Identification of paramyxovirus-specific haemolysis-inhibiting antibodies separate from haemagglutinating-inhibiting and neuraminidase-inhibiting antibodies. 2. NDV and mumps virus haemolysis-inhibiting antibodies

Egg-grown Newcastle disease (NDV) and mumps virus were used for preparation of rabbit hyperimmune sera against purified whole virus and projectionless virus particles. These sera and convalescent sera after natural NDV and mumps infections in chickens and human subjects, respectively, were studied in haemolysis-inhibition (HLI), haemagglutination-inhibition (HI) and neuraminidase-inhibition (NI) tests both before and after absorption with Tween 80-ether (TE) treated virus preparations. In addition, neutralization tests using the different sera were carried out. HI and NI antibodies and the major population of neutralizing antibodies in convalescent sera were removed by absorption with TE treated virus material without changing the titre of non-HI HLI antibodies. Rabbit hyperimmune sera directed against projectionless virus particles exhibited HLI antibody titres in marked excess of HI and NI antibody titres, whereas this was not found in sera against whole virus. Absorption with TE treated virus material resulted in removal of all demonstrable antibody activities in sera against whole virus. The corresponding absorption of sera against projectionless particles eliminated HI antibodies without changing the titre of non-Hi HLI antibodies. In rabbit hyperimmune sera, HI antibodies were of primary importance in neutralization tests. After addition of anti-gamma globulin to the test, an efficient neutralization was observed if mumps non-HI HLI antibodies were used whereas this was not found if NDV non-HI HLI antibodies were used.     

Influenza virus neutralizing antibodies and IgG isotype profiles after immunization of mice with influenza A subunit vaccine using various adjuvants

The influence of various adjuvants on the development of influenza virus neutralizing antibodies and distribution of anti-influenza virus IgG isotypes after immunization of mice with influenza A (H3N2) subunit vaccine was investigated. Serum titres of influenza virus neutralizing antibodies and titres of influenza specific IgG isotypes were determined by a neutralization enzyme immunoassay (N-EIA) and a cell-associated antigen enzyme immunoassay (CA-EIA), respectively. Serum antibody titres as measured by the two tests correlated highly (r = 0.82; P < 0.001). N-EIA titres were enhanced by 38- and 34-fold, when L180.5/RaLPS and FCA, respectively, were administered with 1 microgram of vaccine. The adjuvants Q-VAC, L180.5 [W/O/W], L180.5 alone and Montanide ISA 740 were only moderately or not effective in enhancing the immune response to the 1 microgram dose of vaccine. The Q-VAC and L180.5/RaLPS adjuvants favoured IgG2a and IgG2b isotype responses to influenza compared to the other adjuvants. We suggest that N-EIA and CA-EIA may be valuable tools to monitor the effects of adjuvants on the neutralizing antibody and antibody isotype responses after influenza vaccination.     

Evaluation of an enzyme immunosorbent assay for the diagnosis of Argentine haemorrhagic fever

To elaborate a set of serological tests for the diagnosis of Argentine haemorrhagic fever (AHF), an enzyme-linked immunosorbent assay (ELISA) for detection of specific anti-Junin virus (JV) IgG is described, and its performance is compared with that of the plaque reduction neutralization test (PRNT). The reproducibility, sensitivity, specificity, and confidence limits for positive and negative results for ELISA were statistically analysed. The value of 800 was demonstrated as the lowest positive titer. Titers > or = 800 varied within one (two-fold) dilution in 95.6% of the tests, while the sensitivity and specificity were 99.2% and 98.8%, respectively. The assay yielded 1% of false positives and 0.05% of false negatives. A comparison of ELISA to PRNT in detecting the seroconversion for JV was studied by the chi square test (comparison of proportions in paired samples) and the K parameter for agreement proportion. Comparison of ELISA to PRNT showed no significant difference in the proportions of positive and negative results of these assays (P < 0.01), demonstrating an equivalent performance (K = 0.98) in the diagnosis of AHF. In addition, the simplicity and safety of the procedures involved make this ELISA the most suitable test to detect natural human JV infections.     

