Induction Of Virus-neutralizing Antibodies Research Articles

Preclinical immunogenicity and protective efficacy of a SARS-CoV-2 RBD-based vaccine produced with the thermophilic filamentous fungal expression system Thermothelomyces heterothallica C1.

The emergency use of vaccines has been the most efficient way to control the coronavirus disease 19 (COVID-19) pandemic. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has reduced the efficacy of currently used vaccines. The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein is the main target for virus neutralizing (VN) antibodies. A SARS-CoV-2 RBD vaccine candidate was produced in the Thermothelomyces heterothallica (formerly, Myceliophthora thermophila) C1 protein expression system and coupled to a nanoparticle. Immunogenicity and efficacy of this vaccine candidate was tested using the Syrian golden hamster (Mesocricetus auratus) infection model. One dose of 10-μg RBD vaccine based on SARS-CoV-2 Wuhan strain, coupled to a nanoparticle in combination with aluminum hydroxide as adjuvant, efficiently induced VN antibodies and reduced viral load and lung damage upon SARS-CoV-2 challenge infection. The VN antibodies neutralized SARS-CoV-2 variants of concern: D614G, Alpha, Beta, Gamma, and Delta. Our results support the use of the Thermothelomyces heterothallica C1 protein expression system to produce recombinant vaccines against SARS-CoV-2 and other virus infections to help overcome limitations associated with the use of mammalian expression system.

The induction of virus-neutralizing antibodies in influenza A-infected mice treated with Oseltamivir phosphate: effect of dosage and scheduling.

Oseltamivir phosphate (OS) is currently the most frequently used influenza antiviral drug. It moderates the course of influenza virus type A(IAV) infection, however, its impact on the induction of virus-neutralizing antibodies (VNAbs) is not understood in details. Here, we examined the influence of low (10mg/kg) or high (60mg/kg) doses of OS on the viral titer in lungs of BALB/c mice infected with 0.5LD50 of IAV and on the level of VNAbs. Prophylactic application of OS (6hbefore the infection) delayed the increase of viral titer in lungs with alower peak in comparison to non-treated control mice. After therapeutic OS application (44hafter the infection), maximum of virus titer did not significantly change. However, the induction of VNAbs strongly decreased, to 16.7%-18.1% of the control, after preventive application of high OS dose. Aminimal decrease of VNAbs titers was observed in groups of mice treated with low dose of OS applied therapeutically. They lowered to 91.1% / 14 or to 94.1% / 21 days post infection (p.i.) of VNAbs titers of non-treated control mice. In all other groups, levels of VNAbs titers dropped to 26.5-53.7% of those of non-treated mice. It should be noted that VNAbs titers were in direct proportion to maximal virus titers in mouse lungs of corresponding groups. In summary, after OS application the clinical symptoms of the disease were milder or non-observable in all OS-treated groups, but the lowering of VNAbs titers was dependent on the OS dose and interval between drug app-lication and the start of infection. Keywords: influenza Avirus; Oseltamivir; prophylactic treatment; therapeutic treatment; virus-neutralizing antibodies.

A Potent Anti-Simian Immunodeficiency Virus Neutralizing Antibody Induction Associated with a Germ Line Immunoglobulin Gene Polymorphism in Rhesus Macaques

Virus infection induces B cells with a wide variety of B cell receptor (BCR) repertoires. Patterns of induced BCR repertoires are different in individuals, while the underlying mechanism causing this difference remains largely unclear. In particular, the impact of germ line BCR immunoglobulin (Ig) gene polymorphism on B cell/antibody induction has not fully been determined. In the present study, we found a potent antibody induction associated with a germ line BCR Ig gene polymorphism. B404-class antibodies, which were previously reported as potent anti-simian immunodeficiency virus (SIV) neutralizing antibodies using the germ line VH3.33 gene-derived Ig heavy chain, were induced in five of 10 rhesus macaques after SIVsmH635FC infection. Investigation of VH3.33 genes in B404-class antibody inducers (n = 5) and non-inducers (n = 5) revealed association of B404-class antibody induction with a germ line VH3.33 polymorphism. Analysis of reconstructed antibodies indicated that the VH3.33 residue 38 is the determinant for B404-class antibody induction. B404-class antibodies were induced in all the macaques possessing the B404-associated VH3.33 allele, even under undetectable viremia. Our results show that a single nucleotide polymorphism in germ line VH genes could be a determinant for induction of potent antibodies against virus infection, implying that germ line VH-gene polymorphisms can be a factor restricting effective antibody induction or responsiveness to vaccination.IMPORTANCE Vaccines against a wide variety of infectious diseases have been developed mostly to induce antibodies targeting pathogens. However, small but significant percentage of people fail to mount potent antibody responses after vaccination, while the underlying mechanism of host failure in antibody induction remains largely unclear. In particular, the impact of germ line B cell receptor (BCR)/antibody immunoglobulin (Ig) gene polymorphism on B cell/antibody induction has not fully been determined. In the present study, we found a potent anti-simian immunodeficiency virus neutralizing antibody induction associated with a germ line BCR/antibody Ig gene polymorphism in rhesus macaques. Our results demonstrate that a single nucleotide polymorphism in germ line Ig genes could be a determinant for induction of potent antibodies against virus infection, implying that germ line BCR/antibody Ig gene polymorphisms can be a factor restricting effective antibody induction or responsiveness to vaccination.

