Profiling of hMPV F-specific antibodies isolated from human memory B cells.

Human metapneumovirus (hMPV) belongs to the Pneumoviridae family and is closely related to respiratory syncytial virus (RSV). The surface fusion (F) glycoprotein mediates viral fusion and is the primary target of neutralizing antibodies against hMPV. Here we report 113 hMPV-F specific monoclonal antibodies (mAbs) isolated from memory B cells of human donors. We characterize the antibodies' germline usage, epitopes, neutralization potencies, and binding specificities. We find that unlike RSV-F specific mAbs, antibody responses to hMPV F are less dominant against the apex of the antigen, and the majority of the potent neutralizing mAbs recognize epitopes on the side of hMPV F. Furthermore, neutralizing epitopes that differ from previously defined antigenic sites on RSV F are identified, and multiple binding modes of site V and II mAbs are discovered. Interestingly, mAbs that bind preferentially to the unprocessed prefusion F show poor neutralization potency. These results elucidate the immune recognition of hMPV infection and provide novel insights for future hMPV antibody and vaccine development.

Development of a microneutralization assay for HSV-2.

BACKGROUND: Plaque Reduction Neutralization Test (PRNT) is the standard assay used for measuring neutralizing antibody responses to Herpes simplex virus type-2 (HSV-2). The PRNT is a cumbersome, time-consuming and laborious assay. The development of a faster, high throughput microneutralization assay (MNA) for HSV-2 viruses carried out in a 96-well format will allow for rapid testing of large numbers of samples for drug and vaccine development. METHODS: We describe the generation of a MNA that utilizes a pair of anti-HSV human monoclonal antibodies (mAbs) for virus detection in HSV-2 infected Vero cells. Antibodies were generated by B-cell cloning from PBMC's isolated from HSV-1 negative/HSV-2 positive donors. We describe the selection and characterization of the antibodies used for virus detection by ELISA with purified, recombinant anti-HSV glycoproteins, antibody binding in infected cells, and Western Blot. We determine the anti-HSV-2 neutralizing titers of immune sera from mice by MNA and PRNT and compare these results by linear regression analysis. RESULTS: We show that neutralization titers for HSV-2, determined by the 96-well MNA correlate with titers determined by a PRNT completed in 24-well plates in both the absence (R2 = 0.8250) and presence (R2 = 0.7075) of complement. CONCLUSIONS: We have successfully developed an MNA that can be used in place of the burdensome PRNT to determine anti-HSV-2 neutralizing activity in serum. This MNA has much greater throughput than the PRNT, allowing many more samples to be processed in a shorter time saving ∼90 % of the time required by the laboratory scientist to complete the task as compared to the traditional PRNT.

Immunogenicity generated by mRNA vaccine encoding VZV gE antigen is comparable to adjuvanted subunit vaccine and better than live attenuated vaccine in nonhuman primates.

Shingles is a painful, blistering rash caused by reactivation of latent varicella-zoster virus (VZV) and most frequently occurs in elderly and immunocompromised individuals. Currently, two approved vaccines for the prevention of shingles are on the market, a live attenuated virus vaccine ZOSTAVAX® (Merck & Co., Inc., Kenilworth, NJ, USA) and an AS01B adjuvanted subunit protein vaccine Shingrix™ (Glaxo Smith Kline, Rockville, MD, USA). Human clinical immunogenicity and vaccine efficacy data is available for these two benchmark vaccines, offering a unique opportunity for comparative analyses with novel vaccine platforms and animal model translatability studies. The studies presented here utilized non-human primates (NHP) to evaluate humoral and cellular immune response by three vaccine modalities: the new platform of lipid nanoparticle (LNP) formulated mRNA encoding VZV gE antigen (VZV gE mRNA/LNP) as compared with well-established platforms of live attenuated VZV (VZV LAV) and adjuvanted VZV gE subunit protein (VZV gE protein/adjuvant). The magnitude of response to vaccination with a single 100-200 µg mRNA dose or two 50 µg mRNA doses of VZV gE mRNA/LNP were comparable to two 50 µg protein doses of VZV gE protein/adjuvant, suggesting the VZV gE mRNA/LNP platform has the potential to elicit a robust immune response, and both modalities generated markedly higher responses than VZV LAV. Additionally, the slopes of decay for VZV-specific antibody titers were roughly similar across all three vaccines, indicating the magnitude of peak immunogenicity was the driving force in determining immune response longevity. Finally, vaccine-induced immunogenicity with VZV LAV and VZV gE protein/adjuvant in NHP closely resembled human clinical trials immune response data for ZOSTAVAX® and Shingrix™, helping to validate NHP as an appropriate preclinical model for evaluating these vaccines.

