Influenza Hemagglutinin Research Articles

Intensifying Continuous Production of Gag-HA VLPs at High Cell Density Using Stable Insect Cells Adapted to Low Culture Temperature.

Protein production processes based on stable insect cell lines require intensification to be competitive with the insect cell-baculovirus expression vector system (IC-BEVS). High cell density (HCD) cultures operate continuously, capable of maintaining specific production rates for extended periods of time which may lead to significant improvements in production yields. However, setting up such processes is challenging (e.g., selection of cell retention device and optimization of dilution rate), often demanding the manipulation of large volumes of culture medium with associated high cost. In this study, we developed a process for continuous production of Gag virus–like particles (VLP) pseudotyped with a model membrane protein (influenza hemagglutinin, HA) at HCD using stable insect cells adapted to low culture temperature. The impact of the cell retention device (ATF vs. TFF) and cell-specific perfusion rate (CSPR) on cell growth and protein expression kinetics was evaluated. Continuous production of Gag-HA VLPs was possible using both retention devices and CSPR of 0.04 nL/cell.d; TFF induces higher cell lysis when compared to ATF at later stages of the process (kD = 0.009 vs. 0.005 h−1, for TFF and ATF, respectively). Reducing CSPR to 0.01–0.02 nL/cell.d using ATF had a negligible impact on specific production rates (rHA = 72–68 titer/109 cell.h and rp24 = 12–11 pg/106 cell.h in all CSPR) and on particle morphology (round-shaped structures displaying HA spikes on their surface) and size distribution profile (peaks at approximately 100 nm). Notably, at these CSPRs, the amount of p24 or HA formed per volume of culture medium consumed per unit of process time increases by up to 3-fold when compared to batch and perfusion operation modes. Overall, this work demonstrates the potential of manipulating CSPRs to intensify the continuous production of Gag-HA VLPs at HCD using stable insect cells to make them an attractive alternative platform to IC-BEVS.

P1614: MOLECULAR EPIDEMIOLOGY OF INFLUENZA VIRUS IN PATIENTS WITH HEMATOLOGIC DISORDERS

Background: Whereas influenza virus infections usually present as self-limiting upper respiratory tract infections, they are also one of the most common causes of viral lower respiratory tract infections. Especially immunocompromised patients, such as ones suffering from malignant diseases, are at a higher risk of severe influenza virus infection, thus leading to increased morbidity and mortality. With influenza viruses possessing the ability of antigenic shift and antigenic drift, consequently altering the viral genome frequently and avoiding long-term immunity of the host, the necessity for regular data collection is clear. Aims: With this study we want to retrospectively assess influenza virus epidemiology in patients with hematological malignancies and investigate clinical and virological risk factors of severe infection to be able to apply information about improvement of hygiene and care. Methods: We included clinical data from 159 influenza positive patients from the Department of Haematology of the University Hospital Heidelberg from the seasons of 2014 to 2019 to analyse occurrence of nosocomial transmission, prolonged viral shedding and associated risk factors for severe infection. Virological data was obtained by nasopharyngeal swabs, which were collected routinely, then Sanger sequencing of the hemagglutinin gene was performed for phylogenetic and mutational analysis. Combination of clinical and virological data was assessed to evaluate specific susceptibility to influenza subtype, subgroup or amino acid mutation. Results: 42 patients (26%) developed LRTI. Associated risk factors with development of lower respiratory tract infection (LRTI) (p<0.05) were nosocomial infection (p=0.008), IgG levels lower than 6g/dl (p=0.046), bacterial (p=0.001), fungal (p<0.001) and bacterial and fungal (p=0.017) coinfection. 9 of 31 (29%) of bacterial copathogens belonged to multi-resistant strains. LRTI associated mortality rate in our study population was noticeably high with 38%. 67% of patients with consecutive testing (n=54) presented with prolonged viral shedding (median 9.5 days). Epidemiological comparison with national surveillance data mostly showed no divergence of our patient group regarding influenza type and subtype. In 115 patients, sequencing of influenza hemagglutinin gene was successful, and 13 virus isolates revealed significant substitutions: In A/H1N1 samples P137S, D187N and D222V; in A/H3N2 samples D7N, T10M, D101E, I140M, Q197K, Q197R and I214T; in B/Yamagata samples Y165H, P171Q and N202R and none in B/Victoria samples. Most of these substitutions could be assigned to an antigenic site or receptor binding site. Summary/Conclusion: While no specific susceptibility to influenza subtypes or subgroups could be observed, high LRTI associated mortality rate and occurrence of nosocomial transmission as well as multi-resistant copathogens illustrate the need for better hospital hygiene and early evaluation of disease severity. To understand detected mutations and their impact on the course of infection as well as virus evolution and vaccination efficiency, further collection of data is needed.

