Recombinant Hemagglutinin Research Articles

The position of Spy Tag/Catcher system in hepatitis B core protein particles affects the immunogenicity and stability of the synthetic vaccine

Presenting exogenous antigens on virus-like particles (VLPs) through “plug-and-display” decoration strategies based on SpyTag/SpyCatcher isopeptide bonding have emerged as attractive technology for vaccine synthesis. However, whether the position of ligation site in VLPs will impose effects on immunogenicity and physiochemical properties of the synthetic vaccine remains rarely investigated. Here in the present work, the well-established hepatitis B core (HBc) protein was used as chassis to construct dual-antigen influenza nanovaccines, with the conserved epitope peptides derived from extracellular domain of matrix protein M2 (M2e) and hemagglutinin (HA) as target antigens. The M2e antigen was genetically fused to the HBc in the MIR region, together with the SpyTag peptide, which was fused either in the MIR region or at the N-terminal of the protein, so that a recombinant HA antigen (rHA) linked to SpyCatcher can be displayed on it, at two different localizations. Both synthetic nanovaccines showed ability in inducing strong M2e and rHA-specific antibodies and cellular immunogenicity; nevertheless, the one in which rHA was conjugated by N-terminal Tag ligation, was superior to another one synthesized by linking the rHA to MIR region SpyTagged-HBc in all aspects, including higher antigen-specific immunogenicity responses, lower anti-HBc carrier antibody, as well as better dispersion stability. Surface charge and hydrophobicity properties of the two synthetic nanovaccines were analyzed, results revealed that linking the rHA to MIR region SpyTagged-HBc lead to more significant and disadvantageous alteration in physiochemical properties of the HBc chassis. This study will expand our knowledge on “plug-and-display” decoration strategies and provide helpful guidance for the rational design of HBc-VLPs based modular vaccines by using SpyTag/Catcher synthesis.

Recombinant hemagglutinin displaying on yeast reshapes congenital lymphocyte subsets to prompt optimized systemic immune protection against avian influenza infection.

Prophylactic vaccination is regarded as the most effective means to control avian flu infection. Currently, there is a need for a universal vaccine that provides broad and long-lasting protection against influenza virus. Meanwhile, although yeast-based vaccines have been used in clinic, studies are still required to further understand the molecular mechanism of yeast-based vaccines under physiological conditions. We generated a yeast-based vaccine against influenza hemagglutinin (HA) of H5, H7 and H9 using surface displaying technology and evaluated the protective efficacy of chickens after exposure to H9N2 influenza virus. Oral yeast vaccine provided less clinical syndrome, reduced viral loading and alleviated airway damage significantly. Compared to the commercial inactivated vaccine, yeast vaccine stimulated the activation of splenic NK and APCs cells and boosted TLR7-IRF7-IFN signaling in spleen. Meanwhile, γδ T cells in the bursa of Fabricius were activated and the innate lymphoid cells (ILCs) in the bursa of Fabricius promoted the CILPs to differentiate to ILC3 cells in oral yeast birds. Moreover, the reshaped gut microbiota and a suppressed Th17-IL17-mediated inflammation in intestine was observed in oral yeast chickens, which might facilitate the recovery of intestinal mucosal immunity upon virus infection. Collectively, our findings suggest that oral yeast based multivalent bird flu vaccines provide an attractive strategy to update host defense function via reshapes of multi-systemic immune homeostasis.

The slow-release platform, VacSIM, shapes host immune response against influenza infection after vaccination.

