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Histopathological analyses of S-M1 VLP vaccinated mice. Tissue samples were collected post mortem and embedded in paraffin. The samples were then sectioned and stained with hematoxylineosin. * indicates airway, + indicates blood vessel, black arrows indicate peribronchiolar accumulation of lymphocytes, plasma cells, and histiocytes. White arrows indicate Virchow-Robin spaces expanded by lymphocytes and plasma cells.
Source publication
Virus-like particles (VLPs) are highly immunogenic and versatile subunit vaccines composed of multimeric viral proteins that mimic the whole virus but lack genetic material. Due to the lack of infectivity, VLPs are being developed as safe and effective vaccines against various infectious diseases. In this study, we generated a chimeric VLP-based CO...
Contexts in source publication
Context 1
... of the lungs show perivascular and peribronchiolar accumulations of lymphocytes, plasma cells, and histiocytes. Additionally, alveolar macrophages were scattered within the airways (Figure 7). Notably, it is unusual that the PBS group in this study had such a low lung pathology ( Figure 7). ...
Context 2
... alveolar macrophages were scattered within the airways (Figure 7). Notably, it is unusual that the PBS group in this study had such a low lung pathology ( Figure 7). The clinical observations, ISH results, and laboratory findings correlate with an adequate response to a challenge/disease, but the lung pathology is minimal or even normal in some mice. ...
Context 3
... the lesions are typically regionally variable. Within the central nervous system, the lymphoplasmacytic and histiocytic inflammation were noted in both the PBS and S-M1 VLPs groups, resulting in the expansion of the vascular walls within the multifocal vessels in the cerebrum (Figure 7). The vasculature was obscured by significant numbers of histiocytes, lymphocytes, and plasma cells, as well as rare neutrophils which largely expanded the Virchow-Robin space and infiltrated the adjacent parenchyma. ...
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Chikungunya virus (CHIKV) is a mosquito-borne virus. The emergence of CHIKV infection has raised global concern, and there is a growing need to develop safe and effective vaccines. Here, adenovirus 5 was used as the vaccine vector to construct recombinant adenoviruses expressing CHIKV E2, E1, and E2-6K-E1, respectively. And then the immunogenicity...
Citations
... Of note, in the blood, levels of activated T cells after challenge infections were not significantly different compared to the control-vaccinated animals. Since it has been demonstrated that the systemic SARS-CoV-2 spread can also be mediated via the blood [75][76][77], we hypothesize a role of the blood T cells for the prevention of SARS-CoV-2 infection within the brain. Since the brain manifestation is a non-COVID-19-disease-specific outcome of the K18-hACE2 mouse model and associated to the artificial expression of the human ACE2 receptor molecule in the brain [78], it might be interesting to test the emergency vaccination regimen in another animal model without the dominant brain manifestation and neurological outcome. ...
The sudden emergence of SARS-CoV-2 demonstrates the need for new vaccines that rapidly protect in the case of an emergency. In this study, we developed a recombinant MVA vaccine co-expressing SARS-CoV-2 prefusion-stabilized spike protein (ST) and SARS-CoV-2 nucleoprotein (N, MVA-SARS-2-ST/N) as an approach to further improve vaccine-induced immunogenicity and efficacy. Single MVA-SARS-2-ST/N vaccination in K18-hACE2 mice induced robust protection against lethal respiratory SARS-CoV-2 challenge infection 28 days later. The protective outcome of MVA-SARS-2-ST/N vaccination correlated with the activation of SARS-CoV-2-neutralizing antibodies (nABs) and substantial amounts of SARS-CoV-2-specific T cells especially in the lung of MVA-SARS-2-ST/N-vaccinated mice. Emergency vaccination with MVA-SARS-2-ST/N just 2 days before lethal SARS-CoV-2 challenge infection resulted in a delayed onset of clinical disease outcome in these mice and increased titers of nAB or SARS-CoV-2-specific T cells in the spleen and lung. These data highlight the potential of a multivalent COVID-19 vaccine co-expressing S- and N-protein, which further contributes to the development of rapidly protective vaccination strategies against emerging pathogens.
