Passive Mouse Protection Research Articles

Characterization of a new protective antigen of Streptococcus canis

Streptococcus canis (S. canis), a lancefield group G streptococcus, is an opportunistic pathogen mainly found in dogs and cats. The study on pathogenesis and protective immune mechanism of S. canis is not clear. A new streptococcal protective antigen (SPA) was first identified from a genomic library of S. canis. SPA of S. canis (SPASc) contained a 1224-bp open reading frame which encoded a 407aa protein and a 34-aa signal sequence with a deduced molecular mass of 46.368 kDa. Protein analysis and BLAST result showed that SPASc was homologous to the SPA of Streptococcus. equi subsp. zooepidemicus, M protein Streptococcus. equi., and SPA of Streptococcus pyogenes. The protective response of SPASc antiserum was demonstrated by passive mouse protection. These studies suggested that SPASc might be an important component of vaccines to prevent S. canis infections.

The Fine Structure of Neisseria meningitidis Lipooligosaccharide from the M986 Strain and Three of Its Variants

Neisseria meningitidis is a cause of fatal sepsis and epidemic meningitis. A major virulence factor is cell wall lipooligosaccharide (LOS). The M986 strain has been used extensively in immunological and vaccine research. Yet, the LOS repertoire of this strain is not known. Here we have investigated the LOS structures of M986 and three of its variants OP1, OP2-, and OP2+. This strain and its variants present a series of related LOS families that are increasingly truncated in their listed order. The major structural differences are seen in the lacto-N-neotetraose alpha-chain. The gamma-chain Hep II contains two phosphoethanolamine (PEA) substitutions at C3 and C6/7. These substitutions were seen in all strains except OP2+ where the canonical core Hep II is missing. The PEA disubstitution was present in nearly stoichiometric amounts with only minor amounts of monosubstitution observed, and no glycomers devoid of PEA were seen. This was also the case in LOS with a complete lacto-N-neotetraosyl alpha-chain even though previous reports suggested that the presence of an extended alpha-chain hinders C3 PEA substitution of Hep II. Approximately 50% of gamma-chain GlcNAc was present in its 3-OAc-substituted form. Because Hep II C3 PEA substitution and gamma-chain GlcNAc OAc addition have been reported to negatively interact, the co-existence of these two modifications in these strains is unique. The LOS structures of M986 and three of its variants have been determined, which better defines these strains as tools for immunological and vaccine research.

Intranasal IFNgamma extends passive IgA antibody protection of mice against Mycobacterium tuberculosis lung infection.

Intranasal inoculation of mice with monoclonal IgA against the alpha-crystallin (acr1) antigen can diminish the tuberculous infection in the lungs. As this effect has been observed only over a short-term, we investigated if it could be extended by inoculation of IFNgamma 3 days before infection, and further co-inoculations with IgA, at 2 h before and 2 and 7 days after aerosol infection with Mycobacterium tuberculosis H37Rv. This treatment reduced the lung infection at 4 weeks more than either IgA or IFNgamma alone (i.e. 17-fold, from 4.2 x 10(7) to 2.5 x 10(6) CFU, P = 0.006), accompanied also by lower granulomatous infiltration of the lungs. IFNgamma added prior to infection of mouse peritoneal macrophages with IgA-opsonized bacilli resulted in a synergistic increase of nitric oxide and TNFalpha production and a 2-3 fold decrease in bacterial counts. Our improved results suggest, that combined treatment with IFNgamma and IgA could be developed towards prophylactic treatment of AIDS patients, or as an adjunct to chemotherapy.

Safety, efficacy and cross-protectivity of a live intranasal aerosol haemorrhagic septicaemia vaccine

The safety, efficacy and cross-protectivity of a live intranasal aerosol haemorrhagic septicaemia vaccine containing Pasteurella multocida serotype B:3,4 were tested in young cattle and buffaloes in Myanmar, where more than...

Study on Immunity of an Experimental Oil Adjuvant Haemorrhagic Septicaemia Vaccine in Cattle.