Abstract Influenza virus is a leading cause of respiratory infections known to cause global morbidity and mortality, despite the widespread use of multiple influenza vaccines. Current influenza vaccines target the hemagglutinin (HA) envelope protein responsible for the attachment and entry of influenza into host cells. However, high mutation rates during influenza replication lead to mutations that cause the HA protein to be unrecognizable by host antibodies, increasing levels of infection within both vaccinated and unvaccinated individuals. A promising approach to circumvent this problem is through the use of a slow release delivery platform which has been previously shown to induce the longevity of germinal center reactions, allowing for the generation of higher affinity antibodies and more evolved B cells. Here we show that vaccination with the slow-delivery platform, Vaccine Self-Assembling Immune Matrix (VacSIM), in combination with recombinant HA antigens, increases antibody titers against H1 and H3 HAs in vaccinated groups of mice. When mice immunized with VacSIM/H1 HA were challenged with a lethal H1N1 dose, a 25% increase in survival was seen compared to the non-VacSIM group and 100% increase against mock immunized. Similarly, when immunized with VacSIM/H3 HA and challenged with H3N2, a 1.8 log reduction in lung viral titers was seen. Further analysis of the vaccine-elicited antibodies showed that vaccination with VacSIM and HA increases HAI activity against multiple strains of influenza within both H1N1 and H3N2 subtypes. We anticipate that use of VacSIM will provide further insight into how consistent exposure through a slow delivery platform can shape the host immune response to increase protection against influenza infection. Supported by grants from CIVIC

Method for screening influenza neutralizing antibodies in crude human plasma and its derivatives using SPR

We applied Surface Plasmon Resonance (SPR) technology to develop a method for potency screening and quantification of anti-influenza antibodies in minimally processed human plasma samples and intravenous immunoglobulin (IGIV) products. We found that specific antibodies in human plasma or IGIV capable of inhibiting binding of influenza hemagglutinin to receptor-analogous glycans do so in concentration-dependent manner. We ranked the inhibitory activity of plasma samples from multiple donors and found a good correlation (r = 0.87) of SPR assay measurements and conventional hemagglutination inhibition (HAI) assay results. This method was also applied to screen for specific anti-influenza antibodies in IGIV lots manufactured pre- and post-2009 H1N1 pandemic. The SPR method was also applied to study binding inhibition of the intact A/California/04/2009 H1N1 and B/Victoria/504/2000 influenza viruses to α2,6 or α2,3-linked synthetic glycans. In contrast to recombinant H1 hemagglutinin, which was found to interact primarily with α2,6-linked terminal sialic acids, intact H1N1 or influenza B virus recognized both types of receptor analogs with different observed dissociation rates and the inhibitory activity of plasma antibodies was dependent on the type of sialic acid link. The SPR method can provide a high-throughput, time-saving and semi-automated alternative to conventional assays such as HAI or microneutralization in situations where screening of large numbers of plasma donations to identify high titer units is needed to product highly potent immunoglobulins.

Intradermal Immunization of Soluble Influenza HA Derived from a Lethal Virus Induces High Magnitude and Breadth of Antibody Responses and Provides Complete Protection In Vivo.

Immunogens mimicking the native-like structure of surface-exposed viral antigens are considered promising vaccine candidates. Influenza viruses are important zoonotic respiratory viruses with high pandemic potential. Recombinant soluble hemagglutinin (HA) glycoprotein-based protein subunit vaccines against Influenza have been shown to induce protective efficacy when administered intramuscularly. Here, we have expressed a recombinant soluble trimeric HA protein in Expi 293F cells and purified the protein derived from the Inf A/Guangdong-Maonan/ SWL1536/2019 virus which was found to be highly virulent in the mouse. The trimeric HA protein was found to be in the oligomeric state, highly stable, and the efficacy study in the BALB/c mouse challenge model through intradermal immunization with the prime-boost regimen conferred complete protection against a high lethal dose of homologous and mouse-adapted InfA/PR8 virus challenge. Furthermore, the immunogen induced high hemagglutinin inhibition (HI) titers and showed cross-protection against other Inf A and Inf B subtypes. The results are promising and warrant trimeric HA as a suitable vaccine candidate.

