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This study describes the immunization of a llama with the prefusion\u2010stabilized HIV\u20101 envelope (Env) trimer, BG505 DS\u2010SOSIP, and the identification and improvement of potent neutralizing nanobodies recognizing the CD4\u2010binding site (CD4bs) of vulnerability. Two of the vaccine\u2010elicited CD4bs\u2010targeting nanobodies, G36 and R27, when engineered into a triple tandem format with llama IgG2a\u2010hinge region and human IgG1\u2010constant region (G36\u00d73\u2010IgG2a and R27\u00d73\u2010IgG2a), neutralized 96% of a multiclade 208\u2010strain panel at geometric mean IC<jats:sub>80<\/jats:sub>s of 0.314 and 0.033\u00a0\u00b5g mL<jats:sup>\u22121<\/jats:sup>, respectively. Cryo\u2010EM structures of these nanobodies in complex with Env trimer revealed the two nanobodies to neutralize HIV\u20101 by mimicking the recognition of the CD4 receptor. To enhance their neutralizing potency and breadth, nanobodies are linked to the light chain of the V2\u2010apex\u2010targeting broadly neutralizing antibody, CAP256V2LS. The resultant human\u2010llama bispecific antibody CAP256L\u2010R27\u00d73LS exhibited ultrapotent neutralization and breadth exceeding other published HIV\u20101 broadly neutralizing antibodies, with pharmacokinetics determined in FcRn\u2010Fc mice similar to the parent CAP256V2LS. Vaccine\u2010elicited llama nanobodies, when combined with V2\u2010apex broadly neutralizing antibodies, may therefore be able to fulfill anti\u2010HIV\u20101 therapeutic and prophylactic clinical goals.<\/jats:p>","DOI":"10.1002\/advs.202309268","type":"journal-article","created":{"date-parts":[[2024,5,5]],"date-time":"2024-05-05T05:55:25Z","timestamp":1714888525000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Ultrapotent Broadly Neutralizing Human\u2010llama Bispecific Antibodies against HIV\u20101"],"prefix":"10.1002","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4154-4322","authenticated-orcid":false,"given":"Jianliang","family":"Xu","sequence":"first","affiliation":[{"name":"Vaccine Research Center National Institute of Allergy and Infectious Diseases National Institutes of Health  Bethesda MD 20892 USA"},{"name":"Laboratory of Lymphocyte Nuclear Biology National Institute of Arthritis 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