Non-sterilizing, Infection-Permissive Vaccination With Inactivated Influenza Virus Vaccine Reshapes Subsequent Virus Infection-Induced Protective Heterosubtypic Immunity From Cellular to Humoral Cross-Reactive Immune Responses.

Conventional influenza vaccines aim at the induction of virus-neutralizing antibodies that provide with sterilizing immunity. However, influenza vaccination often confers protection from disease but not from infection. The impact of infection-permissive vaccination on the immune response elicited by subsequent influenza virus infection is not well-understood. Here, we investigated to what extent infection-permissive immunity, in contrast to virus-neutralizing immunity, provided by a trivalent inactivated virus vaccine (TIV) modulates disease and virus-induced host immune responses after sublethal vaccine-matching H1N1 infection in a mouse model. More than one TIV vaccination was needed to induce a serum HI titer and provide sterilizing immunity upon homologous virus infection. However, single TIV administration provided infection-permissive immunity, characterized by lower viral lung titers and faster recovery. Despite the presence of replicating virus, single TIV vaccination prevented induction of pro-inflammatory cyto- and chemokines, alveolar macrophage depletion as well as the establishment of lung-resident B and T cells after infection. To investigate virus infection-induced cross-protective heterosubtypic immune responses in vaccinated and unvaccinated animals, mice were re-infected with a lethal dose of H3N2 virus 4 weeks after H1N1 infection. Single TIV vaccination did not prevent H1N1 virus infection-induced heterosubtypic cross-protection, but shifted the mechanism of cross-protection from the cellular to the humoral branch of the immune system. These results suggest that suboptimal vaccination with conventional influenza vaccines may still positively modulate disease outcome after influenza virus infection, while promoting humoral heterosubtypic immunity after virus infection.

Microencapsulated plasmids expressing Gn and Gc glycoproteins of Rift Valley Fever virus enhance humoral immune response in mice.

The aim of the current study was to develop biodegradable alginate (ALG)/poly-L-lysine (PLL) microcapsules (MC) with entrapped plasmids expressing Gn and Gc glycoproteins of Rift Valley Fever virus (RVFV) and to evaluate the humoral immune response in mice. Expressing phRVF/Gn and phRVF/Gc plasmids which encode full-sized Gn and Gc glycoproteins and contain signal fusion protein F sequences of human parainfluenza (HPIV-1) were constructed. To protect the plasmids from cleavage by extracellular nucleases, they were entrapped into multilayer ALG/PLL microcapsules by layer-by-layer technique. To study the efficacy of humoral immune response, both native and microencapsulated plasmids were injected intramuscular into BALB/c mice. The humoral response in the mice immunized with free plasmids was characterized by virus-neutralizing antibody induction (with titres 1:4 to 1:8), while the injection of microencapsulated plasmids allowed to increase the titre level (from 1:16 to 1:32). The plasmids microencapsulated in biodegradable MC could be promising for development of DNA vaccines against RVFV.