In Silico Analyses of the Role of Codon Usage at the Hemagglutinin Cleavage Site in Highly Pathogenic Avian Influenza Genesis.

A vast diversity of 16 influenza hemagglutinin (HA) subtypes are found in birds. Interestingly, viruses from only two subtypes, H5 and H7, have so far evolved into highly pathogenic avian influenza viruses (HPAIVs) following insertions or substitutions at the HA cleavage site by the viral polymerase. The mechanisms underlying this striking subtype specificity are still unknown. Here, we compiled a comprehensive dataset of 20,488 avian influenza virus HA sequences to investigate differences in nucleotide and amino acid usage at the HA cleavage site between subtypes and how these might impact the genesis of HPAIVs by polymerase stuttering and realignment. We found that sequences of the H5 and H7 subtypes stand out by their high purine content at the HA cleavage site. In addition, fewer substitutions were necessary in H5 and H7 HAs than in HAs from other subtypes to acquire an insertion-prone HA cleavage site sequence, as defined based on in vitro and in vivo data from the literature. Codon usage was more favorable for HPAIV genesis in sequences of viruses isolated from species or geographical regions in which HPAIV genesis is more frequently observed in nature. The results of the present analyses suggest that the subtype restriction of HPAIV genesis to H5 and H7 influenza viruses might be due to the particular codon usage at the HA cleavage site in these subtypes.

Metal-Organic Coordination Polymer for Delivery of a Subunit Broadly Acting Influenza Vaccine.

A zinc-carnosine (ZnCar) metal-organic coordination polymer was fabricated in biologically relevant N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) buffer for use as a vaccine platform. In vitro, ZnCar exhibited significantly less cytotoxicity than a well-established zeolitic imidazolate framework (ZIF-8). Adsorption of CpG on the ZnCar surface resulted in enhanced innate immune activation compared to soluble CpG. The model antigen ovalbumin (OVA) was encapsulated in ZnCar and exhibited acid-sensitive release in vitro. When injected intramuscularly on days 0 and 21 in C57BL/6 mice, OVA-specific serum total IgG and IgG1 were significantly greater in all groups with ZnCar and antigen compared to soluble controls. Th1-skewed IgG2c antibodies were significantly greater in OVA and CpG groups delivered with ZnCar for all time points, regardless of whether the antigen and adjuvant were co-formulated in one material or co-delivered in separate materials. When broadly acting Computationally Optimized Broadly Reactive Antigen (COBRA) P1 influenza hemagglutinin (HA) was ligated to ZnCar via its His-tag, significantly greater antibody levels were observed at all time points compared to soluble antigen and CpG. ZnCar-formulated antigen elicited increased peptide presentation to B3Z T cells in vitro and production of IL-2 after ex vivo antigen recall of splenocytes isolated from vaccinated mice. Overall, this work displays the formation of a zinc-carnosine metal-organic coordination polymer that can be applied as a platform for recombinant protein-based vaccines.

Fc-Mediated Functions of Porcine IgG Subclasses.

The pig is an important agricultural species and powerful biomedical model. We have established the pig, a large natural host animal for influenza with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies. Antibodies provide protection through neutralization and recruitment of innate effector functions through the Fc domain. However very little is known about the Fc-mediated functions of porcine IgG subclasses. We have generated 8 subclasses of two porcine monoclonal anti influenza hemagglutinin antibodies. We characterized their ability to activate complement, trigger cytotoxicity and phagocytosis by immune cells and assayed their binding to monocytes, macrophages, and natural killer cells. We show that IgG1, IgG2a, IgG2b, IgG2c and IgG4 bind well to targeted cell types and mediate complement mediated cellular cytotoxicity (CDCC), antibody dependent cellular cytotoxicity (ADCC) and antibody mediated cell phagocytosis (ADCP). IgG5b and IgG5c exhibited weak binding and variable and poor functional activity. Immune complexes of porcine IgG3 did not show any Fc-mediated functions except for binding to monocytes and macrophages and weak binding to NK cells. Interestingly, functionally similar porcine IgG subclasses clustered together in the genome. These novel findings will enhance the utility of the pig model for investigation of therapeutic antibodies.

