The enormous effects of avian influenza on poultry production and the possible health risks to humans have drawn much attention to this disease. The H9N2 subtype of avian influenza virus is widely prevalent among poultry, posing a direct threat to humans through infection or by contributing internal genes to various zoonotic strains of avian influenza. Despite the widespread use of H9N2 subtype vaccines, outbreaks of the virus persist due to the rapid antigenic drift and shifts in the influenza virus. As a result, it is critical to develop a broader spectrum of H9N2 subtype avian influenza vaccines and evaluate their effectiveness. In this study, a recombinant baculovirus expressing the broad-spectrum HA protein was obtained via bioinformatics analysis and a baculovirus expression system (BES). This recombinant hemagglutinin (HA) protein displayed cross-reactivity to positive sera against several subbranch H9 subtype AIVs. An adjuvant and purified HA protein were then used to create an rHA vaccine candidate. Evaluation of the vaccine demonstrated that subcutaneous immunization of the neck with the rHA vaccine candidate stimulated a robust immune response, providing complete clinical protection against various H9N2 virus challenges. Additionally, virus shedding was more effectively inhibited by rHA than by the commercial vaccine. Thus, our findings illustrate the efficacy of the rHA vaccine candidate in shielding chickens against the H9N2 virus challenge, underscoring its potential as an alternative to conventional vaccines.

As a highly pathogenic avian virus, H5 influenza poses a serious threat to livestock, the poultry industry, and public health security. Hemagglutinin (HA) is both the dominant epitope and the main target of influenza-neutralizing antibodies. Here, we designed a nanoparticle hemagglutinin influenza vaccine to improve the immunogenicity of the influenza vaccine. In this study, HA5 subtype influenza virus was used as the candidate antigen and was combined with the artificially designed double-branch scaffold protein I53_dn5 A and B. A structurally correct and bioactive trimer HA5-I53_dn5B/Y98F was obtained through secretion and purification using an insect baculovirus expression system; I53_dn5A was obtained by purification using a prokaryotic expression system. HA5-I53_dn5B/Y98F and I53_dn5A self-assembled into spherical nanoparticles (HA5-I53_dn5) in vitro with a diameter of about 45 nm. Immunization and serum test results showed that both HA5-I53_dn5B/Y98F and HA5-I53_dn5 could induce HA5-specific antibodies; however, the immunogenicity of HA5-I53_dn5 was better than that of HA5-I53_dn5B/Y98F. Groups treated with HA5-I53_dn5B and HA5-I53_dn5 nanoparticles produced IgG antibody titers that were not statistically different from those of the nanoparticle-containing adjuvant group. This production of trimerized HA5-I53_dn5B and HA5-I53_dn5 nanoparticles using baculovirus expression provides a reference for the development of novel, safe, and efficient influenza vaccines.

Influenza viruses can cause highly infectious respiratory diseases, posing noteworthy epidemic and pandemic threats. Vaccination is the most cost-effective intervention to prevent influenza and its complications. However, reliance on embryonic chicken eggs for commercial influenza vaccine production presents potential risks, including reductions in efficacy due to HA gene mutations and supply delays due to scalability challenges. Thus, alternative platforms are needed urgently to replace egg-based methods and efficiently meet the increasing demand for vaccines. In this study, we employed a baculovirus expression vector system to engineer HA, NA, and M1 genes from seasonal influenza strains A/H1N1, A/H3N2, B/Yamagata, and B/Victoria, generating virus-like particle (VLP) vaccine antigens, H1N1-VLP, H3N2-VLP, Yamagata-VLP, and Victoria-VLP. We then assessed their functional and antigenic characteristics, including hemagglutination assay, protein composition, morphology, stability, and immunogenicity. We found that recombinant VLPs displayed functional activity, resembling influenza virions in morphology and size while maintaining structural integrity. Comparative immunogenicity assessments in mice showed that our quadrivalent VLPs were consistent in inducing hemagglutination inhibition and neutralizing antibody titers against homologous viruses compared to both commercial recombinant HA and egg-based vaccines (Vaxigrip). The findings highlight insect cell-based VLP vaccines as promising candidates for quadrivalent seasonal influenza vaccines. Further studies are worth conducting.

