Antibody Response In Rabbits Research Articles

Immunomodulatory effects of C3bgp on the antibody response to hemocyanin in outbred rabbits and the F1 generation of breeding with siblings

Dynamics and quantitative analyses of monospecific antibody during the primary and secondary humoral responses were determined in outbred rabbits and in the F1 generation of breeding with siblings. The antibody response in rabbits immunized with Keyhole Limpet Hemocyanin (KLH) was studied during a 4-month immunization period. ELISA determination of anti-KLH Ig and anti-KLH IgG alone, in preimmune and immune rabbit sera, was performed. Antibody response in both groups of rabbits was similar when assessed by anti-rabbit Ig but displayed differences when assessed by anti-rabbit IgG. A statistically significant increase in anti-KLH IgG was observed in the F1 inbred rabbits compared to the control group after primary immunization from days 14 to 35. Immunomodulation also elicited differences in the antibody response in the two groups of animals. C3-binding glycoprotein isolated from Cuscuta europea (C3bgp), applied simultaneously with antigen (KLH), produced a much stronger secondary immune response than the antigen alone, in both experimental groups. The enhancement of anti-KLH Ig in C3bgp-treated inbred rabbits was statistically significant in comparison with nontreated inbred rabbits. A significant increase in anti-KLH IgG was observed only for the inbred group after treatment with C3bgp. The results demonstrate that the F1 generation of breeding with sibling leads to significant differences in antibody responses to immunization compared with outbred rabbits, as well as to immunomodulation with C3bgp.

556. Toward the Development of Electroporation for Delivery of DNA Vaccines to Humans

The objectives of this study were: 1) to evaluate the potency of plasmid DNA vaccines encoding two different HIV antigens in Rhesus macaques when delivered intramuscularly with electroporation (EP), and 2) to develop EP electrodes and pulse conditions suitable for human clinical applications. Two groups of monkeys (n=5) were immunized with DNA vaccines encoding HIV Gag and an oligomeric form of HIV Env by three subsequent i.m. injections at weeks 0, 4, and 14. In one group (EP group), injection was followed by six EP pulses delivered with a 6-needle array electrode at a nominal field strength of 233V/cm (Widera et al., J. Immunol. 164, 4635–40, 2000). All animals received a boost with rSF2 Gag protein (week 29) and rSF162 Env protein (weeks 38 and 75). During the DNA priming period (up to week 29) the EP group showed faster onset, higher peak titers (>100-fold for Gag) and longer duration of the antibody response than the non-EP group. Protein boosts restored (Gag) or enhanced (Env) peak Ab titers obtained after the third DNA immunization. Cell-mediated responses were also consistently stronger with EP. Helper T cell responses were measured by a lymphoproliferation assay (LPA) and intracellular cytokine staining (ICS). Anti-Gag LPA responses in animals given EP produced stable stimulation indices (SIs) of 12–19 throughout the DNA priming period, compared with a maximum SI of 5 without EP. For Env the corresponding SIs were 8 –10 vs. a maximum of 4. Also, IFN-gamma production in CD4 T cells, as measured by ICS, was 5 to 9-fold higher in the EP group during the entire DNA priming period for Gag, compared with a 2 to 5-fold increase for Env. Cytolytic T cell activity (CTL response) was measured after in vitro culture of PBMC. EP treatment modestly increased the response rate to anti-Gag CTL (20 vs. 13 cumulative responses). However, anti-Env CTLs were markedly enhanced by EP (16 vs. 2 cumulative responses), with 3 out of 5 animals consistently responding throughout the DNA priming period, vs. only one animal responding transiently in the non-EP group. These data demonstrate that in vivo EP is effective at increasing the potency of DNA vaccines against two different HIV antigens in Rhesus by triggering faster, stronger and longer antibody, helper T cell and CTL responses. Naked DNA injection, although effective in rodents, is known to be ineffective in non-human primates. Since EP is a physical delivery system that consistently results in significantly increased immune responses in a spectrum of species from mice to monkeys, it is reasonable to hypothesize that EP could enable effective DNA vaccination in humans as well. In order to make EP more patient-friendly, we developed EP instrumentation and pulse conditions more compatible with routine clinical applications than those used for the treatments described above. We reduced the number of needle electrodes from 6 to 4, reduced electrode toxicity, and limited the number of pulses to two. Under these clinically superior conditions, gene expression levels and antibody responses in rabbits were unaffected. This encourages further non-human primate studies in preparation for clinical trials.

