Identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against Middle East respiratory syndrome coronavirus

Middle East respiratory syndrome （MERS）, an emerging infectious disease caused by MERS coronavirus （MERS-CoV）, has garnered worldwide attention as a consequence of its continuous spread and pandemic potential, making the development of effective vaccines a high priority. We previously demonstrated that residues 377-588 of MERS-CoV spike （S） protein receptor-binding domain （RBD） is a very promising MERS subunit vaccine candidate, capable of inducing potent neutralization antibody responses. In this study, we sought to identify an adjuvant that optimally enhanced the immunogenicity of S377-588 protein fused with Fc of human IgG （S377-588-Fc）. Specifically, we compared several commercially available adjuvants, including Freund＇s adjuvant, aluminum, Monophosphoryl lipid A, Montanide ISA51 and MF59 with regard to their capacity to enhance the immunogenicity of this subunit vaccine. In the absence of adjuvant, S377-588-Fc alone induced readily detectable neutralizing antibody and T-cell responses in immunized mice. However, incorporating an adjuvant improved its immunogenicity. Particularly, among the aforementioned adjuvants evaluated, MF59 is the most potent as judged by its superior ability to induce the highest titers of IgG, IgG1 and IgG2a subtypes, and neutralizing antibodies. The addition of MF59 significantly augmented the immunogenicity of S377-588-Fcto induce strong IgG and neutralizing antibody responses as well as protection against MERS-CoV infection in mice, suggesting that MF59 is an optimal adjuvant for MERS-CoV RBD-based subunit vaccines.

Reverse vaccinology assisted designing of multiepitope-based subunit vaccine against SARS-CoV-2

Background:Coronavirus disease 2019(COVID-19)linked with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)cause severe illness and life-threatening pneumonia in humans.The current COVID-19 pandemic demands an effective vaccine to acquire protection against the infection.Therefore,the present study was aimed to design a multiepitope-based subunit vaccine(MESV)against COVID-19.Methods:Structural proteins(Surface glycoprotein,Envelope protein,and Membrane glycoprotein)of SARS-CoV-2 are responsible for its prime functions.Sequences of proteins were downloaded from GenBank and several immunoinformatics coupled with computational approaches were employed to forecast B-and T-cell epitopes from the SARS-CoV-2 highly antigenic structural proteins to design an effective MESV.Results:Predicted epitopes suggested high antigenicity,conserveness,substantial interactions with the human leukocyte antigen(HLA)binding alleles,and collective global population coverage of 88.40%.Taken together,276 amino acids long MESV was designed by connecting 3 cytotoxic T lymphocytes(CTL),6 helper T lymphocyte(HTL)and 4 B-cell epitopes with suitable adjuvant and linkers.The MESV construct was non-allergenic,stable,and highly antigenic.Molecular docking showed a stable and high binding affinity of MESV with human pathogenic toll-like receptors-3(TLR3).Furthermore,in silico immune simulation revealed significant immunogenic response of MESV.Finally,MEV codons were optimized for its in silico cloning into the Escherichia coli K-12 system,to ensure its increased expression.Conclusion:The MESV developed in this study is capable of generating immune response against COVID-19.Therefore,if designed MESV further investigated experimentally,it would be an effective vaccine candidate against SARS-CoV-2 to control and prevent COVID-19.

Structural vaccinology: structure-based design of influenza A virus hemagglutinin subtype-specific subunit vaccines

There is a great need for new vaccine development against influenza A viruses due to the drawbacks of traditional vaccines that are mainly prepared using embryonated eggs.The main component of the current split influenza A virus vaccine is viral hemagglutinin(HA)which induces a strong antibody-mediated immune response.To develop a modern vaccine against influenza A viruses,the current research has been focused on the universal vaccines targeting viral M2,NP and HA proteins.Crystallographic studies have shown that HA forms a trimer embedded on the viral envelope surface,and each monomer consists of a globular head(HA1)and a“rod-like”stalk region(HA2),the latter being more conserved among different HA subtypes and being the primary target for universal vaccines.In this study,we rationally designed the HA head based on the crystal structure of the 2009-pandemic influenza A(H1N1)virus HA as a model,tested its immunogenicity in mice,solved its crystal structure and further examined its immunological characteristics.The results show that the HA globular head can be easily prepared by in vitro refolding in an E.coli expression system,which maintains its intact structure and allows for the stimulation of a strong immune response.Together with recent reports on some similar HA globular head preparations we conclude that structure-based rational design of the HA globular head can be used for subtype-specific vaccines against influenza viruses.

