Intranasal immunization with recombinant Vaccinia virus encoding trimeric SARS-CoV-2 spike receptor-binding domain induces neutralizing antibody

Respiratory transmission of SARS-CoV-2 is considered to be the major dissemination route for COVID-19, therefore, mucosal immune responses have great importance in preventing SARS-CoV-2 from infection. In this study, we constructed a recombinant Vaccinia virus (VV) harboring trimeric receptor-binding domain (RBD) of SARS-CoV-2 spike protein (VV_-tRBD), and evaluated the immune responses towards RBD following intranasal immunization against mice and rabbits. In BALB/c mice, intranasal immunization with VV_-tRBD elicited robust humoral and cellular immune responses, with high-level of both neutralizing IgG and IgA in sera against SARS-CoV-2 psudoviruses, and a number of RBD-specific IFN-γ-secreting lymphocytes. Sera from immunized rabbits also exhibited neutralization effects. Notably, RBD-specific secretory IgA (sIgA) in both nasal washes and bronchoalveolar lavage fluids (BALs) were detectable and showed substantial neutralization activities. Collectively, a recombinant VV expressing trimeric RBD confers robust systemic immune response and mucosal neutralizing antibodies, thus warranting further exploration as a mucosal vaccine.     

Recombinant measles virus encoding the spike protein of SARS-CoV-2 efficiently induces Th1 responses and neutralizing antibodies that block SARS-CoV-2 variants

Owing to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, the development of effective and safe vaccines has become a priority. The measles virus (MeV) vaccine is an attractive vaccine platform as it has been administered to children for more than 40 years in over 100 countries. In this study, we developed a recombinant MeV expressing the full-length SARS-CoV-2 spike protein (rMeV-S) and tested its efficacy using mouse and hamster models. In hCD46Tg mice, two-dose rMeV-S vaccination induced higher Th1 secretion and humoral responses than one-dose vaccination. Interestingly, neutralizing antibodies induced by one-dose and two-dose rMeV-S immunization effectively blocked the entry of the α, β, γ, and δ variants of SARS-CoV-2. Furthermore, two-dose rMeV-S immunization provided complete protection against SARS-CoV-2 in the hamster model. These results suggest the potential of rMeV-S as a vaccine candidate for targeting SARS-CoV-2 and its variants.     

Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice

Measles remains a significant problem in both the developed and developing world, and new measles vaccination strategies need to be developed. This paper examines the strategy of utilizing transgenic plants expressing a measles antigen for the development of an oral sub-unit measles vaccine. A 1.8 kb fragment encompassing the coding region of the measles virus hemagglutinin (H) protein was cloned into a plant expression cassette. Three different expression constructs were tested: pBinH (H gene alone), pBinH/KDEL (addition of a C-terminal endoplasmic reticulum-retention sequence SEKDEL) and pBinSP/H/KDEL (further addition of an authentic N-terminal plant signal peptide). The highest levels of recombinant H protein production were observed in plants transformed with pBinH/KDEL. Mice inoculated intraperitoneally with transgenic plant derived recombinant H protein produced serum anti-H protein antibodies that neutralized the measles virus (MV) in vitro. Mice gavaged with transgenic tobacco leaf extracts also developed serum H protein-specific antibodies with neutralizing activity against MV in vitro. These results indicate that the plant-derived measles H protein is immunogenic when administered orally and that, with further development, oral vaccination utilizing transgenic plants may become a viable approach to measles vaccine development.     

Optimized recombinant dense bodies of human cytomegalovirus efficiently prime virus specific lymphocytes and neutralizing antibodies without the addition of adjuvant

Control of human cytomegalovirus (HCMV) infection correlates with the reconstitution of antiviral T lymphocytes in haematopoietic stem cell transplant recipients. A vaccine to foster this reconstitution and to ameliorate the severe consequences of HCMV reactivation is yet unavailable. This work focused on providing a rationale for the amendment of the yields and the antigenic composition of a vaccine, based on subviral dense bodies (DB) of HCMV. Modified DB were generated that contained the HLA-A2 presented IE1 model peptide TMYGGISLL, integrated at different positions in the major DB protein pp65. Insertion at position W175 of pp65 allowed efficient formation of recDB in the cytoplasm of infected cells and resulted in considerable yields of these particles. Even in the absence of adjuvant, these particles proved to be highly immunogenic with respect to CD8 and CD4 T cell and neutralizing antibody responses.     

