H5 Hemagglutinin Research Articles

Evaluation of Tissue Tropism and Horizontal Transmission of a Duck Enteritis Virus Vectored Vaccine in One-Day-Old Chicken

Herpesvirus of turkey (HVT) recombinant vector vaccines are widely used in the poultry industry. However, due to limitations in loading multiple foreign antigens into a single HVT vector, other viral vectors are urgently needed. Since chickens lack maternal immunity to duck enteritis virus (DEV), vector vaccines using DEV as a backbone are currently under study. Even though a recently developed DEV vector vaccine expressing the influenza hemagglutinin H5 of highly pathogenic avian influenza (DEV-H5) induces highly detectable anti-HA antibodies, safety issues hamper further vaccine development. In this work, tissue affinity and horizontal transmission in 1-day-old chickens were systematically evaluated after DEV-H5 vector vaccine inoculation. Sixty percent of DEV-H5-inoculated chickens died between day 2 and day 7 post-inoculation. The displayed clinical signs consisted of lethargy, anorexia, and diarrhea, and virus was shed in feces. Gross and/or histological lesions were recorded in the kidney, heart, intestine, liver, lung, and spleen. Moreover, DEV-H5 replication in intestinal cells caused an increment in interferon-α expression, while occluding junction proteins and ZO-1 expression were significantly upregulated. As a control, birds inoculated with a commercial recombinant turkey herpesvirus expressing the VP2 protein of the infectious bursal disease virus (HVT-VP2) vector vaccine showed neither clinical signs nor mortality. Overall, while the HVT-VP2 vaccine demonstrated complete safety in 1-day-old chickens, our potential DEV-H5 vaccine requires further attenuation for consideration as a vector vaccine candidate in chickens.

Conserved sites on the influenza H1 and H3 hemagglutinin recognized by human antibodies.

Monoclonal antibodies (mAbs) targeting the influenza hemagglutinin (HA) have the potential to be used as prophylactics or templates for next-generation vaccines that provide broad protection. Here, we isolated broad, subtype-neutralizing mAbs from human B cells targeting the H1 or H3 HA head as well as a unique mAb targeting the stem. The H1 mAbs target the previously defined lateral patch epitope on H1 HAs and recognize HAs from 1933 to 2021 in addition to a swine H1N1 virus with pandemic potential. Using directed evolution, we improved the neutralization potency of these H1 mAbs towards a contemporary H1 strain. Using deep mutational scanning of four antigenically distinct H1N1 viruses, we identified potential viral escape pathways. For the H3 mAbs we used cryo-EM to define the targeted epitopes: one mAb recognizes the side of the H3 head, accommodating the N133 glycan and a pocket underneath the receptor binding site. The other H3 mAb recognizes an epitope in the HA stem that overlaps with previously characterized mAbs, but with distinct antibody variable genes and mode of recognition. Collectively, these mAbs identify common sites recognized by broad, subtype-specific mAbs that may be elicited by next-generation vaccines.

Preclinical immunogenicity and safety of hemagglutinin-encoding modRNA influenza vaccines

Seasonal epidemics of influenza viruses are responsible for a significant global public health burden. Vaccination remains the most effective way to prevent infection; however, due to the persistence of antigenic drift, vaccines must be updated annually. The selection of vaccine strains occurs months in advance of the influenza season to allow adequate time for production in eggs. RNA vaccines offer the potential to accelerate production and improve efficacy of influenza vaccines. We leveraged the nucleoside-modified RNA (modRNA) platform technology and lipid nanoparticle formulation process of the COVID-19 mRNA vaccine (BNT162b2; Comirnaty®) to create modRNA vaccines encoding hemagglutinin (HA) (modRNA-HA) for seasonal human influenza strains and evaluated their preclinical immunogenicity and toxicity. In mice, a monovalent modRNA vaccine encoding an H1 HA demonstrated robust antibody responses, HA-specific Th1-type CD4+ T cell responses, and HA-specific CD8+ T cell responses. In rhesus and cynomolgus macaques, the vaccine exhibited durable functional antibody responses and HA-specific IFN-γ+ CD4+ T cell responses. Immunization of mice with monovalent, trivalent, and quadrivalent modRNA-HA vaccines generated functional antibody responses targeting the seasonal influenza virus(es) encoded in the vaccines that were greater than, or similar to, those of a licensed quadrivalent influenza vaccine. Monovalent and quadrivalent modRNA-HA vaccines were well-tolerated by Wistar Han rats, with no evidence of systemic toxicity. These nonclinical immunogenicity and safety data support further evaluation of the modRNA-HA vaccines in clinical studies.