Characterization of potent RSV neutralizing antibodies isolated from human memory B cells and identification of diverse RSV/hMPV cross-neutralizing epitopes.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children and older adults. Currently, no licensed vaccine is available, and therapeutic options are limited. The primary target of neutralizing antibodies to RSV is the surface fusion (F) glycoprotein. Understanding the recognition of antibodies with high neutralization potencies to RSV F antigen will provide critical insights in developing efficacious RSV antibodies and vaccines. In this study, we isolated and characterized a panel of monoclonal antibodies (mAbs) with high binding affinity to RSV prefusion F trimer and neutralization potency to RSV viruses. The mAbs were mapped to previously defined antigenic sites, and some that mapped to the same antigenic sites showed remarkable diversity in specificity, binding, and neutralization potencies. We found that the isolated site III mAbs shared highly conserved germline V-gene usage, but had different cross-reactivities to human metapneumovirus (hMPV), possibly due to the distinct modes/angles of interaction with RSV and hMPV F proteins. Furthermore, we identified a subset of potent RSV/hMPV cross-neutralizing mAbs that target antigenic site IV and the recently defined antigenic site V, while the majority of the mAbs targeting these two sites only neutralize RSV. Additionally, the isolated mAbs targeting site Ø were mono-specific for RSV and showed a wide range of neutralizing potencies on different RSV subtypes. Our data exemplify the diversity of anti-RSV mAbs and provide new insights into the immune recognition of respiratory viruses in the Pneumoviridae family.

Design and characterization of a fusion glycoprotein vaccine for Respiratory Syncytial Virus with improved stability.

Respiratory Syncytial Virus (RSV) infection is the leading cause of lower respiratory tract infection in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. The infectious RSV particle is decorated with a type I viral fusion (F) glycoprotein that structurally rearranges from a metastable prefusion form to a highly stable postfusion form. In people naturally infected with RSV, the neutralizing antibodies primarily recognize the prefusion conformation. Therefore, engineered RSV F protein stabilized in its prefusion conformation has been an attractive strategy for developing RSV F vaccine antigens. Long-term stability at 4 °C or higher is a desirable attribute for a RSV F subunit vaccine antigen. We have previously shown that a prefusion stabilized RSV F construct, DS-Cav1, undergoes conformational changes and forms intermediate structures upon long-term storage at 4 °C. Structure-based design was performed to improve the stability of the RSV F subunit vaccine. We identified additional mutations that further stabilize RSV F protein in its prefusion conformation by using binding to a previously described antigenic site I antibody 4D7 as the screening tool. In addition, we designed and identified variants with increased expression levels, which is another desirable attribute for a subunit vaccine. Our data suggested that an RSV F variant F111 is properly folded, and has improved heat stability as well as stability upon long-term storage at 4 °C. A mouse immunogenicity study demonstrated that no compromise in immunogenicity (both binding and neutralizing antibody levels) was observed with the introduction of these additional mutations.

Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein.

Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7.

Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

Discovery of novel selective norepinephrine reuptake inhibitors: 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols (WYE-103231).

Structural modification of a virtual screening hit led to the identification of a new series of 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols which are potent and selective inhibitors of the norepinephrine transporter over both the serotonin and dopamine transporters. One representative compound S-17b (WYE-103231) had low nanomolar hNET potency (IC(50) = 1.2 nM) and excellent selectivity for hNET over hSERT (>1600-fold) and hDAT (>600-fold). S-17b additionally had a good pharmacokinetic profile and demonstrated oral efficacy in rat models of ovariectomized-induced thermoregulatory dysfunction and morphine dependent flush as well as the hot plate and spinal nerve ligation (SNL) models of acute and neuropathic pain.

Heterocyclic cycloalkanol ethylamines as norepinephrine reuptake inhibitors.

A series of heterocyclic cycloalkanol ethylamines have been prepared to expand our norepinephrine reuptake inhibitor (NRI) program. Synthesis of a variety of heterocycles identified (+)-S-21, a potent NRI efficacious in an animal model for thermoregulatory dysfunction.

1-(Indolin-1-yl)-1-phenyl-3-propan-2-olamines as potent and selective norepinephrine reuptake inhibitors.

