scholarly journals Oligomeric state of the ZIKV E protein defines protective immune responses

2019 ◽  
Author(s):  
Stefan W. Metz ◽  
Ashlie Thomas ◽  
Alex Brackbill ◽  
John Forsberg ◽  
Michael J. Miley ◽  
...  
Keyword(s):  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Quaternary Structure ◽  
Poor Performance ◽  
Protein Subunit ◽  
Oligomeric State ◽  
Subunit Vaccines ◽  
Safety Issues ◽  
E Protein ◽  
Domain Iii

AbstractThe current leading Zika vaccine candidates in clinical testing are based on live or killed virus platforms, which have safety issues, especially in pregnant women. Zika subunit vaccines, however, have shown poor performance in preclinical studies. We hypothesized that Zika Envelope (E) protein subunit vaccines have performed poorly because the antigens tested have been recombinant E monomers that do not display critical quaternary structure epitopes present on Zika E protein homodimers that cover the surface of the virus. To test this hypothesis, we engineered and produced stable recombinant E protein homodimers. Unlike the E monomer, the dimer was recognized by strongly neutralizing monoclonal antibodies isolated from Zika-immune individuals. In a mouse model of vaccination, the dimeric antigen stimulated strongly neutralizing antibodies that targeted epitopes that were similar to epitopes recognized by human antibodies following natural Zika virus infection. In contrast, the monomer antigen stimulated lower levels of neutralizing antibodies directed to simple epitopes on domain III of E protein. In a mouse model of ZIKV challenge, only E dimer antigen stimulated protective antibodies, not the monomer. These results highlight the importance of mimicking the highly structured flavivirus surface when designing subunit vaccines. The flavivirus field has a long history of using E monomers as vaccine antigens with limited success. These results are applicable to developing second generation subunit vaccines against Zika as well as other medically important flaviviruses such as dengue and yellow fever viruses.

scholarly journals Oligomeric state of the ZIKV E protein defines protective immune responses

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Stefan W. Metz ◽  
Ashlie Thomas ◽  
Alex Brackbill ◽  
John Forsberg ◽  
Michael J. Miley ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Quaternary Structure ◽  
Poor Performance ◽  
Oligomeric State ◽  
Subunit Vaccines ◽  
Safety Issues ◽  
E Protein ◽  
Protective Antibodies ◽  
Low Levels ◽  
Domain Iii

Abstract The current leading Zika vaccine candidates in clinical testing are based on live or killed virus platforms, which have safety issues, especially in pregnant women. Zika subunit vaccines, however, have shown poor performance in preclinical studies, most likely because the antigens tested do not display critical quaternary structure epitopes present on Zika E protein homodimers that cover the surface of the virus. Here, we produce stable recombinant E protein homodimers that are recognized by strongly neutralizing Zika specific monoclonal antibodies. In mice, the dimeric antigen stimulate strongly neutralizing antibodies that target epitopes that are similar to epitopes recognized by human antibodies following natural Zika virus infection. The monomer antigen stimulates low levels of E-domain III targeting neutralizing antibodies. In a Zika challenge model, only E dimer antigen stimulates protective antibodies, not the monomer. These results highlight the importance of mimicking the highly structured flavivirus surface when designing subunit vaccines.

scholarly journals Dimerization of Dengue Virus E Subunits Impacts Antibody Function and Domain Focus

2020 ◽  
Vol 94 (18) ◽  
Author(s):  
Ashlie Thomas ◽  
Devina J. Thiono ◽  
Stephan T. Kudlacek ◽  
John Forsberg ◽  
Lakshmanane Premkumar ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Virus Vaccine ◽  
Neutralizing Antibodies ◽  
Quaternary Structure ◽  
Protein Structures ◽  
Protein Subunit ◽  
Subunit Vaccines ◽  
E Protein ◽  
Vaccine Antigens ◽  
Viral Surface

ABSTRACT Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers. IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines.

