Passive Mouse Protection Research Articles

An intranasal attenuated Coxsackievirus B3 vaccine induces strong systemic and mucosal immunity against CVB3 lethal challenge.

Coxsackievirus B3 (CVB3) triggers viral myocarditis, with no effective vaccine yet. This fecal-oral transmitted pathogen has prompted interest in mucosal immunization strategies to impede CVB3 spread. We developed a new attenuated vaccine strain, named CVB3(mu). The potential of CVB3(mu) to stimulate mucosal immune protection remains to be elucidated. This study evaluates the attenuation characteristics of CVB3(mu) via a rapid evolution cellular model and RNA sequencing. Its temperature sensitivity and safety were evaluated through in vitro and in vivo experiments. The mucosal immunity protection of CVB3(mu) was assessed via intranasal immunization in Balb/c mice. The results indicate that CVB3(mu) exhibits temperature sensitivity and forms smaller plaques. It sustains fewer genetic mutations and still possesses certain attenuated traits up to the 25th passage, in comparison to CVB3(WT). Intranasal immunization elicited a significant serum neutralizing antibodies, and a substantial sIgA response in nasal washes. In vivo trials revealed CVB3(mu) protection in adult mice and passive protection in suckling mice against lethal CVB3(WT) challenges. In conclusion, CVB3(mu), a live attenuated intranasal vaccine, provides protection involving humoral and mucosal immunity, making it a promising candidate to control CVB3 spread and infection.

Active Immunization with Pasteurella multocida Lysate Elicits Antibody that Protects Rabbits against Virulent Pasteurella multocida and Protects Mice by Passive Immunization

Background: Pasteurellosis is an economically important disease of livestock worldwide and vaccination is effective mean to control the disease outbreaks. One of alternative method for vaccine preparation could be the use of lytic phages as bacterial inactivating agents and employing “phage lysate immunogen” as an immunizing agent. Therefore, the study was undertaken to explore the protective immune response of novel phage lysate vaccine against Pasteurella multocida. Methods: A phage lysate vaccine of Pasteurella multocida was prepared using isolated lytic phage and subjected to vaccine response study in rabbits. In this experiment, two mouse fixed-dose of lysate was used and the prophylactic efficacies of lysates were evaluated in rabbits by passive mouse protection test (PMPT), western blotting and direct virulent challenge.Result: Passive transfer of antibodies to mice using post-immunization sera of rabbits protected the animals against the challenge with A: 1 strain (75%) and B: 2 strain (50%). In western blotting, a total number of 4 bands were observed in the region between 130 kDa to 25 kDa in the protein of the phage lysate. The study suggested the significance increase in humoral immunity by phage lysate in murine Pasteurellosis.

Immunization with Epstein–Barr Virus Core Fusion Machinery Envelope Proteins Elicit High Titers of Neutralizing Activities and Protect Humanized Mice from Lethal Dose EBV Challenge

Epstein–Barr virus (EBV) is the primary cause of infectious mononucleosis and is strongly implicated in the etiology of multiple lymphoid and epithelial cancers. EBV core fusion machinery envelope proteins gH/gL and gB coordinately mediate EBV fusion and entry into its target cells, B lymphocytes and epithelial cells, suggesting these proteins could induce antibodies that prevent EBV infection. We previously reported that the immunization of rabbits with recombinant EBV gH/gL or trimeric gB each induced markedly higher serum EBV-neutralizing titers for B lymphocytes than that of the leading EBV vaccine candidate gp350. In this study, we demonstrated that immunization of rabbits with EBV core fusion machinery proteins induced high titer EBV neutralizing antibodies for both B lymphocytes and epithelial cells, and EBV gH/gL in combination with EBV trimeric gB elicited strong synergistic EBV neutralizing activities. Furthermore, the immune sera from rabbits immunized with EBV gH/gL or trimeric gB demonstrated strong passive immune protection of humanized mice from lethal dose EBV challenge, partially or completely prevented death respectively, and markedly decreased the EBV load in peripheral blood of humanized mice. These data strongly suggest the combination of EBV core fusion machinery envelope proteins gH/gL and trimeric gB is a promising EBV prophylactic vaccine.

