This study aimed to identify and isolate broadly neutralizing human monoclonal antibodies (NAbs) against HIV-1 by designing antigenically resurfaced glycoproteins that target the conserved CD4-binding site (CD4bs) of the HIV-1 envelope protein gp120. The researchers developed a recombinant form of gp120, RSC3, which specifically interacts with NAbs directed to the CD4bs. Using this probe, they identified and sorted individual B cells from an HIV-1-infected donor whose sera contained broad NAbs. From these cells, three monoclonal antibodies (mAbs) were isolated: VRC01, VRC02, and VRC03. These mAbs showed strong reactivity to RSC3 and weak or no reactivity to a control protein lacking the CD4bs region. Neutralization assays demonstrated that VRC01 and VRC02, which partially mimic the interaction of CD4 with gp120, neutralized 91% and 90% of HIV-1 isolates, respectively, while VRC03 neutralized 57%. The study highlights the potential of targeting the conserved CD4bs region for HIV-1 vaccine design and provides insights into how the human immune system can effectively target this vulnerable site on the viral envelope.This study aimed to identify and isolate broadly neutralizing human monoclonal antibodies (NAbs) against HIV-1 by designing antigenically resurfaced glycoproteins that target the conserved CD4-binding site (CD4bs) of the HIV-1 envelope protein gp120. The researchers developed a recombinant form of gp120, RSC3, which specifically interacts with NAbs directed to the CD4bs. Using this probe, they identified and sorted individual B cells from an HIV-1-infected donor whose sera contained broad NAbs. From these cells, three monoclonal antibodies (mAbs) were isolated: VRC01, VRC02, and VRC03. These mAbs showed strong reactivity to RSC3 and weak or no reactivity to a control protein lacking the CD4bs region. Neutralization assays demonstrated that VRC01 and VRC02, which partially mimic the interaction of CD4 with gp120, neutralized 91% and 90% of HIV-1 isolates, respectively, while VRC03 neutralized 57%. The study highlights the potential of targeting the conserved CD4bs region for HIV-1 vaccine design and provides insights into how the human immune system can effectively target this vulnerable site on the viral envelope.