Characterization of humoral and cell-mediated immunity induced by mRNA vaccines expressing influenza hemagglutinin stem and nucleoprotein in mice and nonhuman primates

In response to immune pressure, influenza viruses evolve, producing drifted variants capable of escaping immune recognition. One strategy for inducing a broad-spectrum immune response capable of recognizing multiple antigenically diverse strains is to target conserved proteins or protein domains. To that end, we assessed the efficacy and immunogenicity of mRNA vaccines encoding either the conserved stem domain of a group 1 hemagglutinin (HA), a group 2 nucleoprotein (NP), or a combination of the two antigens in mice, as well as evaluated immunogenicity in naïve and influenza seropositive nonhuman primates (NHPs). HA stem-immunized animals developed a robust anti-stem antibody binding titer, and serum antibodies recognized antigenically distinct group 1 HA proteins. These antibodies showed little to no neutralizing activity in vitro but were active in an assay measuring induction of antibody-dependent cellular cytotoxicity. HA-directed cell-mediated immunity was weak following HA stem mRNA vaccination; however, robust CD4 and CD8 T cell responses were detected in both mice and NHPs after immunization with mRNA vaccines encoding NP. Both HA stem and NP mRNA vaccines partially protected mice from morbidity following lethal influenza virus challenge, and superior efficacy against two different H1N1 strains was observed when the antigens were combined. In vivo T cell depletion suggested that anti-NP cell-mediated immunity contributed to protection in the mouse model. Taken together, these data show that mRNA vaccines encoding conserved influenza antigens, like HA stem and NP in combination, induce broadly reactive humoral responses as well as cell-mediated immunity in mice and NHPs, providing protection against homologous and heterologous influenza infection in mice.

Magnitude and breadth of antibody cross-reactivity induced by recombinant influenza hemagglutinin trimer vaccine is enhanced by combination adjuvants

The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Most adjuvants induced broad IgG profiles, although the response to a combination of CpG, MPLA and AddaVax (termed ‘IVAX-1’) appeared more quickly and reached a greater magnitude than the other formulations tested. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNγ gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.

Atypical B cells up-regulate costimulatory molecules during malaria and secrete antibodies with T follicular helper cell support.

Several infectious and autoimmune diseases are associated with an expansion of CD21-CD27- atypical B cells (atBCs) that up-regulate inhibitory receptors and exhibit altered B cell receptor (BCR) signaling. The function of atBCs remains unclear, and few studies have investigated the biology of pathogen-specific atBCs during acute infection. Here, we performed longitudinal flow cytometry analyses and RNA sequencing of Plasmodium falciparum (Pf)-specific B cells isolated from study participants before and shortly after febrile malaria, with simultaneous analysis of influenza hemagglutinin (HA)-specific B cells as a comparator. At the healthy baseline before the malaria season, individuals had similar frequencies of Pf- and HA-specific atBCs that did not differ proportionally from atBCs within the total B cell population. BCR sequencing identified clonal relationships between Pf-specific atBCs, activated B cells (actBCs), and classical memory B cells (MBCs) and revealed comparable degrees of somatic hypermutation. At the healthy baseline, Pf-specific atBCs were transcriptionally distinct from Pf-specific actBCs and classical MBCs. In response to acute febrile malaria, Pf-specific atBCs and actBCs up-regulated similar intracellular signaling cascades. Pf-specific atBCs showed activation of pathways involved in differentiation into antibody-secreting cells and up-regulation of molecules that mediate B-T cell interactions, suggesting that atBCs respond to T follicular helper (TFH) cells. In the presence of TFH cells and staphylococcal enterotoxin B, atBCs of malaria-exposed individuals differentiated into CD38+ antibody-secreting cells in vitro, suggesting that atBCs may actively contribute to humoral immunity to infectious pathogens.

