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Avian A(H5N1) influenza virus poses an elevated zoonotic threat to humans, and no pharmacological products are currently registered for fast-acting pre-exposure protection in case of spillover leading to a pandemic. Here, we show that an epitope on the stem domain of H5 hemagglutinin is highly conserved and that the human monoclonal antibody CR9114, targeting that epitope, potently neutralizes all pseudotyped H5 viruses tested, even in the rare case of substitutions in its epitope. Further, intranasal administration of CR9114 fully protects mice against A(H5N1) infection at low dosages, irrespective of pre-existing immunity conferred by the quadrivalent seasonal influenza vaccine. These data provide a proof-of-concept for broad, pre-exposure protection against a potential future pandemic using the intranasal administration route. Studies in humans should assess if autonomous administration of a broadly-neutralizing monoclonal antibody is safe and effective and can thus contribute to pandemic preparedness.

Monoclonal antibodies have two core mechanisms of protection: an antibody’s antigen-binding fragment (Fab) can bind and neutralize viral pathogens and its fragment crystallizable domain (Fc) catalyzes effector functions. We investigated the relative contribution of Fab- versus Fc-mediated mechanisms of protection through passive administration of distinct forms of the pan-reactive anti-influenza antibody CR9114. We demonstrated that the contribution of Fc-independent (Fab-dependent) versus Fc-dependent mechanisms of protection is defined by the route of administration. We used CR9114 variants (wild-type, two Fc-silenced variants, or the bivalent antigen-binding fragment F(ab′) 2 ), administered either intravenously or intranasally. We found that intravenously-administered CR9114 requires the Fc domain to provide potent, pre-exposure protection against influenza A and B viral challenge. In contrast, when CR9114 was administered locally to the nasal mucosa, the main mode of protection was provided by F(ab′) 2 , and was largely Fc-independent. Importantly, this mode of protection following intranasal administration also applied to non-neutralized influenza B strains. Moreover, intranasal administration resulted in an increase in potency against influenza A/H1N1, A/H5N1, A/H3N2, B/Yam and B/Vic compared to intravenous administration up to 50-fold. These results shed new light on the application of monoclonal antibodies such as CR9114 to combat viral infection locally, and will help inform clinical strategies of pre-exposure prophylaxis. More fundamentally, this study uncovers distinct modes of protection for systemic versus intranasally-administered prophylactic antibodies.

Site-specific estimates for various environmentalstress factors were related with measured crowncondition data at a systematic 16 ×: 16 km^sup 2^ gridover Europe, according to previously statedhypotheses, using a multiple regression approach,including interactions, and lagged effects of stressfactors. Methodological differences among countriesaccounted for >30% of the variation in defoliation.Nevertheless, crown condition was found to varynaturally with tree age, altitude, drought stress and,most likely, also pathogenic fungi and insects.Significant impacts of air pollution (specificallyozone but also NO^sub x^, SO^sub x^ and acid deposition)were found at regional levels in parts of centralEurope, particularly for deciduous species. Impactsseemed less significant for conifers, especially forspruce, but this might be affected by confoundingeffects or strong correlations between (a harsh)climate and (low) atmospheric deposition in the areawhere spruce predominates. National studies indicatethat ozone and acid deposition can have a significanteffect on the defoliation of spruce as well. Weconclude that while forest condition varies naturally,continued emissions will contribute further to forestdecline in the long term.[PUBLICATION ABSTRACT]