Molecular Characterization of a Clade 2.3.4.4b H5N1 High Pathogenicity Avian Influenza Virus from a 2022 Outbreak in Layer Chickens in the Philippines.

H5 subtype high-pathogenicity avian influenza (HPAI) viruses continue to devastate the poultry industry and threaten food security and public health. The first outbreak of H5 HPAI in the Philippines was reported in 2017. Since then, H5 HPAI outbreaks have been reported in 2020, 2022, and 2023. Here, we report the first publicly available complete whole-genome sequence of an H5N1 high-pathogenicity avian influenza virus from a case in Central Luzon. Samples were collected from a flock of layer chickens exhibiting signs of lethargy, droopy wings, and ecchymotic hemorrhages in trachea with excessive mucus exudates. A high mortality rate of 96-100% was observed within the week. Days prior to the high mortality event, migratory birds were observed around the chicken farm. Lungs, spleen, cloacal swabs, and oropharyngeal-tracheal swabs were taken from two chickens from this flock. These samples were positive in quantitative RT-PCR assays for influenza matrix and H5 hemagglutinin (HA) genes. To further characterize the virus, the same samples were subjected to whole-virus-genome amplification and sequencing using the Oxford Nanopore method with mean coverages of 19,190 and 2984, respectively. A phylogenetic analysis of the HA genes revealed that the H5N1 HPAI virus from Central Luzon belongs to the Goose/Guangdong lineage clade 2.3.4.4b viruses. Other segments also have high sequence identity and the same genetic lineages as other clade 2.3.4.4b viruses from Asia. Collectively, these data indicate that wild migratory birds are the likely source of H5N1 viruses from the 2022 outbreaks in the Philippines. Thus, biosecurity practices and surveillance for HPAI viruses in both domestic and wild birds should be increased to prevent and mitigate HPAI outbreaks.

Codon optimized influenza H1 HA sequence but not CTLA-4 targeting of HA antigen to enhance the efficacy of DNA vaccines in an animal model

Infections caused by the influenza virus lead to both epidemic and pandemic outbreaks in humans and animals. Owing to their rapid production, safety, and stability, DNA vaccines represent a promising avenue for eliciting immunity and thwarting viral infections. While DNA vaccines have demonstrated substantial efficacy in murine models, their effectiveness in larger animals remains subdued. This limitation may be addressed by augmenting the immunogenicity of DNA-based vaccines. In the investigation here, protein expression was enhanced via codon optimization and then mouse cytotoxic T-lymphocyte antigen 4 (CTLA-4) was harnessed as a modulatory adjunct to bind directly to antigen-presenting cells. Further, the study evaluated the immunogenicity of two variants of the hemagglutinin (HA) antigen, i.e. the full-length and the C-terminal deletion versions. The study findings revealed that the codon-optimized HA gene (pcHA) led to increased protein synthesis, as evidenced by elevated mRNA levels. Codon optimization also significantly bolstered both cellular and humoral immune responses. In cytokine assays, all plasmid constructs, particularly pCTLA4-cHA, induced robust interferon (IFN)-γ production, while interleukin (IL)-4 levels remained uniformly non-significant. Mice immunized with pcHA displayed an augmented presence of IFNγ+ T-cells, underscoring the enhanced potency of the codon-optimized HA vaccine. Contrarily, CTLA-4-fused DNA vaccines did not significantly amplify the immune response.

Phase 1 dose-escalation trial evaluating a group 2 influenza hemagglutinin stabilized stem nanoparticle vaccine

The relative conservation of the influenza hemagglutinin (HA) stem compared to that of the immunodominant HA head makes the HA stem an attractive target for broadly protective influenza vaccines. Here we report the first-in-human, dose-escalation, open-label trial (NCT04579250) evaluating an unadjuvanted group 2 stabilized stem ferritin nanoparticle vaccine based on the H10 A/Jiangxi-Donghu/346/2013 influenza HA, H10ssF, in healthy adults. Participants received a single 20 mcg dose (n = 3) or two 60 mcg doses 16 weeks apart (n = 22). Vaccination with H10ssF was safe and well tolerated with only mild systemic and local reactogenicity reported. No serious adverse events occurred. Vaccination significantly increased homologous H10 HA stem binding and neutralizing antibodies at 2 weeks after both first and second vaccinations, and these responses remained above baseline at 40 weeks. Heterologous H3 and H7 binding antibodies also significantly increased after each vaccination and remained elevated throughout the study. These data indicate that the group 2 HA stem nanoparticle vaccine is safe and induces stem-directed binding and neutralizing antibodies.