Analysis and Prospects of Using Recombinant Vaccinia Virus MVA Strain as a Vector in the Development of the Vaccines against Human and Simian Immunodeficiency Virus Diseases

The review presents the results of preclinical use of vector vaccines against human immunodeficiency virus (HIV) disease and simian immunodeficiency virus (SIV) disease. Application of antiretroviral therapy exclusively is insufficient for elimination of HIV from patient’s body. This dictates the need for an effective vaccine which will reduce the number of new cases of the disease and reduce the risk of virus transmission. Current practice of medicinal product development showed the effectiveness of heterologous prime-boost regimens for the induction of expressed immune response in laboratory animals. Various vector constructs were used as priming vaccines: DNA vaccines, Bacille Calmette-Guerin vaccine, chimpanzee adenovirus, vesicular stomatitis virus, alphavirus repli-clone. Booster vaccine was represented by recombinant MVA strain. In all vector vaccines, different genes of immunodominant antigens of HIV and SIV agents were inserted. On rhesus macaques, murine, rabbit models, it was demonstrated that deployed vaccination schemes were safe and induced immune response. Because membrane HIV protein is highly variable, strongly glycoziled and subjected to structural changes during receptor binding, it cannot be viewed as a target for induction of virus neutralized antibodies. Therefore, we mainly studied the cell immune response that was presented by poly-functional CD8+ T-cells. However, some recent researches are aimed at such modification of envelope HIV immunogene that would provide for virus neutralizing antibody induction. The study of protective efficiency of the induced immunity in rhesus macaques, immunized with recombinant vectors expressing SIV’ s immunodominant antigens, in case of subsequent inoculation with virulent SIV strain has revealed that all monkeys developed illness. Assuming that the constructions with SIV’ s immunodominant antigens under protective efficiency testing on rhesus macaques imitate AIDS in humans, it seems that vaccines, developed up-to-date, will not be effective for collective immunity formation against AIDS. Therefore, the search for novel combinations of expressed immunodominant antigens for the inclusion into the composition of priming and booster vaccines remains a priority area at present time.

Beta-propiolactone inactivated bivalent bluetongue virus vaccine containing Montanide ISA-71VG adjuvant induces long-term immune response in sheep against serotypes 4 and 16 even after 3 years of controlled vaccine storage

In this study, we developed and evaluated the beta-propiolactone inactivated bivalent bluetongue virus (BTV) serotypes 4 and 16 vaccine delivered with Montanide™ ISA-71VG adjuvant. The safety, stability and immunological profile of the fresh and after three years of long-term storage of the vaccine formulation was analyzed. We observed after long-term storage that the vaccine emulsion was stable as indicated by unchanged pH and viscosity. The stored vaccine formulation induced virus neutralizing antibodies (VNA) in sheep against both the bluetongue virus serotypes at 7–10 day post-vaccination (dpv). VNA titers reached the peak by 60 dpv and detectable during the entire study period. Antibodies against bluetongue virus structural protein VP7 were detected by ELISA in all BTV vaccinated experimental animal groups. Partial clinical protection was observed in vaccinates against challenge virulent BTV-4 and BTV-16 serotypes by 10 dpv, while complete protection was observed at 14 dpv. The levels of viremia was decreased in challenged sheep by 10 dpv while the viremia was undetectable by 14 dpv. In summary, our newly formulated bivalent BTV (BTV-4 and BTV-16) vaccine delivered with Montanide™ ISA-71VG adjuvant was found safe and stable for over three years and induced protective response in sheep.

A chimeric glycoprotein formulated with a combination adjuvant induces protective immunity against both human respiratory syncytial virus and parainfluenza virus type 3

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) are major causes of serious lower respiratory tract disease in infants. Currently there is no licensed vaccine against RSV or PIV3. To make an effective bivalent subunit vaccine, a chimeric truncated FRHN protein containing the N-terminal ectodomain of the RSV fusion (F) protein linked to the C-terminal ectodomain of the PIV3 haemagglutinin-neuraminidase (HN) protein was produced in HEK293T cells. Mice, cotton rats and hamsters were immunized intramuscularly (IM) with both RSV F and PIV3 HN (FR+HN) or FRHN, formulated with TriAdj, which consists of poly(I:C), innate defense regulator peptide and poly[di(sodium carboxylatoethylphenoxy)]-phosphazene. Both formulations were immunogenic and elicited full protection from RSV; however, animals vaccinated with FRHN/TriAdj were significantly better protected from PIV3 than animals vaccinated with FR+HN/TriAdj. To develop a potentially more effective subunit vaccine, a chimeric glycoprotein (FRipScHN), encoding the RSV F ectodomain stabilized in the pre-fusion form linked to PIV3 HN was generated. Intramuscular vaccination with FRipScHN/TriAdj induced virus neutralizing antibodies followed by complete protection from RSV and PIV3 replication in the lungs of challenged cotton rats. Furthermore, intranasal vaccination with FRipScHN/TriAdj significantly reduced both RSV and PIV3 replication in cotton rats. Mucosal immunization with FRipScHN/TriAdj also elicited strong antigen-specific mucosal and systemic immune responses in a lamb model. In conclusion, the chimeric FRipScHN protein combined with TriAdj has potential for development of a safe, effective, bivalent vaccine against both RSV and PIV3.