Antibodies to malaria peptide mimics inhibit Plasmodium falciparum invasion of erythrocytes.

Apical membrane antigen 1 (AMA1) is expressed on the surfaces of Plasmodium falciparum merozoites and is thought to play an important role in the invasion of erythrocytes by malaria parasites. To select for peptides that mimic conformational B-cell epitopes on AMA1, we screened a phage display library of >10(8) individual peptides for peptides bound by a monoclonal anti-AMA1 antibody, 4G2dc1, known to inhibit P. falciparum invasion of erythrocytes. The most reactive peptides, J1, J3, and J7, elicited antibody responses in rabbits that recognized the peptide immunogen and both recombinant and parasite AMA1. Human antibodies in plasma samples from individuals exposed to chronic malaria reacted with J1 and J7 peptides and were isolated using immobilized peptide immunoadsorbents. Both rabbit and human antibodies specific for J1 and J7 peptides were able to inhibit the invasion of erythrocytes by P. falciparum merozoites. This is the first example of phage-derived peptides that mimic an important epitope of a blood-stage malaria vaccine candidate, inducing and isolating functional protective antibodies. Our data support the use of J1 and J7 peptide mimics as in vitro correlates of protective immunity in future AMA1 vaccine trials.

Candida albicans cell wall antigens for serological diagnosis of candidemia.

Serological tests for diagnosis of disseminated fungal infections in the immunocompromised host are used with varying results. In the present study, the relative ability of antibodies to specifically recognize Candida albicans cell wall components was evaluated in order to find antigenic markers for serological diagnosis of candidemia. Native C. albicans cell wall fragments (CW), periodate- (CWIO4) and proteinase-K- (CWP) treated CW, a mildly extracted phosphopeptidomannan (PPM), and beta(1-3)(1-6)-glucan were used as antigens in ELISA with sera from rabbits immunized with C. albicans (n = 10), patients with culture proven candidemia (n = 8) and healthy individuals (n = 8). The antibody response in rabbits consisted predominantly of anti-PPM antibodies, a finding that was substantiated by inhibition-ELISA. Consistently, periodate treatment (CW104) destroyed a major proportion of the antigenic epitopes. Low rabbit antibody levels were found against glucan, the major Candida cell wall component. These results supported the conclusion that glucan is localized mainly in the inner part of the C. albicans cell wall. In contrast to rabbits' serum IgG antibody response against PPM, which was at least tenfold higher than that raised against CW, patients with candidemia had similar IgG antibody levels against both antigens. These levels were significantly higher than those seen in healthy controls (CW, P = 0.0005 and PPM, P < 0.0001). Although the human anti-glucan and anti-CWIO4 IgG antibody levels were low overall, they were nonetheless significantly increased in the patient group (P = 0.0159 for antiglucan and P = 0.0491 for anti-CWIO4). In addition, a correlation was noticed between levels of these antibodies. No significant differences were found between patients and controls for IgM antibodies when CW, CWIO4, PPM and Glu were used as antigens. In conclusion, IgG antibodies to PPM and native cell wall fragments (CW) were highly discriminatory for recognition of candidemia and these antigens are thus promising candidates for use in serodiagnosis.

Chimeric bacteriophage fr virus-like particles harboring the immunodominant C-terminal region of hamster polyomavirus VP1 induce a strong VP1-specific antibody response in rabbits and mice.