Theoretical Investigations of a point mutation affecting H5 Hemagglutinin’s receptor binding preference

The avian influenza A H5N1 virus is a subtype of influenza A virus (IAV) that causes a highly infectious and severe respiratory illness in birds and poses significant economic losses in poultry farming. To infect host cell, the virus uses its surface glycoprotein named Hemagglutinin (HA) to recognize and to interact with the host cell receptor containing either α2,6- (SAα2,6 Gal) or α2,3-linked Sialic Acid (SAα2,3 Gal). The H5N1 virus has not yet acquired the capability for efficient human-to-human transmission. However, studies have demonstrated that even a single amino acid substitution in the HA can switch its glycan receptor preference from the avian-type SAα2,3 Gal to the human-type SAα2,6 Gal. The present study aims to explain the underlying mechanism of a mutation (D94N) on the H5 HA that causes the protein to change its glycan receptor-binding preference using molecular dynamics (MD) simulations. Our results reveal that the mutation alters the electrostatic interactions pattern near the HA receptor binding pocket, leading to a reduced stability for the HA-avian-type SAα2,3 Gal complex. On the other hand, the detrimental effect of the mutation D94N is not observed in the HA-human-type SAα2,6 Gal complex due to the glycan's capability to switch its topology.

A Rationally Designed H5 Hemagglutinin Subunit Vaccine Provides Broad-Spectrum Protection against Various H5Nx Highly Pathogenic Avian Influenza Viruses in Chickens.

The evolution of the H5 highly pathogenic avian influenza (HPAI) viruses has led to the emergence of distinct groups with genetically similar clusters of hemagglutinin (HA) sequences. In this study, a consensus H5 HA sequence was cloned into the baculovirus expression system. The HA protein was expressed in baculovirus-infected insect cells and utilized as the antigen for the production of an oil emulsion-based H5 avian influenza vaccine (rBacH5Con5Mut). Twenty-one-day-old SPF chickens were immunized with this vaccine and then challenged at 21 days post-vaccination with clade 2.3.2.1, clade 2.3.4.4, and clade 7.2 of H5 HPAI viruses. The sera of vaccinated chickens exhibited high hemagglutination inhibition (HI) titers against the rBacH5 vaccine antigen, while lower HI titers were observed against the different challenge virus H5 hemagglutinins. Furthermore, the rBacH5Con5Mut vaccine provided 100% protection from mortality and clinical signs. Virus isolation results showed that oropharyngeal and cloacal shedding was prevented in 100% of the vaccinated chickens when challenged with clade 2.3.2.1 and clade 2.3.4.4 H5 viruses. When the rBacH5Con5Mut vaccine candidate was administrated at one day of age, 100% protection was demonstrated against the challenge of clade 2.3.4.4 virus at three weeks of age, indicating the potential of this vaccine for hatchery vaccination. Overall, A single immunization of rBacH5Con5Mut vaccine candidate with a consensus HA antigen can protect chickens against different clades of H5 HPAI viruses throughout the rearing period of broiler chickens without a boost, thus fulfilling the criteria for an efficacious broad-spectrum H5 avian influenza vaccine.

Immunogenicity and biodistribution of lipid nanoparticle formulated self-amplifying mRNA vaccines against H5 avian influenza

This study reports on the immunogenicity and biodistribution of H5 hemagglutinin (HA)-based self-amplifying (sa) mRNA vaccines in mice. Four sa-mRNA vaccines encoding either a secreted full-length HA, a secreted HA head domain, a secreted HA stalk domain, or a full-length membrane-anchored HA were investigated. All vaccines elicited an adaptive immune response. However, the full-length HA sa-RNA vaccines demonstrated superior performance compared to head and stalk domain vaccines. The antibody titers positively correlated with the vaccine dose. Cellular immune responses and antigen-specific IgA antibodies in the lungs were also observed. The comparison of the sa-mRNA vaccines encoding the secreted and membrane-anchored full-length HA revealed that anchoring of the HA to the membrane significantly enhanced the antibody and cellular responses. In addition to the injection site, the intramuscularly injected sa-mRNA-LNPs were also detected in the draining lymph nodes, spleen, and to a lesser extent, in the lung, kidney, liver, and heart.

Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance.

H5 influenza is a potential pandemic threat. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic risk, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. Here we use pseudovirus deep mutational scanning to measure how all mutations to a clade 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind a2-6-linked sialic acids, and show that some viruses already carry mutations that stabilize HA. We also identify recent viral strains with reduced neutralization to sera elicited by candidate vaccine virus. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive characterization of mutations to inform surveillance of H5 influenza.

Synthesis and Biological Activity of New Hydrazones Based on N-Aminomorpholine.

The data on the synthesis of N-aminomorpholine hydrazones are presented. It is shown that the interaction of N-aminomorpholine with functionally substituted benzaldehydes and 4-pyridinaldehyde in isopropyl alcohol leads to the formation of corresponding hydrazones. The structure of the synthesized compounds was studied by 1H and 13C NMR spectroscopy methods, including the COSY (1H-1H), HMQC (1H-13C) and HMBC (1H-13C) methodologies. The values of chemical shifts, multiplicity, and integral intensity of 1H and 13C signals in one-dimensional NMR spectra were determined. The COSY (1H-1H), HMQC (1H-13C), and HMBC (1H-13C) results revealed homo- and heteronuclear interactions, confirming the structure of the studied compounds. The antiviral, cytotoxic, and antimicrobial activity of some synthesized hydrazones were investigated. It is shown that 2-((morpholinoimino)methyl)benzoic acid has a pronounced viral inhibitory property, comparable in its activity to commercial drugs Tamiflu and Remantadine. A docking study was performed using the influenza virus protein models (1930 Swine H1 Hemagglutinin and Neuraminidase of 1918 H1N1 strain). The potential binding sites that are complementary with 2-((morpholinoimino)methyl)benzoic acid were found.

Massively parallel free energy calculations for in silico affinity maturation of designed miniproteins.

Computational protein design efforts continue to make remarkable advances, yet the discovery of high-affinity binders typically requires large-scale experimental screening of site-saturated mutant (SSM) libraries. Here, we explore how massively parallel free energy methods can be used for in silico affinity maturation of de novo designed binding proteins. Using an expanded ensemble (EE) approach, we perform exhaustive relative binding free energy calculations for SSM variants of three miniproteins designed to bind influenza A H1 hemagglutinin by Chevalier et al. (2017). We compare our predictions to experimental ΔΔ G values inferred from a Bayesian analysis of the high-throughput sequencing data, and to state-of-the-art predictions made using the Flex ddG Rosetta protocol. A systematic comparison reveals prediction accuracies around 2 kcal/mol, and identifies net charge changes, large numbers of alchemical atoms, and slow side chain conformational dynamics as key contributors to the uncertainty of the EE predictions. Flex ddG predictions are more accurate on average, but highly conservative. In contrast, EE predictions can better classify stabilizing and destabilizing mutations. We also explored the ability of SSM scans to rationalize known affinity-matured variants containing multiple mutations, which are non-additive due to epistatic effects. Simple electrostatic models fail to explain non-additivity, but observed mutations are found at positions with higher Shannon entropies. Overall, this work suggests that simulation-based free energy methods can provide predictive information for in silico affinity maturation of designed miniproteins, with many feasible improvements to the efficiency and accuracy within reach.

Structural Mapping of Polyclonal IgG Responses to HA After Influenza Virus Vaccination or Infection.

Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing context for future vaccine trials and emphasizing the importance of carefully designing vaccines to boost protective responses towards conserved epitopes.

Anti-hemagglutinin monomeric nanobody provides prophylactic immunity against H1 subtype influenza A viruses.