Identification of broad-spectrum neutralizing antibodies against influenza A virus and evaluation of their prophylactic efficacy in mice

Influenza A virus continuously infects humans and the antigenic shifts of this respiratory virus enable it to cross the species barrier, threatening public health with the risk of pandemics. Broadly neutralizing antibodies (bnAbs) that target the antigenic surface glycoprotein, hemagglutinin (HA), of influenza A virus protect against various subtypes of the virus. Here, we screened a human scFv library, through phage display and panning against recombinant HA proteins, to discover human monoclonal antibodies (mAbs) that are broadly active. Consequently, two human mAbs, named G1 and G2, were identified, which target the HA proteins of the H1N1 and H3N2 subtypes, respectively. G1 was shown to have broad binding ability to different HA subtypes of group 1. By contrast, G2 had higher binding affinity but sensed exclusively H3 subtype-derived HAs. In a cell culture-based virus-neutralizing assay, both G1 and G2 efficiently suppressed infection of the parental influenza A viruses of H1N1 and H3N2 subtypes. Mode-of-action studies showed that the G1 antibody blocked HA2-mediated membrane fusion. Meanwhile, G2 inhibited HA1-mediated viral attachment to host cells. It is noteworthy that both antibodies elicited antibody-dependent cellular cytotoxicity (ADCC) activities by recruiting FcγRIIIA-expressing effector cells. In mouse challenge models, single-shot, intraperitoneal administration of chimeric G1 and G2 antibodies with the mouse IgG constant region completely protected mice from viral infections at doses above 10 and 1 mg/kg, respectively. The newly identified bnAbs, G1 and G2, could provide insight into the development of broad-spectrum antivirals against future pandemic influenza A virus involving group 1- or H3-subtyped strains.

Recombinant hemagglutinin protein and DNA-RNA-combined nucleic acid vaccines harbored by yeast elicit protective immunity against H9N2 avian influenza infection

A safe, convenience, and effective vaccine for controlling avian influenza virus infection is crucial in scale poultry production. Yeasts are considered useful vaccine vehicles for the delivery of antigens, which has been used to protect human and animal health. We report here the development of H9N2 strain hemagglutinin (HA)-based recombinant protein vaccines (rH9HA) and DNA-RNA-combined vaccine (rH9-DNA-RNA) in Saccharomyces cerevisiae for the first time. The immunogenicity assay indicated that both rH9HA and rH9-DNA-RNA could induce robust production of serum IgG, mucosal sIgA, and cellular immune responses. The reshape and diversification of gut microbiota and an enriched Lactobacillus, Debaryomyces were observed after oral immunization with rH9HA or rH9-DNA-RNA yeast vaccine, which might contribute to modulate the intestinal mucosal immunity and antiviral process. Oral immunized birds with either rH9HA or rH9-DNA-RNA were effectively protected from H9N2 virus challenge. Our findings suggested that yeast-derived H9N2 HA-based recombinant protein vaccines and DNA-RNA-combined nucleic acid vaccines are feasible and efficacious, opening up a new avenue for rapid and cost-effective production of avian influenza vaccines to achieve good protection effect.

Maternal influenza vaccination preferentially boosts hemagglutinin stem-specific antibody resulting in efficient transplacental transfer of stem-specific IgG.

The objective of this study was to evaluate hemagglutinin stem-specific antibody response to the influenza vaccine during pregnancy and its transfer to the infant. The authors assessed antibody titers among maternal participants and their paired neonate's cord blood (CB) using enzyme-linked immunoassay. Fifteen pregnant participants pre-2019 and post-2019 seasonal influenza vaccine were compared with 18 prenatally vaccinated participants with paired neonatal CB samples. Total IgG and IgG subclass titers specific for whole vaccine antigens versus recombinant hemagglutinin stem-specific antigen were compared using Wilcoxon exact test. Hemagglutinin stem-specific IgG was boosted more robustly than whole vaccine titers when comparing postvaccine versus prevaccine log2 IgG ratios (P= 0.04). Hemagglutinin stem-specific IgG titers were boosted postvaccination (prevaccine: 14.5 [95% confidence interval, 13.8-15.2] vs. postvaccine: 16 [95% confidence interval, 15.2-16.8], P= 0.004). While IgG to whole vaccine was similar in neonatal CB and maternal plasma (P= 0.09), hemagglutinin stem-specific IgG concentrated in CB (P= 0.002), which was dominated by IgG1 subclass (analysis of variance P< 0.05). These data demonstrate the ability of pregnant women to generate a more robust antibody response to the stem region compared with the head region of hemagglutinin with transplacental transfer of IgG.