BACKGROUND: The COVID-19 pandemic caused by SARS-CoV-2 has created serious health problems worldwide. The most effective way to prevent the occurrence of new epidemic outbreaks is vaccination. One of the modern and effective approaches to vaccine development is the use of virus-like particles (VLPs). The aim of the study is to develop a technology for production of VLP based on recombinant SARS-CoV-2 proteins (E, M, N and S) in insect cells.
METHODS: Synthetic genes encoding coronavirus proteins E, M, N and S were used. VLP with various surface proteins of strains similar to the Wuhan virus, Delta, Alpha and Omicron were developed and cloned into the pFastBac plasmid. The proteins were synthesized in the baculovirus expression system and assembled into VLP in the portable Trichoplusia ni cell. The presence of insertion in the baculovirus genome was determined by PCR. ELISA and immunoblotting were used to study the antigenic activity of VLP. VLP purification was performed by ultracentrifugation using 20% sucrose. Morphology was assessed using electron microscopy and dynamic light scattering.
RESULTS: VLPs consisting of recombinant SARS-CoV-2 proteins (S, M, E and N) were obtained and characterized. The specific binding of antigenic determinants in synthesized VLPs with antibodies to SARS-CoV-2 proteins has been demonstrated. The immunogenic properties of VLPs have been studied.
CONCLUSIONS: The production and purification of recombinant VLPs consisting of full-length SARS-CoV-2 proteins with a universal set of surface antigens have been developed and optimized. Self-assembling particles that mimic the coronavirus virion induce a specific immune response against SARS-CoV-2.
Введение. Пандемия COVID-19, вызванная коронавирусом SARS-CoV-2, породила серьезные проблемы в здравоохранении по всему миру. Ученым в кратчайшие сроки пришлось решать задачи по разработке методов лечения и профилактики этого заболевания. Наиболее эффективным способом прерывания развивающихся новых эпидемических вспышек является вакцинация. Одним из современных и эффективных подходов при разработке вакцин является использование вирусоподобных частиц (Virus like particles, VLP). Цель исследования – разработать технологию получения VLP на основе рекомбинантных белков SARS-CoV-2 (E, M, N и S), продуцируемых в клетках насекомых, и дать их комплексную характеристику. Материалы и методы. Источником вирусных белков послужили синтетические гены, кодирующие белки коронавируса E, M, N и S. Были разработаны VLP с разными поверхностными S-белками 4 штаммов коронавируса: подобный вирусу Ухань, Delta, Alpha и Omicron, клонированные в плазмиду pFastBac. Белки были синтезированы в бакуловирусной системе экспрессии и собраны в VLP в перевиваемой линии клеток Trichoplusia ni (T.ni). Синтез генов, клонирование в трансферные плазмиды и получение рекомбинантных бакуловирусов проводили стандартными методами. Наличие вставки в геноме бакуловируса определяли методом полимеразной цепной реакции. Для исследования антигенной активности VLP применяли иммуноферментный анализ, иммуноблоттинг. Очистку VLP проводили ультрацентрифугированием через 20% сахарозу. Оценку морфологии выполняли с помощью электронной микроскопии и методом динамического светорассеяния. Результаты. Получены и охарактеризованы VLP, состоящие из рекомбинантных белков S, M, E и N, на основе консенсусных последовательностей, циркулирующих в мире геновариантов SARS-CoV-2. Показана специфичность антигенных детерминант синтезированных VLP антителам, формирующимся к белкам SARS-CoV-2, изучены иммуногенные свойства VLP. Заключение. Разработаны способы получения и очистки VLP с универсальным набором поверхностных антигенов, способных к самосборке и индуцирующих специфический иммунитет против SARS-CoV-2.

