Recombinant Antigens Based on Non-Glycosylated Regions from RBD SARS-CoV-2 as Potential Vaccine Candidates against COVID-19

The Receptor-Binding Domain (RBD) of the Spike (S) protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has glycosylation sites which can limit the production of reliable antigens expressed in prokaryotic platforms, due to glycan-mediated evasion of the host immune response. However, protein regions without glycosylated residues capable of inducing neutralizing antibodies could be useful for antigen production in systems that do not carry the glycosylation machinery. To test this hypothesis, the potential antigens NG06 and NG19, located within the non-glycosylated S-RBD region, were selected and expressed in Escherichia coli, purified by FPLC and employed to determine their immunogenic potential through detection of antibodies in serum from immunized rabbits, mice, and COVID-19 patients. IgG antibodies from sera of COVID-19-recovered patients detected the recombinant antigens NG06 and NG19 (A450 nm = 0.80 ± 0.33; 1.13 ± 0.33; and 0.11 ± 0.08 for and negatives controls, respectively). Also, the purified antigens were able to raise polyclonal antibodies in animal models evoking a strong immune response with neutralizing activity in mice model. This research highlights the usefulness of antigens based on the non-N-glycosylated region of RBD from SARS-CoV-2 for candidate vaccine development.

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Annotation of Potential Vaccine Targets and Design of a Multi-Epitope Subunit Vaccine against Yersinia pestis through Reverse Vaccinology and Validation through an Agent-Based Modeling Approach

Vaccines ◽

10.3390/vaccines9111327 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1327

Author(s):

Azaz Ul Haq ◽

Abbas Khan ◽

Jafar Khan ◽

Shamaila Irum ◽

Yasir Waheed ◽

...

Keyword(s):

Immune Response ◽

Yersinia Pestis ◽

Vaccine Development ◽

Dynamic Properties ◽

Dynamics Simulation ◽

Strong Immune Response ◽

Agent Based ◽

Aliphatic Index ◽

Structural Vaccinology ◽

Potential Vaccine

Yersinia pestis is responsible for plague and major pandemics in Asia and Europe. This bacterium has shown resistance to an array of drugs commonly used for the treatment of plague. Therefore, effective therapeutics measurements, such as designing a vaccine that can effectively and safely prevent Y. pestis infection, are of high interest. To fast-track vaccine development against Yersinia pestis, herein, proteome-wide vaccine target annotation was performed, and structural vaccinology-assisted epitopes were predicted. Among the total 3909 proteins, only 5 (rstB, YPO2385, hmuR, flaA1a, and psaB) were shortlisted as essential vaccine targets. These targets were then subjected to multi-epitope vaccine design using different linkers. EAAK, AAY, and GPGPG as linkers were used to link CTL, HTL, and B-cell epitopes, and an adjuvant (beta defensin) was also added at the N-terminal of the MEVC. Physiochemical characterization, such as determination of the instability index, theoretical pI, half-life, aliphatic index, stability profiling, antigenicity, allergenicity, and hydropathy of the ensemble, showed that the vaccine is highly stable, antigenic, and non-allergenic and produces multiple interactions with immune receptors upon docking. In addition, molecular dynamics simulation confirmed the stable binding and good dynamic properties of the vaccine–TLR complex. Furthermore, in silico and immune simulation of the developed MEVC for Y. pestis showed that the vaccine triggered strong immune response after several doses at different intervals. Neutralization of the antigen was observed at the third day of injection. Conclusively, the vaccine designed here for Y. pestis produces an immune response; however, further immunological testing is needed to unveil its real efficacy.

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Removal of the N-Glycosylation Sequon at Position N116 Located in p27 of the Respiratory Syncytial Virus Fusion Protein Elicits Enhanced Antibody Responses after DNA Immunization

Viruses ◽

10.3390/v10080426 ◽

2018 ◽

Vol 10 (8) ◽

pp. 426 ◽

Cited By ~ 7

Author(s):

Annelies Leemans ◽

Marlies Boeren ◽

Winke Van der Gucht ◽

Isabel Pintelon ◽

Kenny Roose ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Natural Infection ◽

Antibody Responses ◽

F Protein ◽

Glycosylation Sites ◽

Potential Vaccine ◽

Syncytial Virus ◽

Tract Infections

Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hRSV) remains a major public health priority. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. The protein conserves 5 N-glycosylation sites, two of which are located in the F2 subunit (N27 and N70), one in the F1 subunit (N500) and two in the p27 peptide (N116 and N126). To study the influence of the loss of one or more N-glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with plasmids encoding RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titres were observed following immunization with F N116Q. Moreover, RSV A2-K-line19F challenge of mice that had been immunized with mutant F N116Q DNA was associated with lower RSV RNA levels compared with those in challenged WT F DNA immunized animals. Since p27 is assumed to be post-translationally released after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this glycan can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates.

