Inactivated Antigen Research Articles

Mixed Th1/Th2/Th17 Responses Induced by Plant Oil Adjuvant-Based B. bronchiseptica Vaccine in Mice, with Mechanisms Unraveled by RNA-Seq, 16S rRNA and Metabolomics.

The current Bordetella bronchiseptica (Bb) vaccine, when adjuvanted with alum, does not elicit adequate robust cellular immunity or effective antibody defense against Bb attacks. Unfortunately, antibiotic treatment generally represents an ineffective strategy due to the development of resistance against a broad range of antibiotics. The present study was designed to investigate the immune response, protective capabilities and underlying mechanisms of a plant oil-based adjuvant E515 formulated with inactivated Bb antigen as a potential vaccine candidate against Bordetella bronchiseptica. Immunization studies revealed that a combination of SO, VE and GS (E515) exhibited a good synergistic adjuvant effect. The E515 adjuvanted Bb vaccine was proven to be highly efficacious and induced a mixed Th1/Th2/Th17 immune response in mice, leading to a significant increase in Bb-specific IgG, IgG1 and IgG2a antibodies, proliferative lymphocyte responses and cytokine levels (by lymphocytes and serum) and effectively induced responses by CD4+ TE, TM cells and B cells. The E515 adjuvant significantly enhanced the immune protection provided by the Bb vaccine in a mice model, as indicated by a reduced bacterial burden in the lungs. Multi-omics sequencing analysis revealed that E515 functions as an adjuvant by modulating critical pathways, including cytokine-cytokine receptor interaction, the IL-17 signaling pathway and the chemokine signaling pathway. This modulation also included interactions with beneficial species of bacteria including Alistipes, Odoribacter and Colidextribacter, as well as energy and lipid-related metabolites, thus highlighting its role as an immunomodulatory agent. Collectively, our results demonstrate the huge potential of E515-Bb vaccine candidates, thus highlighting the vegetable oil original adjuvant E515 as a promising agent for the development of new veterinary vaccines.

Enhanced production of recombinant coxsackievirus A16 using a serum-free HEK293A suspension culture system for bivalent enterovirus vaccine development

Coxsackievirus A16 (CVA16) is one of the primary pathogens that causes hand, foot, and mouth disease (HFMD) in young children. In previous studies, CVA16 vaccine development has encountered several challenges, such as inefficient replication of the CVA16 virus in present culture systems, the induction of only mild neutralizing antibody titers, and neutralizing antibodies induced by certain vaccine candidates that are unable to protect against CVA16 viral challenge. In this study, we constructed a DNA-launched CVA16 infectious clone (CVA16ic) based on the genomic sequence of the CVA16 N5079 strain to minimize interference from viral quasispecies. The biochemical properties of this CVA16ic strain were similar to those of its parental strain. Serum-free HEK293A suspension cells, which produced higher virus titers than Vero cells, were demonstrated to improve CVA16 production yields. In addition, our study showed that inactivated EV-A71 antigens could enhance the immunogenicity of inactivated CVA16 mature/full particles (F-particles), suggesting that a bivalent CVA16 and EV-A71 vaccine may be an effective strategy for CVA16 vaccine development. These findings are expected to provide novel strategies and accelerate the development of bivalent HFMD vaccines.

Pathogenicity of Aeromonas veronii from Nile Tilapia (Oreochromis niloticus) and Efficacy of Fish Oral Vaccine against Motile Aeromonad Septicemia in Tank Trials