Marine prebiotics mediate decolonization of Pseudomonas aeruginosa from gut by inhibiting secreted virulence factor interactions with mucins and enriching Bacteroides population

BackgroundPseudomonas aeruginosa intestinal carriage rates are significantly higher in immunosuppressed individuals and hospitalized patients who therefore have increased risk of infections and antibiotic-associated diarrhea. To combat intestinal dysbiosis and decolonize P. aeruginosa from gastrointestinal tract, we investigated the anti-adherence and gut microbiota modulation properties of marine prebiotic fucoidans.MethodsProteomic analysis of culture supernatant was performed by LC–MS/MS. Using lectin-based enzyme-linked immunosorbent assay, hemagglutinin domain interaction and inhibition with biomolecules were studied. We investigated the role of nutritional grade fucoidans in a mouse model and used 16S ribosomal RNA sequencing to examine fecal microbiota composition.ResultsAnalysis of culture supernatant proteins indicated the secretion of two-partner secretion (TPS) family proteins, including TpsA1/CdiA2 and TpsA2/CdiA1. Lectin like activity at the N-terminal of TpsA due to a conserved hemagglutinin domain (Pfam identifier [ID] PF05860) mediates binding to mucins that carry multiple fucosylated glycans. Fucose-rich sulfated polysaccharides (fucoidans) and sulfated dextrans were found to be potent inhibitors of the recombinant N-terminal hemagglutinin domain of TpsA (TpsA-NT-HAD) binding to mucins. In a mouse model, antibiotic-induced dysbiosis was essential for P. aeruginosa gastrointestinal colonization. After prophylactic oral fucoidans supplementation, a higher proportion (60%) of the mice were decolonized over time and resisted re-colonization, this was associated with remarkable expansion of Bacteroides (post-infection day-3 abundance, 29–50%) and consequential reductions in bloom of Enterobacteriaceae and Enterococcaceae populations. In the non-supplemented group, Parabacteroides mediated recovery from dysbiosis but failed to decolonize P. aeruginosa.ConclusionsSupplementing diet with marine prebiotic fucoidans can mediate earlier recovery from dysbiosis and decolonization of P. aeruginosa from gut by inhibiting secreted virulence factor (TpsA/CdiA) interaction with mucins and promoting the growth of beneficial Bacteroides population. We suggest the prophylactic use of nutritional grade fucoidans to decolonize P. aeruginosa from gastrointestinal tract of at-risk individuals to prevent infection and transmission of colonizing P. aeruginosa.

Molecular engineering of a minimal E-cadherin inhibitor protein derived from Clostridium botulinum hemagglutinin

Hemagglutinin (HA), a nontoxic component of the botulinum neurotoxin (BoNT) complex, binds to E-cadherin and inhibits E-cadherin-mediated cell-cell adhesion. HA is a 470kDa protein complex comprising six HA1, three HA2, and three HA3 subcomponents. Thus, to prepare recombinant full-length HA invitro, it is necessary to reconstitute the macromolecular complex from purified HA subcomponents, which involves multiple purification steps. In this study, we developed NanoHA, a minimal E-cadherin inhibitor protein derived from Clostridium botulinum HA with a simple purification strategy needed for production. NanoHA, containing HA2 and a truncated mutant of HA3 (amino acids 380-626; termed as HA3mini), is a 47kDa single polypeptide (one-tenth the molecular weight of full-length HA, 470kDa) engineered with three types of modifications: (i) a short linker sequence between the C terminus of HA2 and N terminus of HA3; (ii) a chimeric complex composed of HA2 derived from the serotype C BoNT complex and HA3mini from the serotype B BoNT complex; and (iii) three amino acid substitutions from hydrophobic to hydrophilic residues on theprotein surface. We demonstrated that NanoHA inhibits E-cadherin-mediated cell-cell adhesion of epithelial cells (e.g., Caco-2 and Madin-Darby canine kidney cells) and disrupts their epithelial barrier. Finally, unlike full-length HA, NanoHA can be transported from the basolateral side to adherens junctions via passive diffusion. Overall, these results indicate that the rational design of NanoHA provides a minimal E-cadherin inhibitor with a wide variety of applications as a lead molecule and for further molecular engineering.