Comparative Immunogenicity of Bacterially Expressed Soluble Trimers and Nanoparticle Displayed Influenza Hemagglutinin Stem Immunogens.

Current influenza vaccines need to be updated annually due to mutations in the globular head of the viral surface protein, hemagglutinin (HA). To address this, vaccine candidates have been designed based on the relatively conserved HA stem domain and have shown protective efficacy in animal models. Oligomerization of the antigens either by fusion to oligomerization motifs or display on self-assembling nanoparticle scaffolds, can induce more potent immune responses compared to the corresponding monomeric antigen due to multivalent engagement of B-cells. Since nanoparticle display can increase manufacturing complexity, and often involves one or more mammalian cell expressed components, it is important to characterize and compare various display and oligomerization scaffolds. Using a structure guided approach, we successfully displayed multiple copies of a previously designed soluble, trimeric influenza stem domain immunogen, pH1HA10, on the ferritin like protein, MsDps2 (12 copies), Ferritin (24 copies) and Encapsulin (180 copies). All proteins were expressed in Escherichia coli. The nanoparticle fusion immunogens were found to be well folded and bound to the influenza stem directed broadly neutralizing antibodies with high affinity. An 8.5 Å Cryo-EM map of Msdps2-pH1HA10 confirmed the successful design of the nanoparticle fusion immunogen. Mice immunization studies with the soluble trimeric stem and nanoparticle fusion constructs revealed that all of them were immunogenic, and protected mice against homologous (A/Belgium/145-MA/2009) and heterologous (A/Puerto Rico/8/1934) challenge with 10MLD50 mouse adapted virus. Although nanoparticle display conferred a small but statistically significant improvement in protection relative to the soluble trimer in a homologous challenge, heterologous protection was similar in both nanoparticle-stem immunized and trimeric stem immunized groups. Such rapidly producible, bacterially expressed antigens and nanoparticle scaffolds are useful modalities to tackle future influenza pandemics.

Peptide Aggregation Induced Immunogenic Rupture (PAIIR).

Immunogenic cell death (ICD) arises when cells are under stress, and their membranes are damaged. They release damage‐associated molecular patterns (DAMPs) that stimulate and drive the type and magnitude of the immune response. In the presence of an antigen, DAMPs ride the longevity and efficacy of antigen‐specific immunity. Yet, no tool can induce the controlled ICD with predictable results. A peptide‐based tool, [II], is designed that aggregates in the cell and causes cell membrane damage, generates ICD and DAMPs release on various cell types, and hence can act as an adjuvant. An influenza vaccine is prepared by combining [II] with influenza hemagglutinin (HA) subunit antigens. The results show that [II] induced significantly higher HA‐specific immunoglobulin G1 (IgG1) and IgG2a antibodies than HA‐only immunized mice, while the peptide itself did not elicit antibodies. This paper demonstrates the first peptide‐aggregation induced immunogenic rupture (PAIIR) approach as a vaccine adjuvant. PAIIR is a promising adjuvant with a high potential to promote universal protection upon influenza HA vaccination.

Generation and characterization of monoclonal antibodies against the hemagglutinin of H3N2 influenza A viruses

Seasonal influenza viruses are highly contagious, leading to 290,000–650,000 mortalities every year globally. Among the influenza viruses, influenza A virus (H3N2) has attracted much attention due to its high frequency of antigenic variations, resulting in poor protection by vaccination. We generated a panel of murine neutralizing monoclonal antibodies (mAbs) against A/Texas/50/2012 (H3N2) and identified the relevant epitopes that potentially influence the antigenicity by selecting mAb-resistant mutants. The epitopes were mainly in antigenic site A (1/9, 11.1%), B (6/9, 66.7%), and C (1/9, 11.1%), which is consistent with recent reports on the immunodominance of antigenic site B. The amino acid substitutions at positions 156, 157, 159, 160, and 189 at antigenic site B resulted in decreased mAb capability for blocking receptor binding. In addition, the neutralizing spectra of three mAbs (1F8, 1G9 and 1H5) were different, suggesting that their epitopes may be different but partially overlapping, and it required further study. Further, the mAb 3F9 selected a new substitution, D53G/N, at antigenic site C and showed in vitro neutralizing activity against A/Victoria/361/2011 (H3N2), A/Texas/50/2012 (H3N2), and A/Hong Kong/2671/2019 (H3N2), suggesting a potential epitope on H3 hemagglutinin for inducing broad neutralizing antibody responses. Continuous research and regular monitoring of novel epitopes are of great importance for improving vaccine strain selection.