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Comparable Long-Term Persistence of Anti-FVIII Antibodies in Hemophilic E17 Mice on Different Genetic Backgrounds Despite Significant Differences in the Amplitude of the Immune Responses.

Blood ◽

10.1182/blood.v108.11.1027.1027 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1027-1027

Author(s):

Natalie Bauer ◽

Christina Hausl ◽

Rafi U. Ahmad ◽

Bernhard Baumgartner ◽

Hans Peter Schwarz ◽

...

Keyword(s):

Immune Response ◽

B Cells ◽

Genetic Background ◽

Neutralizing Antibodies ◽

Plasma Cells ◽

Memory B Cells ◽

Mouse Strains ◽

Igg Antibodies ◽

Specific Memory

Abstract About 30% of patients with severe hemophilia A develop neutralizing antibodies against FVIII (FVIII inhibitors) following replacement therapy. The type of FVIII gene mutation as well as other predisposing genetic factors contribute to the inhibitor phenotype. Based on these findings, we asked if the genetic background modulates the long-term persistence of anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells in the E17 murine hemophilia model. Furthermore, we asked if the recently described inhibition of memory-B-cell re-stimulation by high doses of FVIII is influenced by the genetic background of the murine model. E17 mice on two different genetic backgrounds (C57Bl/6J and Balb/c) were treated with four doses of 200 ng human FVIII at weekly intervals. Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were followed up to 12 months after the last dose of FVIII. Antibody titers and subclasses of antibodies (IgM, IgG1, IgG2a, IgG2b, IgG3) were measured by ELISA. Antibody secreting plasma cells in spleen and bone marrow were detected by ELISPOT as described (Hausl et al., Thromb Haemost 2002). The re-stimulation of FVIII-specific memory B cells was studied as described recently (Hausl et al., Blood 2005). Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were first detectable in E17 Balb/c mice. IgM antibodies in the circulation and IgM secreting plasma cells in the spleen were observed after the first dose of FVIII, IgG antibodies and IgG secreting plasma cells after the second dose. No anti-FVIII antibodies after the first dose of FVIII were observed in E17 C57BL/6J mice but both IgM and IgG antibodies as well as IgM and IgG producing plasma cells were detectable after the second dose of FVIII. The antibody response involved all IgG subclasses in both mouse strains. However, IgG1 was dominant in E17 Balb/c mice whereas IgG2a was dominant in E17 C57BL/6J mice. When the in vitro restimulation of FVIII-specific memory B cells was examined, similar patterns were observed for both mouse strains. Low concentrations of FVIII between 10 and 100 ng/ml FVIII restimulated memory B cells and induced their differentiation into antibody secreting plasma cells whereas high concentrations of FVIII between 1,000 and 20,000 ng/ml FVIII inhibited memory-B-cell-restimulation. These results indicate that the dose-dependent effect of FVIII on the restimulation of FVIII-specific memory B cells does not depend on the genetic background. The major difference between both hemophilic mouse strains was the amplitude of the anti-FVIII immune response. Peak titers of anti-FVIII antibodies and peak concentrations of anti-FVIII antibody secreting plasma cells in spleen and bone marrow were significantly higher in E17 C57BL/6J mice than in E17 Balb/c mice. Whether or not higher ELISA titers correlate with higher Bethesda titers of neutralizing antibodies is currently being investigated. Despite the substantial differences in the amplitude of the immune response, anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells persisted for the whole observation period of 12 months after the last dose of FVIII in both mouse strains. We conclude that the amplitude of the anti-FVIII immune response in hemophilic mice is significantly different between E17 C57BL/6J and E17 Balb/c mice. However, the persistence of the immune response is comparable.