PREPARATION AND COMPARATIVE EVALUATION OF HAEMORRHAGIC SEPTICAEMIA VACCINES USING EOLANE-150 AND EOLANE-170 AS OIL ADJUVANTS FOR CATTLE AND BUFFALO

Haemorrhagic septicaemia (HS) caused by Pasteurella multocida serotype B:2 is an economically important disease of cattle and buffalo, causes heavy economic losses due to sudden death of animals in developing countries like Pakistan. In this country, animals are vaccinated by alum (adjuvant) precipitated vaccine twice a year. Immunity induced through this prophylactic measure lasts for 3-4 months only. Two new HS oil based vaccines were prepared by using two new oil adjuvants such as Eolane-150 and Eolane-170. The ratio of bacterial antigen and oil adjuvants was 1:1 while bacterial dry weight was adjusted to 2 mg/ml. The addition of enrichments and aeration resulted in dense bacterial growth of Pasteurella multocida. Both new vaccines passed sterility, safety and potency tests as per OIE, 2017. Active and passive Mouse Protection Tests were performed to evaluate its potency. Indirect Haem-Agglutination (IHA) test was conducted on serum samples of two rabbits, groups each of which was vaccinated with HS oil based vaccines adjuvanted with Eolane-150 and Eolane-170. IHA indicated that immune response was higher (GMT=32) initially on 45th day to 75th day post vaccination and then declined (GMT=16) in the rabbits vaccinated with HS vaccine adjuvanted with Eolane-150, while protective immune response remained constant (GMT=16) up to ninety days post vaccination in the rabbits vaccinated with HS vaccine adjuvanted with Eolane-170. Vaccines were easy to inject with no side effects, including swelling at the injection site and longer protection as well. That would hopefully motivate the livestock owners and farmers to use this new product to protect their animals against this fatal HS disease.

Immunogenicity of Non-Living Anthrax Vaccine Candidates in Cattle and Protective Efficacy of Immune Sera in A/J Mouse Model Compared to the Sterne Live Spore Vaccine.

The Sterne live spore vaccine (SLSV, Bacillus anthracis strain 34F2) is the veterinary vaccine of choice against anthrax though contra-indicated for use with antimicrobials. However, the use of non-living anthrax vaccine (NLAV) candidates can overcome the SLSV limitation. In this study, cattle were vaccinated with either of the NLAV (purified recombinant PA (PrPA) or crude rPA (CrPA) and formaldehyde-inactivated spores (FIS of B. anthracis strain 34F2) and emulsigen-D®/alhydrogel® adjuvants) or SLSV. The immunogenicity of the NLAV and SLSV was assessed and the protective efficacies evaluated using a passive immunization mouse model. Polyclonal IgG (including the IgG1 subset) and IgM responses increased significantly across all vaccination groups after the first vaccination. Individual IgG subsets titres peaked significantly with all vaccines used after the second vaccination at week 5 and remained significant at week 12 when compared to week 0. The toxin neutralization (TNA) titres of the NLAV vaccinated cattle groups showed similar trends to those observed with the ELISA titres, except that the former were lower, but still significant, when compared to week 0. The opsonophagocytic assay indicated good antibody opsonizing responses with 75% (PrPA+FIS), 66% (CrPA+FIS) and 80% (SLSV) phagocytosis following spores opsonization. In the passive protection test, A/J mice transfused with purified IgG from cattle vaccinated with PrPA+FIS+Emulsigen-D®/Alhydrogel® and SLSV had 73% and 75% protection from challenge with B. anthracis strain 34F2 spores, respectively, whereas IgG from cattle vaccinated with CrPA+FIS+Emulsigen-D®/Alhydrogel® offered insignificant protection of 20%. There was no difference in protective immune response in cattle vaccinated twice with either the PrPA+FIS or SLSV. Moreover, PrPA+FIS did not show any residual side effects in vaccinated cattle. These results suggest that the immunogenicity and protective efficacy induced by the NLAV (PrPA+FIS) in the cattle and passive mouse protection test, respectively, are comparable to that induced by the standard SLSV.

Oral immunization with rotavirus VP7-CTB fusion expressed in transgenic Arabidopsis thaliana induces antigen-specific IgA and IgG and passive protection in mice.

Human rotavirus (HRV) is the primary cause of severe gastroenteritis in children. However, there is currently no protective virus for rotavirus available. In the present study, an HRVVP7-cholera toxin B subunit (CTB) fusion protein was expressed in Arabidopsis thaliana. To determine the adjuvant effect of HRVVP7-CTB, HRVVP7 without CTB was expressed in the same manner. HRVVP7-CTB accounted for 0.39% of the total soluble protein (TSP) in the transgenic seeds and 52.65 µg/g of HRVVP7 protein was expressed in these seeds. Mice were immunized with TSP from the transformed seeds and produced serum immunoglobulin G (IgG) and mucosal IgA specifically directed against HRVVP7. Antibody titers were highest in mice orally immunized with the plant-expressed HRVVP7-CTB protein, whereas HRVVP7-CTB-specific IgG neutralized the rotavirus. Suckling pups born from dams immunized with the HRVVP7-CTB fusion protein were protected against challenge with virulent rotavirus. The results of the present study suggest that the HRVVP7-CTB fusion protein produced in A. thaliana may be a rotaviral-specific candidate subunit vaccine.