Motile aeromonad septicemia (MAS), caused by the Aeromonas species, has been a serious problem in fish health management, particularly in Nile tilapia (Oreochromis niloticus). This study characterized an Aeromonas species isolated from farmed tilapia fingerlings in Binangonan, Rizal, Philippines, and tested for its pathogenicity in tank trials. The isolate, designated as Aeromonas veronii DFR01 (Diseased Fish Rizal), was identified based on 16S rRNA phylogenetic analysis, 16S rRNA homology, and MALDI-TOF mass spectrometry. Its biochemical profile was generated from API and Biolog Gen III systems. A median lethal dose of A. veronii DFR01 was determined to be 107 CFU/mL in tank trials and was utilized as a whole-cell inactivated antigen for oral vaccine development. The immunized tilapia fingerlings produced elevated levels of immunoglobulin M (IgM) in the blood as determined by an enzyme-linked immunosorbent assay (ELISA). There was a significant increase in IgM levels 14 days post-vaccination. A quantitative polymerase chain reaction (qPCR) showed increasing levels of IgM gene expression after vaccination until 38 days of culture. Vaccinated fish showed 25–35% cumulative mortality after the challenge, while non-vaccinated-challenged fish showed 75% mortality. The findings of this research suggest that the fish oral vaccine may prove beneficial for farmed tilapia populations. The vaccine elicited improved immune responses in the fish and resulted in higher survival rates.

Development of two competitive ELISAs based on monoclonal antibodies for the serological detection of Bovine ephemeral fever virus

Bovine ephemeral fever virus (BEFV) is a member of the genus Ephemerovirus in the family Rhabdoviridae. It is an arthropod-borne virus transmitted by many species of midges and mosquitoes. It can cause severe economic consequences due to losses in milk production and the general condition of cattle and water buffalo. BEF occurs in some tropical, subtropical and warm temperate regions of Africa, Australia, the Middle East and Asia with seasonal outbreaks, but its possible spread to other areas (e.g. Europe) cannot be excluded. Therefore, using and developing rapid diagnostic methods with optimal performance is essential for identifying emerging pathogens and their control. In the present study, we developed two competitive serological ELISAs based on monoclonal antibodies (mAbs), designed by using BEFV inactivated antigen and the BEF recombinant nucleoprotein (N), respectively. A panel of 77 BEF-positive and 338 BEF-negative sera was used to evaluate the two tests. With a diagnostic sensitivity of 97.4 % using the inactivated virus and 98.7 % using the recombinant N, and a diagnostic specificity of 100 % using both antigens, our results suggest that these tests are suitable for the serological diagnosis of BEF.

A Novel Application of Virus Like Particles in the Hemagglutination Inhibition Assay.

The hemagglutination inhibition (HI) assay is a traditional laboratory procedure for detection and quantitation of serum antibodies of hemagglutinating viruses containing the hemagglutinin (HA) gene. The current study aimed to investigate the novel use of virus like particles (VLP) as an antigen for the HI assay. VLPs were prepared from a strain of H5N1 using a baculovirus expression system. The VLPs were characterized using the hemagglutination test, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and transmission electron microscopy. The comparative HI assay was performed using three different seed antigens: A/chicken/Mexico/232/94 (H5N2), A/chicken/Egypt/18-H/09(H5N1) and A/goose/Guangdong/1/1996(H5N1). The HI assay of serum antibody titrations using homologous antigens to these vaccinal seeds were compared to the VLP's antigens for the same serum. The HI titers were logically relevant to the similarity between VLP antigens and vaccinal seeds, indicating the VLPs behave similarly to the standard HI assay which uses inactivated whole virus as an antigen. VLPs could be considered as an alternative to the HI assay antigen as they show a relatedness between the similarity with vaccinal seed and serum antibodies. Compared to typical entire H5N1 viral antigen prepared in SPF eggs that require proper inactivation to avoid any public health risk, VLPs prepared in tissue culture, plants or insect cells are a safe, inexpensive and scalable alternative to inactivated whole virus antigen.

