No abstract

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs.

bSterol import has been characterized under various conditions in three distinct fungal species, the model organism Saccharomyces cerevisiae and two human fungal pathogens Candida glabrata and Candida albicans, employing cholesterol, the sterol of higher eukaryotes, as well as its fungal equivalent, ergosterol. Import was confirmed by the detection of esterified cholesterol within the cells. Comparing the three fungal species, we observe sterol import under three different conditions. First, as previously well characterized, we observe sterol import under low oxygen levels in S. cerevisiae and C. glabrata, which is dependent on the transcription factor Upc2 and/or its orthologs or paralogs. Second, we observe sterol import under aerobic conditions exclusively in the two pathogenic fungi C. glabrata and C. albicans. Uptake emerges during post-exponential-growth phases, is independent of the characterized Upc2-pathway and is slower compared to the anaerobic uptake in S. cerevisiae and C. glabrata. Third, we observe under normoxic conditions in C. glabrata that Upc2-dependent sterol import can be induced in the presence of fetal bovine serum together with fluconazole. In summary, C. glabrata imports sterols both in aerobic and anaerobic conditions, and the limited aerobic uptake can be further stimulated by the presence of serum together with fluconazole. S. cerevisiae imports sterols only in anaerobic conditions, demonstrating aerobic sterol exclusion. Finally, C. albicans imports sterols exclusively aerobically in post-exponential-growth phases, independent of Upc2. For the first time, we provide direct evidence of sterol import into the human fungal pathogen C. albicans, which until now was believed to be incapable of active sterol import.

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs.