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.

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.

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.

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.

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.

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.

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.

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.