Computationally optimized broadly reactive Ags (COBRA) targeting H1 elicit a broad cross-reactive and cross-neutralizing Ab response against multiple H1N1 viral strains. To assess B cell breadth, Mus musculus (BALB/c) Ab-secreting cells elicited by a candidate COBRA hemagglutinin (HA) (termed P1) were compared with Ab-secreting cells elicited by historical H1N1 vaccine strains. In addition, to evaluate the Ab response elicited by P1 HA at increased resolution, a panel of P1 HA-specific B cell hybridomas was generated following immunization of mice with COBRA P1 and the corresponding purified mAbs were characterized for Ag specificity and neutralization activity. Both head-and stem-directed mAbs were elicited by the P1 HA Ag, with some mAbs endowed with Ab-dependent cell-mediated cytotoxicity activity. P1 HA-elicited mAbs exhibited a wide breadth of HA recognition, ranging from narrowly reactive to broadly reactive mAbs. Interestingly, we identified a P1 HA-elicited mAb (1F8) exhibiting broad hemagglutination inhibition activity against both seasonal and pandemic H1N1 influenza strains. Furthermore, mAb 1F8 recognized an overlapping, but distinct, epitope compared with other narrowly hemagglutination inhibition-positive mAbs elicited by the P1 or wild-type HA Ags. Finally, P1 HA-elicited mAbs were encoded by distinct H chain variable and L chain variable gene segment rearrangements and possessed unique CDR3 sequences. Collectively, the functional characterization of P1 HA-elicited mAbs sheds further insights into the underlying mechanism(s) of expanded Ab breadth elicited by a COBRA HA-based immunogen and advances efforts toward design and implementation of a more broadly protective influenza vaccine.

This study examines how site-specific binding to three identified neurosteroid binding sites in the α1β3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds to the canonical β3(+)–α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β3 subunit, promoting receptor desensitization and the α1 subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

The two-state coagonist model has been successfully used to analyze and predict peak current responses of the g-aminobutyric acid type A (GABA A) receptor. The goal of the present study was to provide a model-based description of GABA A receptor activity under steady-state conditions after desensitization has occurred. We describe the derivation and properties of the cyclic three-state resting-active-desensitized (RAD) model. The relationship of the model to receptor behavior was tested using concatemeric a1b2g2 GABA A receptors expressed in Xenopus oocytes. The receptors were activated by the orthosteric agonists GABA or b-alanine, the allosteric agonist propofol, or combinations of GABA, propofol, pentobarbital, and the steroid allopregnanolone, and the observed steady-state responses were compared with those predicted by the model. A modified RAD model was employed to analyze and describe the actions on steady-state current of the inhibitory steroid pregnenolone sulfate. The findings indicate that the steady-state activity in the presence of multiple active agents that interact with distinct binding sites follows standard energetic additivity. The derived equations enable prediction of peak and steady-state activity in the presence of orthosteric and allosteric agonists, and the inhibitory steroid pregnenolone sulfate. SIGNIFICANCE STATEMENT The study describes derivation and properties of a three-state resting-active-desensitized model. The model and associated equations can be used to analyze and predict peak and steady-state activity in the presence of one or more active agents.

Influenza viruses represent a threat to the world population. The currently available standard of care influenza vaccines are offered for each influenza season to prevent infection and spread of influenza viruses. Current vaccine formulations rely on using wild-type Ags, including the hemagglutinin (HA) and neuraminidase (NA) proteins as the primary immune targets of the vaccine. However, vaccine effectiveness varies from season to season, ranging from 10 to 75% depending on season and on age group studied. To improve rates of vaccine effectiveness, a new generation of computationally optimized broadly reactive Ags (COBRA)-based vaccines have been developed as a next-generation influenza vaccine. In this report, mice were intranasally, i.p., or i.m. primed with reassortant influenza viruses expressing different H1N1 COBRA HA proteins. These mice were subsequently boosted i.p. or i.m. with the same viruses. Sera collected from mice that were intranasally infected and i.p. boosted with COBRA-based viruses had broad anti-HA IgG binding, hemagglutination inhibition, and neutralizing activity against a panel of seasonal and pandemic H1N1 viruses. Mice immunized with viruses expressing a seasonal or pandemic H1N1 HA protein had antisera that recognized fewer viruses in the panel. Overall, COBRA-based HA proteins displayed on the surface of a virus elicited a breadth of Abs that recognized and neutralized historical H1N1 strains as well as more contemporary H1N1 viruses. ImmunoHorizons, 2018, 2: 226-237.