Development of next-generation influenza virus vaccines is crucial to improve protection against circulating and emerging viruses. Current vaccine formulations have to be updated annually due to mutations in seasonal strains and do not offer protection against strains with pandemic potential. Computationally optimized broadly reactive antigen (COBRA) methodology has been utilized by our group to generate broadly reactive immunogens for individual influenza subtypes, which elicit protective immune responses against a broad range of strains over numerous seasons. Octavalent mixtures of COBRA hemagglutinin (HA) (H1, H2, H3, H5, H7, and influenza B virus) plus neuraminidase (NA) (N1 and N2) recombinant proteins mixed with c-di-AMP adjuvant were administered intranasally to naive or pre-immune ferrets in prime-boost fashion. Four weeks after final vaccination, collected sera were analyzed for breadth of antibody response, and the animals were challenged with seasonal or pre-pandemic strains. The octavalent COBRA vaccine elicited antibodies that recognized a broad panel of strains representing different subtypes, and these vaccinated animals were protected against influenza virus challenges. Overall, this study demonstrated that the mixture of eight COBRA HA/NA proteins mixed with an intranasal adjuvant is a promising candidate for a universal influenza vaccine.
OBJECTIVE: Influenza is a respiratory virus which infects around a billion people globally every year, with millions experiencing severe illness. Commercial vaccine efficacy varies year to year and can be low due to mismatch of circulating virus strains. Thus, the formulation of current vaccines has to be adapted accordingly every year. The development of a broadly reactive influenza vaccine would lessen the global economic and public health burden caused by the different types of influenza viruses. The significance of our research is producing a promising universal vaccine candidate which provides protection against a wider range of virus strains over a wider range of time.

In humans, seasonal influenza viruses cause epidemics. Avian influenza viruses are of particular concern because they can infect multiple species and lead to unpredictable and severe disease. Therefore, there is an urgent need for a universal influenza vaccine that provides protection against all influenza strains. The cyclic GMP-AMP (cGAMP) is a promising adjuvant for subunit vaccines, which promotes type I interferons\' production through the stimulator of interferon genes (STING) pathway. The encapsulation of cGAMP in acetalated dextran (Ace-DEX) microparticles (MPs) enhances its intracellular delivery. In this study, the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology was used to generate H1, H3, and H5 vaccine candidates. Monovalent and multivalent COBRA HA vaccines formulated with cGAMP Ace-DEX MPs were evaluated in mice for protective antibody responses. cGAMP MPs adjuvanted COBRA HA vaccines elicited robust antigen-specific antibodies following vaccination. Compared with COBRA HA vaccine groups with no adjuvant or blank MPs, the cGAMP MPs enhanced HAI activity elicited by COBRA HA vaccines. The HAI activity was not significantly different between cGAMP MPs adjuvanted monovalent or multivalent COBRA HA vaccines. The cGAMP MPs adjuvanted COBRA vaccine groups had higher antigen-specific IgG2a-binding titers than the COBRA vaccine groups with no adjuvant or blank MPs. The COBRA vaccines formulated with cGAMP MPs mitigated diseases caused by influenza viral challenge and decreased pulmonary viral titers in mice. Therefore, the formulation of COBRA vaccines plus cGAMP MPs is a promising universal influenza vaccine that elicits protective immune responses against human seasonal and pre-pandemic strains.
OBJECTIVE: Influenza viruses cause severe respiratory disease, particularly in the very young and the elderly. Next-generation influenza vaccines are needed to protect against new influenza variants. This report used a promising adjuvant, cyclic GMP-AMP (cGAMP), to enhance the elicited antibodies by an improved influenza hemagglutinin candidate and protect against influenza virus infection. Overall, adding adjuvants to influenza vaccines is an effective method to improve vaccines.