The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural proteins VP1, VP2, and VP3, where VP1 represents the major capsid protein of 384 amino acids. Screening of sera from HaPyV-infected papilloma-bearing and papilloma-free hamsters demonstrated the immunodominant features of all three capsid proteins. For both groups of hamsters in the C-terminal region of VP1 immunodominant B-cell epitopes were identified in the regions between amino acids 305 and 351 and amino acids 351 and 384. The high flexibility of the C-terminal region of VP1 was confirmed by the formation of chimeric virus-like particles based on the coat protein of the RNA bacteriophage fr which was previously found to tolerate only very short-sized foreign insertions. Phage fr coat protein-derived virus-like particles tolerated the N-terminal fusion of amino acids 333-384, 351-384, 351-374, and 364-384, respectively, of VP1. The induction of VP1-specific antibodies in rabbits and mice by immunization with chimeric virus-like particles harboring amino acids 333-384, 351-384, and 364-384, respectively, of VP1 suggested the immunodominant nature of the C-terminal region of VP1.

The stoichiometry of trimeric SIV glycoprotein interaction with CD4 differs from that of anti-envelope antibody Fab fragments.

Human and simian immunodeficiency viruses infect host lymphoid cells by binding CD4 molecules via their gp160 envelope glycoproteins. Biochemical studies on recombinant SIVmac32H (pJ5) envelope ectodomain gp140 precursor protein show that the envelope is a trimer. Using size exclusion chromatography, quantitative amino acid analysis, analytical ultracentrifugation, and CD4-based competition assay, we demonstrate that the stoichiometry of CD4 receptor-oligomeric envelope interaction is 1:1. By contrast, Fab fragments of both neutralizing and non-neutralizing monoclonal antibodies bind at a 3:1 ratio. Thus, despite displaying equivalent CD4 binding sites on each of the three gp140 protomers within an uncleaved trimer, only one site binds the soluble 4-domain human CD4 extracellular segment. The anti-cooperativity and the faster k(off) of gp140 trimer:CD4 versus gp120 monomer:CD4 interaction suggest that CD4-induced conformational change is impeded in the intact envelope. The implications of these findings for immunity against human immunodeficiency virus and simian immunodeficiency virus are discussed.

Towards development of an edible vaccine against bovine pneumonic pasteurellosis using transgenic white clover expressing a Mannheimia haemolytica A1 leukotoxin 50 fusion protein.

Development of vaccines against bovine pneumonia pasteurellosis, or shipping fever, has focused mainly on Mannheimia haemolytica A1 leukotoxin (Lkt). In this study, the feasibility of expressing Lkt in a forage plant for use as an edible vaccine was investigated. Derivatives of the M. haemolytica Lkt in which the hydrophobic transmembrane domains were removed were made. Lkt66 retained its immunogenicity and was capable of eliciting an antibody response in rabbits that recognized and neutralized authentic Lkt. Genes encoding a shorter Lkt derivative, Lkt50, fused to a modified green fluorescent protein (mGFP5), were constructed for plant transformation. Constructs were screened by Western immunoblot analysis for their ability to express the fusion protein after agroinfiltration in tobacco. The fusion construct pBlkt50-mgfp5, which employs the cauliflower mosaic virus 35S promoter for transcription, was selected and introduced into white clover by Agrobacterium tumefaciens-mediated transformation. Transgenic lines of white clover were recovered, and expression of Lkt50-GFP was monitored and confirmed by laser confocal microscopy and Western immunoblot analysis. Lkt50-GFP was found to be stable in clover tissue after drying of the plant material at room temperature for 4 days. An extract containing Lkt50-GFP from white clover was able to induce an immune response in rabbits (via injection), and rabbit antisera recognized and neutralized authentic Lkt. This is the first demonstration of the expression of an M. haemolytica antigen in plants and paves the way for the development of transgenic plants expressing M. haemolytica antigens as an edible vaccine against bovine pneumonic pasteurellosis.