This paper is the first in a series of four,describing the hypothesis and approach of acorrelative study between observed data on crowncondition in Europe, monitored since 1986 at asystematic 16 × 16 km grid, and site-specificestimations of various natural and anthropogenicstress factors. The study was based on the hypothesisthat forests respond to various natural andanthropogenic stress factors, whose contributiondepend on the geographic region considered. In view ofthis hypothesis, major stand and site characteristics,chemical soil composition, meteorological stressfactors (temperature and drought stress indices) andair pollution stress (concentrations and/ordepositions of SO^sub x^, NO^sub y^, NH^sub x^ andO^sub 3^) were included as predictor variables. Theresponse variables considered were actual defoliationand changes/trends in defoliation for five major treespecies. The spatial distribution of the averagedefoliation during the period 1986-1995 shows highdefoliation in Central Europe and in parts ofScandinavia and of Southern Europe. There are,however, sharp changes at country borders, which aredue to methodological differences between countries.The spatial distribution of the calculated trends showa distinct cluster of large deterioration in parts ofCentral and Eastern Europe and in Spain and a ratherscattered pattern of positive and negative trends for most of Europe, indicating that other factors than airpollution only have a strong impact on defoliation.The limitations of the study are discussed in view ofthe quality of the considered response and predictor variables.[PUBLICATION ABSTRACT]

Despite the established benefit of intramuscular (i.m.) influenza vaccination, new adjuvants and delivery methods for comparable or improved immunogenicity are being explored. Intradermal (i.d.) antigen administration is hypothesized to initiate an efficient immune response at reduced antigen doses similar to that observed after i.m. full dose vaccination. In a randomized, partially blinded phase II study 224, healthy adults aged ≥18 to ≤60 years were randomly assigned to four groups and received trivalent influenza vaccine at single doses of 3.0, 4.5 and 6.0 μg hemagglutinin (HA) antigen of each influenza virus strain via i.d. injection or 15.0 μg HA of each influenza strain via i.m. delivery. Serum anti-influenza virus antibodies were determined by hemagglutination inhibition (HI) assay before and 3 weeks after vaccination. Safety assessments were made at baseline and at the follow-up visit by the investigators and for a 4-day period post-vaccination by the subjects themselves. The EMEA requirements for re-licensing of influenza vaccines were fulfilled in all groups 3 weeks after vaccination, irrespective of dose and mode of administration. High seroconversion rates were observed in all study groups and for all strains ranging from 50.9 to 85.5% and 70.4 to 87.0% after i.d. and i.m. vaccination, respectively. Seroprotection rates for the A strains A/Solomon Islands and A/Wisconsin were generally higher compared to the B/Malaysia strain and ranged from 89.1 to 98.2% across the i.d. groups. Similar rates of 96.3% for the A/Solomon Islands and 94.4% for the A/Wisconsin strain were observed in the i.m. group. Seroprotection rates for the B/Malaysia strain were 65.5, 83.0 and 72.7% after i.d. administration of 3.0, 4.5, and 6.0 μg HA of each strain, respectively, compared to a seroprotection rate of 85.2% in the i.m. group. In addition, marked increases in geometric mean titer (GMT) were observed across the groups for all influenza virus strains ranging from 6.9 to 70.5 for i.d. and from 16.9 to 56.5 for i.m. antigen delivery. Both routes of administration were well tolerated. Systemic reactions were broadly similar across the groups. With respect to local reactions the frequency of injection site pain and ecchymosis were significantly lower following i.d. vaccination, while other local reactions such as erythema occurred at higher rates with i.d. than with i.m. vaccine administration, as expected due to the mechanism of action for the i.d. route. The virosomal adjuvanted influenza vaccine (Inflexal ®V) was shown to be overall highly immunogenic and well tolerated when given i.d. at reduced doses to healthy adults, eliciting an immune response similar to that observed with full dose i.m. administration and thus suggesting a promising antigen-sparing strategy for universal influenza vaccination against endemic influenza. ISRCTN registry number: 33950739.