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Homologous and heterologous serological response to the N-terminal domain of SARS-CoV-2

10.1101/2021.02.17.431722 ◽

2021 ◽

Author(s):

Huibin Lv ◽

Owen Tak-Yin Tsang ◽

Ray T. Y. So ◽

Yiquan Wang ◽

Meng Yuan ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Global Economy ◽

Vaccine Development ◽

Serological Response ◽

Receptor Binding Domain ◽

Plasma Samples ◽

Mice Model ◽

Terminal Domain ◽

Major Antigen ◽

Serology Testing

SUMMARYThe increasing numbers of infected cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses serious threats to public health and the global economy. Most SARS-CoV-2 neutralizing antibodies target the receptor binding domain (RBD) and some the N-terminal domain (NTD) of the spike protein, which is the major antigen of SARS-CoV-2. While the antibody response to RBD has been extensively characterized, the antigenicity and immunogenicity of the NTD protein are less well studied. Using 227 plasma samples from COVID-19 patients, we showed that SARS-CoV-2 NTD-specific antibodies could be induced during infection. As compared to the serological response to SARS-CoV-2 RBD, the SARS-CoV-2 NTD response is less cross-reactive with SARS-CoV. Furthermore, neutralizing antibodies are rarely elicited in a mice model when NTD is used as an immunogen. We subsequently demonstrate that NTD has an altered antigenicity when expressed alone. Overall, our results suggest that while NTD offers an alternative strategy for serology testing, it may not be suitable as an immunogen for vaccine development.

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Glycans of SARS-CoV-2 Spike Protein in Virus Infection and Antibody Production

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.629873 ◽

2021 ◽

Vol 8 ◽

Author(s):

Xiaohui Zhao ◽

Huan Chen ◽

Hongliang Wang

Keyword(s):

Immune Response ◽

Antibody Production ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Protein Glycosylation ◽

The Other ◽

Defense System ◽

S Protein ◽

Receptor Interactions

Viral protein glycosylation represents a successful strategy employed by the parasite to take advantage of host–cell machinery for modification of its own proteins. The resulting glycans have unneglectable roles in viral infection and immune response. The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which presents on the surface of matured virion and mediates viral entry into the host, also undergoes extensive glycosylation to shield it from the human defense system. It is believed that the ongoing COVID-19 pandemic with more than 90,000,000 infections and 1,900,000 deaths is partly due to its successful glycosylation strategy. On the other hand, while glycan patches on S protein have been reported to shield the host immune response by masking “nonself” viral peptides with “self-glycans,” the epitopes are also important in eliciting neutralizing antibodies. In this review, we will summarize the roles of S protein glycans in mediating virus–receptor interactions, and in antibody production, as well as indications for vaccine development.

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Immunogenicity of an E1-deleted recombinant human adenovirus against rabies by different routes of administration

Journal of General Virology ◽

10.1099/0022-1317-82-9-2191 ◽

2001 ◽

Vol 82 (9) ◽

pp. 2191-2197 ◽

Cited By ~ 32

Author(s):

Ad Vos ◽

Andreas Neubert ◽

Elke Pommerening ◽

Thomas Müller ◽

Leopold Döhner ◽

...

Keyword(s):

Immune Response ◽

Oral Administration ◽

Neutralizing Antibodies ◽

Direct Injection ◽

Human Adenovirus ◽

Adenovirus Type ◽

Vaccine Virus ◽

Strong Immune Response ◽

Routes Of Administration ◽

Immunogenic Properties

The immunogenic properties of an E1-deleted, human adenovirus type 5 (Ad5) vaccine virus with activity against rabies were examined in mice, foxes and dogs using different routes of administration. NMRI mice received 105·8, 105·3, 104·3, 103·3 and 102·3 TCID50 by peroral or intramuscular (i.m.) administration. Furthermore, six mice received 105·8 TCID50 intracerebrally (i.c.). The construct elicited marked seroconversion in mice after oral administration. Immunoreactivity in mice was even more pronounced i.m. and i.c. After direct oral administration (108·0 TCID50) in foxes, six of eight animals developed rabies virus-neutralizing antibodies (VNA). All foxes immunized by direct injection (107·7 TCID50) in the membrane of the jejunum were shown to seroconvert. Pre-existing immunity against canine adenovirus did not hinder the development of rabies VNA after oral application of the construct (108·0 TCID50). Fox cubs (24–29 days old) born from rabies-immune vixens were shown to develop very high levels of rabies VNA after i.m. administration (108·0 TCID50), indicating that the immunogenicity of the construct could surpass maternally transferred immunity. In dogs, the construct (108·0 TCID50) induced a very strong immune response after i.m. administration. However, no immune response was detectable in dogs after direct oral administration (108·3 TCID50) or after endoscopic deposition in the smaller intestine (108·0 TCID50). Hence, it must be concluded that the construct is not suitable for oral vaccination of dogs against rabies.