Protein Nanoparticle-Mediated Delivery of Recombinant Influenza Hemagglutinin Enhances Immunogenicity and Breadth of the Antibody Response.

The vast majority of seasonal influenza vaccines administered each year are derived from virus propagated in eggs using technology that has changed little since the 1930s. The immunogenicity, durability, and breadth of response would likely benefit from a recombinant nanoparticle-based approach. Although the E2 protein nanoparticle (NP) platform has been previously shown to promote effective cell-mediated responses to peptide epitopes, it has not yet been reported to deliver whole protein antigens. In this study, we synthesized a novel maleimido tris-nitrilotriacetic acid (NTA) linker to couple protein hemagglutinin (HA) from H1N1 influenza virus to the E2 NP, and we evaluated the HA-specific antibody responses using protein microarrays. We found that recombinant H1 protein alone is immunogenic in mice but requires two boosts for IgG to be detected and is strongly IgG1 (Th2) polarized. When conjugated to E2 NPs, IgG2c is produced leading to a more balanced Th1/Th2 response. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) significantly enhances the immunogenicity of H1-E2 NPs while retaining the Th1/Th2 balance. Interestingly, broader homo- and heterosubtypic cross-reactivity is also observed for conjugated H1-E2 with MPLA, compared to unconjugated H1 with or without MPLA. These results highlight the potential of an NP-based delivery of HA for tuning the immunogenicity, breadth, and Th1/Th2 balance generated by recombinant HA-based vaccination. Furthermore, the modularity of this protein-protein conjugation strategy may have utility for future vaccine development against other human pathogens.

93. Novel recombinant measles virus vaccine prevents measles virus disease in rhesus macaques

Abstract Background Measles-Mumps-Rubella (MMR) is an effective live-virus vaccine against measles virus (MeV). However, use of MMR is limited by its inability to boost MeV immunity, lack of immunogenicity in infants, and contraindication in pregnant and immunocompromised persons. Methods We evaluated a novel recombinant dimeric MeV hemagglutinin protein vaccine (rMeV) in a rhesus macaque model. Sixteen macaques were primed at day 0 and boosted at day 42 by experimental group: 1) MMR x2; 2) rMeV x2; 3) MMR prime/rMeV boost; 4) control; n=4. Macaques were challenged intratracheally with Bilthoven strain wild type MeV 8 months later. Blood, bone marrow (BM), and lymph node (LN) samples were collected over 3–28 days after challenge. Replication-competent MeV was measured in peripheral blood mononuclear cells (PBMC), BM cells, and LN cells by infectious assay; MeV RNA in PBMC and BM cells was determined by quantitative reverse transcriptase polymerase chain reaction. Plasma was evaluated for MeV-specific IgG and plaque reduction neutralization titer (PRNT). Results Six months after vaccination, mean PRNT was 2,432 in rMeV x2 (standard deviation (SD) 3,840), 3,584 in MMR-prime/rMeV boost (SD 3,072) and 5,120 in MMR x2 groups (SD 3,547). Upon infectious challenge, macaques who received any MeV-containing vaccine developed no clinical signs of measles and had no detectable infectious virus in PBMC, BM cells, or LN cells. All unvaccinated macaques had virus in PBMC that peaked at day 7 (mean 3,162 TCID50/mL, SD 4.1) and resolved by day 14 post challenge, and one macaque developed an extensive rash. Macaques who received any MeV-containing vaccine had no detectable MeV RNA in PBMC or BM cells, whereas all unvaccinated macaques had detectable MeV RNA that peaked at day 7 (1.6e5 copies, SD 10.5) in PBMC. In all experimental groups, MeV-specific IgG titers increased after MeV challenge. Conclusion Macaques who received rMeV and/or MMR were protected from rash, viremia, and detection of MeV RNA in PBMC and BM cells, unlike unvaccinated macaques. These data suggest that rMeV vaccine generates protective immune responses against measles and may be a novel candidate for future measles vaccine strategies. Study of cellular responses after rMeV vaccination and MeV challenge is warranted. Disclosures Jessica Rubens, MD, Mevox: Grant/Research Support Guillaume Stewart-Jones, PhD, Moderna: Inventor of SARS-CoV-2 vaccine sequences|Moderna: Stocks/Bonds Michael Watson, MD, MEVOX Ltd: Board Member|MEVOX Ltd: Ownership Interest|MEVOX Ltd: Stocks/Bonds Barney S. Graham, MD, PhD, BSG: BSG is an inventor on patents for the stabilization of the RSV F protein (WO2014160463A1, Prefusion RSV F proteins and their use).|National Institutes of Health: Inventor on patents for RSV vaccines|National Institutes of Health: inventor on patents for measles and other paramyxovirus vaccines Diane Griffin, MD PhD, Gilead: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GreenLight Biosciences: Advisor/Consultant|Merck: Advisor/Consultant|MeVox: Grant/Research Support|Takeda Pharmaceuticals: Advisor/Consultant.

