-like immunopathology and clinical disease. HIV-1 infection of humans can be controlled by antiretroviral therapy; however, treatment of wild-living African apes with current drug regimens is not feasible. Nonetheless, it may be possible to curb the spread of SIVcpz in select ape communities using vectored immunoprophylaxis and/or therapy. Here, we show that antibodies and antibody-like inhibitors developed to combat HIV-1 infection in humans are capable of neutralizing genetically diverse SIVcpz and SIVgor strains with considerable breadth and potency, including in primary chimpanzee CD4(+) T cells. These reagents provide an important first step toward translating intervention strategies currently developed to treat and prevent AIDS in humans to SIV-infected apes.

Monoclonal antibodies to host cellular receptors for the treatment and prevention of HIV-1 infection.

PURPOSE OF REVIEW: Clinically relevant monoclonal antibodies (mAb) to host cellular receptors have been generated to both the CD4 receptor and the CCR5 coreceptor, cell surface proteins critical for HIV-1 entry. Ibalizumab is a novel humanized mAb that binds to a conformational epitope on CD4 and blocks entry of HIV-1. It has broad and potent antiviral activity in vitro and in vivo. PRO 140 is a humanized mAb that binds to the CCR5 coreceptor and inhibits CCR5-tropic HIV-1 by interfering with viral entry. Antiviral activity has been demonstrated both in vitro against R5 viruses and in vivo in HIV-1-infected individuals harboring CCR5-tropic virus. RECENT FINDINGS: Both antibodies have been administered intravenously in early-phase clinical trials, and current emphasis is on the development of formulations that can be administered subcutaneously. Most recently, bispecific antibodies combining either ibalizumab or PRO 140 with anti-Env broadly neutralizing antibodies have been constructed with vastly improved in-vitro neutralizing profiles, and may offer substantial advantages in the clinic. SUMMARY: mAb to host cellular receptors particularly when combined with broadly neutralizing antibodies in novel conformations may offer advances in both the treatment and prevention of HIV-1 infection.

Rational design and characterization of the novel, broad and potent bispecific HIV-1 neutralizing antibody iMabm36.

BACKGROUND: Although broadly neutralizing monoclonal antibodies (bNAbs) have always been considered to be a potential therapeutic option for the prophylaxis and treatment of HIV infection, their lack of breadth against all HIV variants has been one of the limiting factors. To provide sufficient neutralization breadth and potency against diverse viruses, including neutralization escape mutants, strategies to combine different bNAbs have been explored recently. METHODS: We rationally designed and engineered a novel bispecific HIV-1-neutralizing antibody (bibNAb), iMabm36. The potency and breadth of iMabm36 against HIV were extensively characterized in vitro. RESULTS: iMabm36 comprises the anti-CD4 Ab ibalizumab (iMab) linked to 2 copies of the single-domain Ab m36, which targets a highly conserved CD4-induced epitope. iMabm36 neutralizes a majority of a large, multiclade panel of pseudoviruses (96%, n = 118) at an IC50 concentration of less than 10 µg/mL, with 83% neutralized at an IC50 concentration of less than 0.1 µg/mL. In addition, iMabm36 neutralizes a small panel of replication-competent transmitted-founder viruses to 100% inhibition at a concentration of less than 0.1 µg/mL in a peripheral blood mononuclear cell-based neutralizing assay. Mechanistically, the improved antiviral activity of iMabm36 is dependent on both the CD4-binding activity of the iMab component and the CD4i-binding activity of the m36 component. After characterizing that viral resistance to iMabm36 neutralization was due to mutations residing in the bridging sheet of gp120, an optimized m36 variant was engineered that, when fused to iMab, improved antiviral activity significantly. CONCLUSIONS: The interdependency of this dual mechanism of action enables iMabm36 to potently inhibit HIV-1 entry. These results demonstrate that mechanistic-based design of bibNAbs can generate potential preventive and therapeutic candidates for HIV/AIDS.

Bispecific antibodies directed to CD4 domain 2 and HIV envelope exhibit exceptional breadth and picomolar potency against HIV-1.

In the absence of an effective HIV-1 vaccine, passive immunization using broadly neutralizing Abs or Ab-like molecules could provide an alternative to the daily administration of oral antiretroviral agents that has recently shown promise as preexposure prophylaxis. Currently, no single broadly neutralizing Ab (bNAb) or combination of bNAbs neutralizes all HIV-1 strains at practically achievable concentrations in vivo. To address this problem, we created bispecific Abs that combine the HIV-1 inhibitory activity of ibalizumab (iMab), a humanized mAb directed to domain 2 of human CD4, with that of anti-gp120 bNAbs. These bispecific bNAbs (BibNAbs) exploit iMab's potent anti-HIV-1 activity and demonstrated clinical efficacy and safety to anchor and thereby concentrate a second broadly neutralizing agent at the site of viral entry. Two BibNabs, PG9-iMab and PG16-iMab, exhibit exceptional breadth and potency, neutralizing 100% of the 118 viruses tested at low picomolar concentrations, including viruses resistant to both parental mAbs. The enhanced potency of these BibNAbs was entirely dependent on CD4 anchoring, not on membrane anchoring per se, and required optimal Ab geometry and linker length. We propose that iMab-based BibNAbs, such as PG9-iMab and PG16-iMab, are promising candidates for passive immunization to prevent HIV-1 infection.