Revisiting rabies virus neutralizing antibodies through infecting BALB/c mice with live rabies virus

This study investigates the production of rabies virus (RABV) neutralizing antibody after virus infection through a mouse model. The BALB/c mice from different age groups (three, five, seven week old) were intramuscularly inoculated with live rabies virus (TX coyote 323R). Without pre-exposure or post-exposure prophylaxis (PEP), we found there is a decreased fatality with increased age of animals, the mortalities are 60%, 50%, and 30%, respectively. Interestingly, through assay of rapid fluorescent focus inhibition test (RFFIT), direct fluorescent antibody (DFA) and quantitative Polymerase Chain Reaction (qPCR), the results showed that all the animals that succumbed to rabies challenge, except one, developed circulating neutralizing antibodies, and all the healthy animals, except two, did not generate virus neutralizing antibodies (VNA). Our animal study suggests that the induction of VNA was an indicator of infection progression in the central nervous system (CNS) and speculate that RABV neutralizing antibodies did not cross the blood-brain barrier of the CNS for those diseased animals. We hypothesize that early release of viral antigens from damaged nerve tissue might potentially be a benefit for survivors, and we also discuss several other aspects of the interaction of RABV and its neutralizing antibodies.

Plant-Produced Subunit Vaccine Candidates against Yellow Fever Induce Virus Neutralizing Antibodies and Confer Protection against Viral Challenge in Animal Models.

Yellow fever (YF) is a viral disease transmitted by mosquitoes and endemic mostly in South America and Africa with 20-50% fatality. All current licensed YF vaccines, including YF-Vax® (Sanofi-Pasteur, Lyon, France) and 17DD-YFV (Bio-Manguinhos, Rio de Janeiro, Brazil), are based on live attenuated virus produced in hens' eggs and have been widely used. The YF vaccines are considered safe and highly effective. However, a recent increase in demand for YF vaccines and reports of rare cases of YF vaccine-associated fatal adverse events have provoked interest in developing a safer YF vaccine that can be easily scaled up to meet this increased global demand. To this point, we have engineered the YF virus envelope protein (YFE) and transiently expressed it in Nicotiana benthamiana as a stand-alone protein (YFE) or as fusion to the bacterial enzyme lichenase (YFE-LicKM). Immunogenicity and challenge studies in mice demonstrated that both YFE and YFE-LicKM elicited virus neutralizing (VN) antibodies and protected over 70% of mice from lethal challenge infection. Furthermore, these two YFE-based vaccine candidates induced VN antibody responses with high serum avidity in nonhuman primates and these VN antibody responses were further enhanced after challenge infection with the 17DD strain of YF virus. These results demonstrate partial protective efficacy in mice of YFE-based subunit vaccines expressed in N. benthamiana. However, their efficacy is inferior to that of the live attenuated 17DD vaccine, indicating that formulation development, such as incorporating a more suitable adjuvant, may be required for product development.

Addition of C3d-P28 adjuvant to a rabies DNA vaccine encoding the G5 linear epitope enhances the humoral immune response and confers protection

