Titers In Mice Research Articles

Delta inulin-derived adjuvants that elicit Th1 phenotype following vaccination reduces respiratory syncytial virus lung titers without a reduction in lung immunopathology

ABSTRACTRespiratory syncytial virus (RSV) is a significant cause of lower respiratory tract infections resulting in bronchiolitis and even mortality in the elderly and young children/infants. Despite the impact of this virus on human health, no licensed vaccine exists. Unlike many other viral infections, RSV infection or vaccination does not induce durable protective antibodies in humans. In order to elicit high titer, neutralizing antibodies against RSV, we investigated the use of the adjuvant Advax™, a novel polysaccharide adjuvant based on delta inulin microparticles, to enhance antibody titers following vaccination. BALB/c mice were vaccinated intramuscularly with live RSV as a vaccine antigen in combination with one of two formulations of Advax™. Advax-1 was comprised of the standard delta inulin adjuvant and Advax-2 was formulated delta inulin plus CpG oligodendronucleotides (ODNs). An additional group of mice were either mock vaccinated, immunized with vaccine only, or administered vaccine plus Imject Alum. Following 3 vaccinations, mice had neutralizing antibody titers that correlated with reduction in viral titers in the lungs. Advax-1 significantly enhanced serum RSV-specific IgG1 levels at week 6 indicative of a Th2 response, similar to titers in mice administered vaccine plus Imject Alum. In contrast, mice vaccinated with vaccine plus Advax-2 had predominately IgG2a titers indicative of a Th1 response that was maintained during the entire study. Interestingly, regardless of which AdvaxTM adjuvant was used, the neutralizing titers were similar between groups, but the viral lung titers were significantly lower (∼10E+3pfu/g) in mice administered vaccine with either AdvaxTM adjuvant compared to mice administered adjuvants only. The lung pathology in vaccinated mice with AdvaxTM was similar to Imject Alum. Overall, RSV vaccine formulated with AdvaxTM had high neutralizing antibody titers with low lung viral titers, but exacerbated lung pathology compared to unvaccinated mice.

Novel HLA-A2-restricted human metapneumovirus epitopes reduce viral titers in mice and are recognized by human T cells

Human metapneumovirus (HMPV) is a major cause of morbidity and mortality from acute lower respiratory tract illness, with most individuals seropositive by age five. Despite the presence of neutralizing antibodies, secondary infections are common and can be severe in young, elderly, and immunocompromised persons. Preclinical vaccine studies for HMPV have suggested a need for a balanced antibody and T cell immune response to enhance protection and avoid lung immunopathology. We infected transgenic mice expressing human HLA-A*0201 with HMPV and used ELISPOT to screen overlapping and predicted epitope peptides. We identified six novel HLA-A2 restricted CD8+ T cell (TCD8) epitopes, with M39–47 (M39) immunodominant. Tetramer staining detected M39-specific TCD8 in lungs and spleen of HMPV-immune mice. Immunization with adjuvant-formulated M39 peptide reduced lung virus titers upon challenge. Finally, we show that TCD8 from HLA-A*0201 positive humans recognize M39 by IFNγ ELISPOT and tetramer staining. These results will facilitate HMPV vaccine development and human studies.

Strategies to improve the immunogenicity of prM+E dengue virus type-2 DNA vaccine.