An inactivated SARS-CoV-2 vaccine based on a Vero cell culture-adapted high-titer virus confers cross-protection in small animals

Rapidly waning immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires continued global access to affordable vaccines. Globally, inactivated SARS-CoV-2 vaccines have been widely used during the SARS-CoV-2 pandemic. In this proof-of-concept study we adapted an original-D614G SARS-CoV-2 virus to Vero cell culture as a strategy to enhance inactivated vaccine manufacturing productivity. A passage 60 (P60) virus showed enhanced fitness and 50-fold increased virus yield in a bioreactor compared to the original-D614G virus. It further remained susceptible to neutralization by plasma from SARS-CoV-2 vaccinated and convalescent individuals, suggesting exposure of relevant epitopes. Monovalent inactivated P60 and bivalent inactivated P60/omicron BA.1 vaccines induced neutralizing responses against original-D614G and BA.1 viruses in mice and hamsters, demonstrating that the P60 virus is a suitable vaccine antigen. Antibodies further cross-neutralized delta and BA.5 viruses. Importantly, the inactivated P60 vaccine protected hamsters against disease upon challenge with original-D614G or BA.1 virus, with minimal lung pathology and lower virus loads in the upper and lower airways. Antigenicity of the P60 virus was thus retained compared to the original virus despite the acquisition of cell culture adaptive mutations. Consequently, cell culture adaptation may be a useful approach to increase yields in inactivated vaccine antigen production.

Boosting the immune response in COVID-19 vaccines via an Alum:CpG complex adjuvant

Selecting appropriate adjuvants is crucial for developing an effective vaccine. However, studies on the immune responses triggered by different adjuvants in COVID-19 inactivated vaccines are scarce. Herein, we evaluated the efficacy of Alum, CpG HP021, Alum combined with CpG HP021 (Alum/CpG), or MF-59 adjuvants with COVID-19 inactivated vaccines in K18-hACE2 mice, and compared the different immune responses between K18-hACE2 and BALB/c mice. In K18-hACE2 mice, the Alum/CpG group produced a 6.5-fold increase in anti-receptor-binding domain (RBD) IgG antibody titers compared to the Alum group, and generated a comparable level of antibodies even when the antigen amount was reduced by two-thirds, possibly due to the significant activation of germinal center (GC) structures in the central region of the spleen. Different adjuvants induced a variety of binding antibody isotypes. CpG HP021 and Alum/CpG were biased towards Th1/IgG2c, while Alum and MF-59 were biased toward Th2/IgG1. Cytokines IFN-γ, IL-2, and TNF-α were significantly increased in the culture supernatants of splenocytes specifically stimulated in the Alum/CpG group. The antibody responses in BALB/c mice were similar to those in K18-hACE2 mice, but with lower levels of neutralizing antibodies (NAbs). Notably, the Alum/CpG-adjuvanted inactivated vaccine induced a higher number of T cells secreting IFN-γ and IL-2, increased the percentage of effector memory T (TEM) cells among CD8+ T cells, and effectively protected K18-hACE2 mice from Delta variant challenge. Our results showed that Alum/CpG complex adjuvant significantly enhanced the immune response to inactivated COVID-19 antigens and could induce a long-lasting immune response.

Characterization, histopathology and immunogenicity of the lumpy skin disease virus isolated during 2019-20 in Bangladesh.