Influenza viruses constitute a major threat to human health globally. The viral surface glycoprotein hemagglutinin (HA) is the immunodominant antigen, contains the site for binding to the cellular receptor (RBS), and it is the major target of neutralizing antibody responses post-infection. We developed llama-derived single chain antibody fragments (VHHs) specific for type A influenza virus. Four VHHs were identified and further characterized. VHH D81 bound residues in the proximity of the C-terminal region of HA1 of H1 and H5 subtypes, and showed weak neutralizing activity, whereas VHH B33 bound residues in the proximity of the N-terminal region of the HA's stem domain (HA2) of H1, H5, and H9 subtypes, and showed no neutralizing activity. Of most relevance, VHHs E13 and G41 recognized highly conserved conformational epitopes on the H1 HA's globular domain (HA1) and showed high virus neutralizing activity (ranging between 0.94 to 0.01μM), when tested against several human H1N1 isolates. Additionally, E13 displayed abrogated virus replication of a panel of H1N1 strains spanning over 80 years of antigenic drift and isolated from human, avian, and swine origin. Interestingly, E13 conferred protection in vivo at a dose as low as 0.05 mg/kg. Mice treated with E13 intranasally resulted in undetectable virus challenge loads in the lungs at day 4 post-challenge. The transfer of sterilizing pan-H1 immunity, by a dose in the range of micrograms given intranasally, is of major significance for a monomeric VHH and supports the further development of E13 as an immunotherapeutic agent for the mitigation of influenza infections.

A Broad Influenza Vaccine Based on a Heat-Activated, Tissue-Restricted Replication-Competent Herpesvirus.

Vaccination with transiently activated replication-competent controlled herpesviruses (RCCVs) expressing influenza A virus hemagglutinins broadly protects mice against lethal influenza virus challenges. The non-replicating RCCVs can be activated to transiently replicate with high efficiency. Activation involves a brief heat treatment to the epidermal administration site in the presence of a drug. The drug co-control is intended as a block to inadvertent reactivation in the nervous system and, secondarily, viremia under adverse conditions. While the broad protective effects observed raise an expectation that RCCVs may be developed as universal flu vaccines, the need for administering a co-activating drug may dampen enthusiasm for such a development. To replace the drug co-control, we isolated keratin gene promoters that were active in skin cells but inactive in nerve cells and other cells in vitro. In a mouse model of lethal central nervous system (CNS) infection, the administration of a recombinant that had the promoter of the infected cell protein 8 (ICP8) gene of a wild-type herpes simplex virus 1 (HSV-1) strain replaced by a keratin promoter did not result in any clinical signs, even at doses of 500 times wild-type virus LD50. Replication of the recombinant was undetectable in brain homogenates. Second-generation RCCVs expressing a subtype H1 hemagglutinin (HA) were generated in which the infected cell protein 4 (ICP4) genes were controlled by a heat switch and the ICP8 gene by the keratin promoter. In mice, these RCCVs replicated efficiently and in a heat-controlled fashion in the epidermal administration site. Immunization with the activated RCCVs induced robust neutralizing antibody responses against influenza viruses and protected against heterologous and cross-group influenza virus challenges.

Synergetic light and cytokinin treatments mitigate the recombinant protein yield depression induced by high-density cultivation of hydroponically-grown Nicotiana benthamiana.

Plant molecular farming is currently operating a transition from soil-based cultures towardhydroponic systems. In this study, we designed a whole-plant NFT (nutrient film technique) platform for the transient expression of influenza virus-like particles harboring hemagglutinin H1 proteins in Nicotiana benthamiana. In particular, we examined the effects of plant density during the post-infiltration expression phase on plant growth and H1 yield in relation to the daily light integral (DLI) received by the crop and the exogenous application of 6-BAP cytokinin (CK). We expected from previous work that high DLI and CK treatments would stimulate the development of highly productive leaves on axillary (secondary) stems and thereby improve the H1 yield at the whole-plant scale. Increasing plant density from 35.7 to 61 plants m-2 during the post-infiltration phase significantly decreased the proportion of axillary leaf biomass by 30% and H1 yield per plant by 39%, resulting in no additional yield gain on a whole-crop area basis. Adding CK to the recirculated nutrient solution decreased the harvested leaf biomass by 31% and did not enhance the relative proportion of S leaves of the plants as previously reported with foliar CK application. There was a 36% increase in H1 yield when doubling the DLI from 14 to 28 mol m-2 s-1, and up to 71% yield gain when combining such an increase in DLI with the hydroponic CK treatment. Contrary to our expectations, leaves located on the main stem, particularly those from the upper half of the plant (i.e.,eighth leaf and above), contributed about 80% of total H1 yield. Our study highlights the significantly different phenotype (~30% less secondary leaf biomass) and divergent responses to light and CK treatments of NFT-grown N. benthamiana plants compared to previous studies conducted on potted plants.