Passive protection against anthrax in mice with plasma derived from horses hyper-immunized against Bacillus anthracis Sterne strain.

In this study, equine source polyclonal anti-Bacillus anthracis immunoglobulins were generated and utilized to demonstrate passive protection of mice in a lethal challenge assay. Four horses were hyper-immunized with B. anthracis Sterne strain for approximately one year. The geometric mean anti-PA titer in the horses at maximal response following immunization was 1:77,936 (Log2 mean titer 16.25, SEM ± 0.25 95% CI [15.5 –17.0]). The geometric mean neutralizing titer at maximal response was 1:128 (Log2 mean titer 7, SEM ± 0.0, 95% CI 7). Treatment with hyper-immune plasma or purified immunoglobulins was successful in passively protecting A/J mice from a lethal B. anthracis Sterne strain challenge. The treatment of mice with hyper-immune plasma at time 0 h and 24 h post-infection had no effect on survival, but did significantly increase mean time to death (p < 0.0001). Mice treated with purified immunoglobulins at time 0 h and 24 h post-infection in showed significant increase in survival rate (p < 0.001). Bacterial loads in lung, liver and spleen tissue were also assessed and were not significantly different in mice treated with hyper-immune plasma from placebo treated control mice. Mice treated with purified antibodies demonstrated mean colony forming units/gram tissue fourfold less than mice receiving placebo treatment (p < 0.0001). Immunotherapeutics harvested from horses immunized against B. anthracis Sterne strain represent a rapidly induced, inexpensive and effective expansion to the arsenal of treatments against anthrax.

Use of the mice passive protection test to evaluate the humoral response in goats vaccinated with Sterne 34F2 live spore vaccine

The Sterne live spore vaccine (34F2) is the most widely used veterinary vaccine against anthrax in animals. Antibody responses to several antigens of Bacillus anthracis have been described with a large focus on those against protective antigen (PA). The focus of this study was to evaluate the protective humoral immune response induced by the live spore anthrax vaccine in goats. Boer goats vaccinated twice (week 0 and week 12) with the Sterne live spore vaccine and naive goats were used to monitor the anti-PA and toxin neutralizing antibodies at week 4 and week 17 (after the second vaccine dose) post vaccination. A/J mice were passively immunized with different dilutions of sera from immune and naive goats and then challenged with spores of B. anthracis strain 34F2 to determine the protective capacity of the goat sera. The goat anti-PA ELISA titres indicated significant sero-conversion at week 17 after the second doses of vaccine (p = 0.009). Mice receiving undiluted sera from goats given two doses of vaccine (twice immunized) showed the highest protection (86%) with only 20% of mice receiving 1:1000 diluted sera surviving lethal challenge. The in vitro toxin neutralization assay (TNA) titres correlated to protection of passively immunized A/J mice against lethal infection with the vaccine strain Sterne 34F2 spores using immune goat sera up to a 1:10 dilution (rs ≥ 0.522, p = 0.046). This study suggests that the passive mouse protection model could be potentially used to evaluate the protective immune response in livestock animals vaccinated with the current live vaccine and new vaccines.

Active immunization with Brucella abortus S19 phage lysate elicits serum IgG that protects guinea pigs against virulent B. abortus and protects mice by passive immunization

Brucellosis is an economically important zoonosis of worldwide significance. Earlier (Jain et al., 2015) we reported methodology for generation of phage lysate preparations against Brucella abortus S19 using brucellaphage ‘ϕLd’. In this study, using a fixed dose (Two mouse PD100) of lysates, the prophylactic efficacies of both plain and alum gel adjuvanted lysates were evaluated in guinea pig by direct virulent challenge and passive mouse protection test (PMPT). Strong humoral and cell mediated immune responses in guinea pigs and protection comparable to S19 vaccine was observed with low dose (1.0 μg protein and 120 μg carbohydrate adsorbed on 0.1% aluminium gel). Passive transfer of antibodies to mice using d 90 post immunization sera of guinea pig protected the animals against challenge. The study suggested the significance of humoral immunity in murine brucellosis. Further, the methodology can be explored to produce a new class of immunotherapeutic agents against bovine brucellosis.