Lumpy skin disease (LSD) is a highly contagious vector-borne viral disease of cattle. LSD has emerged in Bangladesh in 2019, causing significant economic losses due to its high morbidity and mortality. This research was designed to isolate, identify, and assess the immunogenicity of LSD virus (LSDV) using nodular tissue samples obtained from affected cattle during the 2019-20 outbreak across nine districts of Bangladesh. To determine the presence of LSDV in nodular tissues, we initially used iiPCR and PCR, followed by histopathological examination. 151 were positive via iiPCR and PCR among the 180 collected samples. The PCR positive 151 samples were then inoculated into 10-day-old embryonated chicken eggs via the CAM route to isolate LSDV, confirmed through PCR. Subsequently, partial sequencing and phylogenetic analysis of the P32 gene were performed to determine the origin of the circulating LSDV strain. The immunogenicity of selected LSDV strains was assessed through an ELISA test. The PCR results revealed a distinct positive band at 192 bp in both the nodular tissue samples and the LSDV isolated from chicken embryo inoculations. Microscopic analysis of the nodular lesions revealed thickening of the epidermis, ballooning degeneration of keratinocytes, and proliferation of follicular epithelia. Additionally, mononuclear infiltration was observed at the demarcation line between infected and healthy tissue, with necrosis of muscular tissues beneath the epidermis. The LSDV isolate from Bangladesh exhibited a close genetic relationship with LSDV strains isolated from neighboring and other regional countries including India, Myanmar, and Mongolia. This observation strongly suggests the possibility of a transboundary spread of the LSD outbreak in Bangladesh during 2019-2020. The results of the immunogenicity test showed that the serum antibody titer remained at a protective level for up to 18 months following secondary immunization with inactivated LSDV antigen. This finding suggests that the inactivated LSDV antigen could be a potential vaccine candidate to protect cattle in Bangladesh against LSDV. In conclusion, our research successfully isolated, identified, and characterized LSDV in cattle nodular tissues from the 2019-20 outbreak in Bangladesh. Furthermore, it provided insights into the probable origin of the circulating strain and investigated a potential vaccine candidate to protect cattle in the region from LSDV.

Inactivated Split MERS-CoV Antigen Prevents Lethal Middle East Respiratory Syndrome Coronavirus Infections in Mice.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes fatal infections, with about 36% mortality in humans, and is endemic to the Middle East. MERS-CoV uses human dipeptidyl peptidase 4 (hDPP4) as a receptor for infection. Despite continued research efforts, no licensed vaccine is available for protection against this disease in humans. Therefore, this study sought to develop an inactivated fragmented MERS-CoV vaccine grown in Vero cells in an hDPP4-transgenic mouse model. Two-dose immunisation in mice with 15, 20, or 25 μg of spike proteins of inactivated split MERS-CoV antigens induced neutralising antibodies, with titres ranging from NT 80 to 1280. In addition, all immunised mice were completely protected, with no virus detection in tissues, weight loss, or mortality. The immunised splenocytes produced more cytokines that stimulate immune response (IFN-γ and TNF-α) than those that regulate it (IL-4 and IL-10). Taken together, the inactivated fragmented MERS-CoV vaccine is effective for the protection of mice against lethal MERS-CoV. Thus, the inactivated fragmented MERS-CoV vaccine warrants further testing in other hosts.

Assessment of Different Newcastle Disease Virus Antigens and Inactivators of Binary Ethylene Amine and Formalin for the Hemagglutination Inhibition Assay.

Newcastle disease is a severe viral threat to the global poultry industry due to its high prevalence and rapid transmission. Evaluating vaccination timing and effectiveness is crucial, often accomplished through the hemagglutination inhibition (HI) assay. This test relies on the virus's agglutination ability in certain animals, utilizing various inactivated antigens. Our study aimed to assess multiple Newcastle viral antigens ( LaSota, clone, thermo-resistant strain, B1, and V4 ) inactivated by binary ethylene amine (BEA) and formalin, seeking the best antigen and inactivator for the HI assay. We prepared the different ND antigens include; LaSota, Clone, thermo resistant, B1, V4 and the mixture of the antigens then inactivated them using BEA and formalin. The hemagglutination (HA) assay determined mean titers, comparing BEA and formalin inactivation. These antigens were also subjected to the HI test using 112 serum samples from different commercial poultry flocks to assess their performance. BEA-inactivated antigens exhibited significantly higher mean titers in the HA assay than formalin-inactivated antigens. In the evaluation of different antigens in the HI test, the mean titer of antigen B1 followed by clone and LaSota displayed a higher mean titer than others. In conclusion, this study recommends using Hitchner pathotype antigens, specifically the B1 vaccine, for Newcastle disease HI testing. BEA is the preferred inactivator, preserving antigen structure particularly the structure of hemagglutinin antigen while minimizing risks. These findings can enhance serological testing accuracy, contributing to more effective disease control and prevention in the poultry industry.