An important goal for dengue vaccines is to induce a high and durable level of neutralizing antibody. Three strategies were investigated for improving the immunogenicity of a prM+E dengue serotype 2 (DENV-2) DNA vaccine: 1) expression in two different plasmids; 2) adjustment of dose; and, 3) introduction of the E sequence of Japanese encephalitis virus (JEV) at the carboxy-terminal portion of DENV-2 E. Expression cassettes were designed to encode a full-length prM+E sequence of DENV-2 virus employing human-preferred codons (D2prMEopt), or a chimeric prM+E sequence in which the 100-residue carboxy-terminal region of E was derived from JEV (D2prMEJE20opt). pHIS and pCMVkan in the presence and absence of CpG motif, respectively, were used for cassette expression. The immunogenicity was compared in mice. Three injections of full-length-D2prMEopt in pHIS and pCMVkan induced a comparable neutralizing antibody titer at post-week-2-injection and post-week-4-injection. The 100-μg DNA dose induced a numerically but not statistically higher neutralizing antibody titer than the 10-μg dose. The chimeric-D2prMEJE20opt produced higher extracellular prM and E protein levels in transfected Vero cells, but had a tendency to induce a lower neutralizing antibody titer in mice when compared with the full-length-D2prMEopt. To optimize the immunogenicity of D2prMEopt-DNA candidate, both expression plasmids can be used to generate reproducible high neutralizing titer. A higher dose of DNA immunogen may induce a higher neutralizing antibody response. The strategy of the C-terminal region chimeric counterpart with JE20 did not improve but may have reduced the induction of neutralizing antibodies.

Combination nanovaccine enhances influenza vaccine efficacy in aged animals

Event Abstract Back to Event Combination nanovaccine enhances influenza vaccine efficacy in aged animals Kathleen Ross1, Jonathan Goodman1, Matthew Jefferson2, Hyelee Loyd3, Metin Uz1, Michael Wannemuehler4, Surya Mallapragada1, Susan Carpenter3, Marian Kohut5 and Balaji Narasimhan1 1 Iowa State University, Chemical & Biological Engineering, United States 2 Iowa State University, Neuroscience, United States 3 Iowa State University, Animal Science, United States 4 Iowa State University, Veterinary Microbiology & Preventive Medicine, United States 5 Iowa State University, Kinesiology, United States Elderly populations have suboptimal responses to influenza vaccination due to age-associated deficiencies in immune responses[1],[2]. Due to poor antibody responses, measures of cell-mediated immunity may better predict protection in aged individuals[1]. Thus, influenza vaccines that stimulate both antibody and cell-mediated immunity would benefit aged populations. Polyanhydride nanoparticles are capable of both adjuvanting and sustaining the release of influenza antigens[3],[4]. Pentablock copolymer hydrogels form depots of antigen and/or nanoparticles that promote rapid development of antibody titers[5]. In this work, the ability of a combination nanovaccine composed of nanoparticles and hydrogels to enhance neutralizing antibody titers and cell-mediated immunity was studied in aged mice. Aged (17 mo.) and young (2 mo.) mice were immunized thrice (days 0, 21, and 56) with 10 µg of H5 hemagglutinin trimer (sH53), or a combination nanovaccine (2 µg H53 encapsulated into 200 µg nanoparticles + 8 µg H53 entrapped in 25 mg hydrogel). Serum samples were collected at 35, 56, and 66 days post-immunization and the neutralizing antibody titer was determined. To assess T cell induction, both mice received 100 µg of ovalbumin (OVA) either solubly or within the combination nanovaccine at day 66. After 6 days, draining lymph nodes were collected and homogenized. Cells were stimulated with PMA/ionomycin and subsequently labelled with anti-CD8β, anti-CD4, anti-IFNγ, and anti-TNFα. In young mice, robust neutralizing antibody titers were detected at each time point. In contrast, no titers were detected in aged mice receiving either the sH53 or the combination nanovaccine until after administration of a 3rd dose. However, the average titer in mice receiving the combination nanovaccine was five-fold higher than that in the sH53-immunized mice. The combination nanovaccine induced greater numbers of CD4+ and CD8+ T cells in draining lymph nodes when compared to that induced by soluble OVA in both age groups; however, this effect was only statistically significant in young mice. Additionally, the aged mice had greater numbers of CD4+ and CD8+ cells producing IFNγ and TNFα in comparison to young mice (Fig. 1). Fig. 1. Combination nanovaccine immunization enhanced T cell response in aged mice. Mice administered the combination nanovaccine demonstrated greater numbers of CD8+ IFNγ+ TNFα+ T cells than mice administered soluble antigen in both age groups. In this work, a combination nanovaccine was demonstrated to enhance both antibody- and cell-mediated immunity in aged mice. While the neutralizing antibody responses in aged mice were impaired, the combination nanovaccine induced enhanced titers in comparison to that induced by sH53. In addition, the combination nanovaccine induced greater numbers of functional T cells after immunization in both age groups. These data indicate the potential of the combination nanovaccine to increase vaccine efficacy in aged populations.

