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

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.

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Emerging Promise of Immunotherapy for Alzheimer’s Disease: A New Hope for the Development of Alzheimer’s Vaccine

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200422105156 ◽

2020 ◽

Vol 20 (13) ◽

pp. 1214-1234 ◽

Cited By ~ 4

Author(s):

Md. Tanvir Kabir ◽

Md. Sahab Uddin ◽

Bijo Mathew ◽

Pankoj Kumar Das ◽

Asma Perveen ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Immune Response ◽

Neutralizing Antibodies ◽

Neurodegenerative Disorder ◽

Symptomatic Relief ◽

Aβ Oligomers ◽

Th2 Immune Response ◽

Age Related ◽

Anti Inflammatory

Background: Alzheimer's disease (AD) is a chronic neurodegenerative disorder and the characteristics of this devastating disorder include the progressive and disabling deficits in the cognitive functions including reasoning, attention, judgment, comprehension, memory, and language. Objective: In this article, we have focused on the recent progress that has been achieved in the development of an effective AD vaccine. Summary: Currently, available treatment options of AD are limited to deliver short-term symptomatic relief only. A number of strategies targeting amyloid-beta (Aβ) have been developed in order to treat or prevent AD. In order to exert an effective immune response, an AD vaccine should contain adjuvants that can induce an effective anti-inflammatory T helper 2 (Th2) immune response. AD vaccines should also possess the immunogens which have the capacity to stimulate a protective immune response against various cytotoxic Aβ conformers. The induction of an effective vaccine’s immune response would necessitate the parallel delivery of immunogen to dendritic cells (DCs) and their priming to stimulate a Th2-polarized response. The aforesaid immune response is likely to mediate the generation of neutralizing antibodies against the neurotoxic Aβ oligomers (AβOs) and also anti-inflammatory cytokines, thus preventing the AD-related inflammation. Conclusion: Since there is an age-related decline in the immune functions, therefore vaccines are more likely to prevent AD instead of providing treatment. AD vaccines might be an effective and convenient approach to avoid the treatment-related huge expense.

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Oligomeric state of the ZIKV E protein defines protective immune responses

Nature Communications ◽

10.1038/s41467-019-12677-6 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

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

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Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2

10.1101/2021.02.27.433054 ◽

2021 ◽

Author(s):

Chotiwat Seephetdee ◽

Nattawut Buasri ◽

Kanit Bhukhai ◽

Kitima Srisanga ◽

Suwimon Manopwisedjaroen ◽

...

Keyword(s):

Low Income ◽

Neutralizing Antibodies ◽

High Titer ◽

Physical Property ◽

Cost Effective ◽

Low Income Countries ◽

Subunit Vaccines ◽

Neutralization Activity ◽

Boost Regimen ◽

Global Logistics

Updated and revised versions of COVID-19 vaccines are vital due to genetic variations of the SARS-CoV-2 spike antigen. Furthermore, vaccines that are safe, cost-effective, and logistically friendly are critically needed for global equity, especially for middle to low income countries. Recombinant protein-based subunit vaccines against SARS-CoV-2 have been reported with the use of the receptor binding domain (RBD) and the prefusion spike trimers (S-2P). Recently, a new version of prefusion spike trimers, so called "HexaPro", has been shown for its physical property to possess two RBD in the "up" conformation, as opposed to just one exposed RBD found in S-2P. Importantly, this HexaPro spike antigen is more stable than S-2P, raising its feasibility for global logistics and supply chain. Here, we report that the spike protein HexaPro offers a promising candidate for SARS-CoV-2 vaccine. Mice immunized by the recombinant HexaPro adjuvanted with aluminium hydroxide using a prime-boost regimen produced high-titer neutralizing antibodies for up to 56 days after initial immunization against live SARS-CoV-2 infection. In addition, the level of neutralization activity is comparable to that of convalescence sera. Our results indicate that the HexaPro subunit vaccine confers neutralization activity in sera collected from mice receiving the prime-boost regimen.

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

10.1101/2021.02.16.431310 ◽

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.

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

10.1101/2020.08.15.252437 ◽

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016284 ◽

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.