Maternal vaccination with a fimbrial tip adhesin and passive protection of neonatal mice against lethal human enterotoxigenic Escherichia coli challenge.

Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain H10407, which has been extensively studied in volunteers, an inoculum of 2 × 10(7) bacteria resulted in 50% lethal doses (LD50) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD50 challenge with H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation.

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium

The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM197 carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection.

A novel recombinant vaccine protecting mice against abrin intoxication

Abrin toxin (AT) consisting of an A chain and a B chain is a potential agent for bioterrorism and an effective vaccine against AT poisoning is urgently required. In this study, AT B chain (ATB) was successfully expressed in the Escherichia coli (E. coli) and assessed the protection capacity against AT intoxication. The recombinant ATB (rATB) subunit induces a good immune response after 4 immunizations. All BALB/c mice immunized intraperitoneally (i.p.) with the purified rATB protein survived after challenged with 5 × LD50 of AT. Transfusion of sera from immunized mice provided passive protection in naive mice. Furthermore, histological findings showed that immunization with rATB decreased the severity of toxin-related tissue damage. This work indicates that the rATB protein may be a promising vaccine candidate against human exposure to AT.

Passive protection of mice against Streptococcus pneumoniae challenge by naturally occurring and vaccine-induced human anti-PhtD antibodies

Currently marketed Streptococcus pneumoniae vaccines are based on polysaccharide capsular antigens from the most common strains. Pneumococcal histidine triad protein D (PhtD) is a conserved surface protein that is being evaluated as a candidate for a vaccine with improved serotype coverage. Here, we measured the functional activity of human anti-PhtD antibodies in a passive protection model wherein mice were challenged with a lethal dose of S. pneumoniae by intravenous injection. This functional activity was compared with anti-PhtD antibody concentrations measured by enzyme-linked immunosorbent assay (ELISA) to estimate the 50% protective dose (ED50). Anti-PhtD antibodies affinity purified from pooled normal human sera passively protected mice with an ED50 of 1679 ELISA units/ml (95% confidence interval, 1420–1946). Sera from subjects injected with aluminum-adjuvanted PhtD in a phase I trial had similar activity per unit of antibody (ED50 = 1331 ELISA units/ml [95% confidence interval, 762–2038]). Vaccine-induced activity in the passive protection model was blocked by pre-incubation with recombinant PhtD but not by a control S. pneumoniae antigen (LytB). These results show that human anti-PhtD antibodies, whether naturally acquired or induced by the PhtD candidate vaccine, are functional. This supports the development of the PhtD candidate as part of a broadly protective pneumococcal vaccine.

Protection against P. multocida B:2 using Recombinant 87kDa OMP Periplasmic Protein

Protection against P. multocida B: 2 using Recombinant 87kDa OMP Periplasmic Protein To clone and express Pasteurella. multocida (P. multocida) encoding 87 kDa omp gene in prokaryotic (secretory) vector and to study the immunogenicity of expressed protein.The genomic DNA was isolated from P. multocida B:2. Specific primers were designed to suite secretory prokaryotic vector system. PCR amplified gene was cloned in pGEMT-EASY vector (Promega, USA) and subcloned into pBAD/gIIIA prokaryotic expression vector. The protein was purified and Passive mouse protection test challenge was conducted in mice.

Correlation between in vitro complement deposition and passive mouse protection of anti-pneumococcal surface protein A monoclonal antibodies.

The shortcomings of the licensed polysaccharide-based pneumococcal vaccine are driving efforts toward development of a protein-based vaccine that is serotype independent and effective in all age groups. An opsonophagocytic killing assay (OPKA) is used to evaluate the antibody response against polysaccharide-based pneumococcal vaccines. However, the OPKA is not reliable for noncapsular antigens. Thus, there is a need to develop an in vitro surrogate for protection for protein vaccine candidates like pneumococcal surface antigen A (PspA). PspA is a serologically variable cell surface virulence factor. Based on its sequence, PspA has been classified into families 1 (clade 1 and 2), 2 (clades 3, 4 and 5), and 3 (clade 6). Here, we report the characterization of 18 IgG anti-PspA monoclonal antibodies (anti-PspA(hkR36A) MAbs) generated from mice immunized with heat-killed strain R36A (clade 2). An enzyme-linked immunosorbent assay (ELISA)-based analysis of the reactivity of the MAbs with recombinant PspAs from the 6 clades indicated that they were family 1 specific. This was confirmed by flow cytometry using a hyperimmune serum generated against PspA from R36A. Eight MAbs that bind at least one clade 1- and clade 2-expressing strain were evaluated for complement deposition, bactericidal activity, and passive protection. The anti-PspA(hkR36A) MAb-dependent deposition of complement on pneumococci showed a positive correlation with passive protection against strain WU2 (r = 0.8783, P = 0.0041). All of our protective MAbs showed bactericidal activity; however, not all MAbs that exhibited bactericidal activity conferred protection in vivo. The protective MAbs described here can be used to identify conserved protection eliciting B cell epitopes for engineering a superior PspA-based vaccine.

