Summary A subset of individuals infected with human immunodeficiency virus 1 (HIV-1) develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing a diverse repertoire of light chains and predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.

Summary Latent reservoirs of HIV-1 infected cells are refractory to antiretroviral therapies (ART) and remain the major barrier to curing HIV-1. Because latently infected cells are long-lived, immunologically invisible, and may undergo homeostatic proliferation, a “shock and kill” approach has been proposed to eradicate this reservoir by combining ART with inducers of viral transcription. However, all attempts to alter the HIV-1 reservoir in vivo have failed to date. Using humanized mice, we show that broadly neutralizing antibodies (bNAbs) can interfere with establishment of a silent reservoir by Fc-FcR mediated mechanisms. In established infection, bNAbs or bNAbs plus single inducers are ineffective in preventing viral rebound. However, bNAbs plus a combination of inducers that act by independent mechanisms synergize to decrease the reservoir as measured by viral rebound. Thus, combinations of inducers and bNAbs constitute a therapeutic strategy that impacts the establishment and maintenance of the HIV-1 reservoir in humanized mice.

Methods to identify genes encoding immunoglobulin heavy and light chains from single B lymphocytes vary in efficiency, error rate and practicability. Here we describe a protocol to sequence and clone the variable antibody region of single antigen-specific mouse memory B cells for antibody production. After purification, antigen-specific mouse memory B cells are first single-cell-sorted by fluorescence-activated cell sorting (FACS), and V(D)J transcripts are amplified by RT-PCR. Fragments are then combined with linearized expression vectors, assembled in vitro as part of a sequence- and ligation-independent cloning (SLIC) reaction and then transformed into Escherichia coli. Purified vectors can then be used to produce monoclonal antibodies in HEK293E suspension cells. This protocol improves the amplification efficiency of antibody variable genes and accelerates the cloning workflow. Antibody sequences will be available in 3-4 d, and microgram to milligram amounts of antibodies are produced within 14 d. The new protocol should be useful for addressing fundamental questions about antigen-specific memory B cell responses, as well as for characterizing antigen-specific antibodies.

The isolation of broadly neutralizing HIV-1 monoclonal antibodies (MAbs) to distinct epitopes on the viral envelope glycoprotein (Env) provides the potential to use combinations of MAbs for prevention and treatment of HIV-1 infection. Since many of these MAbs have been isolated in the last few years, the potency and breadth of MAb combinations have not been well characterized. In two parallel experiments, we examined the in vitro neutralizing activities of double-, triple-, and quadruple-MAb combinations targeting four distinct epitopes, including the CD4-binding site, the V1V2-glycan region, the V3-glycan supersite, and the gp41 membrane-proximal external region (MPER), using a panel of 125 Env-pseudotyped viruses. All MAb combinations showed substantially improved neutralization breadth compared to the corresponding single MAbs, while the neutralization potency of individual MAbs was maintained. At a 50% inhibitory concentration (IC 50 ) cutoff of 1 g/ml per antibody, doubleMAb combinations neutralized 89 to 98% of viruses, and triple combinations neutralized 98 to 100%. Overall, the improvement of neutralization breadth was closely predicted by an additive-effect model and explained by complementary neutralization profiles of antibodies recognizing distinct epitopes. Subtle but consistent favorable interactions were observed in some MAb combinations, whereas less favorable interactions were observed on a small subset of viruses that are highly sensitive to V3-glycan MAbs. These data demonstrate favorable in vitro combinations of broadly neutralizing HIV-1 MAbs and suggest that such combinations could have utility for HIV-1 prevention and treatment.

Klein et al. find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively transferred bNAbs to prevent the emergence of bNAb viral escape variants.