Elimination of HCV via a non-ISG-mediated mechanism by vaniprevir and BMS-788329 combination therapy in human hepatocyte chimeric mice

We previously reported that interferon (IFN)-free direct-acting antiviral combination treatment succeeded in eradicating genotype 1b hepatitis C virus (HCV) in human hepatocyte chimeric mice. In this study, we examined the effect of vaniprevir (MK7009, NS3/4A protease inhibitor) and BMS-788329 (NS5A inhibitor) combination treatment on HCV genotype 1b and the expression of IFN-stimulated genes (ISGs) using a subgenomic replicon system and the same animal model. Combination treatment with vaniprevir and BMS-788329 significantly reduced HCV replication compared to vaniprevir monotherapy in HCV replicon cells (Huh7/Rep-Feo cells). HCV genotype 1b-infected human hepatocyte chimeric mice were treated with vaniprevir alone or in combination with BMS-788329 for four weeks. Vaniprevir monotherapy reduced serum HCV RNA titers in mice, but viral breakthrough was observed in mice with high HCV titers. Ultra-deep sequence analysis revealed a predominant replacement by drug-resistant substitutions at 168 in HCV NS3 region in these mice. Conversely, in mice with low HCV titers, HCV was eradicated by vaniprevir monotherapy without viral breakthrough. In contrast to monotherapy, combination treatment with vaniprevir and BMS-788329 succeeded in completely eradicating HCV regardless of serum viral titer. IFN-alpha treatment significantly increased ISG expression; however, vaniprevir and BMS-788329 combination treatment caused no increase in ISG expression both in cultured cells and in mouse livers. Therefore, combination treatment with vaniprevir and BMS-788329 eliminated HCV via a non-ISG-mediated mechanism. This oral treatment might offer an alternative DAA combination therapy for patients with chronic hepatitis C.

Methylglycol chitosan and a synthetic TLR4 agonist enhance immune responses to influenza vaccine administered sublingually

Influenza is a vaccine-preventable contagious respiratory illness caused by influenza (flu) viruses which can lead to hospitalization and sometimes even death. Current flu vaccines delivered intramuscularly (IM) or intradermally (ID) are less effective at eliciting protective mucosal immune responses and vaccines delivered intranasally (IN) possess potential safety concerns. Sublingual (SL) vaccination is a promising alternative route for vaccine delivery which has been indicated as safe and effective at inducing protective immune responses in both systemic and mucosal compartments. We evaluated the efficacy of methylglycol chitosan (MGC) and a synthetic toll-like receptor 4 agonist (CRX-601), alone or in combination, for improving systemic and mucosal immune responses to a monovalent detergent-split flu virus vaccine delivered SL. SL vaccination of mice with split-flu vaccine formulated with either MGC or CRX-601 resulted in specific serum IgG and mucosal IgA titers that were significantly greater than titers from non-adjuvanted vaccination and equivalent to or greater than titers in mice vaccinated IM. Our results demonstrate that SL vaccination utilizing MGC or CRX-601 as adjuvants is a viable alternative route of vaccination for flu which can elicit systemic immune responses equivalent to or greater than IM vaccination with the added benefit of stimulating a robust specific mucosal immune response.

Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes.

Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.

