scholarly journals Design of the recombinant influenza neuraminidase antigen is crucial for protective efficacy

2021 ◽  
Author(s):  
Jin Gao ◽  
Laura Klenow ◽  
Lisa M. Parsons ◽  
Tahir Malik ◽  
Jie-Nie Phue ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Protective Efficacy ◽  
Molecular Size ◽  
Cell System ◽  
Enzymatic Properties ◽  
H1n1 Vaccine ◽  
Head Domain ◽  
Size Stability ◽  
The One ◽  
Rna Design

Supplementing influenza vaccines with recombinant neuraminidase (rNA) remains a promising approach for improving the suboptimal efficacy. However, correlations among rNA designs, properties, and protection have not been systematically investigated. Here, we performed a comparative analysis of several rNAs produced from different construct designs using the baculovirus/insect cell system. The rNAs were designed with different tetramerization motifs and NA domains from a recent H1N1 vaccine strain (A/Brisbane/02/2018) and were analyzed for enzymatic properties, antigenicity, thermal and size stability, and protection in mice. We found that rNAs containing the NA head-domain versus the full-ectodomain possess distinct enzymatic properties and that the molecular size stability is tetramerization domain-dependent, whereas protection is more contingent on the combination of the tetramerization and NA domains. Following single-dose immunizations, a rNA possessing the full-ectodomain, non-native enzymatic activity, and the tetramerization motif from the human vasodilator-stimulated phosphoprotein provided substantially higher protection than a rNA possessing the head-domain, native activity and the same tetramerization motif. In contrast, these two rNAs provided comparable protection when the tetramerization motif was exchanged with the one from the tetrabrachion protein. These findings demonstrate that the rNA design is crucial for the protective efficacy and should be thoroughly evaluated for vaccine development, as the unpredictable nature of the heterologous domain combination can result in rNAs with similar key attributes but vastly differ in protection.

Design of the Recombinant Influenza Neuraminidase Antigen is Crucial for its Biochemical Properties and Protective Efficacy

2021 ◽  
Author(s):  
Jin Gao ◽  
Laura Klenow ◽  
Lisa Parsons ◽  
Tahir Malik ◽  
Je-Nie Phue ◽  
...  
Keyword(s):  
Single Dose ◽  
Vaccine Development ◽  
Protective Efficacy ◽  
Biochemical Properties ◽  
Cell System ◽  
Influenza Vaccines ◽  
H1n1 Vaccine ◽  
Protective Properties ◽  
Head Domain ◽  
Tetramerization Domain

Supplementing influenza vaccines with recombinant neuraminidase (rNA) antigens remains a promising approach for improving the suboptimal vaccine efficacy. However, correlations among rNA designs, properties, and protection have not been systematically investigated. Here, we performed a comparative analysis of several rNAs produced using the baculovirus/insect cell system. The rNAs were designed with different tetramerization motifs and NA domains from a recent H1N1 vaccine strain (A/Brisbane/02/2018) and were compared for enzymatic property, antigenicity, stability, and protection in mice. We found that distinct enzymatic properties are associated with rNAs containing the NA head-domain versus the full-ectodomain, formation of higher order rNA oligomers is tetramerization domain-dependent, whereas protective efficacy is more contingent on the combination of the tetramerization and NA domains. Following single-dose immunizations, a rNA possessing the full-ectodomain and the tetramerization motif from the human vasodilator-stimulated phosphoprotein provided much better protection than a rNA with ∼10-fold more enzymatically active molecules that is comprised of the head-domain and the same tetramerization motif. In contrast, these two rNA designs provided comparable protection when the tetramerization motif from the tetrabrachion protein was used instead. These findings demonstrate that individual rNAs should be thoroughly evaluated for vaccine development, as the heterologous domain combination can result in rNAs with similar key attributes but vastly differ in protection. IMPORTANCE For several decades it has been proposed that influenza vaccines could be supplemented with recombinant neuraminidase (rNA) to improve the efficacy. However, some key questions for manufacturing stable and immunogenic rNA remain to be answered. We show here that the tetramerization motifs and NA domains included in the rNA construct design can have a profound impact on the biochemical, immunogenic and protective properties. We also show that the single-dose immunization regimen is more informative for assessing the rNA immune response and protective efficacy, which is surprisingly more dependent on the specific combination of NA and tetramerization domains than common attributes for evaluating NA. Our findings may help to optimize the design of rNAs that can be used to improve or develop influenza vaccines.