An oil adjuvant vaccine (OAV) was prepared from a local strain of Pasteurella multocida. Strain 6:B was cultured and inactivated by formalin. Bacterial pellet was prepared by centrifugation and subsequently adjuvanted by Montannide oil ISA-70. A dose of prepared vaccine containing 3ml (2mg dry weight/ml) was injected into five calves by IM route. Animals were bled before and at 24, 90, 150, and 200 days post-vaccination. Collected sera were used in passive mouse protection test (PMPT). Active mouse protection test (AMPT) was carried out for OAV according to standard method. Results of PMPT showed 100% protection up to 150 days and 66-83% up to 200 days post-vaccination. In AMPT, 4 log of protection was gained. In this experiment the immunity induced by OAV adjuvanted by ISA-70 could protect the calves.

Production of monoclonal antibody to subtype 9 of Neisseria meningitidis and the distribution of this subtype in Brazil

A new monoclonal antibody (5F81A4P1.9), which is specific for subtype 9 antigen of meningococci, was studied. The antibodies were raised against a previously non-typable (NT) serogroup B strain from Brazilian patients and were found to react with the subtype antigen of prototype reference strains for subtype 9 (M982), as well as with those of homologous strains. The subtype 9 epitope was found in 6.8% of serogroup B strains among 602 strains of Neisseria meningitidis case isolates, including representative isolates from Brazilian states. Subtype P1.9 was predominantly related to serogroup B in Brazil among the isolates collected during the N. meningitidis epidemic in 1992. No significant differences were observed in the occurrence of subtype P1.9 among strains isolated from several Brazilian states. Fluorescence-activated cell-sorter analysis showed that 5F81A4 MAb recognized a 46 kDa protein on the surface of a homologous strain of N. meningitidis (B:4:P1.9). These results, in association with a bactericidal activity assay for 5F81A4, and with experimental passive protection in mice, demonstrated the importance of subtype 9 class 1 proteins of N meningitidis in Brazil. Serotyping is essential for the development of vaccination strategies.

Staphylococcus aureus types 5 and 8 capsular polysaccharide-protein conjugate vaccines

Background Staphylococcus aureus, the first or second most common pathogen isolated from patients, is capsulated; there are at least 12 capsular types, and types 5 and 8 comprise approximately 85% of blood. Types 5 and 8, composed of a trisaccharide repeat unit including a mannose uronic acid and 2 fucoses, are non-immunogenic. As protein conjugates, they induce opsonophagoctyic antibodies that confer type-specific active and passive protection in mice. Methods A phase II study of patients with end-stage renal disease showed that these conjugates induced approximately one third of the immunoglobulin G antibody of healthy individuals. Increasing the dose to 100 μg of polysaccharide induced levels similar to that in healthy individuals injected with 25 μg. Results In a double-blinded randomized and controlled study of patients undergoing renal dialysis, the conjugates induced statistically significant protection against bacteremia for as long as 10 months after immunization. The estimated protective level was 80 μg Ab/mL. At re-injection approximately 2 years later, 83 of 83 recipients responded with protective levels. Conclusions Conjugate vaccine-induced antibodies to the types 5 and 8 capsular polysaccharide antibodies of S aureus prevent bacteremia caused by this pathogen. The extent and duration of conjugate-induced immunity can be extended by re-immunization approximately 1 year later. Studies of patients undergoing cardiovascular surgery who would be immunized with the staphylococcus conjugates when they are immunologically intact are planned.

Passive Transfer of Serum Antibodies Induced by BBG2Na, a Subunit Vaccine, in the Elderly Protects SCID Mouse Lungs Against Respiratory Syncytial Virus Challenge

Respiratory syncytial virus (RSV) is responsible for severe low respiratory tract infections in young infants and the elderly. To investigate whether BBG2Na, a recombinant subunit vaccine comprising aa 130–230 of the RSV G protein, induced protective Abs in subjects over 60 years during phase II clinical trial, pre- and postimmunization sera of individuals immunized with BBG2Na or placebo were transferred into SCID mice before RSV challenge. These sera dose-dependently reduced lung RSV titers. However at some points of serial dilutions, postimmunization sera of BBG2Na-immunized subjects only were significantly more efficient than the corresponding preimmunization sera, in agreement with the induction of an increased Ab response against multiple epitopes on RSV-A G protein. Thus, BBG2Na is immunogenic in the elderly and confers passive protection in mice after serum transfer. To our knowledge, this is the first description of protective Abs induced by a subunit vaccine in human.