... The M1 matrix protein assisted in the continuous release of budding VLPs from the cell host, thus escaping the need for multiple transfections. In addition, these enveloped chimeric VLPs were found to be immunogenic in mice models and generated S-specific neutralizing antibodies [134]. ...
Vaccines are integral to human life to protect them from life-threatening diseases. However, conventional vaccines often suffer limitations like inefficiency, safety concerns, unavailability for non-culturable microbes, and genetic variability among pathogens. Chimeric vaccines combine multiple antigen-encoding genes of similar or different microbial strains to protect against hyper-evolving drug-resistant pathogens. The outbreaks of dreadful diseases have led researchers to develop economical chimeric vaccines that can cater to a large population in a shorter time. The process development begins with computationally aided omics-based approaches to design chimeric vaccines. Furthermore, developing these vaccines requires optimizing upstream and downstream processes for mass production at an industrial scale. Owing to the complex structures and complicated bioprocessing of evolving pathogens, various high-throughput process technologies have come up with added advantages. Recent advancements in high-throughput tools, process analytical technology (PAT), quality-by-design (QbD), design of experiments (DoE), modeling and simulations, single-use technology, and integrated continuous bioprocessing have made scalable production more convenient and economical. The paradigm shift to innovative strategies requires significant attention to deal with major health threats at the global scale. This review outlines the challenges and emerging avenues in the bioprocess development of chimeric vaccines.
... As shown in Figure 2C, Western blot analysis revealed that the S-HA and M1 proteins were incorporated into the VLPs with the correct molecular weight (S-HA, 174 kDa and M1, 25 kDa). However, the SHAM1 VLPs were slightly larger in size (~100-200 nm diameter), similar to previously engineered pseudotyped VLPs for both SARS-CoV-1 (~160 nm diameter) [42] and SARS-CoV-2 (~80-200 nm diameter) [44,45]. To examine if pseudotyping improves VLP spike yield, S protein quantification was performed as described above ...
... Pseudotyping using influenza proteins was previously employed for SARS-CoV-1 VLPs in Sf9 cells, leading to >twofold improvement in VLP spike yield (1 mg/L) [42] compared to that of SARS-CoV-1 SEM VLPs [43]. A similar strategy was more recently utilized for SARS-CoV-2 VLPs in mammalian [44] and Sf9 insect cells [33,45], though the VLP spike yields were not reported. To determine if pseudotyping similarly improves SARS-CoV-2 VLP spike yield, a baculovirus vector was created to express the SARS-CoV-2 S ectodomain (aa 1-1213) fused to the transmembrane (TM) and cytoplasmic tail (CT) domains of an H1N1 influenza HA protein (accession #NP_040980) in combination with the influenza matrix protein M1 (denoted SHAM1, Figure 2A). ...
... To determine if pseudotyping similarly improves SARS-CoV-2 VLP spike yield, a baculovirus vector was created to express the SARS-CoV-2 S ectodomain (aa 1-1213) fused to the transmembrane (TM) and cytoplasmic tail (CT) domains of an H1N1 influenza HA protein (accession #NP_040980) in combination with the influenza matrix protein M1 (denoted SHAM1, Figure 2A). For simplicity, we refer to this influenza pseudotyping strategy as "pseudotyping" throughout, including designs from other studies [44,45] that used the same HA domains but from different influenza strains. Similar to SE, SM, and SEM VLPs ( Figure 1B,C), SHAM1 VLPs exhibited a spherical morphology and showed binding with multiple anti-S immunogold particles ( Figure 2B). ...
Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04–0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72–144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.