Discovery of Pentacyclic Triterpenoid PROTACs as a Class of Effective Hemagglutinin Protein Degraders.

Influenza hemagglutinin that drives viral entry into cells via the membrane fusion process is an up-and-coming antiviral drug target. Herein, we described for the first time the design, synthesis, and biological characteristics of a new class of pentacyclic triterpenoid-based proteolysis targeting chimeras (PROTACs) to enhance the degradation of hemagglutinin target. Among these PROTACs, V3 showed the best degradation effect on the hemagglutinin with a median degradation concentration of 1.44 μM in a ubiquitin and proteasome-dependent manner and broad-spectrum anti-influenza A virus activity but not affected the entry of influenza virus. Moreover, intravenous injection of V3 protected mice against influenza A virus-induced toxic effects. Further diazirine-containing photo-crosslinking mass spectrometric analysis of hemagglutinin complexes indicated crosslinking to Asn15, Thr31, and Asn27, a novel target of hemagglutinin. Taken together, our data revealed that oleanolic acid-based PROTACs could degrade hemagglutinin protein, providing a new direction toward the discovery of potential anti-influenza drugs.

Epidemiology and Molecular Analyses of Influenza B Viruses in Senegal from 2010 to 2019.

Influenza virus types A and B are responsible for acute viral infections that affect annually 1 billion people, with 290,000 to 650,000 deaths worldwide. In this study, we investigated the circulation of influenza B viruses over a 10-year period (2010–2019). Specimens from patients suspected of influenza infection were collected. Influenza detection was performed following RNA extraction and real-time RT-PCR. Genes coding for hemagglutinin (HA) and neuraminidase (NA) of influenza B viruses were partially sequenced, and phylogenetic analyses were carried out subsequently. During the study period, we received and tested a total of 15,156 specimens. Influenza B virus was detected in 1322 (8.7%) specimens. The mean age of influenza B positive patients was 10.9 years. When compared to reference viruses, HA genes from Senegalese circulating viruses showed deletions in the HA1 region. Phylogenetic analysis highlighted the co-circulation of B/Victoria and B/Yamagata lineage viruses with reassortant viruses. We also noted a clear seasonal pattern of circulation of influenza B viruses in Senegal.

Changes in the organism of patients with chronic inflammatory diseases of the upper respiratory tract at different periods after administration of influenza vaccine. Message 1. Status of congenital immune parameters after a single anti-influenza vaccination

