Glycan heterogeneity as a cause of the persistent fraction in HIV-1 neutralization
Neutralizing antibodies (NAbs) to multiple epitopes on the HIV-1 envelope glycoprotein (Env) have been isolated from infected persons. The potency of NAbs is more often measured than the size of the persistent fraction of infectivity at maximum neutralization, which may also influence preventive efficacy by active or passive immunization and the therapeutic outcome of the latter. HIV-1 CZA97.012, a clone of a Clade C isolate, is neutralized to [~]100% by many NAbs. But here NAb PGT151, directed to a fusion-peptide epitope, was shown to leave a persistent fraction of 15%. NAb PGT145, ligating the Env-trimer apex, was less potent but more effective. We sought explanations of the different persistent fractions by depleting pseudoviral populations of the most PGT151- and PGT145-reactive virions. Thereby, neutralization by the non-depleting NAb increased; it decreased by the depleting NAb. Furthermore, depletion by PGT151 increased sensitivity to autologous neutralization by sera from rabbits immunized with soluble native-like CZA97.012 trimer: substantial persistent fractions were reduced. NAbs in these sera target epitopes comprising residue D411 at the V4-{beta}19 transition in a defect of the glycan shield on CZA97.012 Env. Affinity-fractionated soluble native-like CZA97.012 trimer showed commensurate antigenic differences in analyses by ELISA and surface plasmon resonance. We then demonstrated glycan differences between PGT151- and PGT145-purified trimer fractions by mass spectrometry, providing one explanation for the differential antigenicity. These differences were interpreted in relation to a new structure at 3.4-[A] resolution of the soluble CZA97.012 trimer determined by cryo-electron microscopy. PGT151-purified trimer showed a closed conformation, refuting apex opening as the cause of reduced PGT145 binding. The evidence suggests that differences in binding and neutralization after trimer purification or PV depletion with PGT145 or PGT151 are caused by variation in glycosylation, and that some glycan variants confer antigenic heterogeneity through direct effects on antibody contacts, whereas others act allosterically. Author SummaryNeutralizing antibodies block the entry of HIV-1 into cells and protect against HIV-1 infection in animal models. Therefore, a goal of vaccination is to elicit antibodies that potently neutralize most HIV-1 variants. Such antibodies suppress virus levels when given to HIV-1-infected patients. Their potency is often measured as the concentration that gives 50% or 80% neutralization. But higher degrees of neutralization are needed to protect an organism from infection. And for some antibodies a ceiling is reached, so that even with increased concentrations a constant fraction of infectious virus persists. We studied the carbohydrate moieties on the envelope glycoprotein, which is the sole target for neutralizing antibodies, of one HIV-1 isolate of the most widespread subtype, Clade C, prevalent in Africa and Asia. We show how differences in carbohydrates can contribute to persistent infectivity, because distinct carbohydrates fit different antibodies. With a new three-dimensional structure of the entry-mediating protein from the Clade-C isolate, we illustrate that some carbohydrate differences occur exactly where the antibodies bind, whereas others are located elsewhere and can act indirectly. When we combined two neutralizing antibodies the persistent infectivity shrank. Our results reinforce the need for multiple specificities of neutralizing antibodies in prevention and therapy.