... Pseudovirus neutralization potencies. Pseudovirus microneutralization assays were performed as previously described 14,15 to assess the neutralization potency of NAS against various SARS-CoV-2 pseudoviruses, i.e., ancestral, Alpha, Delta, Omicron BA.1, BA.2, A.4/5, and BA.2.75. The results revealed that NAS potently neutralized all mentioned pseudoviruses with 50% pseudovirus neutralization titers (PVNT 50 ) ranging from 0.0035 to 3.1997 µg/ml (Table 1). ...
An HPMC-based nasal spray solution containing human IgG1 antibodies against SARS-CoV-2 (nasal antibody spray or NAS) was developed to strengthen COVID-19 management. NAS exhibited potent broadly neutralizing activities against SARS-CoV-2 with PVNT50 values ranging from 0.0035 to 3.1997 μg/ml for the following variants of concern (ranked from lowest to highest): Alpha, Beta, Gamma, ancestral, Delta, Omicron BA.1, BA.2, BA.4/5, and BA.2.75. Biocompatibility assessment showed no potential biological risks. Intranasal NAS administration in rats showed no circulatory presence of human IgG1 anti-SARS-CoV-2 antibodies within 120 h. A double-blind, randomized, placebo-controlled trial (NCT05358873) was conducted on 36 healthy volunteers who received either NAS or a normal saline nasal spray. Safety of the thrice-daily intranasal administration for 7 days was assessed using nasal sinuscopy, adverse event recording, and self-reporting questionnaires. NAS was well tolerated, with no significant adverse effects during the 14 days of the study. The SARS-CoV-2 neutralizing antibodies were detected based on the signal inhibition percent (SIP) in nasal fluids pre- and post-administration using a SARS-CoV-2 surrogate virus neutralization test. SIP values in nasal fluids collected immediately or 6 h after NAS application were significantly increased from baseline for all three variants tested, including ancestral, Delta, and Omicron BA.2. In conclusion, NAS was safe for intranasal use in humans to increase neutralizing antibodies in nasal fluids that lasted at least 6 h.
... The production of lentiviral pseudoviruses carrying CoV spike was described in previous studies (18,20). Briefly, to generate these pseudoviruses, a combination of plasmids was used, including the lentivirus backbone that expresses a firefly luciferase reporter gene (pCSFLW), an expression plasmid that expresses HIV-1 structural/ regulatory proteins (pCMVR8.91), and pCAGGS that expresses spikes of SARS-CoV-2. ...
The emergence and rapid evolution of SARS-CoV-2 variants have posed a major challenge to the global efforts to control the COVID -19 pandemic. In this study, we investigated the potential of two SARS-CoV-2 variants, BA.2 and BA.5, to evade neutralization by a human monoclonal antibody targeting the virus’s spike RBD (mAb 1D1). By subjecting the viruses to serial propagation in the presence of the antibody, we found that BA.2 exhibited poor growth, whereas BA.5 regained robust growth with significantly higher kinetics than the parental virus. Genetic analysis identified a single mutation, A475V, in the spike protein of BA.5 that substantially reduced the neutralizing activities of monoclonal antibodies and convalescent sera. In addition, the A475V mutation alone in BA.2 moderately reduced the neutralizing activity but completely abolished the neutralizing effect of mAb 1D1 when F486V or L452R were also present. Our results shed light on the possible evolutionary development of SARS-CoV-2 variants under selection pressure by monoclonal antibodies and have implications for the development of effective antibody therapies and vaccines against the virus.
... Immunogenicity was assessed by functional neutralizing antibody against SARS-CoV-2 Omicron sublineage BA. Pseudovirus neutralization test (pVNT) against the Omicron variants was performed as described previously [14]. Two-fold serial dilutions of serum samples (starting 1:40 or 1:80) were incubated with pseudoviruses displaying the Omicron (BA.2.75 or BA.4/5) spikes in a 1:1 vol/vol ratio in a 96-well culture plate for 1 h at 37 • C. The pseudovirus input used was normalized to 1 × 10 5 RLU/well. ...