Identification of Unique and Conserved Neutralizing Epitopes of Vestigial Esterase Domain in HA Protein of the H9N2 Subtype of Avian Influenza Virus.

The H9N2 subtype of avian influenza virus (AIV) has been reported to infect not only birds, but also humans. The hemagglutinin (HA) protein is the main surface antigen of AIV and plays an important role in the viral infection. For treatment strategies and vaccine development, HA protein has been an important target for the development of broadly neutralizing antibodies against influenza A virus. To investigate the vital target determinant cluster in HA protein in this work, HA gene was cloned and expressed in the prokaryotic expression vector pET28a. The spleen lymphocytes from BALC/c mice immunized with the purified recombinant HA protein were fused with SP2/0 cells. After Hypoxanthine-Aminopterin-Thymidine (HAT) medium screening and indirect ELISA detection, six hybridoma cell lines producing anti-HA monoclonal antibodies were screened. The gradually truncated HA gene expression and western blotting were used to identify their major locations in epitopes specific to these monoclonal antibodies. It was found that the epitopes were located in three areas: 112NVENLEEL119, 117EELRSLFS124, and 170PIQDAQ175. Epitope 112NVENLEEL119 has a partial amino acid crossover with 117EELRSLFS124, which is located in the vestigial esterase domain "110-helix" of HA, and the monoclonal antibody recognizing these epitopes showed the neutralizing activity, suggesting that the region 112NVENLEELRSLFS124 might be a novel neutralizing epitope. The results of the homology analysis showed that these three epitopes were generally conserved in H9N2 subtype AIV, and will provide valuable insights into H9N2 vaccine design and improvement, as well as antibody-based therapies for treatment of H9N2 AIV infection.

Characterization of Neutralizing Monoclonal Antibodies and Identification of a Novel Conserved C-Terminal Linear Epitope on the Hemagglutinin Protein of the H9N2 Avian Influenza Virus.