Efforts to identify new selective and potent norepinephrine reuptake inhibitors (NRIs) for multiple indications by structural modification of the previous 3-(arylamino)-3-phenylpropan-2-olamine scaffold led to the discovery of a novel series of 1-(indolin-1-yl)-1-phenyl-3-propan-2-olamines (9). Investigation of the structure-activity relationships revealed that small alkyl substitution at the C3 position of the indoline ring enhanced selectivity for the norepinephrine transporter (NET) over the serotonin transporter (SERT). Several compounds bearing a 3,3-dimethyl group on the indoline ring, 9k, 9o,p, and 9s,t, exhibited potent inhibition of NET (IC(50) = 2.7-6.5 nM) and excellent selectivity over both serotonin and dopamine transporters. The best example from this series, 9p, a potent and highly selective NRI, displayed oral efficacy in a telemetric rat model of ovariectomized-induced thermoregulatory dysfunction, a mouse p-phenylquinone (PPQ) model of acute visceral pain, and a rat spinal nerve ligation (SNL) model of neuropathic pain.

Discovery of WAY-260022, a Potent and Selective Inhibitor of the Norepinephrine Transporter.

The potency and selectivity of a series of 1-{(1S)-2-[amino]-1-[3-(trifluoromethoxy)phenyl]ethyl}cyclohexanol analogues are described. These compounds were prepared to improve in vitro metabolic stability and achieve brain penetration. Compound 13 (WAY-260022, NRI-022) was found to be a potent inhibitor of norepinephrine reuptake and demonstrated excellent selectivity over the serotonin and dopamine transporters. Additionally, 13 exhibited oral efficacy in a rat model of thermoregulatory dysfunction.

Dual acting norepinephrine reuptake inhibitors and 5-HT(2A) receptor antagonists: Identification, synthesis and activity of novel 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles.

The discovery of a series of 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles, dual acting norepinephrine reuptake inhibitors (NRIs) and 5-HT(2A) receptor antagonists, is described. The synthesis and structure-activity relationship (SAR) of this novel series of compounds is also presented.

Structure-activity relationships of the 1-amino-3-(1H-indol-1-yl)-3-phenylpropan-2-ol series of monoamine reuptake inhibitors.

The SAR of a series of 1-amino-3-(1H-indol-1-yl)-3-phenylpropan-2-ols as monoamine reuptake inhibitors, with a goal to improve both potency toward inhibiting the norepinephrine transporter and selectivity over the serotonin transporter, is reported. The effect of specific substitution on both the 3-phenyl group and the indole moiety were explored. This study led to the discovery of compound 20 which inhibited the norepinephrine transporter with an IC50 value of 4 nM while exhibiting 86-fold selectivity over the serotonin transporter.

Simultaneous telemetric monitoring of tail-skin and core body temperature in a rat model of thermoregulatory dysfunction.

Temperature dysfunction, clinically described as hot flashes/flushes and night sweats, commonly occur in women transitioning through menopause. Research in this field has yet to fully elucidate the biological underpinnings explaining this dysfunction. The need to develop animal models that can be used to study hormone-dependent temperature regulation is essential to advancing this scientific area. Development of telemetric transmitters for monitoring tail-skin (TST) and core body (CBT) temperatures for animal research has increased the accuracy of data by reducing extraneous factors associated with previous methods. However, until recently, TST and CBT could not be simultaneously measured telemetrically within the same animal. In this report, new dual temperature monitoring transmitters were validated by simultaneously evaluating them with the single measurement transmitters using the ovariectomized (OVX) rat thermoregulatory dysfunction model. A major advantage of measuring TST and CBT in the same animal is the ability to relate temporal changes on these two temperature parameters. Comparative experimentation was performed by single administration of clonidine (alpha(2) adrenergic agonist), MDL-100907 (5-HT(2a) antagonist), or a 7-day treatment of 17alpha-ethinyl estradiol (EE). Clonidine caused decreases in TST and CBT, MDL-100907 caused increases in TST while decreasing CBT, and EE caused decreases in TST with minor CBT decreases only at the higher dose. Data from either probe type showed similar results on temperature parameters regardless of transmitter used. These findings support the use of the new dual temperature transmitters and should enhance the quality and interpretation of data being generated in thermoregulation studies.

ICI 182,780 penetrates brain and hypothalamic tissue and has functional effects in the brain after systemic dosing.