Immunogenicity of porcine P[6], P[7]-specific △VP8* rotavirus subunit vaccines with a tetanus toxoid universal T cell epitope

Currently, commercial porcine rotavirus vaccines remain varied limitations. The objective of this study is to develop an alternative porcine rotavirus subunit vaccine candidate by parenteral administration, which enables to elicit robust immune responses against most prevalence porcine rotavirus strains. The bacterially-expressed porcine rotavirus P[6]- or P[7]-specific truncated VP8* (aa 64–223) recombinant protein with or without a universal tetanus toxoid CD4+ T cell epitope P2 was generated. All the recombinant subunit proteins △VP8*s or P2-△VP8*s were of high solubility and high yields. The immunogenicity of each purified △VP8* and P2-△VP8* was evaluated in mice (10μg/dose) or guinea pigs (20μg/dose) immunized IM with 600μg aluminum hydroxide three times at 2-week interval. The introduction of P2T cell epitope to P[7]-△VP8* elicited significantly higher IgG titer in mice than its absence. Comparatively, P2 epitope slightly enhanced the immunogenicity of P[6]-△VP8*. P2-P[7]△VP8* elicited high titer of neutralizing antibody against heterotypic P[7]-specific rotaviruses with varied G type combination. Our data indicated that two subunit vaccines could be plausible bivalent rotavirus vaccine candidate to provide antigenic coverage of porcine rotavirus strains of global or regional importance.

Nasal plasmid Flt3 ligand and CpG ODN alone enhances immediate, influenza virus-specific recall secretory IgA Ab responses in aging (MUC1P.904)

Abstract The elderly have already experienced various pathogens and thus possess pathogen-specific Abs, whose titers are too low to combat reinfection. Thus, it would be of great benefit to the aged population if one could use an innate adjuvant system alone, without vaccine, in order to enhance mucosal immunity against past respiratory infections. We tested whether a combined plasmid Flt3 ligand (pFL) and CpG ODN adjuvant would enhance influenza virus-specific recall Ab responses without a need for additional vaccination. Young adult mice were nasally immunized with A/Puerto Rico/8/34 (PR8) hemagglutinin (HA) plus cholera toxin as mucosal adjuvant three times at weekly intervals. Seven months later, these mice were given nasal pFL and CpG ODN only or combined adjuvant together with PR8 HA. Saliva and plasma samples were collected 7 days later and PR8 HA-specific IgA Ab responses were evaluated. Both groups of mice showed significantly increased and comparable levels of anti-PR8 HA secretory IgA Ab responses in saliva when compared with those responses seen without innate adjuvant. The increase in total IgG Ab responses in mice given pFL and CpG ODN were limited when compared with those seen after primary nasal immunization. Total plasma IgE Ab titers in mice given nasal pFL and CpG ODN only were the same as IgE levels seen in naïve mice. These results suggest that nasal pFL and CpG ODN alone can enhance pre-existing Ag-specific Ab responses without adverse effects in the elderly.

Adjuvant effect of the human metapneumovirus (HMPV) matrix protein in HMPV subunit vaccines.

The human metapneumovirus (HMPV) fusion (F) protein is the most immunodominant protein, yet subunit vaccines containing only this protein do not confer complete protection. The HMPV matrix (M) protein induces the maturation of antigen-presenting cells in vitro. The inclusion of the M protein into an F protein subunit vaccine might therefore provide an adjuvant effect. We administered the F protein twice intramuscularly, adjuvanted with alum, the M protein or both, to BALB/c mice at 3 week intervals. Three weeks after the boost, mice were infected with HMPV and monitored for 14 days. At day 5 post-challenge, pulmonary viral titres, histopathology and cytokine levels were analysed. Mice immunized with F+alum and F+M+alum generated significantly more neutralizing antibodies than mice immunized with F only [titres of 47 ± 7 (P<0.01) and 147 ± 13 (P<0.001) versus 17 ± 2]. Unlike F only [1.6 ± 0.5 × 10(3) TCID50 (g lung)(-1)], pulmonary viral titres in mice immunized with F+M and F+M+alum were undetectable. Mice immunized with F+M presented the most important reduction in pulmonary inflammation and the lowest T-helper Th2/Th1 cytokine ratio. In conclusion, addition of the HMPV-M protein to an F protein-based vaccine modulated both humoral and cellular immune responses to subsequent infection, thereby increasing the protection conferred by the vaccine.