Research and development of a novel subunit vaccine for the currently circulating pseudorabies virus variant in China

Pseudorabies(PR)is a devastating viral disease which leads to fatal encephalitis and respiratory disorders in pigs.Commercial gE-deleted live pseudorabies virus(PRV)vaccine has been widely used to control this disease in China.However,the new-emerging variants of PRV compromises the protection provided by current vaccines and lead to the outbreak of PR in vaccinated pig herds.Several killed and live vaccine candidates based on current PRV variants have been reported to be effective to control the disease.A subunit vaccine based on gB protein,one major PRV glycoprotein which elicits strong humoral and cellular immune responses,however,was never evaluated for protection against the current circulating PRV variants.In this study,full-length PRV gB protein was successfully expressed in baculovirus/insect cells in the soluble format and was tested on 3-week-old piglets as a subunit vaccine.Compared with unvaccinated pigs,the gB-vaccinated pigs developed specific antibody-mediated responses and were protected from the virulent PRV HN1201 challenge.All vaccinated pigs survived without showing any PRV-specific respiratory and neurological signs,but all unvaccinated pigs died within 7 days after HN1201 challenge.Hence,this novel gB-based vaccine could be applied as an effective subunit vaccine to control PRV variant in China.

Protection against lethal subcutaneous challenge of virulent Y. pestis strain 141 using an F1-V subunit vaccine

In this study, we designed and engineered a two-component recombinant fusion protein antigen as a vaccine candidate against the possible biological threat of Yersinia pestis. The recombinant F1-V pro-tein was formulated with Alhydrogel. A four-time injection with a dosage of 10, 20 and 50 μg/mouse in about two months was adopted for vaccination. Serum antibodies and subclass of T helper cells were measured and analyzed. After the final vaccination, the mice were challenged by 141 strain with 25― 600 LD50. In conclusion, the recombinant vaccine was capable of inducing protective immunity against subcutaneous challenge. The level of serum IgG was supposed to be a main factor that affected the final protection of challenge. 20 μg recombinant protein could induce an endpoint titre of serum IgG as high as 51200, which was enough to afford 100% protection against 400 LD50 virulent 141 challenge. The antibody isotype analysis showed that the vaccine induced predominantly an IgG1 rather than IgG2a response. Flow cytometric analysis revealed that Alhydrogel significantly helped induce a stronger humoral immunity instead of CTL cellular response. These findings suggested that the plague F1-V subunit vaccine is promising for the next plague vaccine.

Protective efficacy evaluation of immunogenic protein AHA_3793 of Aeromonas hydrophila as vaccine candidate for largemouth bass Micropterus salmoides

Aeromonas hydrophila is a Gram-negative pathogen that can infect various fish,including largemouth bass(Micropterus salmoides),which have caused huge economic losses.In present study,largemouth bass anti-A.hydrophila antibodies were produced,then a highly immunogenic outer membrane proteins,AHA_3793,was identified by combined western blotting and mass spectrometry analysis.Moreover,AHA_3793 was expressed,and its immunogenicity was further verified by western blotting.Subsequently,the protective efficacy of AHA_3793 were evaluated in largemouth bass.The results showed that rAHA_3793 could produce a relative percentage survival(RPS)of 61.76% for largemouth bass against A.hydrophila challenge.ELISA analysis showed the specific serum antibodies of largemouth bass against rAHA_3793 and A.hydrophila in vaccinated group in weeks 4 and 5 after immunization were significantly higher than those in control group,which suggested that rAHA_3793 induced production of specific serum antibodies against rAHA_3793 and A.hydrophila.The qRT-PCR analysis showed that expressions of CD4-2 and MHC Ⅱα were also significantly up-regulated after immunization.These results collectively demonstrated that rAHA_3793 could induce a strong humoral immune response of largemouth bass,and then produce high immune protection ef fects against A.hydrophila infection.