Anti-CD4 monoclonal antibody ibalizumab exhibits breadth and potency against HIV-1, with natural resistance mediated by the loss of a V5 glycan in envelope.

OBJECTIVES: Passive immunization for the prevention of HIV-1 infection is currently being reenergized. The anti-CD4 monoclonal antibody ibalizumab has demonstrated safety and efficacy in phase 1 and 2 clinical trials for treatment of HIV-1 infection and is undergoing a phase 1 clinical trial in HIV-1 uninfected individuals for prevention. Here, we sought to assess ibalizumab antiviral breadth and potency and to identify determinants of natural preexisting resistance. METHODS: Ibalizumab breadth and potency was assessed against a large clinically relevant panel of HIV-1 pseudoviruses (n = 116) commonly used to assess vaccine candidates. Determinants of resistance were assessed by sequence analysis. RESULTS: Ibalizumab neutralized 92% and 66% of viruses as defined by 50% and 80% inhibition, respectively. Median in vitro neutralization potency by IC50 was 0.03 µg/mL, substantially lower than the broadly neutralizing mAbs, PG9, or VRC01. The dominant determinant of resistance was the absence of a potential N-linked glycosylation site (PNGS) at the V5 N-terminus (P < 0.001), with the V2 loop length possibly influencing the degree of resistance afforded by the absence of the V5 N-terminal PNGS (P = 0.001). Other significant independent correlates of resistance included PNGS at position 386 and the side chain length of residue 375. Ibalizumab exhibited complementary resistance to VRC01 (P = 0.006) and sCD4 (P < 0.001), in part mediated by the V5 PNGS. CONCLUSIONS: Ibalizumab breadth and potency compared favorably with broadly neutralizing anti-HIV-1 monoclonal antibodies, supporting the clinical development of ibalizumab, alone or in combination, for HIV-1 prevention.

HIV-1 and influenza antigens synthetically linked to IgG2a Fc elicit superior humoral responses compared to unmodified antigens in mice.

Using murine IgG subclass molecules (IgG1 or IgG2a) synthetically fused to HIV-1 or influenza test antigens, we explored the potential for IgG Fc scaffolds to augment immunogenicity. Each antigen (Ag) was grafted onto a hinge-Fc scaffold containing all critical residues necessary for interaction with effector cells, thus retaining effector functions of the native IgG subclass. We hypothesized that the differential affinity of FcγRs for specific IgG subclasses would influence the magnitude of immune responses elicited by immunization with an Ag-IgG Fc fusion vaccine. We demonstrate here that the antigen-specific humoral response elicited by Ag-IgG2a fusion vaccines is at least tenfold greater than that elicited by native antigen, that this response is superior to that elicited by Ag-IgG1, and that the augmented antigen-specific humoral response elicited is Fcγ receptor-dependent.

Population level analysis of human immunodeficiency virus type 1 hypermutation and its relationship with APOBEC3G and vif genetic variation.

APOBEC3G and APOBEC3F restrict human immunodeficiency virus type 1 (HIV-1) replication in vitro through the induction of G-->A hypermutation; however, the relevance of this host antiviral strategy to clinical HIV-1 is currently not known. Here, we describe a population level analysis of HIV-1 hypermutation in [corrected] clade B proviral DNA sequences (n = 127). G-->A hypermutation conforming to expected APOBEC3G polynucleotide sequence preferences was inferred in 9.4% (n = 12) of the HIV-1 sequences, with a further 2.4% (n = 3) conforming to APOBEC3F, and was independently associated with reduced pretreatment viremia (reduction of 0.7 log(10) copies/ml; P = 0.001). Defective vif was strongly associated with HIV-1 hypermutation, with additional evidence for a contribution of vif amino acid polymorphism at residues important for APOBEC3G-vif interactions. A concurrent analysis of APOBEC3G polymorphism revealed this gene to be highly conserved at the amino acid level, although an intronic allele (6,892 C) was marginally associated with HIV-1 hypermutation. These data indicate that APOBEC3G-induced HIV-1 hypermutation represents a potent host antiviral factor in vivo and that the APOBEC3G-vif interaction may represent a valuable therapeutic target.

Potential roles for uncoupling proteins in HIV lipodystrophy.