Rabies DNA vaccines based on full-length glycoprotein (G) induce virus neutralizing antibody (VNA) responses and protect against the virus challenge. Although conformational epitopes of G are the main target of VNAs, some studies have shown that a polypeptide linear epitope G5 is also able to induce VNAs. However, a G5 DNA vaccine has not been explored. While multiple doses of DNA vaccines are required in order to confer a protective immune response, this could be overcome by the inclusion of C3d-P28, a molecular adjuvant is know to improve the antibody response in several anti-viral vaccine models. To induce and enhance the immune response against rabies in mice, we evaluated two DNA vaccines based on the linear epitope G5 of Rabies Virus (RABV) glycoprotein (pVaxG5 vaccine) and another vaccine consisting of G5 fused to the molecular adjuvant C3d-P28 (pVaxF1 vaccine). VNA responses were measured in mice immunized with both vaccines. The VNA levels from the group immunized with pVaxG5 decreased gradually, while those from the group vaccinated with pVaxF1 remained high throughout the experimental study. After challenge with 22 LD50 of the Challenge Virus Strain (CVS), the survival rate of mice immunized with pVaxG5 and pVaxF1 was increased by 27% and 50% respectively, in comparison to the PBS group. Furthermore, the in vitro proliferation of anti-rabies specific spleen CD4+ and CD8+ T cells from mice immunized with pVaxF1 was observed. Collectively, these results suggest that the linear G5 epitope is a potential candidate vaccine. Furthermore, the addition of a C3d-P28 adjuvant contributed to enhanced protection, the sustained production of VNAs, and a specific T-cell proliferative response.

Protective capacity of neutralizing and non-neutralizing antibodies against glycoprotein B of cytomegalovirus.

Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. Antiviral chemotherapy remains problematic due to toxicity of the available compounds and the emergence of viruses resistant to available antiviral therapies. Antiviral antibodies could represent a valuable alternative strategy to limit the clinical consequences of viral disease in patients. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. However, the role of anti-gB antibodies in the course of the infection in-vivo remains unknown. We have used a murine CMV (MCMV) model to generate and study a number of anti-gB monoclonal antibodies (mAbs) with differing virus-neutralizing capacities. The mAbs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB. When mAbs were used in immunodeficient RAG-/- hosts to limit an ongoing infection we observed a reduction in viral load both with mAbs having potent neutralizing capacity in-vitro as well as mAbs classified as non-neutralizing. In a therapeutic setting, neutralizing mAbs showed a greater capacity to reduce the viral burden compared to non-neutralizing antibodies. Efficacy was correlated with sustained concentration of virus neutralizing mAbs in-vivo rather than their in-vitro neutralizing capacity. Combinations of neutralizing mAbs further augmented the antiviral effect and were found to be as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mAbs before infection was also protective and both neutralizing and non-neutralizing mAbs were equally effective in preventing lethal infection of immunodeficient mice. In summary, our data argue that therapeutic application of potently neutralizing mAbs against gB represent a strategy to modify the outcome of CMV infection in immunodeficient hosts. When present before infection, both neutralizing and non-neutralizing anti-gB exhibited protective capacity.

Variability in biological behaviour, pathogenicity, protectotype and induction of virus neutralizing antibodies by different vaccination programmes to infectious bronchitis virus genotype Q1 strains from Chile

ABSTRACTIn the period from July 2008 to 2010, a disease episode resulting in serious economic losses in the major production area of the Chilean poultry industry was reported. These losses were associated with respiratory problems, increase of condemnations, drops in egg production and nephritis in breeders, laying hens and broilers due to infections with infectious bronchitis virus (IBV). Twenty-five IBV isolates were genotyped and four strains were selected for further testing by pathotyping and protectotyping. Twenty-four IBV isolates were of the Q1 genotype. The experiments also included comparing the ability of six vaccination programmes to induce virus neutralizing antibodies (VNA) in layers against four selected Chilean strains. Despite the high genetic homology in the S1 gene between the four strains, the heterogeneity in biological behaviour of these different Q1 strains was substantial. These differences were seen in embryonated eggs, in cell culture, in pathogenicity and in level of cross-protection by IBV Massachusetts (Mass) vaccination. This variability underlines the importance of testing more than one strain per serotype or genotype to determine the characteristics of a certain serotype of genotype. The combination of Mass and 793B vaccine provided a high level of protection to the respiratory tract and the kidney for each strain tested in the young birds. The combination of broad live priming using Mass and 793B vaccines and boosting with multiple inactivated IBV antigens induced the highest level of VNA against Q1 strains, which might be indicative for higher levels of protection against Q1 challenge in laying birds.

Infection-permissive immunity against influenza virus provided by vaccination prevents loss of alveolar macrophages and modulates virus-induced cross-reactive cellular immune responses during subsequent influenza infections.

