The major result of this study is the identification of human MAbs from an HIV-1-infected patient with 2F5-like serum Abs that bind to the D664KW666 core 2F5 epitope but use entirely different germ line V genes from 2F5 and that are significantly less divergent than 2F5 from their germ line-encoded counterparts.
Neutralization of HIV-1 by m66.6 was more effective than that by m66 but weaker than that of 2F5 ( and ; see Table S2 in the supplemental material; also of reference 10
). Similar to 2F5, m66.6 did not neutralize most isolates from clade C or neutralized them only in the presence of the high-affinity Fc gamma receptor I (FcγRI); the expression of this receptor on target cells dramatically increased (by >1,000-fold) the potency of both m66.6 and 2F5. For some isolates, e.g., QH0692.42, the increase in potency (more than 3 orders of magnitude) of m66.6 due to the presence of FcγRI expression was significantly larger than that for 2F5 (approximately 1 order of magnitude); however, it should be noted that the 2F5 potency was much higher than that of m66.6 for this isolate when infecting cells lacking the FcγRI (see also of reference 10
IgG1 m66.6 was highly effective (IC50
of ~0.1 μg/ml) against HIV-1Bal
infection of primary cells (PBMCs and macrophages) (); m66.6 and 4E10 neutralized macrophage infection with IC80
s of 0.3 μg/ml and 1.3 μg/ml, respectively, and the negative controls A32 and m102.4 did not neutralize at any concentration. Similar efficacy was observed for a target cell line with low chemokine (C-C motif) receptor 5 (CCR5) surface concentration (M7-Luc cells) (A). TZM-bl cells express significantly higher levels of CCR5 than M7-Luc cells, PBMCs, and macrophages, which is a likely cause of the much lower activity of m66.6 against infection of these cells. From studying the clones of the same cell line expressing various levels of CCR5 molecules at their surface, we previously observed that decreasing CCR5 surface concentration increases the potency of some MAbs, including 2F5 and 4E10, but not that of others, including the CD4 binding site MAb b12 (4
). Therefore, it appears that m66.6 behaves similarly to 2F5 also in relation to the dependence of its neutralizing activity on the CCR5 surface concentration as indicated by the neutralizing activity measured in these assays.
The binding profile of m66.6 to MPER peptides and gp140 was very similar to that of 2F5 (; see Fig. S2 and S3 and Table S3 in the supplemental material). Moreover, MPER peptide-purified Abs from serum from patient SC44, from whom m66.6 was isolated, also exhibited a very similar binding profile to that of m66.6 (C). The profile of m66.6 binding to three different alanine-scanning mutants (B and C; see Fig. S2 and S3 in the supplemental material) was similar to that of 2F5; the only difference was that mutations at two positions (660L and 663L) upstream of the DKW core of the 2F5 epitope abolished binding of m66.6 but did not significantly affect 2F5 binding (B and Fig. S2). Thus, the m66.6 epitope also includes at least two residues upstream from the DKW epitope core. These results strongly indicate that the m66.6 epitope overlaps that of 2F5, yet the two epitopes are not identical. These differences may contribute to the differences observed in their neutralizing activity. Another potential contributor to the differences in their neutralizing activity may be related to differences in their binding to MPER in the context of lipid membranes, however, arguing against this is the similar binding strength of 2F5 and m66.6 IgG1s to MPER in the cell vesicle assay (Fig. S2).
Similar to 2F5 and 4E10, m66.6 reacted to self-antigens (; see Fig. S4 and S5 in the supplemental material). It bound to a panel of self-antigens although its self- and polyreactivity were reduced in the presence of BSA (Fig. S5). Why these cross-reactive MAbs targeting MPER exhibit polyreactivity is not clear. However, the less mutated clonal variant, m66, did not exhibit any measurable polyreactivity, suggesting that it was acquired from somatic hypermutations (SHMs) in Vκ. One can speculate that IgM-expressing B cells switch to IgG-specific memory B cells before the development of auto- or polyreactivity and that IgG-specific memory B cells might develop auto/polyreactivity during affinity maturation by acquiring additional SHMs and survive a peripheral tolerance checking point(s)—there is a significant percentage of circulating nonpathogenic autoreactive IgGs which serve useful functions, including removing cell debris (13
Despite similarities to 2F5, the m66.6 VH and VL sequences are very different from those of 2F5 in two major aspects. First, its closest germ line genes are different than those for 2F5, e.g., the m66.6 and 2F5 closest corresponding VH germ line genes are IGHV5-51*01 and IGHV2-5*10, respectively, and the closest VL germ line genes are IGKV1-39*01 and IGKV1-13*02, respectively. Second, the extent of SHM in m66 and m66.6 is significantly lower than that of 2F5. There are only 8 substituted amino acids in the heavy-chain V gene product of m66 and m66.6 compared to 14 in the 2F5 heavy-chain V gene product. Other known bn Abs, including 4E10, 2G12, and VRC01/02, have significantly more SHM-encoded amino acid substitutions than 2F5 (). Therefore, m66.6 is unique in terms of SHM in that it is lower than even the bn Ab bearing the smallest number of replacements encoded by SHMs in its heavy-chain V gene product. The difference between 8 and 14 mutations is considerable.