Background:
Children 6 months to < 5 years old are recommended to receive 3-dose regimen of BNT162b2. Children previously infected with Omicron variant of SARS-CoV-2 develop immunity from natural infection, therefore may require fewer doses of vaccine.
Objective:
To compare immunogenicity of 1- or 2-dose BNT162b2 in healthy children post COVID-19 with 3-dose BNT162b2 in COVID-naïve children.
Methods:
Children aged 6 months to < 5 years who developed COVID-19 during the Omicron-predominant period were enrolled; Group A 3-6 months(N = 40) and Group B > 6 months(N = 40) prior to vaccination. Participants in Group A and B received 2-dose BNT162b2 intramuscularly 1 month apart. COVID-naïve children were enrolled as a control group (N = 40) and received 3-dose BNT162b2 at month 0,1,3. Neutralizing antibody against Omicron variant(BA.2.75 and BA.4/5) was determined by pseudovirus assays(pVNT) as reported by neutralization dilution for 50%inhibition (ID50) at 28 days after the 1st and 2nd dose.
Results:
From October-November 2022, 120 children with a median age of 2.8 years (IQR 1.6-4.0) were enrolled. The median duration since COVID-19 to vaccination was 4.4 months(IQR 3.8-5.4) in Group A and 7.9 months(7.0-8.5) in Group B. In Group A, the geometric means(GMs) of pVNT-BA.2.75 ID50 were 553 (95%CI 338-906) and 753(516-1098) after 1 and 2 doses, respectively, and the GMs of pVNT-BA.4/5 ID50 were 1936(1402-2673) and 1885(1414-2512), respectively. In Group B, the GMs of pVNT-BA.2.75 ID50 were 1383(1100-1742) and 1419 (1104-1823), and the GMs of pVNT-BA.4/5 ID50 were 2627(2048-3367) and 2056(1546-2735), respectively. Meanwhile in COVID-naïve group, the GMs of pVNT-BA.2.75 and pVNT-BA.4/5 ID50 were 158(98-255) and 59(31-114) after the 3rd dose, respectively. The geometric mean ratio(GMR) of pVNT-BA.2.75 ID50 after 1 dose in Group A and B compared with after 3 doses in COVID-naïve group were 3.50 (1.93-6.34) and 8.74 (4.79-15.95), respectively. The GMR of pVNT-BA.2.75 ID50 after 1 dose in Group B compared with Group A was 2.50 (1.45-4.31).
Conclusions:
Children previously infected with SARS-CoV-2 Omicron variant, developed robust neutralizing antibody response against Omicron variant after single-dose BNT162b2. Children with an interval of > 6 months since COVID-19 infection developed higher neutralizing antibody response compared to those with a 3-to-6-month interval.
... Different VLP-based vaccines against the SARS-CoV-2 utilizing varied VLP platform and diverse expression systems have been investigated [22,23] reported the development of Chimeric VLP-Based COVID-19 Vaccine utilizing SARS-CoV-2 Spike protein and the influenza virus A matrix (M1) VLP. They used human cell lines eukaryotic expression system for vaccine production. ...
... According to their results, high titers of spike specific IgG and neutralizing antibodies were induced through vaccine injection. In another study, the RBD protein from SARS-Cov2 was chemically fused to modified HBs-Ag protein and formulated [23,24]. The comprehensive immunization results from vaccine injection approved the high protection against SARS-CoV-2 in mice. ...
Background
Virus-like particles are an interesting vector platform for vaccine development. Particularly, Hepatitis B virus core antigen has been used as a promising VLP platform. It is highly expressed in different recombinant expression systems, such as E. coli, and self-assembled in vitro. It effectively improves the immunogenicity of foreign antigenic epitopes on its surface. Various foreign antigens from bacteria, viruses, and protozoa can be genetically inserted into such nanoparticles. The effective immunogenicity due to VLP vaccines has been reported. However, no research has been performed on the SARS-CoV2 vaccine within this unique platform through genetic engineering. Considering the high yield of target proteins, low cost of production, and feasibility of scaling up, E. coli is an outstanding expression platform to develop such vaccines. Therefore, in this investigation, we planned to study and develop a unique HBc VLP-based vaccine against SARS-Cov2 utilizing the E. coli expression system due to its importance.