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Chimeric Vaccines Designed by Immunoinformatics-Activated Polyfunctional and Memory T Cells That Trigger Protection against Experimental Visceral Leishmaniasis

Vaccines ◽

10.3390/vaccines8020252 ◽

2020 ◽

Vol 8 (2) ◽

pp. 252 ◽

Cited By ~ 2

Author(s):

Rory Cristiane Fortes De Brito ◽

Jeronimo Conceição Ruiz ◽

Jamille Mirelle de Oliveira Cardoso ◽

Thais Lopes Valentim Di Paschoale Ostolin ◽

Levi Eduardo Soares Reis ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Visceral Leishmaniasis ◽

Vaccine Development ◽

Memory T Cells ◽

Parasite Burden ◽

Effector Memory ◽

Potential Candidate ◽

Protective Mechanisms ◽

Strong Immune Response

Many vaccine candidates against visceral leishmaniasis (VL) have been proposed; however, to date, none of them have been efficacious for the human or canine disease. On this basis, the design of leishmaniasis vaccines has been constantly changing, and the use of approaches to select specific epitopes seems to be crucial in this scenario. The ability to predict T cell-specific epitopes makes immunoinformatics an even more necessary approach, as in VL an efficient immune response against the parasite is triggered by T lymphocytes in response to Leishmania spp. immunogenic antigens. Moreover, the success of vaccines depends on the capacity to generate long-lasting memory and polyfunctional cells that are able to eliminate the parasite. In this sense, our study used a combination of different approaches to develop potential chimera candidate vaccines against VL. The first point was to identify the most immunogenic epitopes of Leishmania infantum proteins and construct chimeras composed of Major histocompatibility complex (MHC) class I and II epitopes. For this, we used immunoinformatics features. Following this, we validated these chimeras in a murine model in a thorough memory study and multifunctionality of T cells that contribute to a better elucidation of the immunological protective mechanisms of polyepitope vaccines (chimera A and B) using multicolor flow cytometry. Our results showed that in silico-designed chimeras can elicit polyfunctional T cells producing T helper (Th)1 cytokines, a strong immune response against Leishmania antigen, and the generation of central and effector memory T cells in the spleen cells of vaccinated animals that was able to reduce the parasite burden in this organ. These findings contribute two potential candidate vaccines against VL that can be used in further studies, and help in this complex field of vaccine development against this challenging parasite.

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Immune Response to COVID-19: Can We Benefit from the SARS-CoV and MERS-CoV Pandemic Experience?

Pathogens ◽

10.3390/pathogens9090739 ◽

2020 ◽

Vol 9 (9) ◽

pp. 739

Author(s):

Emilia Sinderewicz ◽

Wioleta Czelejewska ◽

Katarzyna Jezierska-Wozniak ◽

Joanna Staszkiewicz-Chodor ◽

Wojciech Maksymowicz

Keyword(s):

Immune Response ◽

T Cell ◽

Vaccine Development ◽

T Cell Response ◽

High Fatality Rate ◽

Scientific World ◽

Specific Antibody Production ◽

Potential Vaccine ◽

Cellular Immune

The global range and high fatality rate of the newest human coronavirus (HCoV) pandemic has made SARS-CoV-2 the focus of the scientific world. Next-generation sequencing of the viral genome and a phylogenetic analysis have shown the high homology of SARS-CoV-2 to other HCoVs that have led to local epidemics in the past. The experience acquired in SARS and MERS epidemics may prove useful in understanding the SARS-CoV-2 pathomechanism and lead to effective treatment and potential vaccine development. This study summarizes the immune response to SARS-CoV, MERS-CoV, and SARS-CoV-2 and focuses on T cell response, humoral immunity, and complement system activation in different stages of HCoVs infections. The study also presents the quantity and frequency of T cell responses, particularly CD4+ and CD8+; the profile of cytokine production and secretion; and its relation to T cell type, disease severity, and utility in prognostics of the course of SARS, MERS, and COVID-19 outbreaks. The role of interferons in the therapy of these infections is also discussed. Moreover, the kinetics of specific antibody production, the correlation between humoral and cellular immune response and the immunogenicity of the structural HCoVs proteins and their utility in the development of a vaccine against SARS, MERS, and COVID-19 has been updated.