Introduction: Studies of the clinical status, production of antibodies to hemagglutinins of influenza A and B viruses and the content of immune complexes in patients with chronic inflammatory diseases (CID) of the upper respiratory tract (URT) before and within 36 weeks of vaccination after parenteral vaccination Vaxigrip leads to a short-term (~ 6 months) increase in anti-influenza hemagglutinin t itres. According to modern ideas about the mechanisms of antiviral protection, in addition to humoral, mainly due to antibodies, antiviral protection is largely due to the activation of innate immune factors, both systemic and local. Aim: to evaluate in the dynamics of observations the influence of a single parenteral influenza vaccination on the factors of innate immunity in patients with chronic inflammatory diseases of the upper respiratory tract. Material and methods: Among the factors of innate immunity a significant role is played by interferons, the activity of natural cytolytic cells (NK) of various histogenesis, phagocytic cells and non-specific proteins such as defensins, elafins. Therefore, quantitative and functional characteristics of NK-cells, phagocytic absorption activity of polymorphonuclear leukocytes (PMN), systemic content of γ-interferon (IFN-γ) were studied in patients at the systemic level. At the local level, the concentrations of defensin-β and α-interferon were studied in mucosa secretes (MS) according to modern ideas about the state of the mechanisms of innate antiviral immunity. From the autumn-winter period of 2019 to November 2020, 32 donors (control group) and 32 patients with CID of both sexes were examined, 11 of whom were diagnosed with chronic rhinosinusitis, 9 with chronic tonsillitis, and 12 with chronic pharyngitis. No vaccinations were given 1 year before the study. Samples of venous serum and oropharyngeal secretes of all clinically healthy patients, as well as persons with CID were obtained at the initial examination. After parenteral vaccination with Vaxigrip (SANOFI PASTEUR, S.A., France), blood and MS were tested twice more in vaccinated patients: 12 and 36 weeks after vaccination. The obtained samples of experimental material were stored at a temperature of -20 C◦ until the simultaneous determination of innate immune factors (interferons), in particular with the help of reagents from Cytokine, Hema Medica (RF), defensin-β (Hucalt-bio, the Netherlands). Measurements were performed on a microplate enzyme-linked immunosorbent assay "STAT FAX 2100 (USA). Statistical processing was performed using the non-parametric criterion "U" - Mann-Whitney or Fisher's exact method. Differences were considered statistically significant at p &lt;0.05. The results were given in the form of arithmetic mean (M), median (Me) and percentiles (P25-P75). Results: In determining the role of innate immunity factors in vaccination with influenza vaccine, we conducted clinical and immunological examination of patients with chronic inflammatory diseases of the upper respiratory tract, which included systemic determination of γ-interferon, quantitative and functional activity of natural cytotoxic cells, phagocytosis activity at the stage of attraction and capture. Levels of α-interferon and defensin-β in oropharyngeal secretion (MS) were studied. It was found that before influenza vaccination in patients with chronic inflammatory diseases of the upper respiratory tract found an increased content of γ-interferon and decreased phagocytic activity of polymorphonuclear leukocytes. Decreased α-interferon and defensin-β concentrations were detected in MS patients before vaccination. After vaccination, there were positive changes in the direction of increasing the concentration of nonspecific local immunity factor – defensing 1β and partly in the observation period of 3 months after vaccination- α-interferon. Сonclusion: Among the factors of innate immunity in patients with CІD at the systemic level, decreased cytolytic activity of NK, increased levels of γ-interferon and decreased phagocytic function of neutrophils. Decreased concentration of α-interferon and defensin-1β is determined in the saliva of patients with CID. Vaccination of patients with chronic inflammatory diseases of the upper respiratory tract with influenza vaccine has a positive effect on the state of innate immune factors of local and systemic level: concentration of γ-interferon, phagocytic activity of neutrophils and enhancing the functional activity of natural killer cells, increase in saliva of defensin-1β.

Antigen bivalency of antigen-presenting cell-targeted vaccines increases B cell responses.

Antibodies are important for vaccine efficacy. Targeting antigens to antigen-presenting cells (APCs) increases antibody levels. Here, we explore the role of antigen valency in MHC class II (MHCII)-targeted vaccines delivered as DNA. We design heterodimeric proteins that carry either two identical (bivalent vaccines), or two different antigens (monovalent vaccines). Bivalent vaccines with two identical influenza hemagglutinins (HA) elicit higher amounts of anti-HA antibodies in mice than monovalent versions with two different HAs. Bivalent vaccines increase the levels of germinal center (GC) B cells and long-lived plasma cells. Only HA-bivalent vaccines completely protect mice against challenge with homologous influenza virus. Similar results are obtained with other antigens by targeting CD11c and Xcr1 on dendritic cells (DCs) or when administering the vaccine as protein with adjuvant. Bivalency probably increases B cell responses by cross-linking BCRs in readily observable DC-B cell synapses. These results are important for generating potent APC-targeted vaccines.

Optimization of A(H1N1)pdm09 vaccine seed viruses: The source of PB1 and HA vRNA as a major determinant for antigen yield