The H9N2 avian influenza virus (AIV) remains a serious threat to the global poultry industry and public health. The hemagglutinin (HA) protein is an essential protective antigen of AIVs and a major target of neutralizing antibodies and vaccines. Therefore, in this study, we used rice-derived HA protein as an immunogen to generate monoclonal antibodies (mAbs) and screened them using an immunoperoxidase monolayer assay and indirect enzyme-linked immunosorbent assay. Eight mAbs reacted well with the recombinant H9N2 AIV and HA protein, four of which exhibited potent inhibitory activity against hemagglutination, while three showed remarkable neutralization capacities. Western blotting confirmed that two mAbs bound to the HA protein. Linear epitopes were identified using the mAbs; a novel linear epitope, 480HKCDDQCM487, was identified. Structural analysis revealed that the novel linear epitope is located at the C-terminus of HA2 near the disulfide bond-linked HA1 and HA2. Alignment of the amino acid sequences showed that the epitope was highly conserved among multiple H9N2 AIV strains. The results of this study provide novel insights for refining vaccine and diagnostic strategies and expand our understanding of the immune response against AIV.

Immunogenicity and Protective Efficacy of a Recombinant Pichinde Viral-Vectored Vaccine Expressing Influenza Virus Hemagglutinin Antigen in Pigs.

Influenza A virus of swine (IAV-S) is an economically important swine pathogen. The IAV-S hemagglutinin (HA) surface protein is the main target for vaccine development. In this study, we evaluated the feasibility of using the recombinant tri-segmented Pichinde virus (rPICV) as a viral vector to deliver HA antigen to protect pigs against IAV-S challenge. Four groups of weaned pigs (T01–T04) were included in the study. T01 was injected with PBS to serve as a non-vaccinated control. T02 was inoculated with rPICV expressing green fluorescence protein (rPICV-GFP). T03 was vaccinated with rPICV expressing the HA antigen of the IAV-S H3N2 strain (rPICV-H3). T04 was vaccinated with the recombinant HA protein antigen of the same H3N2 strain. Pigs were vaccinated twice at day 0 and day 21 and challenged at day 43 by intra-tracheal inoculation with the homologous H3N2 IAV-S strain. After vaccination, all pigs in T03 and T04 groups were seroconverted and exhibited high titers of plasma neutralizing antibodies. After challenge, high levels of IAV-S RNA were detected in the nasal swabs and bronchioalveolar lavage fluid of pigs in T01 and T02 but not in the T03 and T04 groups. Similarly, lung lesions were observed in T01 and T02, but not in the T03 and T04 groups. No significant difference in terms of protection was observed between the T03 and T04 group. Collectively, our results demonstrate that the rPICV-H3 vectored vaccine elicited protective immunity against IAV-S challenge. This study shows that rPICV is a promising viral vector for the development of vaccines against IAV-S.

Shaping Neonatal Immunization by Tuning the Delivery of Synergistic Adjuvants via Nanocarriers.

Adjuvanted nanocarrier-based vaccines hold substantial potential for applications in novel early-life immunization strategies. Here, via mouse and human age-specific in vitro modeling, we identified the combination of a small-molecule STING agonist (2′3′-cyclic GMP-AMP, cGAMP) and a TLR7/8 agonist (CL075) to drive the synergistic activation of neonatal dendritic cells and precision CD4 T-helper (Th) cell expansion via the IL-12/IFNγ axis. We further demonstrate that the vaccination of neonatal mice with quadrivalent influenza recombinant hemagglutinin (rHA) and an admixture of two polymersome (PS) nanocarriers separately encapsulating cGAMP (cGAMP-PS) and CL075 (CL075-PS) drove robust Th1 bias, high frequency of T follicular helper (TFH) cells, and germinal center (GC) B cells along with the IgG2c-skewed humoral response in vivo. Dual-loaded cGAMP/CL075-PSs did not outperform admixed cGAMP-PS and CL075-PS in vivo. These data validate an optimally designed adjuvantation system via age-selected small-molecule synergy and a multicomponent nanocarrier formulation as an effective approach to induce type 1 immune responses in early life.

Kinetic of the Antibody Response Following AddaVax-Adjuvanted Immunization with Recombinant Influenza Antigens.