scholarly journals Biomaterials and oxygen join forces to shape the immune response and boost SARS-CoV-2 vaccines

2020 ◽  
Author(s):  
Thibault Colombani ◽  
Loek Eggermont ◽  
Zachary Rogers ◽  
Lindsay McKay ◽  
Laura Avena ◽  
...  
Keyword(s):  
Immune Response ◽  
Neutralizing Antibodies ◽  
High Titer ◽  
Full Potential ◽  
Protein Subunit ◽  
Subunit Vaccines ◽  
Health Crisis ◽  
Th2 Immune Response ◽  
Protective Antibodies ◽  
Advanced Biomaterials

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an unprecedented global health crisis, resulting in a critical need for effective vaccines that generate protective antibodies. Protein subunit vaccines represent a promising approach but often lack the immunogenicity required for strong immune stimulation. To overcome this challenge, we first demonstrate that advanced biomaterials boost effectiveness of SARS-CoV-2 protein subunit vaccines. Additionally, we report that oxygen is a powerful immunological co-adjuvant, a game-changer in the field for unlocking the full potential of vaccines. Mice immunized with oxygen-generating cryogel vaccines exhibited a robust and balanced Th1 and Th2 immune response, leading to sustained and high titer production of neutralizing antibodies against SARS-CoV-2. Our data indicate that this platform is a revolutionary technology with the potential to reinforce any vaccine.

scholarly journals Neuroadapted Yellow Fever Virus 17D: Determinants in the Envelope Protein Govern Neuroinvasiveness for SCID Mice

2003 ◽  
Vol 77 (22) ◽  
pp. 12232-12242 ◽  
Author(s):  
Michael Nickells ◽  
Thomas J. Chambers
Keyword(s):  
Mouse Model ◽  
Yellow Fever ◽  
Scid Mouse ◽  
Yellow Fever Virus ◽  
Scid Mice ◽  
Survival Times ◽  
E Protein ◽  
Average Survival ◽  
Scid Mouse Model ◽  
Domain Iii

ABSTRACT A molecular clone of mouse-neuroadapted yellow fever 17D virus (SPYF-MN) was used to identify critical determinants of viral neuroinvasiveness in a SCID mouse model. Virus derived from this clone differs from nonneuroinvasive YF5.2iv virus at 29 nucleotide positions, encoding 13 predicted amino acid substitutions and 2 substitutions in the 3′ untranslated region (UTR). The virulence determinants of SPYF-MN for SCID mice were identified by constructing and characterizing intratypic viruses in which the E protein of SPYF-MN was expressed in the YF5.2iv background (SPYF-E) or the E protein of YF5.2iv was expressed in the SPYF-MN background (YF5.2-E). SPYF-E caused lethal encephalitis in young adult SCID mice after intraperitoneal inoculation, with average survival times and tissue virus burdens resembling those of mice inoculated with the parental SPYF-MN virus. To define which domains of the E protein are involved in neuroinvasiveness, two viruses were tested in which the amino acid substitutions in domains I-II and III were segregated. This revealed that substitutions in domain III (residues 305, 326, and 380) were critical for the neuroinvasive phenotype, based on average survival times and tissue burdens of infectious virus. Comparison of growth properties of the various intratypic viruses in cell culture indicated that no inherent defects in replication efficiency were likely to account for the biological differences observed in these experiments. These findings demonstrate that the E protein is a critical factor for yellow fever virus neuropathogenesis in the SCID mouse model and that the neuroinvasive properties depend principally on functions contributed by domain III of this protein. To assess whether critical determinants for neuroinvasion of normal ICR mice by SPYF virus were also in the E protein, sequences of viruses recovered from brains of ICR mice succumbing to encephalitis with the parental SPYF virus were derived. No differences were found in the E protein; however, two substitutions were present in the 3′ UTR compared to that of SPYF-MN, one of which is predicted to alter RNA secondary structure in this region. These findings suggest that the 3′ UTR may also affect neuroinvasiveness of SPYF virus in the mouse model.