A study of maternally derived measles antibody in infants born to naturally infected and vaccinated women

Maternal, cord and infant measles antibody levels were measured and compared in a group of 411 vaccinated mothers and 240 unvaccinated mothers, and their babies, between 1983 and 1991. Maternal and cord sera were tested by haemagglutination inhibition and/or enzyme-linked immunosorbent assay, and plaque reduction neutralization tests were also used to test infant sera. Geometric mean titres were significantly higher in the unvaccinated than in the vaccinated mothers (P < 0.001). Infants born to mothers with a history of measles had higher antibody levels at birth than infants of vaccinated mothers and, although the difference narrowed over time, continued to have higher levels up to 30 weeks of age. Between 5 and 7 months of age significantly more of the children of vaccinated mothers had plaque reduction neutralization antibody levels below that which would interfere with vaccination. As the boosting effect of circulating natural measles disappears, earlier measles vaccination may need to be considered, perhaps as part of a two-dose policy.     

Human diploid cell culture rabies vaccine (HDCV) and purified chick embryo cell culture rabies vaccine (PCECV) both confer protective immunity against infection with the silver-haired bat rabies virus strain (SHBRV)

The demonstration of extensive differences in the antigenic makeups of the silver-haired bat rabies virus (SHBRV) and canine rabies virus (COSRV) strains raised concerns as to whether current licensed rabies vaccines are sufficiently protective against SHBRV. NIH mouse protection test results show that both the human diploid cell culture rabies vaccine (HDCV) and the purified chicken embryo cell rabies vaccine (PCECV) protected against lethal infection with SHBRV as well as the canine rabies strain COSRV. However, in this investigation, the potencies of both vaccines in mice were found to be significantly higher for COSRV than for SHBRV. The in vivo protection data are confirmed by in vitro virus neutralizing antibody (VNA) test results which demonstrate that mice immunized with HDCV or PCECV develop significantly higher VNA titres against COSRV than against SHBRV. In contrast, VNA tests of sera from individuals immunized with HDCV or PCECV showed that humans, as opposed to mice, develop significantly higher VNA titres against SHBRV than against COSRV. These data suggest that HDCV and PCECV will protect humans against infection with the silver-haired but rabies virus strain in addition to canine rabies virus strains.     

Induction of aberrant crypt foci and flat-type adenocarcinoma in the colons of dogs by N-ethyl-N''-nitro-nitrosoguanidine and their sequential changes

Dengue virus infections have been well known for many years; still dengue virus is regarded as an 'emerging' pathogen, as the disease profile is changing. Its geographical range and overall incidence, and the incidence of the associated complications, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), are on the increase. Modern-day travel and increasing urbanization seem to be the main contributing factors. In order to estimate the risk of infection during long-term stays in dengue-endemic countries, we tested sera obtained from 323 development aid workers and their family members who had spent on average 9.8 years in dengue-endemic regions for the presence of dengue virus antibodies. Dengue virus antibody screening was done by a commercially available immunofluorescence test (IF). Reactive samples were re-tested by an in-house IF and also tested for cross-reactivity to yellow fever virus using yellow fever IF and neutralization test (NT). Evaluation of the results revealed that the screening test has a specificity of at least 63.2%. In 12 of 19 initially positive cases crossreacting antibodies against yellow fever virus could be ruled out. Three cases remained indeterminable, whereas four of the reactive and 10 (out of 12) of the borderline reactive cases showed crossreactivity with yellow fever virus, probably due to previous vaccination. We found seroprevalence rates of 4.3% with no significant differences related to gender or area of upbringing. Seroprevalence rates were evaluated according to region of suspected or confirmed infection. In two cases the dengue infection had taken a classical clinical course; in another three cases an extraordinary febrile illness was reported in the history. None of the other seropositive individuals had a history of an illness possibly attributable to dengue virus infection. Our results show that there definitely is a risk for long-term expatriates to acquire (mostly non- or oligo-symptomatic) dengue infection, which might be important especially in the light of the supposed aetiology of DHF or DSS as a secondary infection with another dengue virus serotype.     