Inactivated Flagellin-Containing Vaccine Efficacy against Ovine Enzootic Abortion.

Chlamydia abortus is the etiological agent of abortion and fetal loss in sheep, goats and bovine cattle in many countries. Even though commercially available vaccines can reduce the incidence in sheep, the development of new, safe, and effective vaccines remains high on the agenda. In this study, an evaluation was made of the efficacy of a vaccine candidate, an inactivated antigen based on the extract of outer membrane proteins of a C. abortus strain known as Chlamydia VNITIBP-21, in combination with recombinant flagellin as an adjuvant. Pregnant sheep (n = 43) were divided into three groups: an experimental vaccinated group, a control infected group and a control non-infected group. The sheep were vaccinated twice, with an interval of 3 weeks, then infected with the homologous virulent strain of Chlamydia abortus on pregnancy day 75. The vaccine candidate reduced C. abortus shedding in vaginal swabs considerably, in comparison with the control group. In addition, ewes in the experimental group experienced no abortions, while those in the control group experienced instances of abortion, as well as births of weak and nonviable lambs. The findings show that the vaccine candidate proved itself to be promising in combatting the agent of ovine abortion and fetal loss.

Clinical trial results of an associated vaccine against cattle clostridiosis and escherichiosis

In veterinary practice, gastrointestinal diseases of newborn calves, caused by the association of pathogenic bacteria, in particular Clostridium perfringens and Escherichia coli, are often observed. The results of our research have shown that in the Volga Federal District of the Russian Federation, anaerobic enterotoxemia in newborn calves is most often caused by Clostridium perfringens serotypes A, C, D. For the specific prevention of this form of pathology, an associated vaccine has been developed containing inactivated antigens of Clostridium perfringens serotypes A, C, D and Escherichia coli, which produce adhesion factors K99 and A20. Clinical trials of the effectiveness of the vaccine on cattle were carried out in 3 large agricultural enterprises of the Republic of Tatarstan for the production of milk, permanently dysfunctional for clostridiosis and escherichiosis of newborn calves. Disease prevention began with the immunization of pregnant cows and heifers 60 days before the expected calving. The animals were vaccinated twice with an interval of 14 days at a dose of 10 cm3. Calves obtained from immunized cows were vaccinated twice subcutaneously at a dose of 3 cm3 at the age of 18- 20 days. A total of 3156 cows and heifers were vaccinated in three dairy complexes. It was found that the associated vaccine is harmless for deepwalled cows and heifers, does not cause post-vaccination complications. Immunization of animals in the last months of pregnancy contributes to the accumulation of specific antibodies in colostrum, the intake of which provides passive protection of the newborn calf from infection with Cl. perfringens and E. coli bacteria.

Antigenic similarities and clinical cross-protection between variant and classic non-viscous strains of Moritella viscosa in Atlantic salmon in Norway

Moritella viscosa (M. viscosa) is one of the major etiological agents of winter-ulcers in Atlantic salmon (Salmo salar) in Norway. Outbreaks of ulcerative disease in farmed fish occur across the North Atlantic region, causing reduced animal welfare and economical challenges, and are of hindrance for sustainable growth within the industry. Commercially available multivalent core vaccines containing inactivated bacterin of M. viscosa reduce mortality and clinical signs related to winter ulcer disease. It has previously been described two major genetic clades within M. viscosa, typical (hereafter referred to as classic) and variant, based on gyrB sequencing. In addition, there are phenotypical traits such as viscosity that may differ between different types of isolates. Western blot using salmon plasma showed that classic non-viscous strains are antigenically different from the classic viscous type included in core vaccines. Further, Western blot also showed that there are similarities in binding patterns between Norwegian variant and classic non-viscous isolates, indicating they may be antigenically related. Vaccination-challenge trials using Norwegian gyrB-classic non-viscous isolates of M. viscosa, demonstrate that the isolates from the classic clade that are included in current commercial multivalent core vaccines, provide limited cross protection against the emerging non-viscous strains. However, a vaccine recently approved for marketing authorization in Norway, containing inactivated antigen of a variant M. viscosa strain, demonstrates reduced mortality as well as clinical signs caused by infections with the classic non-viscous M. viscosa isolated from outbreaks in Norwegian salmon farms. The study shows that there are antigenic similarities between variant and classic non-viscous types of M. viscosa, and these similarities are mirrored in the observed cross-protection in vaccination-challenge trials.

