High-resolution mapping of the neutralizing and binding specificities of polyclonal sera post-HIV Env trimer vaccination

Mapping polyclonal serum responses is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual antibodies, which incompletely defines the polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. We used mutational antigenic profiling to determine how all mutations in Env affected viral neutralization and electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize serum Fabs bound to Env trimers. The dominant neutralizing specificities were generally only a subset of the more diverse binding specificities. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Furthermore, we refined residue-level epitope specificity directly from sera, revealing subtle differences across sera. Together, mutational antigenic profiling and EMPEM yield a holistic view of the binding and neutralizing specificity of polyclonal sera.

Comprehensive mapping of mutations to the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human serum antibodies

The evolution of SARS-CoV-2 could impair recognition of the virus by human antibody-mediated immunity. To facilitate prospective surveillance for such evolution, we map how convalescent serum antibodies are impacted by all mutations to the spike’s receptor-binding domain (RBD), the main target of serum neutralizing activity. Binding by polyclonal serum antibodies is affected by mutations in three main epitopes in the RBD, but there is substantial variation in the impact of mutations both among individuals and within the same individual over time. Despite this inter- and intra-person heterogeneity, the mutations that most reduce antibody binding usually occur at just a few sites in the RBD’s receptor binding motif. The most important site is E484, where neutralization by some sera is reduced >10-fold by several mutations, including one in emerging viral lineages in South Africa and Brazil. Going forward, these serum escape maps can inform surveillance of SARS-CoV-2 evolution.

High-resolution mapping of the neutralizing and binding specificities of polyclonal rabbit serum elicited by HIV Env trimer immunization

Mapping the epitope specificities of polyclonal serum is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual monoclonal antibodies, which incompletely defines the full polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. To map the neutralizing specificity, we used mutational antigenic profiling to determine how all amino-acid mutations in Env affected viral neutralization. To map the binding specificity, we used electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize the Fabs in serum bound to Env trimers. Mutational antigenic profiling showed that the dominant neutralizing specificities were the C3/V5 and/or 241/289 glycan hole epitopes, which were generally only a subset of the more diverse binding specificities mapped with EMPEM. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Further, mutational antigenic profiling was able to refine epitope specificity in residue-level detail directly from sera, revealing subtle differences across rabbits. Together, mutational antigenic profiling and EMPEM allow for a holistic view of the binding and neutralizing specificity of polyclonal sera and could be used to finely evaluate and guide vaccine design.

Interactions Between Acanthamoeba culbertsoni and Pathogenic Bacteria and their Inhibition by Lectin-Antibodies

In this study, using pathogenic and non-pathogenic bacteria, it was analyzed whether a polyclonal serum and a monoclonal antibody to A. culbertsoni mannose-binding protein (MBP) could inhibit its interaction. The association of the amoeba with E. coli O157:H7 was very strong at a level of over 100%, but the non-pathogenic E. coli strain was about five times lower at 22%. Pathogenic K. pnueumoniae also showed high association with amoeba by about 92% as compared with pathogenic E. coli O157:H7 and S. agalactiae. The polyclonal serum to MBP inhibited E. coli O157:H7 association to amoeba 2.5 times more than untreated E. coli O157:H7. Monoclonal antibody to MBP also inhibited bacterial association with amoeba but was not stronger than the polyclonal serum. Pathogenic E. coli O157:H7 showed about 88% invasion into amoeba and decreased about 22% as compared with associated E. coli O157:H7. Polyclonal serum to MBP inhibited about 55%, 50%, and 44% in E. coli O157:H7, K. pneumoniae and S. agalactiae, respectively. The invasion of K. pneumoniae and S. agalactiae was not high as polyclonal serum but was about 8% to 10% weaker than polyclonal serum. The pathogenic strains of K. pneumoniae and S. agalactiae showed less decrease in survival as shown at invasion than E. coli O157:H7 without antibody. This study provided the information that the pathogenic bacteria could be more interactive with A. culbertsoni trophozoites as a reservoir host than non-pathogenic E. coli, and the amoeba should interact with bacteria by the MBP lectin.