Degree of antigen adsorption in the vaccine or interstitial fluid and its effect on the antibody response in rabbits

The effect of the degree of adsorption of lysozyme by aluminium hydroxide adjuvant on the immune response in rabbits was studied. The surface charge of the adjuvant was modified by pretreatment with phosphate anion to produce five vaccines having degrees of adsorption ranging from 3 to 90%. The degree of adsorption of vaccines exhibiting 3, 35 or 85% adsorption changed to 40% within 1 h after each vaccine was mixed with sheep interstitial fluid to simulate subcutaneous administration. The mean anti-lysozyme antibody titers produced by the vaccines were the same and were four times greater than that produced by a lysozyme solution. Thus, the degree of adsorption of lysozyme in sheep interstitial fluid rather than the degree of adsorption in the vaccine correlated with the immune response.

Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140.

The biologically active form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric. We previously described a soluble HIV-1 IIIB Env protein, gp140, with a stable oligomeric structure composed of uncleaved gp120 linked to the ectodomain of gp41 (P. L. Earl, C. C. Broder, D. Long, S. A. Lee, J. Peterson, S. Chakrabarti, R. W. Doms, and B. Moss, J. Virol. 68:3015-3026, 1994). Here we compared the antibody responses of rabbits to gp120 and gp140 that had been produced and purified in an identical manner. The gp140 antisera exhibited enhanced cross-reactivity with heterologous Env proteins as well as greater neutralization of HIV-1 compared to the gp120 antisera. To examine both immunogenicity and protective efficacy, we immunized rhesus macaques with oligomeric gp140. Strong neutralizing antibodies against a homologous virus and modest neutralization of heterologous laboratory-adapted isolates were elicited. No neutralization of primary isolates was observed. However, a substantial fraction of the neutralizing activity could not be blocked by a V3 loop peptide. After intravenous challenge with simian-HIV virus SHIV-HXB2, three of the four vaccinated macaques exhibited no evidence of virus replication.

Characterization of protective epitopes in a highly conserved Plasmodium falciparum antigenic protein containing repeats of acidic and basic residues.

The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to approximately 90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum.

Antibody responses in rabbits after single immunization with viral glycoprotein-loaded microspheres or ISCOMs

Antigenic and adjuvant activities of microspheres and ISCOMs (immunity stimulating complexes) with incorporated envelope glycoproteins of bovine herpesvirus-1 (BHV-1) were tested in long-term (31-week) experiments in rabbits. Similar antibody responses were recorded after a single intradermal immunization with BHV-1 glycoproteins incorporated into ISCOM nanoparticles or microspheres prepared from linear co-polymers (50/50) of dl-lactic and glycolic acids. Stronger, longer and markedly biphasic antibody responses were observed in rabbits immunized with BHV-1 M- dl-LA microspheres prepared from branched oligoesters of dl-lactic acid and mannitol. Indirect evidence of the incorporation of the viral envelope proteins into any type of microspheres or ISCOMs was obtained by immunoblotting. A set of precipitation lines was detected in rabbit blood sera collected 7 weeks after the immunization with microsphere- or ISCOM-entrapped glycoproteins. A similar, but more numerous, set was found in the control serum collected from cattle after natural infection by BHV-1.

Chemical synthesis of Haemophilus influenzae glycopeptide conjugates.

A simple procedure for conjugating synthetic fragments of the capsular polysaccharide of Haemophilus influenzae type b, poly-3-beta-D-ribose-(1,1)-D-ribitol-5-phosphate (sPRP) to linear peptides is described. The procedure consists of (i) reacting the amino group of amino-heptyl sPRP with m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) in phosphate buffer, pH 7.5; (ii) selectively coupling the MBS-modified sPRP to the sulfhydryl group of the cysteine residue of peptides containing functional T-helper cell epitope(s). The glycopeptide conjugates were purified by gel filtration chromatography, biochemically characterized, and elicited protective level of anti-PRP antibody responses in rabbits.