Previous reports suggest the antiestrogen ICI 182,780 (ICI) does not cross the blood-brain barrier (BBB). However, this hypothesis has never been directly tested. In the present study, we tested whether ICI crosses the BBB, penetrates into brain and hypothalamic tissues, and affects known neuroendocrine functions in ovariectomized rats. Using HPLC with mass spectrometry, ICI (1.0 mg/kg.d, 3 d) was detected in plasma and brain and hypothalamic tissues for up to 24 h with maximum concentrations of 43.1 ng/ml, and 31.6 and 38.8 ng/g, respectively. To evaluate antiestrogenic effects of ICI in the brain after systemic dosing, we tested its ability to block the effect of 17 alpha-ethinyl estradiol (EE) (0.3 mg/kg, 8 d) on tail-skin temperature abatement in the morphine-dependent model of hot flush and on body weight change. In the morphine-dependent model, EE abated 64% of the naloxone-induced tail-skin temperature increase. ICI pretreatment (1.0, 3.0 mg/kg.d) dose dependently inhibited this effect. ICI (3.0 mg/kg.d) alone showed estrogenic-like actions, abating 30% the naloxone-induced flush. In body weight studies, EE-treated rats weighed 58.5 g less than vehicle-treated rats after 8 d dosing. This effect was partially blocked by ICI (3.0 mg/kg.d) pretreatment. Similar to EE treatment, rats receiving 1.0 or 3.0 mg/kg.d ICI alone showed little weight gain compared with vehicle-treated controls. Thus, ICI crosses the BBB, penetrates into brain and hypothalamic tissues, and has both antiestrogenic and estrogenic-like actions on neuroendocrine-related functions.

Structure-activity relationships of the cycloalkanol ethylamine scaffold: discovery of selective norepinephrine reuptake inhibitors.

Further exploration of the cycloalkanol ethylamine scaffold, of which venlafaxine ( 1) is a member, was undertaken to develop novel and selective norepinephrine reuptake inhibitors (NRIs) for evaluation in a variety of predictive animal models. These efforts led to the discovery of a piperazine-containing analogue, 17g (WY-46824), that exhibited potent norepinephrine reuptake inhibition, excellent selectivity over the serotonin transporter, but no selectivity over the dopamine transporter. Synthesis and testing of a series of cyclohexanol ethylpiperazines identified ( S)-(-)- 17i (WAY-256805), a potent norepinephrine reuptake inhibitor (IC 50 = 82 nM, K i = 50 nM) that exhibited excellent selectivity over both the serotonin and dopamine transporters and was efficacious in animal models of depression, pain, and thermoregulatory dysfunction.

Alleviation of thermoregulatory dysfunction with the new serotonin and norepinephrine reuptake inhibitor desvenlafaxine succinate in ovariectomized rodent models.

Hot flushes and night sweats, referred to as vasomotor symptoms (VMS), are presumed to be a result of declining hormone levels and are the principal menopausal symptoms for which women seek medical treatment. To date, estrogens and/or some progestins are the most effective therapeutics for alleviating VMS; however, these therapies may not be appropriate for all women. Therefore, nonhormonal therapies are being evaluated. The present study investigated a new reuptake inhibitor, desvenlafaxine succinate (DVS), in animal models of temperature dysfunction. Both models used are based on measuring changes in tail-skin temperature (TST) in ovariectomized (OVX) rats. The first relies on naloxone-induced withdrawal in morphine-dependent (MD) OVX rats, resulting in an acute rise in TST. The second depends on an OVX-induced loss of TST decreases during the dark phase as measured by telemetry. An initial evaluation demonstrated abatement of the rise in TST with long-term administration of ethinyl estradiol or with a single oral dose of DVS (130 mg/kg) in the MD model. Further evaluation showed that orally administered DVS acutely and dose dependently (10-100 mg/kg) abated a naloxone-induced rise in TST of MD rats and alleviated OVX-induced temperature dysfunction in the telemetry model. Oral administration of DVS to OVX rats caused significant increases in serotonin and norepinephrine levels in the preoptic area of the hypothalamus, a key region of the brain involved in temperature regulation. These preclinical studies provide evidence that DVS directly impacts thermoregulatory dysfunction in OVX rats and may have utility in alleviating VMS associated with menopause.

The role of the selective serotonin reuptake inhibitor fluoxetine in temperature regulation in ovariectomized rat models.