Inactivated vaccine with glycyrrhizic acid adjuvant elicits potent innate and adaptive immune responses against foot-and-mouth disease.

Foot-and-mouth disease (FMD) is an extremely contagious viral disease that is fatal to young animals and is a major threat to the agricultural economy by reducing production and limiting the movement of livestock. The currently commercially-available FMD vaccine is prepared using an inactivated viral antigen in an oil emulsion, with aluminum hydroxide [Al(OH)3] as an adjuvant. However, oil emulsion-based options possess limitations including slow increases in antibody titers (up to levels adequate for defense against viral infection) and risks of local reactions at the vaccination site. Further, Al(OH)3 only induces a T helper 2 (Th2) cell response. Therefore, novel adjuvants that can address these limitations are urgently needed. Glycyrrhizic acid (extracted from licorice roots) is a triterpenoid saponin and has great advantages in terms of price and availability. To address the limitations of the currently used commercial FMD vaccine, we added glycyrrhizic acid as an adjuvant (immunostimulant) to the FMD bivalent (O PA2 + A YC) vaccine. We then evaluated its efficacy in promoting both innate and adaptive (cellular and humoral) immune reactions in vitro [using murine peritoneal exudate cells (PECs) and porcine peripheral blood mononuclear cells (PBMCs)] and in vivo (using mice and pigs). Glycyrrhizic acid has been revealed to induce an innate immune response and enhance early, mid-, and long-term immunity. The studied bivalent vaccine with glycyrrhizic acid increased the expression of immunoregulatory genes such as pattern-recognition receptors (PRRs), cytokines, transcription factors, and co-stimulatory molecules. Collectively, glycyrrhizic acid could have utility as a novel vaccine adjuvant that can address the limitations of commercialized FMD vaccines by inducing potent innate and adaptive immune responses.

Quantification of complete viral particles in inactivated avian influenza virus antigen by high performance size exclusion chromatography coupled with multi-angle laser light scattering

We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R2=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.

Efficacy and immunogenicity of a veterinary vaccine candidate against tick-borne encephalitis in dogs

Tick-borne encephalitis (TBE) is a severe neuroinfection of humans. Dogs are also commonly infected with tick-borne encephalitis virus (TBEV). These infections are usually asymptomatic, but sometimes show clinical signs similar to those seen in humans and can be fatal. To date, there is no TBEV vaccine available for use in dogs. To address this need, a TBEV vaccine candidate for dogs based on inactivated whole virus antigen was developed. The safety, immunogenicity, and efficacy of the vaccine candidate were tested in mice as the preclinical model and in dogs as the target organism. The vaccine was well tolerated in both species and elicited the production of specific anti-TBEV antibodies with virus neutralising activity. Vaccination of mice provided complete protection against the development of fatal TBE. Immunisation of dogs prevented the development of viremia after challenge infection. Therefore, the developed vaccine candidate is promising to protect dogs from severe TBEV infections.