Antibody response in rabbits to serotype-specific determinants in lipopolysaccharides from Moraxella catarrhalis.

Antibodies against the serotype determinant epitopes of Moraxella catarrhalis lipopolysaccharides (LPS) were demonstrated in sera from rabbits immunised with whole bacterial cells. Purified LPS preparations from eight strains of M. catarrhalis were used as antigens in enzyme-linked immunosorbent assays (ELISA) and immunoblotting. The serotype specificity of the antibodies was shown by neutralisation with LPS and with purified polysaccharide obtained from LPS prepared from strains belonging to different serotypes. In immunoblots, antisera against the A and B LPS serotypes reacted only with LPS of its own type, confirming the presence of type-specific antibodies. A weak band was observed with type A LPS and antibody to type C, indicating cross-reactivity between the A and C serotypes. This cross-reaction can be explained on the basis of the known chemical structure of the LPS of the different serotypes. After heterologous absorption of sera, bands were obtained with homologous LPS antigen. These results suggest that the predominant antibody response in rabbits to LPS from M. catarrhalis is serotype-specific, unlike that previously observed in infected human patients.

Longitudinal study of an epitope-biased serum haemagglutination-inhibition antibody response in rabbits immunized with type A influenza virions

This paper describes a longitudinal study of the antibody specificities generated to the haemagglutinin (HA), the major envelope glycoprotein of type A influenza virus particles, during primary and secondary antibody responses in rabbits. Two New Zealand White rabbits (no. 191, no. 192) and an English Half-Lop rabbit (no. 193) were immunized intravenously with β-propiolactone-inactivated virus at 0, 28 and 56 days. Haemagglutination-inhibition (HI) antibody specificities were measured using neutralizing antibody double escape mutants selected with monoclonal antibodies (mabs) specific for an epitope in antigenic site A, site B and site D. In this regard the epitope reactivity to these mabs was represented as A +B −D −, A −B +D − and A −B −D +, where “+” and “−” represent the non-mutated and mutated epitopes respectively. The HI response was well developed at 7 days after primary immunization and at this time the response was equally divided between all three epitopes. Two rabbits (no. 191 and no. 193), showed a bias to the site B epitope from 14 days onwards, such that about half of the total HI activity was to this epitope and the other half was made up of HI antibody to the other two epitopes in approximately equal proportions. In the other New Zealand White rabbit (no. 192) a non-biased response extended throughout the primary response and for one week after the second immunization. Apart from this, a bias to a single epitope was clearly evident in all rabbits after the second immunization, and this constituted up to 70% of the total HI antibody response. The antibody response did not broaden and remained essentially unchanged even after a third immunizing injection. The observed bias to the site B epitope during the secondary response of HA-specific antibody is in accord with a previous cross-sectional study of nine other rabbits.

Antibody production in rabbits and chickens immunized with human IgG A comparison of titre and avidity development in rabbit serum, chicken serum and egg yolk using three different adjuvants

The antibody response in rabbits and chickens (serum and egg yolk) immunized with human immunoglobulin G was studied during a 1-year immunisation period comparing the efficacy of three adjuvants: Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) and Hunter's TiterMax adjuvant (HTM). Monthly boosters of the antigen in saline were administered. Adjuvants were used in the initial immunisation and in a booster at week 26. FCA was found to result in the highest titre in both species. FIA was more efficient than HTM in mediating an antibody response in rabbits. In chickens, FIA resulted in similar antibody titres in serum and egg yolk. The effect of HTM was similar to FIA in chicken serum but lower in egg yolk. The titres obtained in rabbits were approximately a factor 1.5–2.0 higher than in the corresponding chicken groups. However, the antibody productivity of the chicken, when using the egg yolk as the antibody source, was a factor 5–10 higher than that of the rabbit depending on the adjuvant used. FCA and FIA resulted in similar antibody avidity in rabbits whereas HTM had a lesser effect. In chickens FCA initially resulted in the highest antibody avidity in both serum and egg yolk, but after the booster with adjuvant the antibody avidity became similar for all three adjuvants.