Vaccination prevents and reduces the severity of influenza virus infections. Continuous evolution of influenza hemagglutinin (HA) and neuraminidase (NA) supports the virus to evade pre-existing immunity, which demands vaccines to be reformulated every year. Incorporation of polymerase basic protein 1 (PB1) viral RNA (vRNA) of the same origin of HA and NA vRNA has been observed in previous pandemic viruses and occasionally reported for influenza A vaccine prototype strains of prior seasons. At this point, it remains to be explored whether this phenomenon translates into an improved growth phenotype. In this work, we showed that the HA vRNA of A(H1N1)pdm09 is generally incorporated with the PB1 vRNA of the same origin, establishing the beneficial effect of the presence of PB1 and the pattern of the PB1-HA co-incorporation in the A(H1N1)pdm09 model. We further investigated the putative interplay between PB1 and antigenic proteins regarding the vRNA composition of the progeny and observed that vRNA segregation does not appear to be mainly determined by protein-protein interactions; while vRNA-vRNA interactions can be suggested as the main driving force. Our data also indicate an increase in the hemagglutination capacity and neuraminidase activity due to incorporation of PB1, HA and NA from A(H1N1)pdm09, in comparison with the recombinant virus incorporating only HA and NA from A(H1N1)pdm09 – which have the potential to improve current limitations regarding antigenicity and immunogenicity of influenza vaccines. Further knowledge of the complex vRNA-vRNA interaction network between PB1 and HA will additionally contribute to improve current vaccine formulation, and to gradually optimize the production of A(H1N1)pdm09 reverse genetics vaccine seed virus towards a higher cost-effectiveness.

The determination of haemagglutinin influenza antibodies in the Polish population in the epidemic season 2020/2021 during the SARS-CoV-2 pandemic

The aim of the study was to prove the level of antibodies against haemagglutinin in the sera of people from seven age groups in the epidemic season 2020/2021 in Poland to determine the differentiation of the antibody level and the protection rate depending on age. The level of anti-haemagglutinin antibodies was established by haemagglutinin inhibition test (HAI). A total of 700 randomly selected sera from people belonging to 7 different age groups were tested. The results confirmed the presence of antibodies against the following influenza antigens: A/Guangdong-Maonan/SWL1536/2019 (H1N1)pdm09-like virus, A/Hong Kong/2671/2019 (H3N2)-like virus, B/Washington/02/2019 (B/Victoria lineage)-like virus and B/Phuket/3073/2013 (B/Yamagata lineage)-like virus. The level of haemagglutinin antibodies varied between the studied age groups, with the highest values in the 5-9 age group and the lowest in the 0-4 age group. It was also proven that the protection rate was the highest for the A/Hong Kong/2671/2019(H3N2)-like virus antigen, which exceeded the protection level in the 5 age groups. Considering the very low percentage of people vaccinated in the epidemic season 2020/2021 in Poland, which amounted to only 6.1%, the results should be interpreted as the immune system's response to an infection with influenza virus.

Reactogenicity, safety, and immunogenicity of chimeric haemagglutinin influenza split-virion vaccines, adjuvanted with AS01 or AS03 or non-adjuvanted: a phase 1–2 randomised controlled trial

One strategy to develop a universal influenza virus vaccine is to redirect the immune system to the highly conserved haemagglutinin stalk domain by sequentially administering vaccines expressing chimeric (c) haemagglutinins with a conserved stalk domain and divergent head domain, to which humans are naive. We aimed to assess the reactogenicity, safety, and immunogenicity of adjuvanted and unadjuvanted investigational supra-seasonal universal influenza virus vaccines (SUIVs) in healthy young adults. In this observer-masked, randomised, controlled, phase 1-2 trial, we recruited adults aged 18-39 years with no clinically significant conditions from six centres in Belgium and the USA. Participants were randomly assigned to ten equally sized groups via an online system with the MATerial Excellence programme. Vaccines contained heterosubtypic group 1 H8, H5, or H11 haemagglutinin heads, an H1 haemagglutinin stalk, and an N1 neuraminidase (cH8/1N1, cH5/1N1, and cH11/1N1; haemagglutinin dose 15 μg/0·5 mL), administered on days 1 and 57, with a month 14 booster. SUIVs were evaluated in the sequences: cH8/1N1-placebo-cH5/1N1, cH5/1N1-placebo-cH8/1N1, or cH8/1N1-cH5/1N1-cH11/1N1, adjuvanted with either AS03 or AS01, or not adjuvanted. The last group received inactivated quadrivalent influenza vaccine (IIV4)-placebo-IIV4. Primary outcomes were safety (analysed in the exposed population) and immunogenicity in terms of the anti-H1 stalk humoral response at 28 days after vaccination (analysed in the per-protocol population, defined as participants who received the study vaccines according to the protocol). This trial is registered with ClinicalTrials.gov, NCT03275389. Between Sept 25, 2017, and March 26, 2020, 507 eligible participants were enrolled. 468 (92%) participants received at least one dose of study vaccine (exposed population), of whom 244 (52%) were included in the per-protocol population at final analysis at month 26. The safety profiles of all chimeric vaccines were clinically acceptable, with no safety concerns identified. Injection-site pain was the most common adverse event, occurring in 84-96% of participants receiving an adjuvanted SUIV or non-adjuvanted IIV4 and in 40-50% of participants receiving a non-adjuvanted SUIV. Spontaneously reported adverse events up to 28 days after vaccination occurred in 36-60% of participants, with no trends observed for any group. 17 participants had a serious adverse event, none of which were considered to be causally related to the vaccine. Anti-H1 stalk antibody titres were highest in AS03-adjuvanted groups, followed by AS01-adjuvanted and non-adjuvanted groups, and were higher after cH8/1N1 than after cH5/1N1 and after a two-dose primary schedule than after a one-dose schedule. Geometric mean concentrations by ELISA ranged from 21 938·1 ELISA units/mL (95% CI 18 037·8-26 681·8) in the IIV4-placebo-IIV4 group to 116 596·8 ELISA units/mL (93 869·6-144 826·6) in the AS03-adjuvanted cH8/1N1-cH5/1N1-cH11/1N1 group 28 days after the first dose and from 15 105·9 ELISA units/mL (12 007·7-19 003·6) in the non-adjuvanted cH5/1N1-placebo-cH8/1N1 group to 74 639·7 ELISA units/mL (59 986·3-92 872·6) in the AS03-adjuvanted cH8/1N1-cH5/1N1-cH11/1N1 group 28 days after the second dose. The stalk domain seems to be a rational target for development of a universal influenza virus vaccine via administration of chimeric haemagglutinins with head domains to which humans are naive. GlaxoSmithKline Biologicals.