Inactivated influenza virus vaccines expressing COBRA hemagglutinin elicited broadly reactive, long-lived protective antibodies

ABSTRACT The influenza viruses cause seasonal respiratory illness that affect millions of people globally every year. Prophylactic vaccines are the recommended method to prevent the breakout of influenza epidemics. One of the current commercial influenza vaccines consists of inactivated viruses that are selected months prior to the start of a new influenza season. In many seasons, the vaccine effectiveness (VE) of these vaccines can be relatively low. Therefore, there is an urgent need to develop an improved, more universal influenza vaccine (UIV) that can provide broad protection against various drifted strains in all age groups. To meet this need, the computationally optimized broadly reactive antigen (COBRA) methodology was developed to design a hemagglutinin (HA) molecule as a new influenza vaccine. In this study, COBRA HA-based inactivated influenza viruses (IIV) expressing the COBRA HA from H1 or H3 influenza viruses were developed and characterized for the elicitation of immediate and long-term protective immunity in both immunologically naïve or influenza pre-immune animal models. These results were compared to animals vaccinated with IIV vaccines expressing wild-type H1 or H3 HA proteins (WT-IIV). The COBRA-IIV elicited long-lasting broadly reactive antibodies that had hemagglutination-inhibition (HAI) activity against drifted influenza variants.

Detection of Hemagglutinin H5 Influenza A Virus Sequence in Municipal Wastewater Solids at Wastewater Treatment Plants with Increases in Influenza A in Spring, 2024

Detection of Hemagglutinin H5 Influenza A Virus Sequence in Municipal Wastewater Solids at Wastewater Treatment Plants with Increases in Influenza A in Spring, 2024

Very Broadly Effective Hemagglutinin-Directed Influenza Vaccines with Anti-Herpetic Activity.

A universal vaccine that generally prevents influenza virus infection and/or illness remains elusive. We have been exploring a novel approach to vaccination involving replication-competent controlled herpesviruses (RCCVs) that can be deliberately activated to replicate efficiently but only transiently in an administration site in the skin of a subject. The RCCVs are derived from a virulent wild-type herpesvirus strain that has been engineered to contain a heat shock promoter-based gene switch that controls the expression of, typically, two replication-essential viral genes. Additional safety against inadvertent replication is provided by an appropriate secondary mechanism. Our first-generation RCCVs can be activated at the administration site by a mild local heat treatment in the presence of an antiprogestin. Here, we report that epidermal vaccination with such RCCVs expressing a hemagglutinin or neuraminidase of an H1N1 influenza virus strain protected mice against lethal challenges by H1N1 virus strains representing 75 years of evolution. Moreover, immunization with an RCCV expressing a subtype H1 hemagglutinin afforded full protection against a lethal challenge by an H3N2 influenza strain, and an RCCV expressing a subtype H3 hemagglutinin protected against a lethal challenge by an H1N1 strain. Vaccinated animals continued to gain weight normally after the challenge. Protective effects were even observed in a lethal influenza B virus challenge. The RCCV-based vaccines induced robust titers of in-group, cross-group and even cross-type neutralizing antibodies. Passive immunization suggested that observed vaccine effects were at least partially antibody-mediated. In summary, RCCVs expressing a hemagglutinin induce robust and very broad cross-protective immunity against influenza.

Broadly Reactive Nanobody Targeting the H3 Hemagglutinin of the Influenza A Virus.

Monoclonal antibodies and recombinant antibody fragments are a very promising therapeutic tool to combat infectious diseases. Due to their unique paratope structure, nanobodies (VHHs) hold several advantages over conventional monoclonal antibodies, especially in relation to viral infections. Influenza A viruses (IAVs) remain a major threat to public health. The hemagglutinin (HA) protein is the main protective and immunodominant antigen of IAVs. In this study, three broadly reactive nanobodies (D9.2, E12.2, and D4.2) to H3N2 influenza strains were isolated and Fc-fusion proteins (VHH-Fcs) were obtained and characterized in vitro. This modification improved the nanobodies' binding activity and allowed for their interaction with a wider range of strains. The D9.2-Fc antibody showed a 100% protection rate against mortality in vivo in a mouse lethal model. Furthermore, we demonstrated that the observed protection has to do with Fc-FcγR interactions. These results indicate that D9.2-Fc can serve as an effective antiviral agent against the H3N2 influenza infection.