Immunization with HIV-1 trimeric SOSIP.664 BG505 or founder virus C (FVC Env ) covalently complexed to two-domain CD4 S60C elicits cross-clade neutralizing antibodies in New Zealand white rabbits Nancy L. Tumba ⇑ , Gavin R. Owen, Mark A. Killick, Maria A. Papathanasopoulos HIV Pathogenesis Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa article info Article history: Received 7 March 2022 Received in revised form 21 September 2022 Accepted 28 September 2022 Available online 30 September 2022 Keywords: HIV-1 vaccine immunogens Envelope glycoprotein trimers Covalent complexes Broadly neutralizing antibodies New Zealand White rabbits Immunogen design abstract Background: An ongoing challenge in HIV-1 vaccine research is finding a novel HIV-1 envelope glycopro- tein (Env)-based immunogen that elicits broadly cross-neutralizing antibodies (bnAbs) without requiring complex sequential immunization regimens to drive the required antibody affinity maturation. Previous vaccination studies have shown monomeric Env and Env trimers which contain the GCN4 leucine zipper trimerization domain and are covalently bound to the first two domains of CD4 (2dCD4 S60C ) generate potent bnAbs in small animals. Since SOSIP.664 trimers are considered the most accurate, conformation- ally intact representation of HIV-1 Env generated to date, this study further evaluated the immunogeni- city of SOSIP.664 HIV Env trimers (the well characterized BG505 and FVC Env ) covalently complexed to 2dCD4 S60C . Methods: Recombinant BG505 SOSIP.664 and FVC Env SOSIP.664 were expressed in mamma- lian cells, purified, covalently coupled to 2dCD4 S60C and antigenically characterized for their interaction with HIV-1 bnAbs. The immunogenicity of BG505 SOSIP.664-2dCD4 S60C and FVC Env SOSIP.664-2dCD4 S60C was investigated in New Zealand white rabbits and compared to unliganded FVC Env and 2dCD4 S60C . Rabbit sera were tested for the presence of neutralizing antibodies against a panel of 17 pseudoviruses. Results: Both BG505 SOSIP.664-2dCD4 S60C and FVC Env SOSIP.664-2dCD4 S60C elicited a potent, HIV- specific response in rabbits with antibodies having considerable potency and breadth (70.5% and 76%, respectively) when tested against a global panel of 17 pseudoviruses mainly composed of harder-to- neutralize multiple clade tier-2 pseudoviruses. Conclusion: BG505 SOSIP.664-2dCD4 S60C and FVC Env SOSIP.664-2dCD4 S60C are highly immunogenic and elicit potent, broadly neutralizing antibodies, the extent of which has never been reported previously for SOSIP.664 trimers. Adding to our previous results, the ability to consistently elicit these types of potent, cross-neutralizing antibody responses is dependent on novel epitopes exposed following the covalent binding of Env (independent of sequence and conformation) to 2dCD4 S60C . These findings justify further investment into research exploring mod- ified open, CD4-bound Env conformations as novel vaccine immunogens. Ó 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction The HIV/AIDS pandemic has now continued for four decades, with approximately 37.7 million individuals living with HIV glob- ally, and 1.5 million new infections reported in 2020 [1]. While these numbers have vastly decreased from 1997 when the new infections peaked at 2.9 million [1], a decisive halt to this spread can only be achieved with an effective prophylactic vaccine. The broad diversity of HIV-1 has contributed to the difficulty in identi- fying an Env-based vaccine immunogen capable of inducing immu- nity against all circulating HIV-1 groups and clades. To address viral diversity, researchers have tried to design immunogens that direct the immune responses towards regions of the viral envelope glycoprotein (Env) that are highly conserved, such as the CD4 receptor binding site (CD4bs), the gp120-gp41 interface, the V1V2 trimer apex, and the membrane proximal external region (MPER) [2]. A number of broadly neutralizing antibodies (bnAbs) that target these conserved sites have been isolated from HIV- infected individuals thereby showing the possibility of their induc- tion in humans. However, if HIV-infected individuals develop bnAbs, it is too late to impact on disease progression. By contrast, https://doi.org/10.1016/j.jvacx.2022.100222 2590-1362/Ó 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ⇑ Corresponding author at: HIV Pathogenesis Research Unit, Faculty of Health Sciences, University of the Witwartersrand, 7 th floor, Rm 7Q17, 7 York Road Parktown, 2193, South Africa. E-mail addresses: Nancy.tumba@wits.ac.za (N.L. Tumba), gavin.owen@wits.ac.za (G.R. Owen), mark.killick@wits.ac.za (M.A. Killick), maria.papathanasopoulos@wits. ac.za (M.A. Papathanasopoulos). Vaccine: X 12 (2022) 100222 Contents lists available at ScienceDirect Vaccine: X journal homepage: www.elsevier.com/locate/jvacx