Thermoregulation is an integrated network of neuroendocrine, autonomic and somatosensory responses. Thermoregulatory dysfunction occurs during fluctuations or decline of gonadal hormone levels and results in vasomotor symptoms such as hot flushes and/or night-time sweating. The neurotransmitter serotonin (5-HT), has been reported to play a role in thermoregulation via changes in extracellular 5-HT levels and/or activation of various 5-HT receptors. The purpose of this study was to evaluate the role of the selective 5-HT reuptake inhibitor (SSRI), fluoxetine (FLX), on temperature regulation using ovariectomized (OVX) rodent models of thermoregulation. Single, subcutaneous (s.c.) administration of FLX (3, 10, 30 and 60 mg/kg) dose-dependently reduced core body temperature (CBT). FLX at 3 and 10 mg/kg s.c. showed no statistically significant decrease on tail-skin temperature (TST), whereas at higher doses (30 and 60 mg/kg) a significant decrease in TST was noted in the telemetry model. To mimic chronic SSRI treatment, a 5-HT(1A) antagonist (WAY-100635; 0.3 mg/kg) was administered 20 min prior to FLX (10 mg/kg). This combination showed no significant improvement on temperature dysfunction compared to FLX alone. Similarly, in a morphine-dependent model of temperature dysfunction FLX, was inactive at 10 mg/kg whereas the 30 and 60 mg/kg s.c. dose abated the naloxone-induced increase in TST by 55 and 81%, respectively. In summary, FLX affected CBT at all doses, but alleviated thermoregulatory dysfunction only at higher doses that are non-selective for the 5-HT system.

Effects of the 5-HT2A antagonist mirtazapine in rat models of thermoregulation.

Thermoregulation is a complex intercommunicative function requiring coordination between core body temperature (CBT), the central nervous system, and peripheral vasculature. In menopausal women, dysregulation of thermoregulatory mechanisms leads to hot flushes and night sweats. A previous study in ovariectomized (OVX) rats has suggested that mirtazapine can alleviate thermoregulatory dysfunction by blocking 5-HT(2A) receptor signaling. This is in opposition to other work in which 5-HT(2A) receptor blockade appeared to exacerbate thermoregulatory dysfunction in OVX rats. Thus, the goals of the present study were to reexamine the effects of mirtazapine on temperature regulation in OVX rat models and explore further the role of 5-HT(2A) receptor blockade. Mirtazapine exhibited potent functional antagonism (EC(50)=0.62 nM) at the cloned human 5-HT(2A) receptor. In the morphine-dependent model of thermoregulatory dysfunction, mirtazapine (10 mg/kg, i.p.) induced an increase in tail-skin temperature (TST) prior to naloxone administration. In the telemetry model, mirtazapine (0.3-3 mg/kg, i.p.) caused an increase in TST. However, at the highest dose tested (10 mg/kg, i.p.), mirtazapine induced a small but significant decrease in TST followed by an increase in TST. To examine this finding further, mirtazapine's effect on CBT was determined. Administration of mirtazapine (1-3 mg/kg, i.p.) resulted in a slight decrease in CBT but at the 10 mg/kg dose a dramatic decrease (-3.6 degrees C) in CBT was observed. These data support the concept that 5-HT(2A) receptors play a role in temperature regulation but that functional blockade of these receptors by mirtazapine is not a likely mechanism for restoring thermoregulatory processes in OVX rats.

Effect of calcium channel modulators on temperature regulation in ovariectomized rats.

Clinical studies evaluating a calcium channel modulator, gabapentin, for the treatment of vasomotor symptoms have been reported. The present studies evaluated three calcium channel modulators in ovariectomized (OVX) rodent models of temperature regulation. Gabapentin, reported to interact with the alpha(2)delta subunit of voltage-sensitive calcium channels and the L-type voltage-gated calcium channel blockers, verapamil and nifedipine, were examined. These series of experiments demonstrated that orally administered gabapentin, verapamil and nifedipine all acutely and dose-dependently lower tail skin temperature in both models of OVX-induced thermoregulatory dysfunction. These compounds all had a rapid onset of action, however, the efficacy of all three calcium channel modulators is less than that observed following chronic estrogen treatment. Additionally, these compounds were also tested in a telemetric rat model measuring core body temperature to evaluate any temperature effects on internal core temperature. The present data suggests that gabapentin, verapamil and nifedipine all act to globally alter temperature regulation in steroid-dependent models of thermoregulatory function.