Wayne Volkmuth

Antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2.

Similarity between influenza nucleoprotein and hypocretin receptor 2 may trigger vaccine-associated narcolepsy. Immunological mistaken identity New reports of narcolepsy increased after the vaccination campaign against the 2009 A(H1N1) influenza pandemic in some countries but not others. Now Ahmed et al. examine differences between the vaccines used and find a potential mechanistic explanation for the vaccine-specific effect. They found a peptide in influenza nucleopeptide A that shared protein residues with human hypocretin receptor 2, which has been linked to narcolepsy. The vaccine used in unaffected countries contained less influenza nucleoprotein. Indeed, patients with putative vaccine-associated narcolepsy produced antibodies that cross-reacted to both the influenza and the hypocretin receptor 2 epitopes. Although these data do not demonstrate causation, they provide a possible explanation for the association of this particular influenza vaccination with increased reports of narcolepsy. The sleep disorder narcolepsy is linked to the HLA-DQB1*0602 haplotype and dysregulation of the hypocretin ligand-hypocretin receptor pathway. Narcolepsy was associated with Pandemrix vaccination (an adjuvanted, influenza pandemic vaccine) and also with infection by influenza virus during the 2009 A(H1N1) influenza pandemic. In contrast, very few cases were reported after Focetria vaccination (a differently manufactured adjuvanted influenza pandemic vaccine). We hypothesized that differences between these vaccines (which are derived from inactivated influenza viral proteins) explain the association of narcolepsy with Pandemrix-vaccinated subjects. A mimic peptide was identified from a surface-exposed region of influenza nucleoprotein A that shared protein residues in common with a fragment of the first extracellular domain of hypocretin receptor 2. A significant proportion of sera from HLA-DQB1*0602 haplotype–positive narcoleptic Finnish patients with a history of Pandemrix vaccination (vaccine-associated narcolepsy) contained antibodies to hypocretin receptor 2 compared to sera from nonnarcoleptic individuals with either 2009 A(H1N1) pandemic influenza infection or history of Focetria vaccination. Antibodies from vaccine-associated narcolepsy sera cross-reacted with both influenza nucleoprotein and hypocretin receptor 2, which was demonstrated by competitive binding using 21-mer peptide (containing the identified nucleoprotein mimic) and 55-mer recombinant peptide (first extracellular domain of hypocretin receptor 2) on cell lines expressing human hypocretin receptor 2. Mass spectrometry indicated that relative to Pandemrix, Focetria contained 72.7% less influenza nucleoprotein. In accord, no durable antibody responses to nucleoprotein were detected in sera from Focetria-vaccinated nonnarcoleptic subjects. Thus, differences in vaccine nucleoprotein content and respective immune response may explain the narcolepsy association with Pandemrix.

Fractional Third and Fourth Dose of RTS,S/AS01 Malaria Candidate Vaccine: A Phase 2a Controlled Human Malaria Parasite Infection and Immunogenicity Study

BACKGROUND Three full doses of RTS,S/AS01 malaria vaccine provides partial protection against controlled human malaria parasite infection (CHMI) and natural exposure. Immunization regimens, including a delayed fractional third dose, were assessed for potential increased protection against malaria and immunologic responses. METHODS In a phase 2a, controlled, open-label, study of healthy malaria-naive adults, 16 subjects vaccinated with a 0-, 1-, and 2-month full-dose regimen (012M) and 30 subjects who received a 0-, 1-, and 7-month regimen, including a fractional third dose (Fx017M), underwent CHMI 3 weeks after the last dose. Plasmablast heavy and light chain immunoglobulin messenger RNA sequencing and antibody avidity were evaluated. Protection against repeat CHMI was evaluated after 8 months. RESULTS A total of 26 of 30 subjects in the Fx017M group (vaccine efficacy [VE], 86.7% [95% confidence interval [CI], 66.8%-94.6%]; P < .0001) and 10 of 16 in the 012M group (VE, 62.5% [95% CI, 29.4%-80.1%]; P = .0009) were protected against infection, and protection differed between schedules (P = .040, by the log rank test). The fractional dose boosting increased antibody somatic hypermutation and avidity and sustained high protection upon rechallenge. DISCUSSIONS A delayed third fractional vaccine dose improved immunogenicity and protection against infection. Optimization of the RTS,S/AS01 immunization regimen may lead to improved approaches against malaria. CLINICAL TRIALS REGISTRATION NCT01857869.

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

Significance The 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. Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development in vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA)3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Together, these structural insights may aid in the design of a next-generation malaria vaccine.

Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens

There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.

Erratum: Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein (Proceedings of the National Academy of Sciences of the United States of America (2017) 114 (E10438–E10445) DOI: 10.1073/pnas.1715812114)

MICROBIOLOGY, BIOPHYSICS AND COMPUTATIONAL BIOLOGY Correction for “Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein,” by David Oyen, Jonathan L. Torres, Ulrike Wille-Reece, Christian F. Ockenhouse, Daniel Emerling, Jacob Glanville, Wayne Volkmuth, Yevel Flores-Garcia, Fidel Zavala, Andrew B. Ward, C. Richter King, and Ian A. Wilson, which was first published November 14, 2017; 10.1073/pnas.1715812114 (Proc Natl Acad Sci USA 114: E10438–E10445). The authors note that they inadvertently omitted references that reported on computational models of helical structures for the NANP repeats of circumsporozoite protein (CSP) (1–3), one of which is a recent publication that used a helical model of (NANP)6 to explain the multivalent CSP binding of an antiNANP antibody (3). The model described in Oyen et al., however, differs extensively from these previous models in that the NANP repeats adopt a wide, elongated spiral, compared for example to the tightly packed helix as described in ref. 3. The full references 1–3 appear below. The references should have been cited in an additional sentence, which should have appeared on page E10443, left column, first paragraph, to precede the sentence that begins on line 3 with “Many questions remain. . . .” The additional sentence should have appeared as follows: “Helical models have been predicted previously for the NANP repeats (1–3), but these differ substantially from the spiral conformation presented here.” The authors also note that Fig. S7 appeared incorrectly. The corrected Supporting figure and its legend appear below. The SI has been corrected online.