Neutralization of Virus Infectivity by Antibodies: Old Problems in New Perspectives

Abstract

Neutralizing antibodies (NAbs) can be both sufficient and necessary for protection against viral infections, although they sometimes act in concert with cellular immunity. Successful vaccines against viruses induce NAbs but vaccine candidates against some major viral pathogens, including HIV-1, have failed to induce potent and effective such responses. Theories of how antibodies neutralize virus infectivity have been formulated and experimentally tested since the 1930s; and controversies about the mechanistic and quantitative bases for neutralization have continually arisen. Soluble versions of native oligomeric viral proteins that mimic the functional targets of neutralizing antibodies now allow the measurement of the relevant affinities of NAbs. Thereby the neutralizing occupancies on virions can be estimated and related to the potency of the NAbs. Furthermore, the kinetics and stoichiometry of NAb binding can be compared with neutralizing efficacy. Recently, the fundamental discovery that the intracellular factor TRIM21 determines the degree of neutralization of adenovirus has provided new mechanistic and quantitative insights. Since TRIM21 resides in the cytoplasm, it would not affect the neutralization of enveloped viruses, but its range of activity against naked viruses will be important to uncover. These developments bring together the old problems of virus neutralization-mechanism, stoichiometry, kinetics, and efficacy-from surprising new angles.

Figure 1

The mechanism of neutralization. Neutralization of enveloped viruses blocks viral attachment and entry. No other mechanisms are yet known; but entry can be blocked at different stages. The three blue virions to the right represent enveloped virus particles. The first has an IgG bound to its receptor-binding protein (green, for simplicity shown as a single copy). The bound NAb blocks the docking onto the receptor (grey) on the cell surface. The second virion has already established contact between its receptor-binding protein and the cell-surface receptor. The NAb binds to an epitope on the envelope glycoprotein (viral proteins with this function and topology are usually glycosylated) that may have become exposed after the receptor binding and blocks subsequent steps; these could be interactions with a second receptor or the fusogenic refolding of the envelope glycoprotein. The third blue virion is about to fuse with the cell membrane, but NAbs bound to membrane proximal epitopes on fusogenic proteins (not shown) prevent the completion of this process. The latter two interferences with entry could also occur in endosomes, but hardly the first, unless there are alternative attachment proteins the virus can bind to and thereby get internalized. The purple virion in the endosome is prevented by NAbs from fusing its envelope with the vesicular membrane. Alternatively, this purple virion could represent a naked virus particle, the penetration of which is prevented by the NAbs. The block of infection in the endosome could properly be called a postinternalization block of entry; for clarity, entry should refer to transfer of the viral core or capsid, or possibly only genome, into the cytoplasm. The red virion on the cell surface depicts a naked virion that binds to a cell surface receptor and injects the genome into the cytoplasm. This process may occur in vesicles or semisealed invaginations of the cell surface. If the NAbs have not prevented receptor interactions, they may interfere with the extrusion of the genome. The red virion in the cytoplasm has penetrated an endosomal membrane in complex with the NAb, allowing binding to TRIM21 (yellow box with arrow), which mediates the ubiquitination of the complex, targeting it for proteasomal degradation. This fairly recently discovered effect constitutes the clearest example so far of a postentry mechanism of neutralization.

Figure 2

Neutralizing occupancies over heterogeneous populations of enveloped viruses. Two enveloped virions are pictured. Each has twelve glycoprotein spikes schematically displayed, for clarity only at the circumference. Functional spikes are shown in blue, decayed or otherwise nonfunctional ones in grey. Both virions have seven functional and five nonfunctional spikes but with different distributions over the two virion surfaces. If a virion requires a certain number of spikes in contiguity to form an entry complex and the spikes cannot move freely over the virion surface, the two different distributions will confer different neutralization sensitivities. The virion to the left is neutralized: three NAb molecules inactivate the constellation of active spikes and one binds redundantly to an inactive spike. The virion to the right is also neutralized but by only two NAb molecules, one inactivating a group of three spikes (three adjacent ones being postulated here to be the bare minimum for entry) and one binding redundantly to a spike that is functional but inert through lack of active neighbors. Effects of this sort could blur critical occupancy thresholds and reduce the steepness of neutralization curves in experiments with phenotypically mixed virus, of which the virions carry random assortments of antigenic and nonantigenic subunits of the envelope glycoprotein oligomers. Heterogeneity of the number (not shown) and distribution (shown) of functional entry-mediating viral proteins may explain how different occupancies are required for blocking viral entry. Some of these considerations apply also to naked viruses.