Polyspecific malaria antibodies present at the time of infection inhibit the development of immunity to malaria but antibodies specific for the malaria merozoite surface protein, MSP1, facilitate immunity

Serum taken from mice immune to malaria as a result of infection and drug cure, or from mice immunized with a recombinant form of the merozoite surface protein, MSP1, can provide passive protection of recipient mice against the lethal parasite, Plasmodium yoelii YM. However, recipients of MSP1-immune serum go on to develop long-term immunity, whereas recipients of serum from mice naturally immune to malaria rapidly lose their resistance to infection. We demonstrate that 'infection/cure' serum suppresses the development of both antibody and cell-mediated parasite-specific responses in recipients, whereas these develop in recipients of MSP1-specific antibodies. These data have profound implications for our understanding of the development of malaria immunity in babies who passively acquire antibodies from their mothers.

Development of a Monoclonal Antibody Specific to a Recombinant Envelope Protein from Dengue Virus Type 4 Expressed inPichia pastoris

A mouse monoclonal antibody (MAb, 4B6) was able to recognize dengue virus type 4 envelope (E) protein both as a recombinant protein in Pichia pastoris and when it was present in infected brains of suckling mice. 4B6 was characterized by enzyme-linked immunoadsorbent assay (ELISA), hemaglutination inhibition, neutralization, and immunoblot. The MAb was isotyped as IgG2a. It was serotype 4 specific and it inhibited hemaglutination and neutralized homologous virus. It did not enhance infection of P338D1 cells by dengue type 4 virus strain H-241 strain. This MAb was reactive with recombinant E protein and dengue 4 virus, as revealed by Western blot. In vivo, MAb 4B6 conferred passive protection in mice challenged with homologous virus. Currently, this MAb is being used to purify recombinant E protein for further studies.

Production and characterization of a new monoclonal antibody against Neisseria meningitidis: study of the cross-reactivity with different bacterial genera.

We have generated a hybridoma cell line which produces an 8C7Br1 clone of the IgM antibody isotype. It recognizes the 50-, 65-, and 60-kDa antigens and is reactive with strains of N. meningitidis in the 98% of local Neisseria genera by Dot-ELISA assays. Two percent of the strains of N. meningitidis B do not present reactivity with the 8C7Br1 monoclonal antibody (MAb). The antibody reacted against N. meningitidis of serogroups A, B, C, X, Y, Z, and different serotypes and subtypes of N. meningitidis B and C by means of Dot-ELISA and Immunoblot. It cross-reacted with Neisseria gonorrhoeae, Neisseria lactamica, Haemophilus influenzae type b, Escherichia coli, Salmonella typhimurium, Salmonella typhi, Shigella flexneri, Bordetella pertussis, and Bacillus subtilis. The 8C7Br1 MAb reacted with the 65-kDa protein present in the prototype meningococcal strains B:16:B6(B2a:P1.5.2) and 2996 (B2b:P1.5.2). In H. influenzae type b, E. coli and B. subtilis, the MAb recognized the protein of 60, 65, and 70 kDa, respectively. FACS analysis showed that 8C7Brl MAb could recognize the 50-kDa protein on the surface of N. meningitidis homologous (B:4:P1.9) strain. These results, together with the bactericidal activity of 8C7Br1, and an experiment of passive protection in mice, demonstrated the potential importance of the cross-reactive protein as a candidate antigen for N. meningitidis B vaccine composition.

Reduction in functional antibody activity against Streptococcus pneumoniae in vaccinated elderly individuals highly correlates with decreased IgG antibody avidity.

The pneumococcal polysaccharide vaccine is recommended as a means of preventing invasive disease in the elderly. We compared responses to the 23-valent polysaccharide vaccine in 46 previously unvaccinated, healthy, institutionalized elderly persons (mean age, 85.5 years) with those in 12 healthy younger adults (mean age, 37 years) by measuring prevaccination and postvaccination serum IgG antibody concentrations (by ELISA), functional antibody activity (by opsonophagocytosis), IgG antibody avidity, and passive protection in mice. Postvaccination IgG antibody concentrations for two serotypes (6B and 19F) of the five studied (4, 6B, 14, 19F, and 23F) were significantly lower in elderly than in younger adults; however, opsonophagocytic activity was significantly reduced for all serotypes in the elderly. Sera with reduced opsonophagocytic activity (titer, <64) correlated with low IgG antibody avidity and protected mice poorly against pneumococcal challenge. In elderly persons receiving polysaccharide vaccination, there was a significant reduction in the functionality of postvaccination antibodies, and this appeared to increase with advanced age.