A highly conserved epitope-vaccine candidate against varicella-zoster virus induces neutralizing antibodies in mice

Varicella-zoster virus (VZV) is a highly infectious agent of varicella and herpes zoster (HZ). Vaccination is by far the most effective way to prevent these diseases. More safe, stable and efficient vaccines, such as epitope-based vaccines, now have been increasingly investigated by many researchers. However, only a few VZV neutralizing epitopes have been identified to date. We have previously identified a linear epitope between amino acid residues 121 and 135 of gE. In this study, we validated that this epitope is highly conserved amongst different VZV strains that covered five existing phylogenetic clades with an identity of 100%. We evaluated the immunogenicity of the recombinant hepatitis B virus core (HBc) virus-like particles (VLPs) which included amino acids (121–135). VZV-gE-specific antibodies were detected in immunized mouse serum using ELISA. The anti-peptide antiserum positively detected VZV via Western blot and immunofluorescent staining assays. More importantly, these peptides could neutralize VZV, indicating that these peptides represented neutralizing epitopes. These findings have important implications for the development of epitope-based protective VZV vaccines.     

A recombinant baculovirus-expressed S glycoprotein vaccine elicits high titers of SARS-associated coronavirus (SARS-CoV) neutralizing antibodies in mice

A recombinant SARS-CoV spike (S) glycoprotein vaccine produced in insect cells in a pre-clinical development stage is described. A truncated version of S glycoprotein, containing only the ecto-domain, as well as a His-tagged full-length version were cloned and expressed in a serum-free insect cell line, ExpresSF+. The proteins, purified to apparent homogeneity by liquid column chromatography, were formulated without adjuvant at 3, 9, 27, and 50 microg per dose in phosphate saline and used to immunize mice. Both antigens in each formulation elicited a strong immune response after two or three vaccinations with the antigen. Neutralizing antibody titers correlated closely with standard ELISA reactivity against the S glycoprotein. The truncated S protein was also formulated with an adjuvant, aluminum hydroxide, at 1 microg per dose (+/-adjuvant), and 5 microg per dose (+/-adjuvant). Significantly enhanced immune responses, manifested by higher titers of serum ELISA and viral neutralizing antibodies, were achieved in adjuvanted groups with fewer doses and lower concentration of S glycoprotein. These findings indicate that the ecto-domain of SARS-CoV S glycoprotein vaccine, with or without adjuvant, is immunogenic and induces high titers of virus neutralizing antibodies to levels similar to those achieved with the full S glycoprotein vaccine.     

SARS coronavirus spike polypeptide DNA vaccine priming with recombinant spike polypeptide from Escherichia coli as booster induces high titer of neutralizing antibody against SARS coronavirus

Different forms of SARS coronavirus (SARS-CoV) spike protein-based vaccines for generation of neutralizing antibody response against SARS-CoV were compared using a mouse model. High IgG levels were detected in mice immunized with intraperitoneal (i.p.) recombinant spike polypeptide generated by Escherichia coli (S-peptide), mice primed with intramuscular (i.m.) tPA-optimize800 DNA vaccine (tPA-S-DNA) and boosted with i.p. S-peptide, mice primed with i.m. CTLA4HingeSARS800 DNA vaccine (CTLA4-S-DNA) and boosted with i.p. S-peptide, mice primed with oral live-attenuated Salmonella typhimurium (Salmonella-S-DNA-control) and boosted with i.p. S-peptide, mice primed with oral live-attenuated S. typhimurium that contained tPA-optimize800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide, and mice primed with oral live-attenuated S. typhimurium that contained CTLA4HingeSARS800 DNA vaccine (Salmonella-tPA-S-DNA) and boosted with i.p. S-peptide. No statistical significant difference was observed among the Th1/Th2 index among these six groups of mice with high IgG levels. Sera of all six mice immunized with i.p. S-peptide, i.m. DNA vaccine control and oral Salmonella-S-DNA-control showed no neutralizing antibody against SARS-CoV. Sera of the mice immunized with i.m. tPA-S-DNA, i.m. CTLA4-S-DNA, oral Salmonella-S-DNA-control boosted with i.p. S-peptide, oral Salmonella-tPA-S-DNA, oral Salmonella-tPA-S-DNA boosted with i.p S-peptide, oral Salmonella-CTLA4-S-DNA and oral Salmonella-CTLA4-S-DNA boosted with i.p. S-peptide showed neutralizing antibody titers of <1:20-1:160. Sera of all the mice immunized with i.m. tPA-S-DNA boosted with i.p. S-peptide and i.m. CTLA4-S-DNA boosted with i.p. S-peptide showed neutralizing antibody titers of >or=1:1280. The present observation may have major practical value, such as immunization of civet cats, since production of recombinant proteins from E. coli is far less expensive than production of recombinant proteins using eukaryotic systems.     

Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice

Development of vaccination strategies for emerging pathogens are particularly challenging because of the sudden nature of their emergence and the long process needed for traditional vaccine development. Therefore, there is a need for development of a rapid method of vaccine development that can respond to emerging pathogens in a short time frame.     

Targeting the vaccinia virus L1 protein to the cell surface enhances production of neutralizing antibodies

The current live-orthopoxvirus vaccine is associated with minor to serious adverse affects, and is contraindicated for use in a significant portion of the population. As an alternative vaccine, we have previously shown that a DNA subunit vaccine (4pox) based on four orthopoxvirus immunogens (L1R, B5R, A27L and A33R) can produce protective immunity against lethal orthopoxvirus challenges in mice and nonhuman primates. Because antibodies are critical for protection against secondary orthopoxvirus infections, we are now interested in strategies that will enhance the humoral immune response against vaccine targets. Here, we tested the immunogenicity of an L1R construct to which a tissue plasminogen activator signal sequence was placed in frame with the full-length L1R gene. The tPA-L1R construct produced a more robust neutralizing antibody response in vaccinated mice when the DNA vaccine was administered by gene-gun as a prime/single boost. When the tPA-L1R construct was substituted for the unmodified L1R gene in the 4pox vaccine, given as a prime and single boost, animals were better protected from lethal challenge with vaccinia virus (VACV). These findings indicate that adding a tPA-leader sequence can enhance the immunogenicity of the L1R gene when given as a DNA vaccine. Furthermore, our results demonstrate that a DNA-based vaccine is capable of establishing protection from lethal orthopoxvirus challenges when administered as a prime and single boost without requiring adjuvant.     

Hepatitis C virus soluble E2 in combination with QuilA and CpG ODN induces neutralizing antibodies in mice

Several studies have emphasized the importance of an early, highly neutralizing antibody response in the clearance of Hepatitis C virus (HCV) infection. The envelope glycoprotein E2 is a major target for HCV neutralizing antibodies. Here, we compared antibody responses in mice immunized with native soluble E2 (sE2) from the H77 1a isolate coupled with different adjuvants or combinations of adjuvants. Adjuvanting sE2 with Freund's, monophosphoryl lipid A (MPL), cytosine phosphorothioate guanine oligodeoxynucleotide (CpG ODN), or alpha-galactosylceramide (αGalCer) derivatives elicited only moderate antibody responses. In contrast, immunizations with sE2 and QuilA elicited exceptionally high anti-E2 antibody titers. Sera from these mice effectively neutralized HCV pseudoparticles (HCVpp) 1a entry. Moreover, the combination of QuilA and CpG ODN further enhanced neutralizing antibody titers wherein cross-neutralization of HCVpp 4 was observed. We conclude that the combination of QuilA and CpG ODN is a promising adjuvant combination that should be further explored for the development of an HCV subunit vaccine. Our work also emphasizes that the ideal combination of adjuvant and immunogen has to be determined empirically.     

Receptor-binding domain of SARS-CoV spike protein induces long-term protective immunity in an animal model

Development of effective vaccines against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is still a priority in prevention of re-emergence of SARS. Our previous studies have shown that the receptor-binding domain (RBD) of SARS-CoV spike (S) protein elicits highly potent neutralizing antibody responses in the immunized animals. But it is unknown whether RBD can also induce protective immunity in an animal model, a key aspect for vaccine development. In this study, BALB/c mice were vaccinated intramuscularly (i.m.) with 10microg of RBD-Fc (RBD fused with human IgG1 Fc) and boosted twice at 3-week intervals and one more time at 12th month. Humoral immune responses of vaccinated mice were investigated for up to 12 months at a 1-month interval and the neutralizing titers of produced antibodies were reported at months 0, 3, 6 and 12 post-vaccination. Mice were challenged with the homologous strain of SARS-CoV 5 days after the last boost, and sacrificed 5 days after the challenge. Mouse lung tissues were collected for detection of viral load, virus replication and histopathological effects. Our results showed that RBD-Fc vaccination induced high titer of S-specific antibodies with long-term and potent SARS-CoV neutralizing activity. Four of five vaccinated mice were protected from subsequent SARS-CoV challenge because no significant virus replication, and no obvious histopathological changes were found in the lung tissues of the vaccinated mice challenged with SARS-CoV. Only one vaccinated mouse had mild alveolar damage in the lung tissues. In contrast, high copies of SARS-CoV RNA and virus replication were detected, and pathological changes were observed in the lung tissues of the control mice. In conclusion, our findings suggest that RBD, which can induce protective antibodies to SARS-CoV, may be further developed as a safe and effective SARS subunit vaccine.     

