Immunization with attenuated Plasmodium falciparum sporozoites (PfSPZ) has been shown to be protective, but the features of the antibody response induced by this treatment remain unclear. To investigate this response at high resolution, we isolated IgM and IgG monoclonal antibodies from Tanzanian volunteers who were immunized by repeated injection of irradiated PfSPZ and who were found to be protected from controlled human malaria infection (CHMI) with infectious homologous PfSPZ. All IgG monoclonals isolated bound to P. falciparum circumsporozoite protein (PfCSP) and recognized distinct epitopes in the N-terminus, NANP repeat region, and C-terminus. Strikingly, the most effective antibodies, as assessed in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the PfCSP N-terminal junction that is not in the RTS,S vaccine. These dual-specific antibodies were isolated from different donors and used VH3-30 or VH3-33 alleles carrying tryptophan or arginine at position 52. Using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. Our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies.

SignificanceThe Plasmodium falciparum circumsporozoite protein (CSP) has been studied for decades as a potential immunogen, but little structural information is available on how antibodies recognize the immunodominant NANP repeats within CSP. The most advanced vaccine candidate is RTS,S, which includes multiple NANP repeats. Here, we analyzed two functional antibodies from an RTS,S trial and determined the number of repeats that interact with the antibody Fab fragments using isothermal titration calorimetry and X-ray crystallography. Using negative-stain electron microscopy, we also established how the antibody binds to the NANP repeat region in a recombinant CSP construct. The structural features outlined here provide a rationale for structure-based immunogen design to improve upon the efficacy of the current RTS,S vaccine.

SummaryCharacterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies.

The effectiveness of the annual influenza vaccine has declined in recent years, especially for the H3N2 component, and is a concern for global public health. A major cause for this lack in effectiveness has been attributed to the egg-based vaccine production process. Substitutions on the hemagglutinin glycoprotein (HA) often arise during virus passaging that change its antigenicity and hence vaccine effectiveness. Here, we characterize the effect of a prevalent substitution, L194P, in egg-passaged H3N2 viruses. X-ray structural analysis reveals that this substitution surprisingly increases the mobility of the 190-helix and neighboring regions in antigenic site B, which forms one side of the receptor binding site (RBS) and is immunodominant in recent human H3N2 viruses. Importantly, the L194P substitution decreases binding and neutralization by an RBS-targeted broadly neutralizing antibody by three orders of magnitude and significantly changes the HA antigenicity as measured by binding of human serum antibodies. The receptor binding mode and specificity are also altered to adapt to avian receptors during egg passaging. Overall, these findings help explain the low effectiveness of the seasonal vaccine against H3N2 viruses, and suggest that alternative approaches should be accelerated for producing influenza vaccines as well as isolating clinical isolates.

An unprecedented spiral structure of the malarial CSP protein in complex with a protective antibody is revealed by cryo-EM.

The enzyme dihydrofolate reductase (DHFR, E) from Escherichia coli is a paradigm for the role of protein dynamics in enzyme catalysis. Previous studies have shown that the enzyme progresses through the kinetic cycle by modulating the dynamic conformational landscape in the presence of substrate dihydrofolate (DHF), product tetrahydrofolate (THF), and cofactor (NADPH or NADP+). This study focuses on the quantitative description of the relationship between protein fluctuations and product release, the rate-limiting step of DHFR catalysis. NMR relaxation dispersion measurements of millisecond time scale motions for the E:THF:NADP+ and E:THF:NADPH complexes of wild-type and the Leu28Phe (L28F) point mutant reveal conformational exchange between an occluded ground state and a low population of a closed state. The backbone structures of the occluded ground states of the wild-type and mutant proteins are very similar, but the rates of exchange with the closed excited states are very different. Integrated analysis of relaxation dispersion data and THF dissociation rates measured by stopped-flow spectroscopy shows that product release can occur by two pathways. The intrinsic pathway consists of spontaneous product dissociation and occurs for all THF-bound complexes of DHFR. The allosteric pathway features cofactor-assisted product release from the closed excited state and is utilized only in the E:THF:NADPH complexes. The L28F mutation alters the partitioning between the pathways and results in increased flux through the intrinsic pathway relative to the wild-type enzyme. This repartitioning could represent a general mechanism to explain changes in product release rates in other E. coli DHFR mutants.

34The induction of broad and potent immunity by vaccines is the key focus of research efforts 35 aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins 36 have shown promise as vaccine candidates as they can induce potent autologous neutralizing 37 responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated 38 and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better 39 understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of 40 antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-41 epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 42 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and 43 have characteristics akin to several human-derived broadly neutralizing antibodies. 44 (nucleotide level) was 6.4% (range: 2.1%-10.2%) with an average HC complementarity-126 determining region 3 (CDR-H3) length of 15 amino acids (aa) (range: 7-23) ( Table S2). The rh1987 127 KC mAbs utilized HC variable genes from the IGHV3 and IGHV4 families and predominantly used 128 KC V genes from the IGKV1 family (Table S2). All of the rh1987 KC mAbs had a CDR-L3 length of 9 129 aa and their average KC SHM (nucleotide level) was 4.7% (range: 2.6%-6.0%) (Table S2). A single 130 clonal family with 4 members (RM19A) was detected among rh1987 KC mAbs with members 131 isolated from both week 22 and week 25 samples (Table S2). The rh1987 LC mAbs used HC V 132 genes from the IGHV1, IGHV3 and IGHV4 families and LC V genes mainly from the IGLV2 gene 133 family ( Table S2). The rh1987 LC mAbs had an average CDR-L3 length of 10 aa (range: 9-11) with 134 an average LC SHM (nucleotide level) of 3.8% (range: 0.9%-10.6%) (Table S2). Two clonal families 135

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several humanderived broadly neutralizing antibodies.