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100–1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay.

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100-1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.

New protective antigen of group A streptococci.

It is widely believed that the surface M protein of group A streptococci is the predominant surface protein of these organisms containing opsonic epitopes. In the present study, we identified a new surface protein, distinct from M protein, that evokes protective antibodies. A type 18 M-negative mutant was found to be both resistant to phagocytosis in human blood and virulent in mice. The wild-type strain, but not the M-negative mutant, was opsonized by antisera against purified recombinant M18 protein or a synthetic peptide copying the NH2-terminus of M18. However, antisera raised against a crude pepsin extract of the M-negative mutant opsonized both strains, indicating the presence of a protective antigen in addition to type 18 M protein. This antiserum was used to identify and purify a 24-kDa protein fragment (Spa, streptococcal protective antigen) that evoked antibodies that opsonized the M18 parent and the M-negative mutant. The results of passive mouse protection tests confirmed the presence of protective epitopes within Spa. The deduced amino acid sequence of a 636-bp 5' fragment of the spa18 gene showed no homology with sequences in GenBank. These studies reveal the presence of a new protective antigen of certain strains of group A streptococci that may prove to be an important component of vaccines to prevent streptococcal infections.

A recombinant single chain antibody neutralizes coronavirus infectivity but only slightly delays lethal infection of mice.

The variable region genes of a murine anti‐coronavirus monoclonal antibody (mAb) were joined by assembly polymerase chain reaction and expressed in Escherichia coli in a single chain variable fragment (scFv) configuration. After induction of expression, the expected 32‐kDa protein was identified by Western immunoblotting with specific rabbit anti‐idiotype antibodies. The scFv fragments were purified from soluble cytoplasmic preparations by affinity chromatography on nickel agarose, which was possible with an N‐terminal but not with a C‐terminal histidine tag. Purified scFv fragments retained the antigen‐binding properties of the parental antibody, could inhibit its binding to viral antigens with apparently higher efficiency than monovalent antigen‐binding (Fab) fragments, but neutralized viral infectivity with lower efficiency (about sevenfold at a molar level). To evaluate the usefulness of these smaller and less immunogenic molecules in the treatment of viral diseases, mice were treated with purified recombinant scFv fragments and challenged with a lethal viral dose. A small delay in mortality was observed for the scFv‐treated animals. Therefore, even though the scFv could neutralize viral infectivity in vitro, the same quantity of fragments that partially protected mice in the form of Fab only slightly delayed virus‐induced lethality when injected as scFv fragments, probably because of a much faster in vivo clearance: the biologic half‐life was estimated to be about 6 min. Since a scFv derived from a highly neutralizing and protective mAb is only marginally effective in the passive protection of mice from lethal viral infection, the use of such reagents for viral immunotherapy will require strategies to overcome stability limitations.

Immunogenicity of outer membrane protein of Pasteurella multocida in buffalo calves

Outer membrane protein (OMP) from Pasteurella multocida serotype B:2 was extracted and characterized using SDS-PAGE. Ten major polypeptide bands of MW 88 to 25 kDa were observed. Immunoblotting suggested that the polypeptides of MW 44, 37 and 30 kDa were the major immunogens. Buffalo calves vaccinated with the OMP vaccine or a commercial haemorrhagic septicaemia oil adjuvant vaccine developed highest mean log 10 ELISA titres day 21 postvaccination (pv). Antibody titres detectable in these animals using an indirect haemagglutination assay were lower than the ELISA titres but the pattern of the antibody response was similar. A passive mouse protection assay revealed that the maximum protection against the challenge infection was conferred by the serum collected from both the groups on day 21 pv and 26 pv. Following challenge with virulent P. multocida cells, all the five OMP vaccinated animals survived whereas only 2 out of the 3 HS oil adjuvant vaccinated animals withstood the challenge. Results suggested that OMP was protective and could be used in vaccines against haemorrhagic septicaemia.