Red blood cell-mediated delivery of recombinant HIV-1 Tat protein in mice induces anti-Tat neutralizing antibodies and CTL

The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model. HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs). The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin-avidin-biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat.Balb/c mice were then immunized with 10 microg of soluble Tat in complete Freund's adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5 microg soluble Tat in incomplete Freund's adjuvant or with 20 ng of RBC-coupled Tat, respectively. Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies. In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat. These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies.     

Identification and characterization of novel neutralizing epitopes in the receptor-binding domain of SARS-CoV spike protein: revealing the critical antigenic determinants in inactivated SARS-CoV vaccine

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is considered as a major antigen for vaccine design. We previously demonstrated that the receptor-binding domain (RBD: residues 318-510) of S protein contains multiple conformation-dependent neutralizing epitopes (Conf I to VI) and serves as a major target of SARS-CoV neutralization. Here, we further characterized the antigenic structure in the RBD by a panel of novel mAbs isolated from the mice immunized with an inactivated SARS-CoV vaccine. Ten of the RBD-specific mAbs were mapped to four distinct groups of conformational epitopes (designated Group A to D), and all of which had potent neutralizing activity against S protein-pseudotyped SARS viruses. Group A, B, C mAbs target the epitopes that may overlap with the previously characterized Conf I, III, and VI respectively, but they display different capacity to block the receptor binding. Group D mAb (S25) was directed against a unique epitope by its competitive binding. Two anti-RBD mAbs recognizing the linear epitopes (Group E) were mapped to the RBD residues 335-352 and 442-458, respectively, and none of them inhibited the receptor binding and virus entry. Surprisingly, most neutralizing epitopes (Groups A to C) could be completely disrupted by single amino acid substitutions (e.g., D429A, R441A or D454A) or by deletions of several amino acids at the N-terminal or C-terminal region of the RBD; however, the Group D epitope was not sensitive to the mutations, highlighting its importance for vaccine development. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV, and this panel of novel mAbs can be used as tools for studying the structure of S protein and for guiding SARS vaccine design.     

A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys

A DNA vaccine that expresses the premembrane/membrane (prM) and envelope (E) genes of dengue virus serotype-1 was tested for immunogenicity and protection against dengue-1 virus challenge in Aotus nancymae monkeys. The vaccine, in 1 mg doses, was administered intradermally (i.d.) to three monkeys and intramuscularly (i.m.) to three others. For controls, a 1 mg dose of vector DNA was administered i.d. to two monkeys and i.m. to one. All animals were primed and then boosted at one and five months post priming. Sera were collected monthly and analyzed for dengue-1 antibodies by enzyme linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). Dengue-1 antibodies were detectable in the sera from i.d. and i.m. vaccine inoculated animals one month after the first boost and peaked one month after the second boost. The antibody levels from sera of animals that received the vaccine via the i.d. route were twice those from sera of animals that received the vaccine via the i.m. route. Six months after the second boost all inoculated and two naive monkeys were challenged with 1.25x10(4) plaque forming units (PFU) of dengue-1 virus. Two vaccine immunized animals were protected from viremia while the others showed a reduction in viremia. The mean days of viremia were 1 and 1.3 for the animals that were immunized with the vaccine via the i.d. or i.m. route, respectively vs 4 and 2 mean days of viremia in the animals inoculated with control DNA. Naive animals were viremic for an average of 4 days. All of the three control monkeys that received control DNA inoculum by either the i.d. or i.m. route had an intermittent viremia pattern with one or more negative days interspersed between the positive days. This pattern was not observed in any of the vaccine recipients or the na?ve control monkeys. These results demonstrate that DNA immunization is a promising approach for the development of dengue vaccines and that A. nancymae monkeys are suitable for dengue vaccine trials.     