Peptides derived from the unique region of B19 parvovirus minor capsid protein elicitneutralizing antibodies in rabbits

B19 parvovirus is pathogenic in humans. The virus propagates in the bone marrow, where it is cytotoxic to erythroid progenitor cells. Antibodies appear in blood after infection and neutralize virus in vitro; infection appears to confer lasting immunity. The predominant immune response on immunoblot is to the minor capsid protein (VP1), which differs from the major capsid protein (VP2) by an additional 227 amino acids. We previously demonstrated that antisera directed to a fusion protein containing this unique region or to more limited fusion peptides of 50–100 amino acids each neutralized virus. In the current work, we tested synthetic peptides of about 20 amino acids derived from the VP1 unique region for their ability to elicit a neutralizing antibody response in rabbits. Individual peptides were covalently linked to a lysine core to produce a multivalent antigen. Animals produced antibodies to all 13 synthetic peptides, as determined by ELISA. At 12 weeks, animals injected with one of three peptides — two from the far amino terminus and the third from the center of the unique region — had produced antibodies that completely neutralized virus; by 16 weeks, antisera elicited with another four peptides also were effective. In summary, we identified regions containing neutralizing epitopes within the first 80 amino acids and amino acids 148–205 of the unique region. Our data suggest that synthetic peptides might be useful vaccine reagents for protection against parvovirus infection in humans.

Multibranched peptide constructs derived from the V3 loop of envelope glycoprotein gp120 inhibit human immunodeficiency virus type 1 infection through interaction with CD4

The V3 loop of the gp120 of human immunodeficiency virus type 1 (HIV-1) is assumed to be involved in HIV-1-mediated membrane fusion. V3-derived peptides have been shown either to enhance or to prevent HIV-1 infection. Multibranched peptide constructs (MBPCs) derived from the V3 North American/European consensus sequence were designed to sort out these conflicting findings. At 5 μM, MBPC1 (8-branched GPGRAF) totally, and MBPC2 ([RKSIHIGPGRAFYT]4) partially, inhibited HIV-1 LAI infection, whereas the GPGRAF monomer had only a limited effect. A peptide of the entire V3 consensus loop and a control MBPC had no detectable activity. The 5 μM MBPC1 HIV-1-inhibiting concentration was not cytotoxic, nor did it alter T lymphocyte allogeneic, antigen-, or mitogen-induced reactivities, and it was about 5- to 50-fold lower (MBPC2 and MBPC1, respectively) than that resulting in 50% cell death. Analysis of MBPC immunoreactivity showed that MBPC2, but not MBPC1, strongly reacted with human HIV-1 positive sera. Only MBPC2 elicited significant antibody responses in rabbits. The V3-derived MBPCs bound to CD4 + cells, as determined by immunofluorescence analysis. The binding was inhibited either by soluble CD4 or by CD4 monoclonal antibody (mAb) MT151, which recognizes the CDR3 region of the D1 domain of CD4, but not by other CD4 mAbs Leu3a, OKT4A, Q4021, 13B8-2, 5A8, RFT4, nor by the CD26 mAb BA5. Therefore, it appears likely that MBPCs inhibit HIV-1 infection by interacting with the CDR3 region of CD4 or with a region in its vicinity.

Evaluation of the immunogenicity of polymerized hemoglobin solutions in a rabbit model.