Evaluation of dynamics of immune responses and protective efficacy in piglets immunized with an inactivated porcine reproductive and respiratory syndrome vaccine candidate

Porcine Reproductive and Respiratory Syndrome (PRRS) is an important viral disease of swine that causes significant mortality in piglets and production losses in adult pigs. In this study, we investigated the protective efficacy of an inactivated PRRS virus vaccine candidate and evaluated the differences in PRRSV specific anamnestic response in piglets when challenged with live PRRSV at two different intervals post-immunization. Six-week-old piglets were immunized intramuscularly with an inactivated, Montanide ISA-206 adjuvanted Indian PRRSV isolate, followed by a booster dose at 21 days post-immunization. Homologous live PRRS virus challenge was done on 60 and 180 days post-booster (dpb). We assessed humoral and cell-mediated immune responses at various intervals post-immunization and after challenge. Viraemia, virus shedding in nasal secretions and lung lesion scores were studied to assess the efficacy of the vaccine candidate. All the immunized pigs developed PRRSV-specific antibodies upon booster dose administration. Neutralizing antibody (NA) titres before challenge, in most animals, ranged between 0 and 4. Potentially protective NA titre of 8 was observed in serum of seven out of the 12 immunized piglets after challenge, across the immunized groups. A significant increase in the mean T-helper, T-cytotoxic, memory or activated T-helper and NK cell populations was observed in immunized piglets challenged at 180 dpb, from 4 to 11 dpc, 5 to 11 dpc, 5 to 7 dpc and 6 to 11 dpc, respectively as compared to the challenge controls. Protective efficacy of the inactivated PRRSV antigen against the homologous virus challenge was evidenced by earlier onset of PRRSV specific virus neutralizing antibodies and cell mediated immune responses, reduced viremia, nasal virus shedding and severity of lung lesions in immunized piglets as compared to unimmunized controls post-challenge. Our results indicated that the inactivated PRRSV antigen elicited better virus specific anamnestic immune responses in piglets when challenged at six months after the single booster dose, due to age related increase in antigen-specific memory T helper cell responses, as compared to those challenged at 2 months post booster.

An Advax-CpG55.2 adjuvanted recombinant hemagglutinin vaccine provides immunity against H7N9 influenza in adult and neonatal mice

There is a major unmet need for strategies to improve the immunogenicity and effectiveness of pandemic influenza vaccines, particularly in poor responder populations such as neonates. Recombinant protein approaches to pandemic influenza offer advantages over more traditional inactivated virus approaches, as they are free of problems such as egg adaptation or need for high level biosecurity containment for manufacture. However, a weakness of recombinant proteins is their low immunogenicity. We asked whether the use of an inulin polysaccharide adjuvant (Advax) alone or combined with a TLR9 agonist (CpG55.2) would enhance the immunogenicity and protection of a recombinant hemagglutinin vaccine against H7N9 influenza (rH7HA), including in neonatal mice. Advax adjuvant induced predominantly IgG1 responses against H7HA, whereas Advax-CpG55.2 adjuvant also induced IgG2a, IgG2b and IgG3 responses, consistent with the TLR9 agonist component inducing a Th1 bias. Advax-CpG55.2 adjuvanted rH7HA induced high serum neutralizing antibody titers in adult mice. In newborns it similarly overcame immune hypo-responsiveness and enhanced serum anti-rH7HA IgG levels in 7-day-old BALB/C and C57BL/6 mice. Immunized adult mice were protected against a lethal H7N9 virus challenge. When formulated with Advax-CpG55.2 adjuvant, greater protection was seen with rH7HA than with inactivated H7 whole virus antigen. Advax-CpG55.2 adjuvanted rH7HA represents a promising influenza vaccine platform for further development.

Development of an Inactivated Camelpox Vaccine from Attenuated Camelpox Virus Strain: Safety and Protection in Camels.