Polycation-decorated PLA microspheres induce robust immune responses via commonly used parenteral administration routes.

Recombinant viral subunit-based vaccines have gained increasing attention due to their enhanced safety over the classic live-attenuated or inactivated vaccines. The low immunogenicity of the subunit antigen alone, however, requires the addition of an adjuvant to induce immunity. Particulate-based delivery systems have great potential for developing new vaccine adjuvants, compared to traditional aluminum-based saline adjuvants. The physicochemical properties of particulate vaccines have been extensively investigated; however, few studies have focused on how the administration route of various adjuvant-antigen combinations impacts the efficacy of the immune response. Here, for the first time, the viral Hepatitis B surface antigen (HBsAg) was combined with aluminum-based or cationic-microsphere (MP) based adjuvants to investigate the characteristics of immune responses elicited after immunization via the subcutaneous, intramuscular, or intraperitoneal routes respectively. In vitro, the MP-based vaccine significantly increased dendritic cell (DC) activation with up-regulated CD40 and CD80 expression and IL-12 production compared to alum-based vaccine. After immunization, both MP and alum-based vaccines produced increased IgG titers in mice. The administration route of these vaccines did influenced immune responses. The MP-based vaccine delivered via the intramuscular route yielded the highest levels of the IgG2a isotype. The alum-based vaccine, delivered via the same route, produced an IgG1-dominated humoral immune response. Moreover, subcutaneous and intramuscular immunizations with MP-based vaccine augmented Granzyme B, Th1-type cytokines (IL-2, IL-12, and IFN-γ), and Th2 cytokine IL-4 secretions. These results demonstrate that MP-based vaccines have the capacity to induce higher cellular and humoral immune response especially via an intramuscular administration route than an alum-based vaccine.

EVALUATION OF IMMUNOMODULATORY ACTIVITY OF SESBANIA GRANDIFLORA FLOWERS EXTRACT IN MICE

The aim of the present study was to investigate the immunomodulatory activity of Sesbania grandiflora on cellular and humoral immunity. Oral administration of the methanolic extract (200 and 400mg/kg) of S. grandiflora flowers, in mice, dose-dependently significantly enhanced the production of circulating antibody titre in mice in response to SRBC. It significantly potentiated the delayed-type hypersensitivity reaction induced by sheep red blood cells (SRBC). Good response was also found towards phagocytosis in carbon clearance assay and prevented myelosuppression in cyclophosphamide drug. Aqueous extract at 250mg/kg dose level failed to show immunomodulatory activity but 500mg/kg of aqueous extract potentiated the activity however less significantly compared with both dose of methanolic extract. The results obtained in this study indicate that methanolic extract (400mg/kg) of S . grandiflora possesses potential immunomodulatory activity.

Abstract 2586: Bortezomib reduces preexisting antibodies to recombinant immunotoxins in mice