Figure 3

The kinetics of NAb binding. Soluble envelope glycoprotein oligomers can be immobilized on SPR chips via His or epitope tags. If the trimers are good structural mimics of native functional oligomers, and the density of trimers approximate that on the virion surface, then the NAb binding involved in neutralization can be simulated and its kinetic constants can be determined by SPR. Here, a soluble, stabilized trimer of the envelope glycoprotein of a Clade A isolate of HIV-1 was studied. The subunits of the trimer are labeled in the schematic to the lower right; the black bars represent engineered disulfide bonds that were introduced to stabilize each protomer of the trimer. The trimer was immobilized and the binding of NAbs and non-NAbs was compared. The sensorgrams show the response (RU) over time (s) during an association phase (upward curve) and a dissociation phase (downward curve: in several cases dissociation is very slow and barely measurable). Antibodies directed to different groups of epitopes, as indicated for the three diagrams, are compared. The nonneutralizing antibodies are marked with arrows. Thus, neutralization correlates eminently with binding to trimers that are native-like according to electron microscopy. The figure is reproduced from Sanders et al. [103] with modifications.

Figure 4

NAbs bind with different stoichiometries to oligomeric antigens. The images show electron-microscopy-based reconstructions of soluble trimers derived from the same Clade A isolate as in Figure 2 at ∼23 Å resolution. (a) The propeller-like top view and the side view of the trimer with one Fab directed to the CD4-binding site (PGV04) on each of the three gp120 subunits. (b) The same trimer is shown in complex with a soluble form of CD4 and the Fab of an antibody that binds to the coreceptor-binding site (17b). This NAb neutralizes poorly as IgG and better as a Fab, because the epitope is not constitutively present and is relatively inaccessible in the viral entry complex when induced by CD4 interactions. Three copies each of CD4 and Fab molecules can be seen in the top view. (c) The same combinations as in (a) and (b) are shown first and last in the row. The second and fourth panels represent trimers in complex with the Fabs of two NAbs (PGT122 and PGT135) that bind to different epitopes and with different angles from each other and from PGV04 and 17b. In the middle is a NAb with the unusual stoichiometry of one Fab per trimer, PG9, which binds preferentially to trimers and at an oblique angle to an epitope with direct contributions from two subunits. The blue mesh delineates the trimer itself. The figure is reproduced from Sanders et al. [103].

Figure 5

Neutralization potency and efficacy. (a) Potency: conventional neutralization curves (relative infectivity as a function of the logarithmic antibody concentration) for simulated data are shown. The green and red curves describe neutralization with identical potency, that is, IC₅₀ values, although the green curve has a higher slope coefficient. Red, black, and blue curves represent decreasing potencies in that order while having the same slope. Neutralization represented by the grey curve falls between the black and blue in potency but has markedly higher slope coefficient than both. Antigenic heterogeneity reduces the slope coefficient and so does negative cooperativity. Positive cooperativity would raise the slope coefficient. (b) Efficacy: exactly the same simulated data as in (a) are plotted in a log-log plot to illustrate the importance of the persistent fraction (PF) of infectivity after neutralization. The two most potent NAbs from (a) (red and green) are shown to have widely different efficacies: the persistent fraction differs by about three logs. Furthermore, the curves for the less potent NAbs (black and blue) cross the red curve and tend towards a greater efficacy by one or half a log, respectively. The greater slope of the grey curve than of the others is apparent also in this plot but only here is the greater efficacy of the neutralization represented by the grey than by the green curve evident, another case of lower potency and greater efficacy.