The 'HIV lipodystrophy syndrome' consists of several distinct components, including lipoatrophy (pathological subcutaneous fat loss), lipohypertrophy (abdominal/visceral adiposity), and metabolic complications including insulin resistance and dyslipidemia. Lipoatrophy appears to represent an adipose tissue-specific form of mitochondrial toxicity associated strongly with stavudine NRTI therapy, whilst the 'metabolic syndrome' phenotype is associated with HIV protease inhibitor therapy. In this context, the role of uncoupling proteins (UCPs) in modulating resting energy expenditure in response to elevated fatty acid flux associated with the 'metabolic syndrome' is supported by clinical data as well as findings of elevated adipose tissue UCP expression. The role of UCPs in this syndrome therefore exemplifies the multifactorial nature of these antiretroviral therapy complications.

Mitochondrial proliferation, DNA depletion and adipocyte differentiation in subcutaneous adipose tissue of HIV-positive HAART recipients.

OBJECTIVES: To examine the in vivo effects of highly active antiretroviral therapy (HAART) regimens on adipose tissue mitochondrial DNA (mtDNA) depletion, mitochondrial organellar proliferation, and markers of adipocyte differentiation and phenotype. DESIGN AND METHODS: DNA and mRNA quantification using real-time PCR methods was performed on adipose tissue samples from 31 HIV-infected individuals, of whom 11 were treatment-naive and 20 were receiving HAART. mtDNA depletion was measured as mtDNA copies/cell, and mitochondrial proliferation by quantification of mitochondrial protein mass. Regulation of mitochondrial biogenesis was assessed by NRF-1 and mtTFA mRNA. PPARgamma, UCP2 and UCP1 mRNA expression was used to assess adipocyte differentiation and phenotype. RESULTS: Stavudine-based HAART recipients (n=10) displayed significant mtDNA depletion (12.8% of control, P<0.001), mildly increased mitochondrial protein mass (2.6-fold of control, P=0.032) and decreased expression of PPARgamma (53.9% of control, P=0.021), UCP2 (62.2% of control, P=0.024) and UCP3 (51.8% of control, P=0.047) mRNA compared with controls. Zidovudine-based HAART recipients (n=7) also displayed significant mtDNA depletion (34.45% of control, P=0.031), increased mitochondrial protein mass (5.7-fold of control, P=0.009), and markedly increased UCP1 (18-fold of control, P=0.009) mRNA. Elevated UCP1 mRNA expression was found to be associated with non-stavudine (zidovudine or abacavir), protease inhibitor (PI)-containing HAART (95-fold of non-stavudine, non-PI-containing HAART, P=0.006). CONCLUSION: Differential effects of stavudine and zidovudine therapy on mtDNA depletion and expression of adipocyte differentiation markers PPARgamma and UCP2 were observed, consistent with increased adipose tissue toxicity associated with stavudine therapy. Increased UCP1 mRNA, a marker of brown adipose tissue phenotype, was associated with non-stavudine, PI-containing HAART, and may represent an adaptive response to the increased fatty acid flux associated with PI therapy, and may contribute to the increased resting energy expenditure reported in such patients.

Accumulation of mitochondrial DNA mutations in human immunodeficiency virus-infected patients treated with nucleoside-analogue reverse-transcriptase inhibitors.

Nucleoside reverse-transcriptase inhibitor (NRTI) therapy for human immunodeficiency virus (HIV) infection has been associated with mitochondrial DNA (mtDNA) polymerase-gamma inhibition and subsequent mtDNA depletion. Effects on mtDNA mutation, although suggested by critical involvement of polymerase-gamma in DNA-repair reactions, are unknown. In the present study, we assessed the nature and frequency of mitochondrial genome sequence differences in peripheral-blood samples taken prior to NRTI therapy and after 6-77 mo of treatment in 16 NRTI-treated patients. Samples from 10 HIV-infected, treatment-naive control individuals were taken at similar time intervals. Single-stranded conformation polymorphism (SSCP) and DNA-sequencing analysis techniques were used to detect mitochondrial genome sequence variants between paired longitudinal samples, and heteroplasmic populations were quantified after cloning and repeat SSCP/sequencing. Of 16 individuals treated with NRTIs, 5 exhibited altered SSCP profiles associated with the development of novel heteroplasmic DNA sequence changes, whereas no SSCP pattern change within these regions was observed in the control individuals. Heteroplasmic sequence changes were distributed across four regions of the genome: the noncoding region to 12S ribosomal RNA, reduced-nicotinamide-adenine-dinucleotide dehydrogenase 1, and cytochrome oxidase subunits I and III. Of the total of 26 patients who were examined in the present study, 4 of 5 patients with detectable mtDNA sequence changes since commencement of therapy developed evidence of peripheral fat wasting (lipoatrophy) between sample intervals (P=.031). One patient, without detectable sequence changes on NRTI therapy, also developed lipoatrophy. Levels of mtDNA copies/cell in blood samples were determined by quantitative PCR for 11 of the 16 NRTI-exposed patients; 7 of these 11 patients showed reduced levels of mtDNA in blood after therapy, including all 3 patients tested with evidence of mtDNA sequence changes on therapy. These data indicate that NRTI therapy provides conditions permissive for the development of peripheral-blood mtDNA mutations in vivo.