Results
Insertion of the selected epitope was done into the major immunodominant region (MIR) of truncated (149 residues) hepatitis B core capsid protein. The chimeric protein was constructed in PET28a⁺ and expressed through the bacterial E. coli BL21 expression system. However, the protein was expressed in inclusion body forms and extracted following urea denaturation from the insoluble phase. Following the extraction, the vaccine protein was purified using Ni2 + iminodiacetic acid (IDA) affinity chromatography. SDS-PAGE and western blotting were used to confirm the protein expression. Regarding the denaturation step, the unavoidable refolding process was carried out, so that the chimeric VLP reassembled in native conformation. Based on the transmission electron microscopy (TEM) analysis, the HBC VLP was successfully assembled. Confirming the assembled chimeric VLP, we explored the immunogenic effectivity of the vaccine through mice immunization with two-dose vaccination with and without adjuvant. The utilization of adjuvant was suggested to assess the effect of adjuvant on improving the immune elicitation of chimeric VLP-based vaccine. Immunization analysis based on anti-spike specific IgG antibody showed a significant increase in antibody production in harvested serum from immunized mice with HBc-VLP harboring antigenic epitope compared to HBc-VLP- and PBS-injected mice.
Conclusions
The results approved the successful production and the effectiveness of the vaccine in terms of humoral IgG antibody production. Therefore, this platform can be considered a promising strategy for developing safe and reasonable vaccines; however, more complementary immunological evaluations are needed.
... The pVNT against the Omicron variant (BA.2) was performed as described previously [17][18][19] . Briefly, two-fold serial dilutions of the sera (starting 1: 40) were incubated with pseudoviruses displaying the Omicron (BA.2) spike in a 1: 1 volume/volume ratio in a 96-well culture plate for 1 hour at 37 °C. ...
Objectives:
To evaluate the immunogenicity of an extended interval regimen of BNT162b2 among healthy school-age children.
Methods:
A randomized-control trial conducted among healthy Thai children aged 5-11 years. Participants received 2 doses of BNT162b2 with an 8-week (extended dosing) versus 3-week interval. Immunogenicity was determined by neutralization test (NT) against the Omicron variant; surrogate virus NT (sVNT;BA.1,%inhibition) and pseudovirus NT (pVNT;BA.2,ID50). The third dose was offered to participants who had sVNT <68%inhibition. The immunogenicity outcome was evaluated at 14 days after the second and third dose.
Results:
During February to April 2022, 382 children with a median age (IQR) of 8.4 years (6.6-10.0) were enrolled. At 14 days after 2-doses of BNT162b2, geometric means (GMs) of sVNT in 8-week versus 3-week interval groups were 49.6 (95%CI 44.8-54.9), versus 16.5 (95%CI 13.0-20.9), with a geometric means ratio of 3.0 (95%CI 2.4-3.8). Among 102 participants who received the third dose at a median of 15 weeks from the second dose, the GMs of sVNT increased to 73.3 (95%CI 69.0-77.8) and pVNT was 326 (95%CI 256-415).
Conclusion:
Extended 8-week interval regimen of BNT162b2 induced higher neutralizing antibodies than a standard 3-week interval regimen. The third dose induced high neutralizing antibodies against the Omicron variant.
... Influenza VLPs expressing the codon-optimized full-length S, S1, or S2 subunits were reported to induce virus-specific antibody responses in mice, but the neutralizing antibody responses were mediocre and failed to fully inhibit the virus receptor and ligand binding interaction [127]. Immunizing K18-hACE2 mice with HEK293T-derived chimeric influenza VLPs reduced the lung virus titers and promoted their survival post-challenge infection with SARS-CoV-2, albeit with moderate levels of pulmonary inflammation and brain lesion [128]. An interesting approach was recently reported. ...