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Fusion to C3d Enhances the Immunogenicity of the E2 Glycoprotein of Type 2 Bovine Viral Diarrhea Virus

Journal of Virology ◽

10.1128/jvi.78.4.1616-1622.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 1616-1622 ◽

Cited By ~ 23

Author(s):

Lingshu Wang ◽

J. Oriol Sunyer ◽

Leonard J. Bello

Keyword(s):

Immune Response ◽

Bovine Viral Diarrhea Virus ◽

Neutralizing Antibodies ◽

Primary Immune Response ◽

Enzyme Linked Immunosorbent Assay ◽

Bovine Viral Diarrhea ◽

Protein Subunit ◽

Diarrhea Virus ◽

Viral Diarrhea ◽

Viral Diarrhea Virus

ABSTRACT The use of DNA and protein subunit vaccines in animals provides an opportunity to introduce vaccines that are arguably the safest that can be developed. For that reason, considerable effort is under way to devise methods of enhancing the immunogenicity of such vaccines. Seven years ago it was shown that fusing complement fragment C3d to hen egg lysozyme (HEL) enhanced the immunogenicity of HEL 10,000-fold. Based on this observation, we decided to evaluate the effect of C3d on the immunogenicity of the E2 protein of bovine viral diarrhea virus (BVDV). E2 is the major target of neutralizing antibody during BVDV infection. To test the effect of C3d on E2 immunogenicity, expression cassettes encoding a secreted form of E2 alone (E2s) or E2 fused to three copies of murine C3d (E2s-C3d) were constructed. The proteins were purified from the supernatants of transfected cells and used to immunize mice. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA) for E2s-specific antibody and by a virus neutralization test. The ELISA results indicated that the E2s-C3d protein is 10,000-fold more immunogenic than the E2s protein alone. The maximum primary immune response was elicited with <0.1 μg of E2s-C3d protein without an adjuvant. In addition, we have shown for the first time that high levels of anti-E2s and neutralizing antibodies can be elicited when this same low concentration of E2s-C3d is used to both prime and boost the immune response. We conclude that the E2s-C3d fusion protein has significant potential as a subunit vaccine against BVDV infection.

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

Journal of Virology ◽

10.1128/jvi.00745-20 ◽

2020 ◽

Vol 94 (18) ◽

Cited By ~ 1

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.

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Mice Immunized with the Vaccine Candidate HexaPro Spike Produce Neutralizing Antibodies against SARS-CoV-2

Vaccines ◽

10.3390/vaccines9050498 ◽

2021 ◽

Vol 9 (5) ◽

pp. 498

Author(s):

Chotiwat Seephetdee ◽

Nattawut Buasri ◽

Kanit Bhukhai ◽

Kitima Srisanga ◽

Suwimon Manopwisedjaroen ◽

...

Keyword(s):

Low Income ◽

Neutralizing Antibodies ◽

High Titer ◽

Physical Property ◽

Cost Effective ◽

Low Income Countries ◽

Subunit Vaccines ◽

Neutralization Activity ◽

Boost Regimen ◽

Global Logistics

Updated and revised versions of COVID-19 vaccines are vital due to genetic variations of the SARS-CoV-2 spike antigen. Furthermore, vaccines that are safe, cost-effective, and logistic-friendly are critically needed for global equity, especially for middle- to low-income countries. Recombinant protein-based subunit vaccines against SARS-CoV-2 have been reported using the receptor-binding domain (RBD) and the prefusion spike trimers (S-2P). Recently, a new version of prefusion spike trimers, named HexaPro, has been shown to possess two RBD in the “up” conformation, due to its physical property, as opposed to just one exposed RBD found in S-2P. Importantly, this HexaPro spike antigen is more stable than S-2P, raising its feasibility for global logistics and supply chain. Here, we report that the spike protein HexaPro offers a promising candidate for the SARS-CoV-2 vaccine. Mice immunized by the recombinant HexaPro adjuvanted with aluminum hydroxide using a prime-boost regimen produced high-titer neutralizing antibodies for up to 56 days after initial immunization against live SARS-CoV-2 infection. Also, the level of neutralization activity is comparable to that of convalescence sera. Our results indicate that the HexaPro subunit vaccine confers neutralization activity in sera collected from mice receiving the prime-boost regimen.

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Oligomeric state of the ZIKV E protein defines protective immune responses

10.1101/674424 ◽

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.

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