Chemical modification of proteins carries the risk that neo-antigens are introduced. To investigate the potential immunogenicity of human glutaraldehyde-polymerized hemoglobin (polyHbX1), we analyzed the antibody responses of rabbits after hyperimmunization with complete Freund's adjuvant. In view of the species difference, we also tested rabbit hemoglobin that was modified in the same way as human polyHbX1. Thereafter, we studied the antibody response after weekly intravenous infusion of clinically relevant doses of polyHbX1 to evaluate whether an immune response is likely to occur when modified hemoglobin is used as blood substitute. The occurrence of an antibody response was tested by using an enzyme immunoassay (ELISA). To find out whether antibodies were directed against neo-epitopes on human polyHbX1 we used a competitive ELISA. The results showed that polymerized hemoglobin may weakly activate the immune system in special conditions, but is unlikely to do so when it is used as blood substitute.

Induction of antibody responses that neutralize human T-cell leukemia virus type I infection in vitro and in vivo by peptide immunization.

In order to define neutralization regions on the envelope antigen of human T-cell leukemia virus type I (HTLV-I), we have generated a number of new anti-envelope gp46 monoclonal antibodies from rats and mice. Epitopes recognized by new monoclonal antibodies which could neutralize HTLV-I in syncytium and transformation inhibition assays were localized to sequences in gp46 from amino acids 186 to 193, 190 to 195, 191 to 195, 191 to 196, and 194 to 199. Ovalbumin-conjugated synthetic gp46 peptides containing these neutralization epitopes, pep190-199 (a synthetic gp46 peptide containing amino acids 190 to 199) and pep180-204, but not pep185-194 or pep194-203, could give rise to HTLV-I-neutralizing antibody responses in rabbits. These immune or nonimmune rabbits were then challenged with HTLV-I by intravenous inoculation with 5 x 10(7) live HTLV-I-producing ILT-8M2 cells. By a PCR assay, it was revealed that HTLV-I provirus was detected in peripheral blood lymphocytes from nonimmune and pep288-312-immunized rabbits, whereas the provirus was not detected in peripheral blood lymphocytes from pep190-199- and pep180-204-immunized rabbits over an extended period. These results suggest that the induction of anti-gp46 neutralizing antibody responses by immunization with synthetic peptides has the potential to protect animals against HTLV-I infection in vivo.

BvgAS-mediated signal transduction: analysis of phase-locked regulatory mutants of Bordetella bronchiseptica in a rabbit model

Members of the Bordetella genus alternate between two distinct phenotypic phases in response to changes in their environment. This switch, termed phenotypic modulation, is mediated by the BvgAS sensory transduction system. We developed an animal model based on the interaction of Bordetella bronchiseptica with one of its natural hosts, the rabbit. To investigate the importance of BvgAS signal transduction, we constructed constitutive (RB53) and Bvg- (RB54) phase-locked derivatives of a wild-type strain, RB50. RB50 and RB53, but not RB54, established respiratory infections in B. bronchiseptica-free rabbits with an intranasal 50% infective dose of less than 200 organisms, and the course of the infection closely resembled that observed with naturally infected rabbits. Bacteria were recovered from the nasal cavity, larynx, trachea, and lungs in similar numbers from RB50- and RB53-infected rabbits, yet no pathology was detected by histological examination of lung and tracheal sections. The antibody responses in rabbits inoculated with RB50 or RB53 were quantitatively and qualitatively indistinguishable; high titers of antibodies were generated primarily against Bvg(+)-phase-specific antigens. No response against flagella, a Bvg- phase factor, was detected. Assessment of bacteria associated with alveolar macrophages indicated that only a small percentage of bacteria, if any, appear to be residing within lung macrophages. We also tested the ability of these strains to survive in a nutrient poor environment, conditions which may be encountered within certain niches in the host or in an environmental reservoir. The Bvg- phase was advantageous for growth under these conditions. Our results indicate the Bvg+ phase is sufficient for establishment of respiratory tract infection in the rabbit and the normal BvgAS-mediated response to environmental signals is not required during initial colonization. The Bvg- phase may play a role at later stages of infection, including persistence, transmission, or survival in the environment.