H7N9 subtype avian influenza virus (AIV) poses a great challenge to poultry industry. Virus-like particle (VLP) is a prospective alternative for the traditional egg-based influenza vaccines. N-linked glycosylation (NLG) regulates the efficacy of influenza vaccines, whereas the impact of NLG modifications on the efficacy of influenza VLP vaccines remains unclear. Here, H7N9 VLPs were assembled in insect cells through co-infection with the baculoviruses expressing the NLG-modified hemagglutinin (HA), neuraminidase and matrix proteins, and the VLP vaccines were assessed in chickens and mice. NLG modifications significantly enhanced hemagglutination-inhibition and virus neutralization antibody responses in mice, rather than in chickens, because different immunization strategies were used in these animal models. The presence of dual NLG at residues 133 and 158 significantly elevated HA-binding IgG titers in chickens and mice. The VLP vaccines conferred complete protection and significantly suppressed virus replication and lung pathology post challenge with H7N9 viruses in chickens and mice. VLP immunization activated T cell immunity-related cytokine response and inhibited inflammatory cytokine response in mouse lung. Of note, the presence of dual NLG at residues 133 and 158 optimized the capacity of the VLP vaccine to stimulate interleukin-4 expression, inhibit virus shedding or alleviate lung pathology in chickens or mice. Intriguingly, the VLP vaccine with NLG addition at residue 133 provided partial cross-protection against the H5Nx subtype AIVs in chickens and mice. In conclusion, dual NLG at residues 133 and 158 in HA can be potentially used to enhance the efficacy of H7N9 VLP vaccines in chickens and mammals.

Many protein expression systems are primarily utilised to produce a single, specific recombinant protein. In contrast, most biological processes such as virus assembly rely upon a complex of several interacting proteins rather than the activity of a sole protein. The high complexity of the baculovirus genome, coupled with a multiphase replication cycle incorporating distinct transcriptional steps, made it the ideal system to manipulate for high-level expression of a single, or co-expression of multiple, foreign proteins within a single cell. We have developed and utilised a series of recombinant baculovirus systems to unravel the sequential assembly process of a complex non-enveloped model virus, bluetongue virus (BTV). The high protein yields expressed by the baculovirus system not only facilitated structure-function analysis of each viral protein but were also advantageous to crystallography studies and supported the first atomic-level resolution of a recombinant viral protein, the major BTV capsid protein. Further, the formation of recombinant double-shelled virus-like particles (VLPs) provided insights into the structure-function relationships among the four major structural proteins of the BTV whilst also representing a potential candidate for a viral vaccine. The baculovirus multi-gene expression system facilitated the study of structurally complex viruses (both non-enveloped and enveloped viruses) and heralded a new generation of viral vaccines.

UNASSIGNED: Equine influenza (EI) is a transmissible viral respiratory sickness of the Equidae family. Two viruses, H7N7 and H3N8 caused EI; however, H7N7 has not been detected for decades. H3N8 has circulated and bifurcated into Eurasian and American lineages. The latter subsequently diversified into Kentucky, South America, and Florida sub-lineages. Florida clade 1 (FC1) and Florida clade 2 (FC2) strains are the only circulating EI viruses (EIVs) in the meantime. Immunization is considered the major means for the prevention and control of EI infection. Using disparate technologies and platforms, several vaccines have been developed and commercialized. According to the recommendations of the World Organization for Animal Health (WOAH), all commercial vaccines shall comprise representatives of both FC1 and FC2 strains. Unfortunately, most of the commercially available vaccines were not updated to incorporate a representative of FC2 strains.
UNASSIGNED: The purpose of this research was to develop a new EI vaccine candidate that incorporates the hemagglutinin (HA) antigen from the currently circulating FC2.
UNASSIGNED: In this study, we report the expression of the full-length recombinant HA gene of FC2 in the baculovirus expression system.
UNASSIGNED: The HA recombinant protein has been proven to maintain its biological characteristics by hemadsorption (HAD) and hemagglutination tests. Moreover, using a reference-specific serum, the specificity of the HA has been confirmed through the implementation of immunoperoxidase and western immunoblotting assays.
UNASSIGNED: In conclusion, we report the expression of specific biologically active recombinant HA of FC2, which would act as a foundation for the generation of an updated EI subunit or virus vector vaccine candidates.