Immunological Effect of Subunit Influenza Vaccine Entrapped by Liposomes

Objective To elevate the immunological effect of subunit influenza vaccine in infants and aged people （over 60） using liposomal adjuvant in the context of its relatively low immunity and to investigate the relation between vaccine antigens and liposomal characteristics. Methods Several formulations of liposomal subunit influenza vaccine were prepared. Their relevant characteristics were investigated to optimize the preparation method. Antisera obtained from immunizinged mice were used to evaluate the antibody titers of various samples by HI and EL1SA. Results Liposomal trivalent influenza vaccine prepared by film evaporation in combinedation with freeze-drying significantly increased its immunological effect in SPF Balb/e mice. Liposomal vaccine stimulated the antibody titer of H3N2, H1N1, and B much stronger than conventional influenza vaccine. As a result, liposomal vaccine （mean size： 4.5-5.5 pm, entrapment efficiency： 30%-40%） significantly increased the immunological effect of subunit influenza vaccine. Conclusion The immune effect of liposomal vaccine depends on different antigens, and enhanced immunity is not positively correlated with the mean size of liposome or its entrapped efficiency.

Neutralizing Antibody Titer Test of Ebola Recombinant Protein Vaccine and Gene Vector Vaccine pVR-GP-FC

Objective In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. Methods A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. Results Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1：105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. Conclusion An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.

SpyStapler-mediated assembly of nanoparticle vaccines

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has wreaked havoc around the globe,with no end in sight.The rapid emergence of viral mutants,marked by rapid transmission and effective immune evasion,has also posed unprecedented challenges for vaccine development,not least in its speed,mass production,and distribution.Here we report a versatile“plug-and-display”strategy for creating protein vaccines,including those against malaria parasites and SARS-CoV-2,through the combined use of the intrinsically disordered protein ligase SpyStapler and computationally designed viral-like particles.The resulting protein nanoparticles harboring multiple antigens induce potent neutralizing antibody responses in mice,substantially stronger than those induced by the corresponding free antigens.This modular vaccine design enabled by SpyStapler furnishes us with a new weapon for combatting infectious diseases.

Immune Responses to Varicella-Zoster Virus Glycoprotein E Formulated with Poly(Lactic-co-Glycolic Acid) Nanoparticles and Nucleic Acid Adjuvants in Mice

The subunit herpes zoster vaccine Shingrix is superior to attenuated vaccine Zostavax in both safety and efficacy,yet its unlyophilizable liposome delivery system and the limited supply of naturally sourced immunological adjuvant QS-21 still need to be improved.Based on poly(lactic-co-glycolic acid)(PLGA)delivery systems that are stable during the lyophilization and rehydration process and using a double-emulsion(w/o/w)solvent evaporation method,we designed a series of nanoparticles with varicella-zoster virus antigen glycoprotein E(VZV-g E)as an antigen and nucleic acids including polyinosinic-polycytidylic acid(Poly I:C)and phosphodiester Cp G oligodeoxynucleotide(Cp G ODN),encapsulated as immune stimulators.While cationic lipids(DOTAP)have more potential than neutral lipids(DOPC)for activating g E-specific cell-mediated immunity(CMI)in immunized mice,especially when g E is encapsulated in and presented on the surface of nanoparticles,PLGA particles without lipids have the greatest potential to induce not only the highest g Especific Ig G titers but also the strongest g E-specific CMI responses,including the highest proportions of interferon-c(IFNc)-and interleukin-2(IL-2)-producing CD4?/CD8?T cells according to a flow cytometry assay and the greatest numbers of IFN-c-and IL-2-producing splenocytes according to an enzyme-linked immunospot(ELISPOT)assay.These results showed that immune-stimulating nucleic acids together with the PLGA delivery system showed promise as a safe and economical varicella and zoster vaccine candidate.