Abstract Conventional influenza vaccines aim at the induction of virus-neutralizing antibodies. Influenza viruses can escape neutralization by antibodies through antigenic drift or shift or because antibody levels diminish. We wondered to what extent infection-permissive immunity provided by a trivalent inactivated influenza virus vaccine (TIV) could modulate disease and virus-induced host immune responses after homologous H1N1 infection. We first focused on alveolar macrophages, cells that are transiently lost during influenza infection in the mouse-influenza challenge model. Later we focused on how vaccine-induced immunity modulates induction of cross-reactive CD8+ T cells by virus infection. TIV vaccination did not result in detectable HI titers but correlated with lower viral lung titers and faster recovery after challlenge. Alveolar macrophages were abolished at 7dpi in negative control mice, but not in TIV-vaccinated mice. TIV vaccination allowed the induction but also affected levels of cross-reactive NP-specific CD8+ T cell responses, which correlated with protection against heterosubtypic H3N2 virus. The effect of TIV vaccination on TCR Vbeta-region bias of CD8+ T cells after secondary challenge with H3N2 virus was also investigated. These results suggest that suboptimal vaccination with conventional influenza vaccines may still positively modulate disease outcome, thereby allowing induction of heterosubtypic immunity by virus infection.

Expression of bovine rotavirus VP8 and preparation of IgY antibodies against recombinant VP8.

Group A rotavirus is the leading cause of acute gastroenteritis in cattle and swine. Although, vaccination against this virus is an effective strategy for prevention, additional strategy to control disease is necessary. Egg yolk immunoglobulin (IgY)-based passive immunization could be a better option in preventing this disease. Bovine rotavirus (BRV) is group A rotavirus and possesses a genome of 11 segments of double-stranded RNA. The outer layer of capsid is composed of two proteins (VP7 and VP4), which induce virus neutralizing antibodies. Trypsin cleavage of VP4 produces VP8 (28 kDa) and VP5 (60 kDa) fragments. Since a number of studies have demonstrated the induction of neutralizing antibodies using VP8 subunit vaccines, we have produced IgY against the recombinant VP8. The cDNA spanning the VP8 subunit was amplified from bovine rotavirus-infected cells and cloned into pET21d(+) expression vector to generate recombinant VP8. The resulting carboxy-terminal His-tagged VP8 proteins were expressed in Escherichia coli strain BL21(DE3) by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified protein was used as the immunizing agent to produce polyclonal antibodies in chicken. The resulting polyclonal antisera specifically recognized VP8 in Western blot assay and were able to neutralize BRV replication in cell cultures. These results demonstrate that IgY can be used in immunological assays and, in addition, in passive immunization of newborn calves against BRV.

Baculovirus Displaying Hemagglutinin Elicits Broad Cross-Protection against Influenza in Mice.

The widespread influenza virus infection further emphasizes the need for novel vaccine strategies that effectively reduce the impact of epidemic as well as pandemic influenza. Conventional influenza vaccines generally induce virus neutralizing antibody responses which are specific for a few antigenically related strains within the same subtype. However, antibodies directed against the conserved stalk domain of HA could neutralize multiple subtypes of influenza virus and thus provide broad-spectrum protection. In this study, we designed and constructed a recombinant baculovirus-based vaccine, rBac-HA virus, that expresses full-length HA of pandemic H1N1 influenza virus (A/California/04/09) on the viral envelope. We demonstrated that repeated intranasal immunizations with rBac-HA virus induced HA stalk-specific antibody responses and protective immunity against homologous as well as heterosubtypic virus challenge. The adoptive transfer experiment shows that the cross-protection is conferred by the immune sera which contain HA stalk-specific antibodies. These results warrant further development of rBac-HA virus as a broad-protective vaccine against influenza. The vaccine induced protection against infection with the same subtype as well as different subtype, promising a potential universal vaccine for broad protection against different subtypes to control influenza outbreaks including pandemic.

Molecular diagnosis and adaptation of highly virulent infectious bursal disease virus on chicken embryo fibroblast cell