Abstract Recombinant immunotoxin (RIT) therapy is limited in patients by neutralizing antibody responses. Most patients with normal immune systems make neutralizing antibodies after one cycle (three doses) of RIT, preventing repeated dosing. Furthermore, some patients have preexisting antibodies from environmental exposure to Pseudomonas exotoxin (PE), the component of the RIT that elicits the neutralizing antibody response. Bortezomib is an FDA-approved proteasome inhibitor which selectively targets and kills plasma cells which are necessary for a neutralizing antibody response. Bortezomib, in combination with methotrexate, rituximab, and cyclophosphamide, was shown to reduce antibody levels &amp;gt;64-fold following enzyme replacement therapy in the clinical setting of Pompe disease. We hypothesized bortezomib may abrogate the neutralizing antibody levels, making dosing of RIT possible in mice already immune to RIT. We immunized BALB/c mice with multiple doses of SS1P, a RIT whose antibody portion targets mesothelin. Antibody titers were determined by immune-capture ELISA. Mice with elevated levels were separated into groups to receive bortezomib (1mg/kg i.v., twice a week for 5.5 weeks) or saline control. Bortezomib treatment lowered the starting antibody titer in mice previously immunized with RIT by an average of 50%, while antibody titers in control mice increased by an average of 20%. Despite success in lowering titers, bortezomib did not abrogate titer in any mice. To further diminish preexisting antibodies, we performed a separate experiment in which mice with preexisting anti-SS1P antibodies received bortezomib with concomitant pentostatin and cyclophosphamide (P/C) therapy. P/C is an immune-depleting regimen already shown to be effective in preventing the onset of new neutralizing antibodies. Combination treatment lowered the starting antibody titer in mice previously immunized with RIT by an average of 73%, while antibody titers in control mice increased by an average of 80%. We have shown that bortezomib reduces, but does not eliminate, antibody titer in mice with preexisting antibodies to RIT. Addition of the immune-depleting P/C regimen further diminishes antibody titers. We are investigating remaining plasma cell populations as a potential source of remaining antibody. Ongoing experiments also include incorporating other FDA-approved agents into our study. Citation Format: Michael L. Manning, Emily Mason-Osann, Masanori Onda, Ira Pastan. Bortezomib reduces preexisting antibodies to recombinant immunotoxins in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2586. doi:10.1158/1538-7445.AM2014-2586

Evaluation of a fully human monoclonal antibody against multiple influenza A viral strains in mice and a pandemic H1N1 strain in nonhuman primates

Influenza virus is a global health concern due to its unpredictable pandemic potential. Frequent mutations of surface molecules, hemagglutinin (HA) and neuraminidase (NA), contribute to low efficacy of the annual flu vaccine and therapeutic resistance to standard antiviral agents. The populations at high risk of influenza virus infection, such as the elderly and infants, generally mount low immune responses to vaccines, and develop severe disease after infection. Novel therapeutics with high effectiveness and mutation resistance are needed. Previously, we described the generation of a fully human influenza virus matrix protein 2 (M2) specific monoclonal antibody (mAb), Z3G1, which recognized the majority of M2 variants from natural viral isolates, including highly pathogenic avian strains. Passive immunotherapy with Z3G1 significantly protected mice from the infection when administered either prophylactically or 1–2days post infection. In the present study, we showed that Z3G1 significantly protected mice from lethal infection when treatment was initiated 3days post infection. In addition, therapeutic administration of Z3G1 reduced lung viral titers in mice infected with different viral strains, including amantadine and oseltamivir-resistant strains. Furthermore, prophylactic and therapeutic administration of Z3G1 sustained O2 saturation and reduced lung pathology in monkeys infected with a pandemic H1N1 strain. Finally, de-fucosylated Z3G1 with an IgG1/IgG3 chimeric Fc region was generated (AccretaMab® Z3G1), and showed increased ADCC and CDC in vitro. Our data suggest that the anti-M2 mAb Z3G1 has great potential as a novel anti-flu therapeutic agent.

Development and pre-clinical evaluation of two LAIV strains against potentially pandemic H2N2 influenza virus.