LNP-CpG ODN-adjuvanted varicella-zoster virus glycoprotein E induced comparable levels of immunity with Shingrix^(TM) in VZV-primed mice

Latent varicella-zoster virus(VZV)may be reactivated to cause herpes zoster,which affects one in three people during their lifetime.The currently available subunit vaccine Shingrix^(TM) is superior to the attenuated vaccine Zostavax®in terms of both safety and efficacy,but the supply of its key adjuvant component QS21 is limited.With ionizable lipid nanoparticles(LNPs)that were recently approved by the FDA for COVID-19 mRNA vaccines as carriers,and oligodeoxynucleotides containing CpG motifs(CpG ODNs)approved by the FDA for a subunit hepatitis B vaccine as immunostimulators,we developed a LNP vaccine encapsulating VZV-glycoprotein E(gE)and CpG ODN,and compared its immunogenicity with Shingrix^(TM) in C57BL/6J mice.The results showed that the LNP vaccine induced comparable levels of gE-specific IgG antibodies to Shingrix^(TM) as determined by enzymelinked immunosorbent assay(ELISA).Most importantly,the LNP vaccine induced comparable levels of cellmediated immunity(CMI)that plays decisive roles in the efficacy of zoster vaccines to Shingrix^(TM) in a VZVprimed mouse model that was adopted for preclinical studies of Shingrix^(TM) .Number of IL-2 and IFN-γsecreting splenocytes and proportion of T helper 1(Th1)cytokine-expressing CD4^(+)T cells in LNP-CpG-adjuvanted VZV-gE vaccinated mice were similar to that of Shingrix^(TM) boosted mice.All of the components in this LNP vaccine can be artificially and economically synthesized in large quantities,indicating the potential of LNP-CpGadjuvanted VZV-gE as a more cost-effective zoster vaccine.

Vaccination of Goats with 31 kDa and 32 kDa Schistosoma japonicum Antigens by DNA Priming and Protein Boosting

Two Schistosomajaponicum vaccine candidate antigens Sj 31 and Sj 32, which have shown particular promise to induce protective immunity in mice, were used to immunize goats by using a DNA priming-protein boosting strategy in present work. DNA vaccine formulations of the two antigens （VRSj31 and VRSj32） were produced and injected intramuscularly twice at a 2-week interval and then recombinant proteins （rSj31 and rSj32） together with Freund Complete Adjuvant （FCA） were used to boost the goats. The experiment was repeated in different batche cercariae. A strong anamnestic antibody response was induced after boost. A significant reduction of liver egg counts and miracidial hatching was showed in both experiments. Significant protections against challenge infection were elicited with 31.6% of percentage reduction for worm recovery in the second experiment and 20.9% in the first experiment, respectively.

A recombinant receptor-binding domain in trimeric form generates protective immunity against SARSCoV-2 infection in nonhuman primates

A safe and effective vaccine is critical to combat the COVID-19 pandemic.Here,we developed a trimeric SARS-CoV-2 receptor-binding domain(RBD)subunit vaccine candidate that simulates the natural structure of the spike(S)trimer glycoprotein.Immunization with the RBD trimer-induced robust humoral and cellular immune responses,and a high level of neutralizing antibodies was maintained for at least 4.5 months.Moreover,the antibodies that were produced in response to the vaccine effectively cross-neutralized the SARS-CoV-2501Y.V2 variant(B.1.351).Of note,when the vaccine-induced antibodies dropped to a sufficiently low level,only one boost quickly activated the anamnestic immune response,conferring full protection against a SARSCoV-2 challenge in rhesus macaques without typical histopathological changes in the lung tissues.These results demonstrated that the SARS-CoV-2 RBD trimer vaccine candidate is highly immunogenic and safe,providing long-lasting,broad,and significant immunity protection in nonhuman primates,thereby offering an optimal vaccination strategy against COVID-19.