This article describes the preparation of an inactivated vaccine from an attenuated strain of camelpox. The attenuated camelpox virus (CMLV) was grown in lamb kidney cells and in Vero cells. CMLV was accumulated to a significantly higher (p ≤ 0.05) titer in lamb kidney cells (7.75 ± 0.08 log TCID50/mL) than in Vero cells (4.00 ± 0.14 log TCID50/mL). During virus inactivation, a concentration of 0.05% beta-propiolactone (BPL) completely inactivated the virus in 6 h at a temperature of 22 ± 1 °C, while a concentration of 0.2% formaldehyde inactivated the virus in 8 h. However, a viral antigen inactivated by BPL was used for vaccine preparation. The inactivated viral antigen was adsorbed with aluminum hydroxide gel, and as a result, an inactivated candidate vaccine was prepared. While the safety of the candidate vaccine was tested in camels and white mice, the protective efficacy of the vaccine was tested only in camels. In the safety evaluation of the inactivated vaccine, the vaccine was not observed to cause any adverse effects in mice and camels. During the immunogenicity study in camels, antibody formation started (0.2 ± 0.16 log2) at Day 21 post-vaccination (PV), and the antibody titer peaked (1.33 ± 0.21 log2) at Day 60 PV and decreased at Day 90 PV (0.50 ± 0.22 log2). Furthermore, no antibodies were detected in vaccinated camels from Days 180 to 365 PV. Camels that received vaccination and were subsequently exposed to wild-type virus evinced a healthy state despite lacking antibodies. In contrast, unvaccinated camels exhibited susceptibility to camelpox upon challenge.

Investigating antigenic features of the SARS-CoV-2 isolated in Russian Federation in 2021–2022 by hyperimmune mouse serum neutralisation

Introduction. The rapid spread of a new coronavirus infection among populations in many countries worldwide has contributed to the genetic evolution of the virus, resulting in the emergence of multiple genetic variants of the SARS-CoV-2 coronavirus. Mutations in the viral genome can affect the ability of the virus to bypass the immune system and complicate development of diagnostic and prophylactic drugs. Data on the neutralizing activity of the sera obtained against previously circulating genetic variants of the virus in relation to current SARS-CoV-2 strains may serve as a scientific basis for the selection of the antigens in vaccine development. The aim of this work was to study cross-reactivity of SARS-CoV-2 coronavirus strains belonging to different genetic variants, which were isolated in the territory of the Russian Federation during 20202022 in the neutralization reaction using mouse hyperimmune sera. Materials and methods. Ten strains of SARS-CoV-2 coronavirus belonging to different genetic variants were used (three non-VOC strains, alpha, beta, gamma, delta, delta+AY, omicron 1 and omicron 2). The hCoV-19/Australia/VIC01/2020 strain (Wuhan) was included in the study as a prototypical variant. BALBc mice were immunized with inactivated concentrated antigen mixed with a 1:1 adjuvant, which was a virus-like immunostimulatory complex based on Quillaja saponaria (Quillaja saponaria). The antibody titer was determined in the neutralization reaction. Results. Essential decrease of neutralizing ability of antibodies specific to non-vOC genetic variants of SARS-CoV-2 coronavirus was revealed against beta VOC and to a lesser degree against alpha and gamma VOC variants. The differences in the neutralizing activity level of antibodies for alpha and beta VOC variants are not significant among themselves, and with gamma VOC variants there are no significant differences. Neutralizing ability of antibodies specific to delta VOC against alpha and beta VOC variants decreased 4-fold. Neutralizing activity of sera obtained to omicron 1 and 2 variants in relation to the prototype coronavirus variant was reduced 18-fold, to the gamma variant 12-fold, to delta variants more than 30-fold; for other variants it was even lower. Conclusions. The results obtained testify to the presence of cross-reactivity between strains of coronavirus belonging to genetic lines Wuhan, alpha, beta, gamma; it is weaker for delta variants. Mutations in the genome of VOC omicron variants led to a significant decrease in antigenic cross-links with earlier genetic variants of the coronavirus. These findings explain the low efficacy of vaccines based on the Wuhan strain, synthetic immunogens, and recombinant proteins based on it against omicron VOC variants, which have caused a rise in morbidity since early 2022, as well as cases of re-infection of humans with new genetic variants of the coronavirus.