Isolation and characterisation of novel broadly neutralising antibodies from rabbits immunised with the HIV-1 Env/2dCD4S60C immune complex
Date
2020
Authors
Smith, Simone
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Abstract
Developing a safe and effective prophylactic vaccine against HIV-1 still remains the most effective
strategy for controlling and eliminating the worldwide health burden of HIV infection. In order for
an HIV-1 vaccine to be effective, it would need to elicit “long-lasting” broadly neutralizing
antibody (bNAb) responses capable of providing protection against a large number of globally
circulating HIV-1 subtypes. The development of effective vaccines by previous human clinical
trials have failed to elicit protective immunity, and this may be due to the many challenges of HIV 1 vaccine design including the conformation-dependent exposure of critical epitopes in an
immunogen. The viral envelope glycoprotein (Env; the target of bNAbs) binding to CD4 exposes
epitopes which are masked in the unbound Env conformation, such as CD4 induced epitopes that
are subsequently exposed during the open Env conformation. A preliminary study performed by
our laboratory testing various HIV-1 Env/2dCD4S60C immune complexes in rabbit immunisations
have demonstrated that it is possible to generate antibodies capable of neutralising tier-2 (harder
to neutralise) HIV-1 pseudoviruses in vitro. The aim of this study was to delineate the neutralising
responses observed in these Env/2dCD4S60C immunised rabbits by isolating and characterising the
monoclonal antibodies from these sera. The first objective was to confirm the presence of
neutralising antibody responses elicited by the immunized New Zealand white rabbits in response
to a booster with the immunogen complex using the TZM-bl based neutralising assays. The
trimeric gp140GCN4 and 2dCD4S60C were expressed, purified, and complexed for use in the rabbit
booster immunizations. Next, we sought to sort for single antigen-specific memory B cells using
a multi-parameter Flow Cytometry and Fluorescence Activated Cell Sorting (FACS) technique.
Following single cell sorting, transcriptionally active linear expression cassettes (TAPs) were
assembled by PCR. The linear cassettes containing a promoter, the antibodies variable and constant
regions and a terminator sequence were assembled in a mammalian expression vector system as
TAPs. Matched linear cassettes pairs (heavy- and light-chains) were co-transfected in HEK 293T
mammalian cells and tissue culture supernatants were collected, concentrated and evaluated for
monoclonal antibody expression. Concentrated culture supernatants were tested to determine the
binding activity of the expressed antibodies, and confirm size and identity using ELISAs and SDS PAGE/western blot analyses, respectively. Using the TZM-bl neutralization assay, our results
confirmed the persistence of circulating neutralizing antibody responses two years after the last
v
immunization (new baseline) against a heterologous, tier-2 clinically relevant pseudovirus,
ZM53M.PB12. In addition, we demonstrated that these specific neutralization titres (ID50) could
once again be elevated in three rabbits following a single boosting immunization with the
Env/2dCD4S60C complex. Anti-HIV-1 specific B cells from rabbit PMBCs post booster were
successfully isolated and sorted as single cells using multi-parameter Flow Cytometry and FACS
techniques. All elements of the TAP cassette were successfully PCR amplified from the sorted B
cells, however, subsequent analysis showed that the antibody variable fragments were missing
from the assembled TAP cassettes. As such, the expressed proteins failed to bind the Env,
2dCD4S60C and Env/2dCD4S60C antigens in an ELISA, and the presence of truncated antibody
fragments was confirmed in the immunoblot assays. Overall, the durability of the bNAb response
elicited by immunization with the Env-2dCD4S60C complex were confirmed, highlighting the
importance of isolating and characterizing the monoclonal antibodies responsible for
neutralization, and future strategies can optimize the protocols described here
Description
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Faculty of Health Sciences, School of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, 2020