Experimental immunization against Lyme borreliosis with recombinant Osp proteins: an overview.

Interest in human and veterinary vaccines against Lyme borreliosis is growing. Both whole cell immunization and subunit vaccines can protect against infection with Borrelia burgdorferi. For development of a human vaccine the focus has been on a subunit vaccine. The most promising candidate is OspA, a major outer membrane lipoprotein of B. burgdorferi sensu lato. Of Osp proteins A through D, OspA shows the least variability between strains in its sequence and in the level of its expression. Borreliae in ticks express OspA. Antibodies to OspA kill borreliae in vitro and provide passive protection in mice. Active immunization of mice with OspA provides protection against challenge by syringe inoculation or tick bite. The lipid moiety of the OspA is necessary for immunogenicity in the absence of a potent adjuvant. A recombinant OspA-based vaccine is already in clinical trials. Although there is compelling evidence that immunization with OspA will provide protection, questions remain regarding the duration of protection from such immunization, the necessity to have a minimum level of neutralizing antibodies at all times for protection, and the relationship of an immune response to OspA and autoimmune features of Lyme borreliosis. The experimental aspects of immunization with Osp-A based constructs and other Lyme vaccine candidates are reviewed and discussed.

Immunity and vaccine development in Pasteurella multocida infections

The role of LPS in immunity was studied using monoclonal antibodies (MAbs) and active immunisation experiments. A panel of six MAbs produced against Pasteurella multocida serotype B:2 reacted with the LPS of serotypes B:2 and B:5, but not with other serotypes. The MAbs could opsonise P. multocida for phagocytosis by mouse macrophages, but were not bactericidal in the presence of complement. They conferred only partial passive protection in mice. Similar results showing only partial protection were obtained when purified LPS was used to actively immunise mice prior to challenge, suggesting that LPS plays a partial role in immunity to infection. The aroA gene from P. multocida serotypes A:1 and A:3 was cloned and inactivated by insertion of a kanamycin resistance gene. The mutated gene was re-introduced onto the chromosome by allelic exchange. The resultant aroA mutants were highly attenuated in a mouse model system, with a 6-log decrease in ID 50. Virulence could be restored by complementation with a functional aroA gene. Mice immunised with two doses of the live mutants were protected against lethal challenge with the homologous parental strain, but not against the heterologous strain. P. multocida A:l and A:3 expressed unique proteins when grown in iron-restricted medium. Moreover, the outer membrane (OM) fractions of these cells contained novel proteins of 75 kDa, 85 kDa and 94 kDa molecular mass. Mice were immunised with OM fractions prepared from serotype A:3 grown in iron-restricted (OM Fe −) or iron-replete (OM Fe +) media. When low challenge doses were used, both immunogens protected mice against serotype A:3, but only the OM Fe − fraction protected mice against heterologous challenge with serotype A:l. When higher challenge doses were used, only partial protection was observed.

Protection from lethal coronavirus infection by immunoglobulin fragments.

Molecular mechanisms of in vitro and in vivo virus neutralization by specific Ab remain largely undefined. Murine coronaviruses provide an excellent animal model for such studies. To determine the role of Ab bivalency and the contribution of its Fc portion in the neutralization of viral infectivity and passive protection of mice by an in vitro neutralizing and in vivo protective mAb (7-10A), F(ab')2 and Fab fragments were generated and their biologic properties were examined. The two fragments reacted in ELISA like the whole Ab against viral Ag or specific anti-idiotypic Abs. The affinity constants of the different Ab preparations were determined by surface plasmon resonance using immobilized anti-idiotypic Abs. The apparent affinity constant of the whole Ab molecule was 7.0 x 10(9) M-1 and was reduced 2-fold for F(ab')2 fragments and 14-fold for Fab molecules. Like whole Ab, both F(ab')2 and Fab fragments could neutralize virus in vitro and passively protect mice in vivo. However, the efficiency of in vivo neutralization by Fab fragments was reduced compared with the bivalent molecules, despite almost identical half-lives of both types of Ab fragments. These results demonstrate that in vitro and in vivo virus neutralization mechanisms by this Ab are independent of Fc-mediated functions and bivalency, but are probably influenced by Ab avidity. Also, this is the first report of in vivo protection against a viral infection by Fab fragments of antiviral Ab.