scholarly journals Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization

2021 ◽  
Author(s):  
Hannah L. Turner ◽  
Raiees Andrabi ◽  
Christopher A. Cottrell ◽  
Sara T. Richey ◽  
Ge Song ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Envelope Glycoprotein ◽  
Neutralizing Antibodies ◽  
Epitope Mapping ◽  
Antibody Responses ◽  
Viral Fusion ◽  
Protein Subunit ◽  
Subunit Vaccines ◽  
Hiv Envelope ◽  
Viral Surface

AbstractRationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2 and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non or weakly neutralizing (“off-target”) antibodies. Using our recently developed electron microscopy epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.

scholarly journals Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment immunogen

2020 ◽  
Author(s):  
Sameer Kumar Malladi ◽  
Randhir Singh ◽  
Suman Pandey ◽  
Savitha Gayathri ◽  
Kawkab Kanjo ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Viral Vectors ◽  
Mammalian Cell Culture ◽  
Geometric Mean ◽  
High Yield ◽  
Great Promise ◽  
Protein Subunit ◽  
Protein Fragment ◽  
Subunit Vaccines ◽  
Major Impediment

AbstractVirtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel, nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100°C when lyophilized, upto 70°C in solution and stable for over four weeks at 37°C. In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant AddaVax™, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

scholarly journals Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

2020 ◽  
pp. jbc.RA120.016284
Author(s):  
Sameer Kumar Malladi ◽  
Randhir Singh ◽  
Suman Pandey ◽  
Savitha Gayathri ◽  
Kawkab Kanjo ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Viral Vectors ◽  
Mammalian Cell Culture ◽  
Geometric Mean ◽  
High Yield ◽  
Great Promise ◽  
Protein Subunit ◽  
Protein Fragment ◽  
Subunit Vaccines ◽  
Major Impediment

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214 mg/L in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over four weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax™, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

scholarly journals Genotype-Specific Neutralization and Protection by Antibodies against Dengue Virus Type 3

2010 ◽  
Vol 84 (20) ◽  
pp. 10630-10643 ◽  
Author(s):  
James D. Brien ◽  
S. Kyle Austin ◽  
Soila Sukupolvi-Petty ◽  
Katie M. O'Brien ◽  
Syd Johnson ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Surface Display ◽  
Viral Envelope ◽  
Yeast Surface Display ◽  
E Protein ◽  
Lateral Ridge ◽  
Dengue Virus Type 3 ◽  
Domain Iii ◽  
Positive Strand Rna ◽  
Type 3

ABSTRACT Dengue viruses (DENV) comprise a family of related positive-strand RNA viruses that infect up to 100 million people annually. Currently, there is no approved vaccine or therapy to prevent infection or diminish disease severity. Protection against DENV is associated with the development of neutralizing antibodies that recognize the viral envelope (E) protein. Here, with the goal of identifying monoclonal antibodies (MAbs) that can function as postexposure therapy, we generated a panel of 82 new MAbs against DENV-3, including 24 highly neutralizing MAbs. Using yeast surface display, we localized the epitopes of the most strongly neutralizing MAbs to the lateral ridge of domain III (DIII) of the DENV type 3 (DENV-3) E protein. While several MAbs functioned prophylactically to prevent DENV-3-induced lethality in a stringent intracranial-challenge model of mice, only three MAbs exhibited therapeutic activity against a homologous strain when administered 2 days after infection. Remarkably, no MAb in our panel protected prophylactically against challenge by a strain from a heterologous DENV-3 genotype. Consistent with this, no single MAb neutralized efficiently the nine different DENV-3 strains used in this study, likely because of the sequence variation in DIII within and between genotypes. Our studies suggest that strain diversity may limit the efficacy of MAb therapy or tetravalent vaccines against DENV, as neutralization potency generally correlated with a narrowed genotype specificity.