Paracoccidioides HSP90 Can Be Found in the Cell Surface and Is a Target for Antibodies with Therapeutic Potential

Paracoccidioidomycosis (PCM) is one of the most frequent systemic mycoses in Latin America. It affects mainly male rural workers in impoverished regions, and the therapy can last up to two years or use drugs that are very toxic. Given the need for novel safe and effective approaches to treat PCM, we have been developing monoclonal antibodies (mAbs) that could be used not only to block specific fungal targets, but also modulate the host’s antifungal immunity. In this work we show the generation of and promising results with an mAb against Heat Shock Protein (HSP)90, a molecular chaperone that is an important virulence factor in fungi. Using recombinant Paracoccidioides lutzii (Pb01) and P. brasiliensis (Pb18) HSP90 proteins produced in E. coli, we immunized mice and generated polyclonal antibodies and an IgG1 hybridoma mAb. The proteins were very immunogenic and both the polyclonal serum and mAb were used in immunofluorescence experiments, which showed binding of antibodies to the yeast cell surface. The mAb successfully opsonized P. lutzii and P. brasiliensis cells in co-incubations with J774.16 macrophage-like cells. Our results suggest that this mAb could serve as the basis for new immunotherapy regimens for PCM.

A stabilized glycomimetic conjugate vaccine inducing protective antibodies against Neisseria meningitidis serogroup A

Neisseria meningitidis serogroup A capsular polysaccharide (MenA CPS) consists of (1 → 6)-2-acetamido-2-deoxy-α-D-mannopyranosyl phosphate repeating units, O-acetylated at position C3 or C4. Glycomimetics appear attractive to overcome the CPS intrinsic lability in physiological media, due to cleavage of the phosphodiester bridge, and to develop a stable vaccine with longer shelf life in liquid formulation. Here, we generate a series of non-acetylated carbaMenA oligomers which are proven more stable than the CPS. An octamer (DP8) inhibits the binding of a MenA specific bactericidal mAb and polyclonal serum to the CPS, and is selected for further in vivo testing. However, its CRM197 conjugate raises murine antibodies towards the non-acetylated CPS backbone, but not the natural acetylated form. Accordingly, random O-acetylation of the DP8 is performed, resulting in a structure (Ac-carbaMenA) showing improved inhibition of anti-MenA CPS antibody binding and, after conjugation to CRM197, eliciting anti-MenA protective murine antibodies, comparably to the vaccine benchmark.

Strain-Dependent Activity of Zika Virus and Exposure History in Serological Diagnostics

Zika virus (ZIKV) circulates as two separate lineages, with significant genetic variability between strains. Strain-dependent activity has been reported for dengue virus, herpes simplex virus and influenza. Strain-dependent activity of subject specimens to a virus could be an impediment to serological diagnosis and vaccine development. In order to determine whether ZIKV exhibits strain-dependent activity when exposed to antibodies, we measured the neutralizing properties of polyclonal serum and three monoclonal antibodies (ZKA185, 753(3)C10, and 4G2) against three strains of ZIKV (MR−766, PRVABC59, and R103454). Here, MR−766 was inhibited almost 60% less by ZKA185 than PRVABC59 and R103454 (p = 0.008). ZKA185 enhanced dengue 4 infection up to 50% (p = 0.0058). PRVABC59 was not inhibited by mAb 753(3)C10 while MR−766 and R103453 were inhibited up to 90% (p = 0.04 and 0.036, respectively). Patient serum, regardless of exposure history, neutralized MR−766 ~30%−40% better than PRVABC56 or R103454 (p = 0.005−0.00007). The most troubling finding was the significant neutralization of MR−766 by patients with no ZIKV exposure. We also evaluated ZIKV antibody cross reactivity with various flaviviruses and found that more patients developed cross-reactive antibodies to Japanese encephalitis virus than the dengue viruses. The data here show that serological diagnosis of ZIKV is complicated and that qualitative neutralization assays cannot discriminate between flaviviruses.

Mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin

A longstanding question is how influenza evolves to escape human immunity, which is polyclonal and can target many distinct epitopes on the virus. Here we map how all amino-acid mutations to influenza’s major surface protein affect viral neutralization by polyclonal human sera. The serum of some individuals is so focused that it selects single mutations that reduce viral neutralization by over an order of magnitude. However, different viral mutations escape the sera of different individuals. This individual-to-individual variation in viral escape mutations isnotpresent among ferrets, which are frequently used as a model in influenza studies. Our results show how different single mutations help influenza escape the immunity of different members of the human population, a phenomenon that could shape viral evolution and disease susceptibility.