Elimination of receptor binding by influenza hemagglutinin improves vaccine-induced immunity

The binding of influenza hemagglutinin (HA) to sialic acid (SA) receptors plays a well-defined role in shaping infection but the impact of such binding on vaccine responses has not yet been explored. We generated a virus-like particle (VLP) vaccine bearing the HA of H1N1 A/California/07/09 that is unable to bind to its α(2,6)-linked SA receptor (H1Y98F-VLP) and compared its immunogenicity and efficacy to a wild-type H1-VLP (H1WT-VLP) in mice. The H1Y98F-VLP elicited significantly stronger and more durable antibody responses (hemagglutination inhibition and microneutralization titers) and greater avidity maturation, likely attributable to improved germinal center formation. H1Y98F-VLP also resulted in a robust population of IL-2+TNFα+IFNγ− CD4+ T cells that correlated with antibody responses. Compared to H1WT-VLP vaccination, mice immunized with H1Y98F-VLP had 2.3-log lower lung viral loads and significantly lower pulmonary inflammatory cytokine levels 5 days post-challenge. These findings suggest that abrogation of HA-SA interactions may be a promising strategy to improve the quality and durability of influenza vaccine-induced humoral responses.

Identifying vaccine escape sites via statistical comparisons of short-term molecular dynamics

The identification of viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a molecular dynamics (MD)-based approach that goes beyond contact mapping, scales well to a desktop computer with a modern graphics processor, and enables the user to identify functional protein sites that are prone to vaccine escape in a viral antigen. We first implement our MD pipeline to employ site-wise calculation of Kullback-Leibler divergence in atom fluctuation over replicate sets of short-term MD production runs thus enabling a statistical comparison of the rapid motion of influenza hemagglutinin (HA) in both the presence and absence of three well-known neutralizing antibodies. Using this simple comparative method applied to motions of viral proteins, we successfully identified in silico all previously empirically confirmed sites of escape in influenza HA, predetermined via selection experiments and neutralization assays. Upon the validation of our computational approach, we then surveyed potential hotspot residues in the receptor binding domain of the SARS-CoV-2 virus in the presence of COVOX-222 and S2H97 antibodies. We identified many single sites in the antigen-antibody interface that are similarly prone to potential antibody escape and that match many of the known sites of mutations arising in the SARS-CoV-2 variants of concern. In the Omicron variant, we find only minimal adaptive evolutionary shifts in the functional binding profiles of both antibodies. In summary, we provide an inexpensive and accurate computational method to monitor hotspots of functional evolution in antibody binding footprints.