scholarly journals Neuroadapted Yellow Fever Virus Strain 17D: a Charged Locus in Domain III of the E Protein Governs Heparin Binding Activity and Neuroinvasiveness in the SCID Mouse Model

2008 ◽  
Vol 82 (24) ◽  
pp. 12510-12519 ◽  
Author(s):  
Janice Nickells ◽  
Maria Cannella ◽  
Deborah A. Droll ◽  
Yan Liang ◽  
William S. M. Wold ◽  
...  
Keyword(s):  
Mouse Model ◽  
Yellow Fever ◽  
Envelope Protein ◽  
Scid Mouse ◽  
Yellow Fever Virus ◽  
Binding Activity ◽  
Heparin Binding ◽  
E Protein ◽  
Scid Mouse Model ◽  
Domain Iii

ABSTRACT A molecular clone of yellow fever virus (YFV) strain 17D was used to identify critical determinants of mouse neuroinvasiveness previously localized to domain III of the neuroadapted SPYF-MN virus envelope protein. Three candidate virulence substitutions (305F→V, 326K→E, and 380R→T) were individually evaluated for their roles in this phenotype in a SCID mouse model. The virus containing a glutamic acid residue at position 326 of the envelope protein (326E) caused rapidly lethal encephalitis, with a mortality rate and average survival time resembling those of the parental SPYF-MN virus. Determinants at positions 380 (380T) and 305 (305V) did not independently affect neuroinvasiveness. Testing a panel of viruses with various amino acid substitutions at position 326 revealed that attenuation of neuroinvasiveness required a positively charged residue (lysine or arginine) at this position. Molecular-modeling studies suggest that residues 326 and 380 contribute to charge clusters on the lateral surface of domain III that constitute putative heparin binding sites, as confirmed by studies of heparin inhibition of plaque formation. The neuroinvasiveness of YFVs in the SCID model correlated inversely with sensitivity to heparin. These findings establish that residue 326 in domain III of the E protein is a critical determinant of YFV neuroinvasiveness in the SCID mouse model. Together with modeling of domain III from virulent YFV strains, the data suggest that heparin binding activity involving lysine at position 326 may be a modulator of YFV virulence phenotypes.

Eliciting cross-neutralizing antibodies in mice challenged with a dengue virus envelope domain III expressed inEscherichia coli

2012 ◽  
Vol 58 (4) ◽  
pp. 369-380 ◽  
Author(s):  
Jie Yang ◽  
Junlei Zhang ◽  
Wei Chen ◽  
Zhen Hu ◽  
Junmin Zhu ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Exchange Chromatography ◽  
Global Public Health ◽  
Virus Envelope ◽  
Virus Challenge ◽  
Denv Serotypes ◽  
E Protein ◽  
Domain Iii ◽  
Neutralizing Epitopes

Dengue viruses (DENVs) are mosquito-borne infectious pathogens that pose a serious global public health threat, and at present, no therapy or effective vaccines are available. Choosing suitable units as candidates is fundamental for the development of a dengue subunit vaccine. Domain III of the DENV-2 E protein (EDIII) was chosen in the present study and expressed in Escherichia coli by N-terminal fusion to a bacterial leader (pelB), and C-terminal fusion with a 6×His tag based on the functions of DENV structure proteins, especially the neutralizing epitopes on the envelope E protein. After two-step purification using Ni–NTA affinity and cation-exchange chromatography, the His-tagged EDIII was purified up to 98% homogenicity. This recombinant EDIII was able to trigger high levels of neutralizing antibodies in both BALB/c and C57BL/6 mice. Both the recombinant EDIII and its murine antibodies protected Vero cells from DENV-2 infection. Interestingly, the recombinant EDIII provides at least partial cross-protection against DENV-1 infection. In addition, the EDIII antibodies were able to protect suckling mice from virus challenge in vivo. These data suggest that a candidate molecule based on the small EDIII protein, which has neutralizing epitopes conserved among all 4 DENV serotypes, has important implications.

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