Recent progress in the freeze-etching technique

1971 ◽  
Vol 261 (837) ◽  
pp. 121-131 ◽  
Keyword(s):  
High Vacuum ◽  
Molecular Size ◽  
Etching Technique ◽  
Structural Distortions ◽  
Shadow Casting ◽  
Structural Details ◽  
Freeze Etching ◽  
The One ◽  
Insight Into ◽  
Made In

The freeze-etching technique must be improved if structures at the molecular size level are to be seen. The limitations of the technique are discussed here together with the progress made in alleviating them. The vitrification of living specimens is limited by the fact that very high freezing rates are needed. The critical freezing rate can be lowered on the one hand by the introduction of antifreeze agents, on the other hand by the application of high hydrostatic pressure. The fracture process may cause structural distortions in the fracture face of the frozen specimen. The ‘double-replica’ method allows one to evaluate such artefacts and provides an insight into the way that membranes split. During etching there exists the danger of contaminating the fracture faces with condensable gases. Because of specimen temperatures below —110 °C, special care has to be taken in eliminating water vapour from the high vacuum. An improvement in coating freeze-etched specimens has resulted from the application of electron guns for evaporation of the highest melting-point metals. If heat transfer from gun to specimen is reduced to a minimum, Pt, Ir, Ta, W and C can be used for shadow casting. Best results are obtained with Pt-C and Ta-W . With the help of decoration effects Pt-C shadow castings give the most information about the fine structural details of the specimen.

Broad neutralization of H1 and H3 viruses by adjuvanted influenza HA stem vaccines in nonhuman primates

2021 ◽  
Vol 13 (583) ◽  
pp. eabe5449
Author(s):  
Nicole Darricarrère ◽  
Yu Qiu ◽  
Masaru Kanekiyo ◽  
Adrian Creanga ◽  
Rebecca A. Gillespie ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Nonhuman Primates ◽  
Vaccine Development ◽  
Protective Efficacy ◽  
Neutralizing Antibody ◽  
Gene Families ◽  
Influenza Viruses ◽  
Public Health Importance ◽  
Water Emulsion

Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus–associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.

scholarly journals Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice

2017 ◽  
Vol 24 (11) ◽  
Author(s):  
Ahreum Kim ◽  
Yun-Gyoung Hur ◽  
Sunwha Gu ◽  
Sang-Nae Cho
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Vaccine Development ◽  
Protective Efficacy ◽  
Interleukin 2 ◽  
T Cell Epitopes ◽  
Content Type ◽  
Complete Form ◽  
Ifn Γ

ABSTRACT The aim of this study was to evaluate the protective efficacy of MTBK_24820, a complete form of PPE39 protein derived from a predominant Beijing/K strain of Mycobacterium tuberculosis in South Korea. Mice were immunized with MTKB_24820, M. bovis Bacilli Calmette-Guérin (BCG), or adjuvant prior to a high-dosed Beijing/K strain aerosol infection. After 4 and 9 weeks, bacterial loads were determined and histopathologic and immunologic features in the lungs and spleens of the M. tuberculosis-infected mice were analyzed. Putative immunogenic T-cell epitopes were examined using synthetic overlapping peptides. Successful immunization of MTBK_24820 in mice was confirmed by increased IgG responses (P < 0.05) and recalled gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-6, and IL-17 responses (P < 0.05 or P < 0.01) to MTBK_24820. After challenge with the Beijing/K strain, an approximately 0.5 to 1.0 log10 reduction in CFU in lungs and fewer lung inflammation lesions were observed in MTBK_24820-immunized mice compared to those for control mice. Moreover, MTBK_24820 immunization elicited significantly higher numbers of CD4+ T cells producing protective cytokines, such as IFN-γ and IL-17, in lungs and spleens (P < 0.01) and CD4+ multifunctional T cells producing IFN-γ, tumor necrosis factor alpha (TNF-α), and/or IL-17 (P < 0.01) than in control mice, suggesting protection comparable to that of BCG against the hypervirulent Beijing/K strain. The dominant immunogenic T-cell epitopes that induced IFN-γ production were at the N terminus (amino acids 85 to 102 and 217 to 234). Its vaccine potential, along with protective immune responses in vivo, may be informative for vaccine development, particularly in regions where the M. tuberculosis Beijing/K-strain is frequently isolated from TB patients.

scholarly journals CsrRS Regulates Group B Streptococcus Virulence Gene Expression in Response to Environmental pH: a New Perspective on Vaccine Development

2009 ◽  
Vol 191 (17) ◽  
pp. 5387-5397 ◽  
Author(s):  
Isabella Santi ◽  
Renata Grifantini ◽  
Sheng-Mei Jiang ◽  
Cecilia Brettoni ◽  
Guido Grandi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Factors ◽  
Vaccine Development ◽  
Ph Regulation ◽  
Protective Efficacy ◽  
Virulence Gene ◽  
Ph Sensing ◽  
Group B Streptococci ◽  
Virulence Gene Expression ◽  
Group B