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Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines

10.1101/2021.03.15.435528 ◽

2021 ◽

Author(s):

Alexandra C Walls ◽

Marcos C Miranda ◽

Minh N Pham ◽

Alexandra Schaefer ◽

Allison Greaney ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Polyclonal Antibodies ◽

Clinical Stage ◽

Binding Domain ◽

Single Shot ◽

Receptor Binding Domain ◽

Negative Consequences ◽

Neutralizing Activity

Understanding the ability of SARS-CoV-2 vaccine-elicited antibodies to neutralize and protect against emerging variants of concern and other sarbecoviruses is key for guiding vaccine development decisions and public health policies. We show that a clinical stage multivalent SARS-CoV-2 receptor-binding domain nanoparticle vaccine (SARS-CoV-2 RBD-NP) protects mice from SARS-CoV-2-induced disease after a single shot, indicating that the vaccine could allow dose-sparing. SARS-CoV-2 RBD-NP elicits high antibody titers in two non-human primate (NHP) models against multiple distinct RBD antigenic sites known to be recognized by neutralizing antibodies. We benchmarked NHP serum neutralizing activity elicited by RBD-NP against a lead prefusion-stabilized SARS-CoV-2 spike immunogen using a panel of single-residue spike mutants detected in clinical isolates as well as the B.1.1.7 and B.1.351 variants of concern. Polyclonal antibodies elicited by both vaccines are resilient to most RBD mutations tested, but the E484K substitution has similar negative consequences for neutralization, and exhibit modest but comparable neutralization breadth against distantly related sarbecoviruses. We demonstrate that mosaic and cocktail sarbecovirus RBD-NPs elicit broad sarbecovirus neutralizing activity, including against the SARS-CoV-2 B.1.351 variant, and protect mice against severe SARS-CoV challenge even in the absence of the SARS-CoV RBD in the vaccine. This study provides proof of principle that sarbecovirus RBD-NPs induce heterotypic protection and enables advancement of broadly protective sarbecovirus vaccines to the clinic.

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Comparative Immunomodulatory Evaluation of the Receptor Binding Domain of the SARS-CoV-2 Spike Protein; a Potential Vaccine Candidate Which Imparts Potent Humoral and Th1 Type Immune Response in a Mouse Model

Frontiers in Immunology ◽

10.3389/fimmu.2021.641447 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tripti Shrivastava ◽

Balwant Singh ◽

Zaigham Abbas Rizvi ◽

Rohit Verma ◽

Sandeep Goswami ◽

...

Keyword(s):

Immune Response ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Vaccine Candidate ◽

Unmet Need ◽

Binding Domain ◽

Spike Protein ◽

Cell Immune Response ◽

Receptor Binding Domain

The newly emerged novel coronavirus, SARS-CoV-2, the causative agent of COVID-19 has proven to be a threat to the human race globally, thus, vaccine development against SARS-CoV-2 is an unmet need driving mass vaccination efforts. The receptor binding domain of the spike protein of this coronavirus has multiple neutralizing epitopes and is associated with viral entry. Here we have designed and characterized the SARS-CoV-2 spike protein fragment 330-526 as receptor binding domain 330-526 (RBD330-526) with two native glycosylation sites (N331 and N343); as a potential subunit vaccine candidate. We initially characterized RBD330-526 biochemically and investigated its thermal stability, humoral and T cell immune response of various RBD protein formulations (with or without adjuvant) to evaluate the inherent immunogenicity and immunomodulatory effect. Our result showed that the purified RBD immunogen is stable up to 72 h, without any apparent loss in affinity or specificity of interaction with the ACE2 receptor. Upon immunization in mice, RBD generates a high titer humoral response, elevated IFN-γ producing CD4+ cells, cytotoxic T cells, and robust neutralizing antibodies against live SARS-CoV-2 virus. Our results collectively support the potential of RBD330-526 as a promising vaccine candidate against SARS-CoV-2.

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