With technological advancements enabling globalization, the intercontinental transmission of pathogens has become much easier. Respiratory viruses are one such group of pathogens that require constant monitoring since their outbreak leads to massive public health crises, as exemplified by the influenza virus, respiratory syncytial virus (RSV), and the recent coronavirus disease 2019 (COVID-19) outbreak caused by the SARS-CoV-2. To prevent the transmission of these highly contagious viruses, developing prophylactic tools, such as vaccines, is of considerable interest to the scientific community. Virus-like particles (VLPs) are highly sought after as vaccine platforms for their safety and immunogenicity profiles. Although several VLP-based vaccines against hepatitis B and human papillomavirus have been approved for clinical use by the United States Food and Drug Administration, VLP vaccines against the three aforementioned respiratory viruses are lacking. Here, we summarize the most recent progress in pre-clinical and clinical VLP vaccine development. We also outline various strategies that contributed to improving the efficacy of vaccines against each virus and briefly discuss the stability aspect of VLPs that makes it a highly desired vaccine platform.
... Pseudovirus microneutralization assays were performed as previously described 13,14 to assess the neutralization potency of COVITRAP™ against various SARS-CoV-2 pseudoviruses, i.e., Ancestral, Alpha, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.4/5, and Omicron BA.2.75. The results revealed that COVITRAP™ potently neutralized all mentioned pseudoviruses with 50% pseudovirus neutralization titers (PVNT50) ranging from 0.0035 to 3.1997 µg/ml (Table 1). ...
Successful COVID-19 prevention requires additional measures beyond vaccination, social distancing, and masking. A nasal spray solution containing human IgG1 antibodies against SARS-CoV-2 (COVITRAP™) was developed to strengthen other COVID-19 preventive arsenals. Here, we evaluated its pseudovirus neutralization potencies, preclinical and clinical safety profiles, and intranasal SARS-CoV-2 inhibitory effects in healthy volunteers ( NCT05358873 ). COVITRAP™ exhibited broadly potent neutralizing activities against SARS-CoV-2 with PVNT50 values ranging from 0.0035 to 3.1997 μg/ml for the following variants of concern (ranked from lowest to highest): Alpha, Beta, Gamma, Ancestral, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.4/5, and Omicron BA.2.75. It demonstrated satisfactory preclinical safety profiles based on evaluations of in vitro cytotoxicity, skin sensitization, intracutaneous reactivity, and systemic toxicity. Its intranasal administration in rats did not yield any detected circulatory levels of the human IgG1 anti-SARS-CoV-2 antibodies at any time point during the 120 hours of follow-up. A double-blind, randomized, placebo-controlled trial (RCT) was conducted on 36 healthy volunteers who received either COVITRAP™ or a normal saline nasal spray at a 3:1 ratio. Safety of the thrice-daily intranasal administration for 7 days was assessed using nasal sinuscopy, adverse event recording, and self-reporting questionnaires. COVITRAP™ was well tolerated, with no significant adverse effects in healthy volunteers for the entire 14 days of the study. The intranasal SARS-CoV-2 inhibitory effects of COVITRAP™ were evaluated in nasal fluids taken from volunteers pre- and post-administration using a SARS-CoV-2 surrogate virus neutralization test. SARS-CoV-2 inhibitory effects in nasal fluids collected immediately or six hours after COVITRAP™ application were significantly increased from baseline for all three variants tested, including Ancestral, Delta, and Omicron BA.2. In conclusion, COVITRAP™ was safe for intranasal use in humans to provide SARS-CoV-2 inhibitory effects in nasal fluids that lasted at least six hours. Therefore, COVITRAP™ can be considered an integral instrument for COVID-19 prevention.