H2N2 Influenza A caused the Asian flu pandemic in 1957, circulated for more than 10 years and disappeared from the human population after 1968. Given that people born after 1968 are naïve to H2N2, that the virus still circulates in wild birds and that this influenza subtype has a proven pandemic track record, H2N2 is regarded as a potential pandemic threat. To prepare for an H2N2 pandemic, here we developed and tested in mice and ferrets two live attenuated influenza vaccines based on the haemagglutinins of the two different H2N2 lineages that circulated at the end of the cycle, using the well characterized A/Leningrad/134/17/57 (H2N2) master donor virus as the backbone. The vaccine strains containing the HA and NA of A/California/1/66 (clade 1) or A/Tokyo/3/67 (clade 2) showed a temperature sensitive and cold adapted phenotype and a reduced reproduction that was limited to the respiratory tract of mice, suggesting that the vaccines may be safe for use in humans. Both vaccine strains induced haemagglutination inhibition titers in mice. Vaccination abolished virus replication in the nose and lung and protected mice from weight loss after homologous and heterologous challenge with the respective donor wild type strains. In ferrets, the live attenuated vaccines induced high virus neutralizing, haemagglutination and neuraminidase inhibition titers, however; the vaccine based on the A/California/1/66 wt virus induced higher homologous and better cross-reactive antibody responses than the A/Tokyo/3/67 based vaccine. In line with this observation, was the higher virus reduction observed in the throat and nose of ferrets vaccinated with this vaccine after challenge with either of the wild type donor viruses. Moreover, both vaccines clearly reduced the infection-induced rhinitis observed in placebo-vaccinated ferrets. The results favor the vaccine based on the A/California/1/66 isolate, which will be evaluated in a clinical study.

Genetic and pathogenic characteristics of H1 avian and swine influenza A viruses.

This study examined the potential for cross-species transmission of influenza viruses by comparing the genetic and pathogenic characteristics of H1 avian influenza viruses (AIVs) with different host origins in Korea. Antigenic and phylogenetic analyses of H1 AIVs circulating in Korea provided evidence of genetic similarity between viruses that infect domestic ducks and those that infect wild birds, although there was no relationship between avian and swine viruses. However, there were some relationships between swine and human viral genes. The replication and pathogenicity of the H1 viruses was assessed in chickens, domestic ducks and mice. Viral shedding in chickens was relatively high. Virus was recovered from both oropharyngeal and cloacal swabs up to 5-10 days post-inoculation. The titres of domestic duck viruses in chickens were much higher than those of wild-bird viruses. Both domestic duck and wild-bird viruses replicated poorly in domestic ducks. None of the swine viruses replicated in chickens or domestic ducks; however, six viruses showed relatively high titres in mice, regardless of host origin, and induced clinical signs such as ruffled fur, squatting and weight loss. Thus, although the phylogenetic and antigenic analyses showed no evidence of interspecies transmission between birds and swine, the results suggest that Korean H1 viruses have the potential to cause disease in mammals. Therefore, we should intensify continuous monitoring of avian H1 viruses in mammals and seek to prevent interspecies transmission.

Effect of transfer factor on immune efficacy of PCV2 subunit vaccine

To investigate the effect of transfer factor(TF) on the immune efficacy of porcine circovirus type 2(PCV2) subunit vaccine,spleen from healthy porcine was grinded and the homogenate was frozen-thawed six times.TF was obtained after the homogenate was centrifuged and ultrafiltered.After physical and chemical inspection,TF was added to PCV2 subunit vaccine(Cap vaccine),named TF-Cap vaccine.TF-Cap vaccine and Cap vaccine were used to immunize mice with physiological saline as control.The mice were separated into two groups: one-time vaccination and two-time vaccination.Blood was collected at 10-d interval through caudal vein after the first vaccination and PCV2 antibody was tested using indirect enzyme-linked immuno-sorbent assay.The result showed that the concentration of the ultrafiltered TF is 1.83 mg/mL.Detection of PCV2 specific antibody showed that there are significant differences(P0.01) between vaccine group and control group while there is no significant differences(P0.05) among vaccine groups.However,PCV2 antibody titer in mice immunized TF-Cap vaccine only once was comparable to mice immunized Cap-vaccine twice,indicating PCV2 subunit vaccine with TF can enhance the antibody production.