ABSTRACT To identify factors involved in the response of group B streptococci (GBS) to environmental pH, we performed a comparative global gene expression analysis of GBS at acidic and neutral pHs. We found that the transcription of 317 genes was increased at pH 5.5 relative to that at pH 7.0, while 61 genes were downregulated. The global response to acid stress included the differential expression of genes involved in transport, metabolism, stress response, and virulence. Known vaccine candidates, such as BibA and pilus components, were also regulated by pH. We observed that many of the genes involved in the GBS response to pH are known to be controlled by the CsrRS two-component system. Comparison of the regulon of wild-type strain 2603 V/R with that of a csrRS deletion mutant strain revealed that the pH-dependent regulation of 90% of the downregulated genes and 59.3% of the up-regulated genes in strain 2603 V/R was CsrRS dependent and that many virulence factors were overexpressed at high pH. Beta-hemolysin regulation was abrogated by selective inactivation of csrS, suggesting the implication of the CsrS protein in pH sensing. These results imply that the translocation of GBS from the acidic milieu of the vagina to the neutral pH of the neonatal lung signals the up-regulation of GBS virulence factors and conversion from a colonizing to an invasive phenotype. In addition, the fact that increased exposure of BibA on the bacterial surface at pH 7.0 induced opsonophagocytic killing of GBS in immune serum highlights the importance of pH regulation in the protective efficacy of specific antibodies to surface-exposed GBS proteins.

scholarly journals Development and Applications of Viral Vectored Vaccines to Combat Zoonotic and Emerging Public Health Threats

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 680
Author(s):  
Sophia M. Vrba ◽  
Natalie M. Kirk ◽  
Morgan E. Brisse ◽  
Yuying Liang ◽  
Hinh Ly
Keyword(s):  
Public Health ◽  
Vaccine Development ◽  
Yellow Fever Virus ◽  
Protective Efficacy ◽  
Cost Effective ◽  
Vaccine Vectors ◽  
Advantages And Disadvantages ◽  
Viral Vaccine ◽  
Health Threats ◽  
Syncytial Virus

Vaccination is arguably the most cost-effective preventative measure against infectious diseases. While vaccines have been successfully developed against certain viruses (e.g., yellow fever virus, polio virus, and human papilloma virus HPV), those against a number of other important public health threats, such as HIV-1, hepatitis C, and respiratory syncytial virus (RSV), have so far had very limited success. The global pandemic of COVID-19, caused by the SARS-CoV-2 virus, highlights the urgency of vaccine development against this and other constant threats of zoonotic infection. While some traditional methods of producing vaccines have proven to be successful, new concepts have emerged in recent years to produce more cost-effective and less time-consuming vaccines that rely on viral vectors to deliver the desired immunogens. This review discusses the advantages and disadvantages of different viral vaccine vectors and their general strategies and applications in both human and veterinary medicines. A careful review of these issues is necessary as they can provide important insights into how some of these viral vaccine vectors can induce robust and long-lasting immune responses in order to provide protective efficacy against a variety of infectious disease threats to humans and animals, including those with zoonotic potential to cause global pandemics.

scholarly journals Multiparameter Selection of Helicobacter pylori Antigens Identifies Two Novel Antigens with High Protective Efficacy

2002 ◽  
Vol 70 (11) ◽  
pp. 6499-6503 ◽  
Author(s):  
N. Sabarth ◽  
R. Hurwitz ◽  
T. F. Meyer ◽  
D. Bumann
Keyword(s):  
Helicobacter Pylori ◽  
Protective Immunity ◽  
Vaccine Development ◽  
Protective Efficacy ◽  
Infection Model ◽  
Protective Antigens ◽  
High Yields ◽  
Selection Of

ABSTRACT A multiparameter selection of Helicobacter pylori antigens for vaccine development identified 15 candidates, 6 of which are known protective antigens. Two novel antigens with low homology to other organisms (HP0231 and HP0410) were overexpressed and purified with high yields. Both confer protective immunity in the mouse Helicobacter infection model.

scholarly journals Expression and Solubilization of Insect Cell-Based Rabies Virus Glycoprotein and Assessment of Its Immunogenicity and Protective Efficacy in Mice

2011 ◽  
Vol 18 (10) ◽  
pp. 1673-1679 ◽  
Author(s):  
R. Ramya ◽  
B. Mohana Subramanian ◽  
V. Sivakumar ◽  
R. L. Senthilkumar ◽  
K. R. S. Sambasiva Rao ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Insect Cell ◽  
Neutralizing Antibodies ◽  
Subunit Vaccine ◽  
Vaccine Development ◽  
Protective Efficacy ◽  
Vector System ◽  
Soluble Form ◽  
Rabies Virus Glycoprotein ◽  
Virus Glycoprotein

ABSTRACTRabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed inSpodoptera frugiperda(Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.

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