Materials and Methods Infectious bursal disease (IBD) is a highly contagious disease of young chicken caused by infectious bursal disease virus (IBDV), characterized by immunosuppression and mortality generally at 3 to 6 weeks of age [1]. It is economically important to the poultry industry worldwide, due to increased susceptibility to other diseases and negative interference with effective vaccination. IBDV is a double stranded RNA virus with bi-segmented genome and belongs to the genus of family [2]. There are two distinct serotypes of the virus, but only serotype 1 viruses cause disease in poultry and six antigenic types are identified by virus neutralisation test. Although viral antigen has been detected in other organs within the first few hours of infection, the most extensive virus replication takes place primarily in the bursa of fabricius. Activated dividing B lymphocytes that secrete IgM serve as target cells for the virus. Viral infection results in lymphoid depletion of B cells and thedestructionofbursal tissues, leading to an increased susceptibility to other infectious diseases and poor immune response to vaccines. is considered to be the major host protective antigen and contains the major antigenic site responsible for eliciting neutralizing antibodies [3]. induces virus neutralizing antibodies that protect chickens from IBDV [4]. It is responsible for antigenic variation [5], tissue culture adaptation and virulence [6]. Study of virological characterization and molecular diagnosis of IBD will strengthen the antiviral therapy and disease control. IBD virus can infect and grow on various primary cell cultures of avian origin. Commonly used cell lines are chicken embryo fibroblast (CEF), chicken embryo kidney, chicken embryo bursa, vero, baby hamster kidney etc. The virulence of IBDV is lost during the adaptation on CEF cell culture but antigenicity is retained [7]. The present study aims at RT-PCR based detection and adaptation of IBDV field isolates.

Immunological evaluation of mannosylated chitosan nanoparticles based foot and mouth disease virus DNA vaccine, pVAC FMDV VP1–OmpA in guinea pigs

A DNA vaccine for foot and mouth disease (FMD) based on mannosylated chitosan nanoparticles was evaluated in guinea pigs. The DNA construct was comprised of FMD virus full length-VP1 gene and outer membrane protein A (Omp A) gene of Salmonella typhimurium as a Toll-like receptor (TLR)-ligand in pVAC vector. Groups of guinea pigs immunized either intramuscularly or intra-nasally were evaluated for induction of virus neutralizing antibodies, Th1(IgG2) and Th2 (IgG1) responses, lymphocyte proliferation, reactive nitrogen intermediate production, secretory IgA for naso-mucosal immune response and protection upon homotypic type O virulent FMD virus challenge. The results indicate the synergistic effect of OmpA on the immunogenic potential of FMD DNA vaccine construct delivered using mannosylated chitosan nano-particles by different routes of administration. These observations suggest the substantial improvement in all the immunological parameters with enhanced protection in guinea pigs.

A recombinant rabies virus encoding two copies of the glycoprotein gene confers protection in dogs against a virulent challenge.

The rabies virus (RABV) glycoprotein (G) is the principal antigen responsible for the induction of virus neutralizing antibodies (VNA) and is the major modality of protective immunity in animals. A recombinant RABV HEP-Flury strain was generated by reverse genetics to encode two copies of the G-gene (referred to as HEP-dG). The biological properties of HEP-dG were compared to those of the parental virus (HEP-Flury strain). The HEP-dG recombinant virus grew 100 times more efficiently in BHK-21 cell than the parental virus, yet the virulence of the dG recombinant virus in suckling mice was lower than the parental virus. The HEP-dG virus can improve the expression of G-gene mRNA and the G protein and produce more offspring viruses in cells. The amount of G protein revealed a positive relationship with immunogenicity in mice and dogs. The inactivated HEP-dG recombinant virus induced higher levels of VNA and conferred better protection against virulent RABV in mice and dogs than the inactivated parental virus and a commercial vaccine. The protective antibody persisted for at least 12 months. These data demonstrate that the HEP-dG is stable, induces a strong VNA response and confers protective immunity more effectively than the RABV HEP-Flury strain. HEP-dG could be a potential candidate in the development of novel inactivated rabies vaccines

HA2 glycopolypeptide of influenza A virus and antiviral immunity

Influenza A viruses (IAVs) cause acute respiratory infections in humans against which an effective prevention has not yet been developed due to their high variability and broad host specificity. The permanent threat of arising new influenza pandemic is represented by avian viruses which after their interspecies transmission can cause a disease with a devastating impact on humans lacking the specific immunity. Since the current vaccines inducing virus-neutralizing (VN) antibodies are targeted at a variable globular part of hemagglutinin (HA), their efficacy is limited and they need permanent updating. On the other hand, conserved IAV antigens such as proton channel M2, membrane protein M1 or nucleoprotein (NP) do not induce VN antibodies, but they do induce heterosubtypic protection resulting in the reduction of virus replication and an improved recovery from the disease. From this point of view recent attention has also been focused on the conserved part of HA, its HA2 glycoprotein (HA2). The main aspects revealing a contribution of HA2 gp to protective immunity are discussed in this review.