Avian influenza H9N2 viruses are considered as a pandemic threat. We assessed the safety and immunogenicity of fourteen H9N2 vaccine formulations. A randomized, phase I trial was done in 353 adults, aged 18–82 years. Subjects received two doses of A/Hong Kong/1073/99 (H9N2) whole-virus, alum-adjuvanted whole-virus, virosomal, or intradermal whole-virus vaccine at four doses (1.7, 5, 15 or 45 μg haemagglutinin). Sera were obtained before and three weeks after each vaccination (days 0, 21, and 42) for haemagglutination–inhibition (HAI) and neutralization assays. All formulations were well tolerated. Pre-vaccination sera from subjects aged below or above 40 years had baseline antibody to H9N2 in 1% and 16% of samples. Compared to intramuscular whole-virus vaccine, alum-adjuvanted vaccine was more immunogenic, intradermal vaccine was comparable, and virosomal vaccine less immunogenic. Among subjects under 40 years, two doses (45, 15, and 5 μg) of alum-adjuvanted vaccine achieved seroprotective HAI titres in 50%, 41%, and 39% respectively, and neutralization seroconversions in 83%, 82%, and 78% of recipients. Among subjects over 40 years, one dose (45, 15, and 5 μg) of alum-adjuvanted vaccine achieved seroprotective HAI titres in 50%, 25% and 0% respectively, and neutralization seroconversions in 88%, 63% and 63% of recipients. Among immunologically naive subjects under 40 years, two doses of vaccine are required and alum-adjuvanted vaccines were most immunogenic. Among immunologically primed subjects over 40 years, one dose of whole-virus or alum-adjuvanted vaccine induced immune responses; the second dose provided less additional benefit. However, no vaccine formulation satisfied all European regulatory criteria for pandemic vaccines.

Many of us rely on seasonal vaccines for protection against influenza and are only too aware of their limited breadth. Broadly neutralizing antibodies (bnAbs) that target the conserved hemagglutinin (HA) stem of the influenza virus provide hope for the development of universal vaccines and are being evaluated in clinical trials. Van Dongen et al. selected and optimized a small-molecule lead compound that recapitulates key interactions of the bnAb with HA. Like the bnAb, the compound inhibited viral fusion in the endosomes of target cells. The compound protected mice from influenza after oral administration and neutralized virus infection in a 3D cell culture of human bronchial epithelial cells. Science , this issue p. eaar6221 A drug-like molecule that mimics the binding and functionality of a broadly neutralizing antibody inhibits influenza virus infection. Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions.

Previously, we have shown the potency of recombinant Adenovirus serotype 35 viral vaccines (rAd35) to induce strong immune response against the circumsporozoite protein (CS) of the plasmodium parasite. To further optimize immunogenicity of Ad35-based malaria vaccines we formulated rAd35.CS vaccine with aluminium phosphate adjuvant (AlPO 4). In contrast to the conventional protein based vaccines no absorption to aluminium adjuvant was observed and rAd35 viral in vitro infectivity in mammalian cells was preserved. Immunization with Ad35.CS formulated with AlPO 4 resulted in significantly higher CS specific T and B cell responses in mice upon either single or prime-boost vaccination regimens as compared to rAd35.CS alone. With these results we report for the first time the feasibility of using an AlPO 4 adjuvant to increase the potency of a live adenovirus serotype 35-based vaccine.

Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted 3D-cell culture of fully differentiated human bronchial epithelial cells. Co-crystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions. A small molecule that mimics the binding and functionality of a broadly neutralizing antibody as an effective fusion inhibitor of influenza virus.

The stress by air pollution at the systematic Pan-European 16 x 16 km super(2) forest (crown) condition monitoring network, is discussed by comparing site-specific estimates of critical and present concentration and deposition levels for S and N compounds and ozone. Results indicate that the exceedance of critical levels, related to direct above-ground impacts, decrease going from O sub(3) > SO sub(2) > N compounds. Critical N loads related to effects on the forest understorey are exceeded at approximately 25% of the plots, located mainly in Western and Central Europe. Critical N loads related to effects on trees are hardly ever exceeded, but most likely, this is an under estimate. Critical acid deposition levels are exceeded at approximately 30% of the plots with a low base saturation, where acid inputs may release toxic Al. This is especially the case in Central and Eastern Europe, where present loads are high and in boreal forest in Southern Scandinavia where critical loads are low. Although the uncertainties in the calculated exceedances is large, the spatial pattern, which is most important for a correlative study, seems reliable, implying that the critical load concept is suitable for regional risk assessments.