Modified Opsonization, Phagocytosis, and Killing Assays To Measure Potentially Protective Antibodies against Pneumococcal Surface Protein A

The standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface protein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant α-helical or proline-rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels of antibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonal antibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibodies to PspA.

Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice

Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni–NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.

Display of enterovirus 71 VP1 on baculovirus as a type II transmembrane protein elicits protective B and T cell responses in immunized mice

Human enterovirus 71 (EV71) has become a major public health threat across Asia Pacific. The virus causes hand, foot, and mouth disease which can lead to neurological complications in young children. There are no specific antivirals or vaccines against EV71 infection. The major neutralizing epitope of EV71 is located in the carboxy-terminal half of the VP1 protein at amino acid positions 215–219 (Lim et al., 2012). To study the immunogenicity of VP1 we have developed a baculovirus vector which displays VP1 as a type II transmembrane protein, providing an accessible C-terminus. Immunization of mice with this recombinant baculovirus elicited neutralizing antibodies against heterologous EV71 in an in vitro microneutralization assay. Passive protection of neonatal mice confirmed the prophylactic efficacy of the antisera. Additionally, EV71 specific T cell responses were stimulated. Taken together, our results demonstrate that the display of VP1 as a type II transmembrane protein efficiently stimulated both humoral and cellular immunities.

Histophilus somni IbpA Fic cytotoxin is conserved in disease strains and most carrier strains from cattle, sheep and bison

Histophilus somni causes bovine pneumonia, septicemia, myocarditis, thrombotic meningoencephalitis and arthritis, as well as a genital or upper respiratory carrier state in normal animals. However, differences in virulence factors among strains are not well studied. The surface and secreted immunoglobulin binding protein A (IbpA) Fic motif of H. somni causes bovine alveolar type 2 (BAT2) cells to retract, allowing virulent bacteria to cross the alveolar monolayer. Because H. somni IbpA is an important virulence factor, its presence was evaluated in different strains from cattle, sheep and bison to define whether there are syndrome specific markers and whether antigenic/molecular/functional conservation occurs. A few preputial carrier strains lacked IbpA by Western blotting but all other tested disease or carrier strains were IbpA positive. These positive strains had either both IbpA DR1/Fic and IbpA DR2/Fic or only IbpA DR2/Fic by PCR. IbpA Fic mediated cytotoxicity for BAT2 cells and sequence analysis of IbpA DR2/Fic from selected strains revealed conservation of sequence and function in disease and IbpA positive carrier strains. Passive protection of mice against H. somni septicemia with antibody to IbpA DR2/Fic, along with previous data, indicates that the IbpA DR1/Fic and/or DR2/Fic domains are candidate vaccine antigens for protection against many strains of H. somni. Since IbpA DR2/Fic is conserved in most carrier strains, they may be virulent if introduced to susceptible animals at susceptible sites. Conservation of the protective IbpA antigen in all disease isolates tested is encouraging for development of protective vaccines and diagnostic assays.

Oral administration of plant-based rotavirus VP6 induces antigen-specific IgAs, IgGs and passive protection in mice

Plant viruses show significant potential as expression vectors for producing foreign proteins in plants. In this study, codon-optimized VP6 gene (sVP6) of human rotavirus was engineered as a replacement to the coat protein (CP) open reading frame of Beet black scorch virus (BBSV). In vitro-generated RNA transcripts corresponding to the engineered virus were infectious when inoculated onto the leaves of Chenopodium amaranticolor. Molecular analysis revealed that sVP6 was efficiently expressed and accounted for 0.25% of the total soluble protein (TSP) in plant leaves on 7 dpi. On average, a high level 1.54 μg of sVP6 was expressed in each gram of infected leaves. Oral immunization of female BALB/c mice with the plant-based sVP6 protein induced high titers of anti-VP6 mucosal IgA and serum IgG. 60% of suckling pups born from immunized dams were protected against the virulent rotavirus challenge and those infected pups developed less severe diarrhea. These results suggested that it is feasible to induce lactogenic immunity against an enteric pathogen through oral vaccination, by using the antigen produced in a new BBSV-based plant protein expression system.