UNASSIGNED: COVID-19 is rampant throughout the world, which has caused great damage to human lives and seriously hindered the development of the global economy. Aiming at the treatment of SARS-CoV-2, in this study, we proposed a novel fenobody strategy based on ferritin (Fe) self-assembly technology.
UNASSIGNED: The neutralizing nanobody H11-D4 of SARS-CoV-2 fused to the C-terminus of end-modified human ferritin was expressed in E. coli and silkworm baculovirus expression systems. A large number of nanoparticles were successfully self-assembled in silkworms, while relatively few nanoparticles can be observed in the treated products from E. coli by electron microscopy. Subsequently, the fenobody\'s expression level and neutralizing activity were then evaluated.
UNASSIGNED: The results showed that the IC50 of H11-D4 and fenobody Fe-H11-D4 expressed in E. coli were 171.1 nmol L-1 and 20.87 nmol L-1, respectively. However, the IC50 of Fe-HD11-D4 expressed in silkworms was 1.46 nmol L-1 showing better neutralization activity.
UNASSIGNED: Therefore, fenobodies can be well self-assembled in silkworm baculovirus expression system, and ferritin self-assembly technology can effectively improve nanobody neutralization activity.

H7N9 avian influenza virus (AIV) has caused huge losses in the poultry industry and impacted human public health security, and still poses a potential threat. Currently, immune prevention and control of avian influenza relies on traditional inactivated vaccines; however, they have some limitations and genetically engineered avian influenza subunit vaccines may be potential candidate vaccines. In this study, a T169A mutation in the HA protein derived from H7N9 AIV A/Chicken/Guangdong/16876 (H7N9-16876) was generated using the baculovirus expression system (BVES). The results showed that the mutant (HAm) had significantly increased thermostability compared with the wild-type HA protein (HA-WT). Importantly, immunizing chickens with HAm combined with ISA 71VG elicited higher cross-reactive hemagglutination inhibition (HI) antibody responses and cytokine (IFN-γ and IL-4) secretion. After a lethal challenge with heterologous H7N9 AIV, the vaccine conferred chickens with 100% (10/10) clinical protection and effectively inhibited viral shedding, with 90% (9/10) of the chickens showing no virus shedding. The thermostability of HAm may represent an advantage in practical vaccine manufacture and application. In general, the HAm generated in this study represents a promising subunit vaccine candidate for the prevention and control of H7N9 avian influenza.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV), which can cause severe clinical disease and even death in humans. In recent years, the disease has spread to a wider area, posing a major public health threat to China as well as the Middle East, Europe and Africa, and there is no safe and effective vaccine to prevent the disease. Recently, it has been shown that using the Zera fusion to target proteins can enhance immunogenicity and improve the potential for developing viral vaccines. Based on this finding, in this study, two vaccine candidates, Zera-Gn and Zera-Np, were prepared using an insect baculovirus system expressing CCHFV glycoprotein (Gn) and nucleocapsid protein (Np) fused with Zera tags, and evaluated for immunogenicity in BALB/c mice. The obtainedresults showed that both Zera-Gn and Zera-Np recombinant nanoparticles were successfully expressed, and Zera-Gn had good induction of humoral and cellular immunity in mice, and its immunogenicity was significantly higher than that of Zera-Np. The results indicated that Zera-Gn self-assembled nanoparticles prepared by fusing Zera tags with CCHFV spike-in protein Gn have the potential to be a candidate vaccine for CCHF, and this study provides a reference for the development of Zera self-assembled nanoparticle vaccine for CCHF.