Adjuvants enhance, prolong, and modulate immune responses by vaccine antigens to maximize protective immunity and enable more effective immunization in the young and elderly. Most adjuvants are formulated with injectable vaccines. However, an intranasal route of vaccination may induce mucosal and systemic immune responses for enhancing protective immunity in individuals and be easier to administer compared to injectable vaccines. In this study, a next generation of broadly-reactive influenza hemagglutinin (HA) vaccines were developed using the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology. These HA vaccines were formulated with Mastoparan 7 (M7-NH2) mast cell degranulating peptide adjuvant and administered intranasally to determine vaccine-induced seroconversion of antibodies against a panel of influenza viruses and protection following infection with H1N1 and H3N2 viruses in mice. Mice vaccinated intranasally with M7-NH2-adjuvanted COBRA HA vaccines had high HAIs against a panel of H1N1 and H3N2 influenza viruses and were protected against both morbidity and mortality, with reduced viral lung titers, following challenge with an H1N1 influenza virus. Additionally, M7-NH2 adjuvanted COBRA HA vaccines induced Th2 skewed immune responses with robust IgG and isotype antibodies in the serum and mucosal lung lavages. Overall, this intranasally delivered M7-NH2 -adjuvanted COBRA HA vaccine provides effective protection against drifted H1N1 and H3N2 viruses.

Angiosperm trees usually develop tension wood (TW) in response to gravitational stimulation. TW comprises abundant gelatinous (G-) fibers with thick G-layers primarily composed of crystalline cellulose. Understanding the pivotal factors governing G-layer formation in TW fiber remains elusive. This study elucidates the role of a Populus trichocarpa COBRA family protein, PtrCOB3, in the G-layer formation of TW fibers. PtrCOB3 expression was upregulated, and its promoter activity was enhanced during TW formation. Comparative analysis with wild-type trees revealed that ptrcob3 mutants, mediated by Cas9/gRNA gene editing, were incapable of producing G-layers within TW fibers and showed severely impaired stem lift. Fluorescence immunolabeling data revealed a dearth of crystalline cellulose in the tertiary cell wall (TCW) of ptrcob3 TW fibers. The role of PtrCOB3 in G-layer formation is contingent upon its native promoter, as evidenced by the comparative phenotypic assessments of pCOB11::PtrCOB3, pCOB3::PtrCOB3, and pCOB3::PtrCOB11 transgenic lines in the ptrcob3 background. Overexpression of PtrCOB3 under the control of its native promoter expedited G-layer formation within TW fibers. We further identified 3 transcription factors that bind to the PtrCOB3 promoter and positively regulate its transcriptional levels. Alongside the primary TCW synthesis genes, these findings enable the construction of a 2-layer transcriptional regulatory network for the G-layer formation of TW fibers. Overall, this study uncovers mechanistic insight into TW formation, whereby a specific COB protein executes the deposition of cellulose, and consequently, G-layer formation within TW fibers.

Influenza outbreaks are a major burden worldwide annually. While seasonal vaccines do provide protection against infection, they are limited in that they need to be updated every year to account for the constantly mutating virus. Recently, lipid nanoparticles (LNPs) encapsulating mRNA have seen major success as a vaccine platform for SARS-CoV-2. Herein, we applied LNPs to deliver an mRNA encoding a computationally optimized broadly active (COBRA) influenza immunogen. These COBRA mRNA LNPs induced a broadly active neutralizing antibody response and protection after lethal influenza challenge. To further increase the immunogenicity of the COBRA mRNA LNPs, we combined them with acetalated dextran microparticles encapsulating a STING agonist. Contrary to recent findings, the STING agonist decreased the immunogenicity of the COBRA mRNA LNPs which was likely due to a decrease in mRNA translation as shown in vitro. Overall, this work aids in future selection of adjuvants to use with mRNA LNP vaccines.

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.

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine.

Adjuvants enhance immune responses stimulated by vaccines. To date, many seasonal influenza vaccines are not formulated with an adjuvant. In the present study, the adjuvant Advax-SM™ was combined with next generation, broadly reactive influenza hemagglutinin (HA) vaccines that were designed using a computationally optimized broadly reactive antigen (COBRA) methodology. Advax-SM™ is a novel adjuvant comprising inulin polysaccharide and CpG55.2, a TLR9 agonist. COBRA HA vaccines were combined with Advax-SM™ or a comparator squalene emulsion (SE) adjuvant and administered to mice intramuscularly. Mice vaccinated with Advax-SM™ adjuvanted COBRA HA vaccines had increased serum levels of anti-influenza IgG and IgA, high hemagglutination inhibition activity against a panel of H1N1 and H3N2 influenza viruses, and increased anti-influenza antibody secreting cells isolated from spleens. COBRA HA plus Advax-SM™ immunized mice were protected against both morbidity and mortality following viral challenge and, at postmortem, had no detectable lung viral titers or lung inflammation. Overall, the Advax-SM™-adjuvanted COBRA HA formulation provided effective protection against drifted H1N1 and H3N2 influenza viruses.