Recombinant modified vaccinia virus Ankara efficiently restimulates human cytotoxic T lymphocytes in vitro

The immunogenicity of recombinant modified vaccinia Ankara, a highly attenuated vaccinia virus, expressing influenza nucleoprotein (MVA-NP) and HIV-1 gag (MVA-gag) was investigated. Restimulation of peripheral blood lymphocytes of healthy subjects with MVA-NP led to expansion of CTL with specificity for known NP epitopes. These CTL efficiently lysed NP peptide-pulsed targets and released interferon-gamma (IFN-gamma) on contact with epitope peptide. MVA-NP-stimulated CTL specific for the HLA-B8 epitope, NP380-88, stained with a tetrameric complex of HLA-B8 refolded with the NP380-88 peptide and anti-CD8 antibody on flow cytometry. CTL were also elicited from two HIV-1 seropositive donors by restimulation with MVA-HIV-1 gag and showed specificity for immunodominant gag epitopes. These data indicate that restimulation of human CTL with recombinant MVA is effective and suggest that MVA will elicit CTL responses in humans in vivo.     

Zeocin和GFP双筛选标记重组痘苗病毒载体的构建

目的 为研究重组痘苗病毒通用载体,构建融合Zeocin和GFP双筛选标记质粒pCB-ZeoGFP.方法 质粒LeGo-G/Zeo含有Zeo-GFP融合基因片段,通过PCR反应、BamHI酶切后连接替换空白质粒pCB的筛选基因gpt,通过菌落PCR,酶切图谱分析及测序分析鉴定重组质粒,构建成功后将其与野生型痘苗病毒进行位点特异性同源重组,经Zeocin药物筛选2代后,用流式细胞仪观察重组痘苗病毒GFP表达.结果 经菌落PCR,1号、5号和10号菌落中扩增出426 bp 的目的条带,与预期的大小完全一致,经酶切分析和DNA测序进一步验证了重组质粒pCB-Zeo-GFP;该质粒与野生型痘苗病毒同源重组,得到了重组的痘苗病毒；Zeocin 筛选2代后,病毒上清感染细胞,流式细胞分析7.18％的细胞中有CFP的表达,而阴性对照仅有1.43％；Zeocin 药物筛选5代后,通过激光共聚焦可在80％以上的细胞中观察带GFP;提取的重组痘苗基因组DNA也扩增出预期大小目的条带.结论 含Zeocin和GFP双筛选标记的新型重组痘苗病毒不仅具有可观察性且具有药物抗性,非常容易进行筛选鉴定.     

Rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of DNA, inactivated virus, or recombinant vaccinia virus vaccines

Long-term levels of neutralizing antibody were evaluated in mice after a single immunization with experimental DNA or recombinant vaccinia virus (RVV) vaccines encoding the rabies virus glycoprotein (G), or the commercially available inactivated virus human diploid cell vaccine (HDCV). Anamnestic antibody titers were also evaluated after two booster immunizations with vaccines that were identical to or different from the priming vaccine. Five hundred and forty days (1.5 year) after a single immunization with any of the three vaccines, neutralizing antibody titers remained greater than the minimal acceptable human level of antibody titer (0.5 International Units (IU)/ml). In addition, either an HDCV or DNA booster elicited early and elevated anamnestic antibody responses in mice that had been primed with any of the three vaccines. In contrast, RVV boosters failed to elevate titers in mice that had been previously primed with RVV, and elicited slowly rising titers in mice that had been primed with either DNA or HDCV. Thus, a single vaccination with any of the three different vaccines elicited long-term levels of neutralizing antibody that exceeded 0.5 IU/ml. In contrast, different prime-booster vaccine combinations elicited anamnestic neutralizing antibody responses that increased quickly, increased slowly or failed to increase.     

Laser-facilitated epicutaneous immunization of mice with SARS-CoV-2 spike protein induces antibodies inhibiting spike/ACE2 binding

The skin represents an attractive target tissue for vaccination against respiratory viruses such as SARS-CoV-2. Laser-facilitated epicutaneous immunization (EPI) has been established as a novel technology to overcome the skin barrier, which combines efficient delivery via micropores with an inherent adjuvant effect due to the release of danger-associated molecular patterns. Here we delivered the S1 subunit of the Spike protein of SARS-CoV-2 to the skin of BALB/c mice via laser-generated micropores with or without CpG-ODN1826 or the B subunit of heat-labile enterotoxin of E.coli (LT-B). EPI induced serum IgG titers of 1:3200 that could be boosted 5 to 10-fold by co-administration of LT-B and CpG, respectively. Sera were able to inhibit binding of the spike protein to its receptor ACE2. Our data indicate that delivery of recombinant spike protein via the skin may represent an alternative route for vaccines against Covid-19.