Antibody- and cell-mediated immunity is known to be key to overcoming the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent that has caused the coronavirus disease 2019 (COVID-19) pandemic. The role of antibodies in neutralizing the virus is unquestioned. However, much remains unclear about how these molecules offer protection against new emerging variants of this virus, and even of additional viruses from this family.
A new study in the journal Immunity shows in fascinating detail how antibodies elicited by this virus show affinity maturation, a process occurring by somatic hypermutation (SHM) in the lymphoid germinal centers, to become more specific in their recognition of antigens.
The researchers looked at antibodies to the viral spike, specifically the receptor-binding domain (RBD), from five individuals, one month after infection as well as six months later. These were classified into six groups by clone and analyzed.
Neutralizing antibodies elicited by SARS-CoV-2 target the spike RBD, which binds to the angiotensin-converting enzyme 2 (ACE2), and as a result, many convalescent patients show powerful RBD-directed neutralizing activity. These are therefore used to confer passive immunity.
What were the findings?
The neutralizing antibodies targeting the RBD during early convalescence show low levels of SHM, with antibodies from different individuals with a history of either natural infection and being near-identical, encoded by the same set of rearranged genes. Similarly, high titers of neutralizing antibodies are induced by vaccines.
In some patients, however, infection may not induce an adequate antibody response leading to low neutralizing titers, even when the frequently isolated potent antibodies can be cloned.
The RBD-targeting antibodies belong to three structural classes. Class 1 is encoded from VH3-53 or VH3-63 gene segments, and class 2 from multiple VH gene segments. Class 1 has short CDRH2 regions, but binds to the RBD’s ACE2 binding site in the ‘up’ conformation, whereas class 2 and class 3 antibodies bind to RBDs irrespective of their conformation.
Certain class 2 antibodies form a bridge between neighboring RBDs and thus lock them down into the prefusion conformation. Class 3 antibodies do not bind to the ACE2 binding site.
Antibodies put positive selection pressure on the viral population, but their decline in titer over time suggests that reinfections are likely to occur. This is borne out by recent reports that cases and reinfections caused by viral variants are correlated with antibody resistance.
Antibodies from a set of over 1,000 cloned from convalescent patients at approximately 1 and 6 months after diagnosis. These patients had a spectrum of diseases from mild to moderately severe. Over this period, antibodies developed in a divergent manner, indicating their evolution and maturation.
Class 2 antibodies were dominant in convalescents, though the other two classes were also found. Antibody maturation enhanced class 2 antibody potency. For instance, C144 isolated at 1 month had high potency, with half-maximal inhibitory concentration (IC50) <10 ng/mL, and is being developed clinically. At six months, C051 and C052 were isolated from the same patient, with slightly less and similar potency to the earlier C144. However, the later antibodies had fourfold higher SHM frequencies.
Affinity maturation and antibody potency/resilience
A pseudotype neutralizing assay showed that several RBD mutations, including E484K/A/G, caused the virus to develop resistance to C144. Some of these substitutions spared neutralization by the later antibodies. The E484K substitution led to resistance to two, or all three antibodies, by preventing antibody binding to the RBD.
The Q493R/K substitutions were also associated with C144 resistance. When the pseudovirus was allowed to replicate in the presence of the later antibodies, the E484K mutant became dominant due to its resistance to the latter.
“Thus, affinity maturation of this family of potently neutralizing antibodies enabled the retention of activity against a subset of naturally occurring potential escape variants.” Yet, E484K caused each of these antibodies to become useless as a neutralizing antibody.
Similarly, the other groups showed increased potency along with a change in the spike substitutions selected by the antibody, causing neutralization escape. With some groups, such as the C548/C549 and C098/99 antibody pairs, affinity maturation appeared to make it more difficult for the virus to acquire antibody resistance by making two or more substitutions necessary at the RBD to confer a significant reduction in C549 potency. It also markedly increased antibody potency.
Class 3 antibody maturation and early antibody resistance
Class 3 antibodies do not prevent ACE2-RBD binding but show potent neutralizing activity. Some have very low IC50 values, but conversely, fail to produce complete neutralization.
One pair, C132/C512, isolated at one month, had a low SHM rate and was weakly neutralizing and high SHM rate/potent neutralizing activity, respectively. In this case, maturation both increased neutralizing potency and changed the selected resistance-conferring mutations as well. This was seen with another pair, with the earlier C032 antibody showing two-fold less potency than the six-month C080, but the latter remained active against mutants that conferred resistance to the earlier one.
Antibody activity against SARS-CoV-2 VOC
Finally, all class 2 antibodies showed less activity in the presence of the E484K mutation along with the K417N/E484K/N501Y combination. The latter is often observed in recent VOCs. Though C144/C051/C052 all failed binding affinity for the E484K variant, the six-month antibodies C055 and C549 showed higher affinity and activity against this substitution.
Compared to E484K alone, the presence of the latter combination did reduce affinity and activity in the case of C549. Meanwhile, the L455R/E484K combination suppressed antibody binding and activity altogether, confirming that the presence of two mutations are needed to allow maximal escape from the C549 antibody.
For class 1 antibodies, these mutations did not cause any loss of potency, but the L455R/E484K combination caused some loss, due to the former substitution alone. With class 3, none of this caused resistance, and in fact, some enhanced antibody potency. This suggests that the beta VOC B.1.351 does not reduce class 1 and class 3 mature antibody activity.
Resistance to other Sarbecoviruses
Moreover, many of the resistance mutations occur at positions that show variable residues among the sarbecovirus family. This explains why these viruses are not neutralized by the one-month antibodies, but are neutralized by two of the three six-month antibodies that can be overcome only by two or more simultaneous mutations.
Thus, in some cases, antibody evolution enabled neutralization of heterologous sarbecoviruses.”
What were the conclusions?
The findings of this study demonstrate that antibodies to the SARS-CoV-2 spike and RBD not only increased their binding affinity but gained potency in viral neutralization. Moreover, antibodies evolved to produce changes in the pathways by which mutations occurred in the virus, blocking the acquisition of viral resistance to neutralization and immune escape.
In some cases, antibody maturation made it necessary for multiple substitutions to occur before the virus could escape neutralization. With others, increased specific affinity increased the breadth of neutralization, covering circulating variants of concern of SARS-CoV-2 and heterologous viruses belonging to the sarbecovirus family.
These characteristics were due to the occurrence of mutations that increased the antibody-RBD junction variability. The enhancement of antibody diversity that occurred as a result of repeated or chronic exposure to SARS-CoV-2 antigens thus leads to an increased breadth of neutralization, not only against emerging VOCs but other coronaviruses with pandemic potential.
This could point the way to developing vaccines that induce higher levels of antibody maturation, resulting in greater SHM and diversification of antibody range to recognize closely related pathogens as well.