An estimate for 6 amino acid replacements spread across any of 100 positions in a given germ line VH gene product (and assuming 20 possible amino acid replacements at each position) leads to (100 × 20)6 ~ 1020 possible Abs bearing 6 replacements compared to the original VH amino acid sequence; each Ab variant corresponds to one unique SHM pathway at the protein level, and far more at the DNA level. Some of the generated Abs are identical, and some cannot fold correctly. This huge number is only an estimate of the number of possible maturation pathways for an Ab to acquire 6 additional mutations in a VH gene product. It illustrates as a major point the high complexity involved with even a few amino acid substitutions.
The Vκ gene products for m66 and m66.6 also have fewer substitutions than those for 2F5-3, 10 and 14 mutations, respectively, resulting in far more complex possible maturation pathways for 2F5 compared to those for m66.6. The SHM pathways for other bn Abs, especially VRC01, are much more complex than those for m66.6 and 2F5 because they have significantly more (VRC01 has 66) amino acid substitutions from the corresponding VH and VL germ line gene products (), which supports our hypothesis that all bn Abs must be highly mutated (3
). Whether and how the degree of somatic hypermutation depend on the epitope need further research.
On the basis of the degree of required SHM alone, one can speculate that m66 and m66.6 would be more straightforward to elicit than 2F5 and other known bn Abs. However, one should note that 8 and 10 mutations in the VH and VL gene products of m66.6 still suggest a high level of SHM, which could lead to highly complex maturation pathways and a low probability of eliciting m66.6 itself. An estimate similar to that described above for an average of 9 [(8 + 10)/2] mutations in about 100 positions leads to about 1030
possible Abs. Even if most of those Abs do not fold properly and only the CDRs are mutated, still the number of possible pathways to elicit such a large number of Abs is staggering. In contrast, some known bn Abs against severe acute respiratory syndrome (SARS) coronavirus (CoV) and Hendra and Nipah viruses (henipaviruses) have only a few substitutions from their closest germ line gene products (e.g., m396 against the SARS CoV has 5 substitutions and 1 substitution from the closest germ line VH and VL gene products, respectively, and m106 against henipaviruses has 1 and 0 substitution, respectively [3
]). One should note that some cross-reactive neutralizing Abs against these viruses could have a higher number of SHMs; however, the important point is that the MAbs that neutralize these viruses (which do not cause long-term chronic infections) can have only a few amino acid substitutions from its germ line counterpart yet can still exhibit cross-reactive neutralization, whereas for HIV-1, there are no such examples reported. One could hypothesize that Abs with low levels of SHM on average are more straightforward to elicit because of the relatively low number of possible maturation pathways needed for their maturation, and therefore, Envs of these viruses (e.g., henipaviruses and SARS CoV) could be highly immunogenic in terms of eliciting bn Abs.
We have demonstrated two new, HIV-1-cross-reactive neutralizing Abs that are specific for the 2F5 epitope yet are entirely different from 2F5 in their germ line V gene composition. Understanding the mutational pathways by which m66.6 and related Abs are elicited may facilitate design strategies for eliciting highly somatically mutated and more potent neutralizing Abs. The recent success of a chimeric immunogen, based on the fusion of the HA1 domain of influenza virus to HIV-1 gp41, in eliciting HIV-1-neutralizing Abs targeting the MPER (19
), could provide a basis for testing our hypothesis that increasing SHM-encoded substitutions in the Ab response will increase the potency and breadth of the neutralizing Ab response, and the degree to which SHM can improve these features. Moreover, the successful elicitation of m66.6 and related Abs as a proof of concept justifying the significance of the MPER as a vaccine target could also pave the way for elicitation of other known highly somatically mutated and more potent bn Abs. Further experiments in humans or appropriate animal models, e.g., transgenic mice bearing human germ line Ab genes, could determine whether m66.6 and related Abs targeting the m66.6/2F5 epitope can be elicited by rationally designed vaccine immunogens.