Notwithstanding the current SARS-CoV-2 pandemic, influenza virus infection still represents a global health concern in terms of hospitalizations and possible pandemic threats. The objective of next-generation influenza vaccines is not only to increase the breadth of response but also to improve the elicitation of an effective and robust immune response, especially in high-risk populations. To achieve this second objective, the administration of adjuvanted influenza vaccines has been considered. In this regard, the monitoring and characterization of the antibody response associated with the administration of adjuvanted vaccines has been evaluated in this study in order to shed light on the kinetic, magnitude and subclass usage of antibody secreting cells (ASCs) as well as of circulating antigen-specific serum antibodies. Specifically, we utilized the DBA/2J mouse model to assess the kinetic, magnitude and IgG subclass usage of the antibody response following an intramuscular (IM) or intraperitoneal (IP) immunization regimen with AddaVax-adjuvanted bivalent H1N1 and H3N2 computationally optimized broadly reactive antigen (COBRA) influenza recombinant hemagglutinins (rHAs). While the serological evaluation revealed a homogeneous kinetic of the antibody response, the detection of the ASCs through a FluoroSpot platform revealed a different magnitude, subclass usage and kinetic of the antigen-specific IgG secreting cells peaking at day 5 and day 9 following the IP and IM immunization, respectively.

Formulation of dry powders of vaccines containing MF59 or AddaVax by Thin-Film Freeze-Drying: Towards a dry powder universal flu vaccine

MF59® is an oil-in-water (O/W) nanoemulsion-based vaccine adjuvant that is often used in seasonal and pandemic influenza vaccines. We explored the feasibility of developing dry powders of vaccines adjuvanted with MF59 or AddaVax™, a preclinical grade equivalent of MF59 with the same composition and droplet size as MF59, by thin-film freeze-drying (TFFD). Liquid AddaVax alone was successfully converted to a dry powder by TFFD using trehalose as a stabilizing agent while maintaining the droplet size distribution of AddaVax after it was reconstituted. TFFD was then applied to convert liquid AddaVax-adjuvanted vaccines containing either a model antigen (e.g., ovalbumin) or mono-, bi-, and tri-valent recombinant hemagglutinin (rHA) protein-based H1 and/or H3 (universal) influenza vaccine candidates, as well as the MF59-containing Fluad® Quadrivalent influenza vaccine to dry powders. Both antigens and stabilizing agents affected the physical properties of the vaccines (e.g., mean particle size and particle size distribution) after the vaccines were subjected to TFFD. Importantly, the integrity and hemagglutination activity of the rHA antigens did not significantly change and the immunogenicity of reconstituted influenza vaccine candidates was maintained when evaluated in a mouse model. The vaccine dry powder was not sensitive to repeated freezing-and-thawing, in contrast to its liquid counterpart. It is concluded that TFFD can be applied to convert liquid vaccines containing MF59 or AddaVax to dry powders while maintaining the immunogenicity of the vaccines. Ultimately, TFFD technology may be used to prepare dry powders of multivalent universal influenza vaccines.

STING agonist-containing microparticles improve seasonal influenza vaccine efficacy and durability in ferrets over standard adjuvant.

The current pandemic highlights the need for effective vaccines against respiratory viruses. An ideal vaccine should induce robust and long-lasting responses with high manufacturing scalability. We use an adjuvant comprised of a Stimulator of Interferon Genes (STING) agonist incorporated in a scalable microparticle platform to achieve durable protection against the influenza virus. This formulation overcomes the challenges presented by the cytosolic localization of STING and the hydrophilicity of its agonists. We evaluated a monoaxial formulation of polymeric acetalated dextran microparticles (MPs) to deliver the STING agonist cyclic GMP-AMP (cGAMP) which achieved >10× dose-sparing effects compared to other published work. Efficacy was evaluated in ferrets, a larger animal model of choice for influenza vaccines. cGAMP MPs with recombinant hemagglutinin reduced viral shedding and improved vaccine outcomes compared to a seasonal influenza vaccine. Importantly, sustained protection against a lethal influenza infection was detected a year after a single dose of the vaccine-adjuvant.

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.