Ibalizumab (formerly TNX-355) is a first-in-class, monoclonal antibody inhibitor 
of CD4-mediated human immunodeficiency type 1 (HIV-1) entry. Multiple clinical 
trials with HIV-infected patients have demonstrated the antiviral activity, 
safety, and tolerability of ibalizumab treatment. A 9-week phase Ib study adding 
ibalizumab monotherapy to failing drug regimens led to transient reductions in 
HIV viral loads and the evolution of HIV-1 variants with reduced susceptibility 
to ibalizumab. This report characterizes these variants by comparing the 
phenotypic susceptibilities and envelope (env) sequences of (i) paired baseline 
and on-treatment virus populations, (ii) individual env clones from selected 
paired samples, and (iii) env clones containing site-directed mutations. Viruses 
with reduced susceptibility to ibalizumab were found to exhibit reduced 
susceptibility to the anti-CD4 antibody RPA-T4. Conversely, susceptibility to 
soluble CD4, which targets the HIV-1 gp120 envelope protein, was enhanced. No 
changes in susceptibility to the fusion inhibitor enfuvirtide or the CCR5 
antagonist maraviroc were observed. Functionally, viruses with reduced 
ibalizumab susceptibility also displayed high levels of infectivity relative to 
those of paired baseline viruses. Individual env clones exhibiting reduced 
ibalizumab susceptibility contained multiple amino acid changes in different 
regions relative to the paired baseline clones. In particular, clones with 
reduced susceptibility to ibalizumab contained fewer potential asparagine-linked 
glycosylation sites (PNGSs) in variable region 5 (V5) than did paired 
ibalizumab-susceptible clones. The reduction in ibalizumab susceptibility due to 
the loss of V5 PNGSs was confirmed by site-directed mutagenesis. Taken together, 
these findings provide important insights into resistance to this new class of 
antiretroviral drug.