Ginsenoside Re as an adjuvant to enhance the immune response to the inactivated rabies virus vaccine in mice

The inactivated rabies virus vaccine (RV) is a relatively expensive vaccine, prone to failure in some cases. Ginsenoside Re (Re) is a saponin isolated from Panax ginseng, and has an adjuvant property. Here the adjuvant effect of Re to improve the immune response to the RV is evaluated in mice. ICR mice were immunized with saline, 2.50mg/kg Re, 20μl RV, 100μl RV, or 20μl of RV adjuvanted with Re (1.25, 2.50 or 5.00mg/kg). Different time points after boosting, we measured serum antibodies in blood samples and separated splenocytes to detect lymphocyte proliferation and the production of IL-4, IL-10, IL-12, and IFN-γ in vitro. We also compared immunizations containing 20μl RV and 20μl RV adjuvanted with Re (5.00mg/kg) for the expression of CD4(+) and CD8(+) T-cell subsets at different time points. Results indicated that co-administration of Re significantly enhanced serum antibody titers, increased the CD4(+):CD8(+) ratio, and enhanced both proliferation responses and IL-4, IL-10, IL-12 and IFN-γ secretions. Both Th1 and Th2 immune responses were activated. The supplementation of the Re (5.00mg/kg) to 20μl of RV significantly amplified serum antibody responses and Th1/Th2 responses inducing similar protection as did 100μl of RV. This suggests that Re could be used to reduce the dose, and therefore the cost, of the RV to achieve the same effective protection. Re merits further studies for use with vaccines of mixed Th1/Th2 immune responses.

Serum Bactericidal Assays To Evaluate Typhoidal and Nontyphoidal Salmonella Vaccines

Invasive Salmonella infections for which improved or new vaccines are being developed include enteric fever caused by Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B and sepsis and meningitis in young children in sub-Saharan Africa caused by nontyphoidal Salmonella (NTS) serovars, particularly S. enterica serovars Typhimurium and Enteritidis. Assays are needed to measure functional antibodies elicited by the new vaccines to assess their immunogenicities and potential protective capacities. We developed in vitro assays to quantify serum bactericidal antibody (SBA) activity induced by S. Typhi, S. Paratyphi A, S. Typhimurium, and S. Enteritidis vaccines in preclinical studies. Complement from various sources was tested in assays designed to measure antibody-dependent complement-mediated killing. Serum from rabbits 3 to 4 weeks of age provided the best complement source compared to serum from pigs, goats, horses, bovine calves, or rabbits 8 to 12 weeks of age. For S. Enteritidis, S. Typhimurium, and S. Typhi SBA assays to be effective, bacteria had to be harvested at log phase. In contrast, S. Paratyphi A was equally susceptible to killing whether it was grown to the stationary or log phase. The typhoidal serovars were more susceptible to complement-mediated killing than were the nontyphoidal serovars. Lastly, the SBA endpoint titers correlated with serum IgG anti-lipopolysaccharide (LPS) titers in mice immunized with mucosally administered S. Typhimurium, S. Enteritidis, and S. Paratyphi A but not S. Typhi live attenuated vaccines. The SBA assay described here is a useful tool for measuring functional antibodies elicited by Salmonella vaccine candidates.

Pretreatment of Mice with Oligonucleotide prop5 Protects Them from Influenza Virus Infections

Influenza A virus is a successful parasite and requires host factors to complete its life cycle. Prop5 is an antisense oligonucleotide, targeting programmed cell death protein 5 (PDCD5). In this study, we tested the antiviral activity of prop5 against mouse-adapted A/FM/1/47 strain of influenza A virus in a mouse model. Prop5 intranasally administered the mice at dosages of 10 and 20 mg/kg/d at 24 h and 30 min before infection, provided 80% and 100% survival rates and prolonged mean survival days in comparison with influenza virus-infected mice (both p < 0.01). Moreover, viral titres in mice pretreated with prop5, at dose of 10 and 20 mg/kg/d, had declined significantly on day two, four, and six post-infection compared with the yields in infected mice (p < 0.05 or p < 0.01); lung index in mice pretreated with prop5 (20 mg/kg/d) had been inhibited on day six post-infection (p < 0.05). Western blotting and immunohistochemistry showed that prop5 could down-regulate the PDCD5 protein expression levels in lung tissues of infected mice. These data indicate that antisense oligonucleotide prop5 is a promising drug for prophylaxis and control influenza virus infections and provides an insight into the host-pathogen interaction.