Exploring the potential of engineered antibodies to prevent HIV cell-free and cell-cell transmission
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Date
2021
Authors
Van Dorsten, Rebecca Toumi
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Abstract
Broadly neutralizing antibodies (bNAbs), isolated from some HIV-infected individuals, can
prevent infection in animal models and are being evaluated for the prevention and treatment
of HIV infection in humans. To further explore the potential of bNAbs, we designed and
expressed single-chain variable fragments (scFv) that artificially link the variable regions of
antibody heavy and light chains and which may have advantages due to their smaller size. We
tested individual scFv derived from bNAbs targeting the major sites of vulnerability on the
HIV-1 envelope glycoprotein, namely CAP256-VRC26.25 (V2 apex), PGT121 (N332-supersite),
3BNC117 (CD4bs), and 10E8v4 (MPER) using a pseudovirus neutralization assay. Each scFv
retained good neutralizing activity against a large multi-subtype virus panel, although there
was variable loss of function compared to the parental IgG antibodies. For CAP256-VRC26.25
(hereafter CAP256.25), there was a significant loss of potency (138-fold), in part related to
differential interaction with charged amino acids in the V2 epitope at residues 166 and 169.
There was also a reduction in potency for the 3BNC117 scFv (13-fold) among viruses lacking
the N276 glycan. Similarly, a reduction in potency for the PGT121 scFv (4-fold) was found
among viruses lacking an N332 glycan and in viruses with a longer V1 loop. This variation at
key residues affected scFv neutralization more than the matched IgGs, suggesting that scFv
interacted with their epitopes in subtly different ways. Remarkably, the scFv of 10E8v4
maintained breadth of 100% in our panel with only a minor reduction in potency. Overall,
these scFv of clinically relevant bNAbs retained significant neutralizing activity, indicating that
they may be suitable for passive immunization to prevent HIV-1 infection.
HIV envelope diversity represents a significant challenge to the efficacy of bNAbs and it is
widely acknowledged that double or triple combinations will be needed to ensure complete
coverage. To assess whether this would also apply to scFv, we tested five scFv of bNAbs in
equimolar combinations against a small multiclade panel of viruses. This included the four
bNAbs described above plus 8ANC195, an antibody that targets the gp120-gp41 interface.
Similar to IgG, experimental combinations of two and three scFv showed a significant
improvement in potency and breadth compared to single scFv. Using the Loewe additive
model, combination titres were predicted using single scFv data and compared to the
experimental combination data. Most combinations followed this additive model of potency
and no significant antagonism was observed for any combination of two or three scFv. Low
levels of synergy were observed within several combinations specifically those containing
CAP256.25 and 10E8v4 scFv. This was shown by the improved potency of the experimental
titres compared to the predicted titres based on this model. These data and model were used
to extrapolate and predict neutralization titres for a larger 45-virus panel. At therapeutic
levels (1μg/mL), 100% coverage was reached for one of the dual and three of the triple
combinations. These combinations always contained 10E8v4 scFv but never 8ANC195.
Moreover, the geometric mean potency for the best triple combination consisting of
CAP256.25, 10E8v4, and 3BNC117, was significantly improved to 0.047μg/mL compared to
the most potent single scFv (0.12μg/mL, p<0.0001). These results show that combinations of
scFv generally follow an additive model of potency, with modest levels of synergy and that
their breadth and potency are significantly improved when applied in combinations.
Transmission of HIV through cell-associated HIV particles may play an important role in sexual
and perinatal transmission. bNAbs have been shown to have reduced activity in this mode of
transmission, which could limit their ability to prevent infection. Furthermore, cell-associated
virus is thought to play an important role in maintaining HIV infection by establishing and
maintaining the HIV reservoir. To explore the ability of smaller molecules to prevent this mode
of transmission, we tested the scFv and IgG of four bNAbs, namely CAP256.25, PGT121,
3BNC117, and 10E8v4, in a cell-free and cell-cell transmission assay. We found that the IgG
lost potency against cell-associated virus compared to the neutralization of free virus, with a
9-fold geometric mean potency difference for all IgG. This potency loss was most pronounced
in PGT121 IgG (19-fold) and 3BNC117 IgG (15-fold) but was also noted for CAP256.25 IgG (3.4-
fold) and 10Ev4 IgG (4.9-fold). However, the scFv neutralized both cell-free and cell associated viruses with similar potency (geometric mean potency difference between 1.3 and
2.3 fold). Thus, despite the differential potency losses between IgG and scFv for three of the
four bNAbs in the free virus assay, scFv retain an advantage in blocking this mode of
transmission. The net result was that 3BNC117 and PGT121 IgG and scFv neutralized cell associated virus with similar potency, though the CAP256.25 scFv was slightly less potent than
the IgG (13-fold). In contrast, 10E8v4 was slightly better at preventing cell-cell transmission
as an scFv than its IgG counterpart (2-fold), possibly due to the location of its epitope close to
the cell membrane. These data demonstrated that scFv may show potential for the
prevention of cell-associated virus neutralization, as their small size likely allows them to
better diffuse between cells as well as into the mucosal tissues where cell-cell transmission
occurs.
In conclusion, the scFv of HIV-directed bNAbs retained much of their function in both cell-free
neutralization and cell-cell neutralization assays. Furthermore, they showed the expected
level of activity when tested in combination and in some cases displayed modest synergy. Due
to their smaller size, scFv are more easily incorporated into novel expression vectors and
could be particularly useful for vectored immunoprophylaxis (VIP) for the prevention of HIV
infection. Taken together, the findings from this thesis support the further research and
development of scFv derived from HIV bNAbs for potential use in clinical applications.
Description
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2021