Snakebite is a neglected public health issue, with many scientific and medical issues to be solved. Cobras are among the most common venomous snakes in Myanmar and are responsible for a considerable number of severe snakebite envenoming. There are three species of cobra (Naja kaouthia, Naja mandalayensis and Ophiophagus hannah) in Myanmar. The study aims to characterize the N. kaouthia and N. mandalayensis venoms and to investigate the efficacy of anti-cobra antivenom (BPI) against the two venoms. Protein components and fibrinogenolytic activity were determined by SDS-PAGE. Enzymatic activities for PLA2, protease and acetylcholinesterase were determined by spectrophotometric method. Anticoagulant activity was determined by recalcification time of citrated human plasma. Myotoxicity, necrotizing activity, median lethal dose (LD50) and median effective dose (ED50) were determined by WHO recommended methods. The SDS-PAGE displayed the proteins and enzymes containing in two venoms were different. N. kaouthia venom exhibited more in PLA2, acetylcholinesterase, anticoagulant, fibrinogenolytic and necrotizing activities than N. mandalayensis venom. N. mandalayensis venom had more protease activity and myotoxicity than N. kaouthia venom. The median lethal dose (LD50) of N. kaouthia and N. mandalayensis venom was 4.33 μg/mouse and 5.04 μg/mouse respectively. Both venoms induced fibrinogen Aα chain degradation in 30 min (N. kaouthia) and in 6 h (N. mandalayensis). The same median effective dose (ED50) (19.56 μg/mouse) showed that anti-NK antivenom can neutralize against lethal effect of N. mandalayensis venom. It can also neutralize the protease activity, anticoagulant activity and fibrinogenolytic activity of both venoms. Immunodiffusion and immunoblotting studies showed that the antivenom recognized its homologous venom (N. kaouthia) and cross-reacted against the heterologous venom (N. mandalayensis). The anti-NK antivenom is suitable to use for N. mandalayensis bite if monospecific antivenom is not available.

Influenza viruses cause a common respiratory disease known as influenza. In humans, seasonal influenza viruses can lead to epidemics, with avian influenza viruses of particular concern because they can infect multiple species and lead to unpredictable and severe disease. Therefore, there is an urgent need for a universal influenza vaccine that provides protection against seasonal and pre-pandemic influenza virus strains. The cyclic GMP-AMP (cGAMP) is a promising adjuvant for subunit vaccines that promotes type I interferons production through the stimulator of interferon genes (STING) pathway. The encapsulation of cGAMP in acetalated dextran (Ace-DEX) microparticles (MPs) enhances its intracellular delivery. In this study, the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology was used to generate H1, H3, and H5 vaccine candidates. Monovalent and multivalent COBRA HA vaccines formulated with cGAMP Ace-DEX MPs were evaluated in a mouse model for antibody responses and protection against viral challenge. Serological analysis showed that cGAMP MPs adjuvanted monovalent and multivalent COBRA vaccines elicited robust antigen-specific antibody responses after a prime-boost vaccination and antibody titers were further enhanced after second boost. Compared to COBRA vaccine groups with no adjuvant or blank MPs, the cGAMP MPs enhanced HAI antibody responses against COBRA vaccination. The HAI antibody titers were not significantly different between cGAMP MPs adjuvanted monovalent and multivalent COBRA vaccine groups for most of the viruses tested in panels. The cGAMP MPs adjuvanted COBRA vaccines groups had higher antigen-specific IgG2a binding titers than the COBRA vaccine groups with no adjuvant or blank MPs. The COBRA vaccines formulated with cGAMP MPs mitigated disease caused by influenza viral challenge and decreased pulmonary viral titers in mice. Therefore, the formulation of COBRA vaccines plus cGAMP MPs is a promising universal influenza vaccine that elicits protective immune responses against human seasonal and pre-pandemic strains.