Strain-specific neutralization of human cytomegalovirus isolates by human sera

Induction of an effective antibody response against human cytomegalovirus (HCMV) is an important defense mechanism since it is potentially capable of neutralizing infectious viruses. We have analyzed the extent of HCMV strain-specific neutralization capacity in human sera. Nine recent HCMV isolates and their corresponding sera were investigated in cross-neutralization assays. We observed differences, independent of the overall neutralization capacity, in the 50% neutralization titers of the sera against individual strains, differences that ranged from 8-fold to more than 60-fold. For one isolate, complete resistance to neutralization by two human sera was observed. The neutralization capacity of human sera was not influenced by the presence of various concentrations (up to 100-fold excess) of noninfectious envelope glycoproteins, an inherent contamination of virus preparations from recent HCMV isolates. This indicated that the decisive parameter for neutralization is the titer of the neutralizing antibodies and that neutralization is largely independent of the concentration of virus. Analysis with transplant patients revealed that during primary infection strain-specific and strain-common antibodies are produced asynchronously. Thus, our data demonstrate that the induction of strain-specific neutralizing antibodies is a common event during infection with HCMV and that it might have important implications for the course of the infection and the development of anti-HCMV vaccines.     

Study of the V3 loop as a target epitope for antibodies involved in the neutralization of primary isolates versus T-cell-line-adapted strains of human immunodeficiency virus type 1

Previous studies characterized the third variable (V3) loop of the envelope gp120 as the principal neutralizing determinant for laboratory T-cell-line-adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1). However, primary viruses isolated from infected individuals are more refractory to neutralization than TCLA strains, suggesting that qualitatively different neutralizing antibodies may be involved. In this study, we investigated whether the V3 loop constitutes a linear target epitope for antibodies neutralizing primary isolates. By using peptides representative of the V3 regions of various primary isolates, an early, relatively specific and persistent antibody response was detected in sera from HIV-infected patients. To assess the relationship between these antibodies and neutralization, the same peptides were used in competition and depletion experiments. Addition of homologous V3 peptides led to a competitive inhibition in the neutralization of the TCLA strain HIVMN/MT-4 but had no effect on the neutralization of the autologous primary isolate. Similarly, the removal of antibodies that bind to linear V3 epitopes resulted in a loss of HIVMN/MT-4 neutralization, whereas no decrease in the autologous neutralization was measured. The different roles of V3-specific antibodies according to the virus considered were thereby brought to light. This confirmed the involvement of V3 antibodies in the neutralization of a TCLA strain but emphasized a more pronounced contribution of either conformational epitopes or epitopes outside the V3 loop as targets for antibodies neutralizing primary HIV-1 isolates. This result underlines the need to focus on new vaccinal immunogens with epitopes able to induce broadly reactive and efficient antibodies that neutralize a wide range of primary HIV-1 isolates.     

Real time observation of the binding of herpes simplex virus type 1 (HSV-1) to immobilized heparan sulfate and the neutralization of HSV-1 by sulfonated human immunoglobulin

A real time biomolecular interaction assay system involving an optical sensor was applied to quantitative analysis of the binding of herpes simplex virus type 1 (HSV-1) to immobilized heparan sulfate, a cellular receptor component of HSV-1, and the neutralization antibody titer against this virus with a commercially available sulfonated human immunoglobulin preparation. The virus titer in a viral solution and the neutralizing antibody titer in the human immunoglobulin preparation could be successfully estimated in a short time with this system without any difficult cell culture.     

Characterization of simian-human immunodeficiency virus envelope glycoprotein epitopes recognized by neutralizing antibodies from infected monkeys

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.     

Conserved N-linked oligosaccharides of the C-terminal portion of human immunodeficiency virus type 1 gp120 and viral susceptibility to neutralizing antibodies

We have constructed a mutated infectious HIV variant lacking the signals for addition of three N-linked glycans situated in the V4, C4 and V5 regions of HIV gp120. When comparing mutated virus with wildtype virus we found essentially no differences in the phenotypic characteristics of the two viruses except for the expected electrophoretic mobility shift of radioimmuno-precipitated mutated gp120, resulting from the missing N-glycans. Thus, the infectivity titer and the capacity to induce syncytia were similar for the two viruses. The sensitivity of mutant and wildtype virus to a number of neutralizing agents was determined. As expected, the mutant virus was significantly less sensitive to neutralization by Con A, with affinity for the N-glycans eliminated. We found, however, that antibodies to the V3 loop and sCD4 neutralized wild-type virus as efficiently as mutant virus, whereas 2G12, a monoclonal antibody, binding to a discontinuous neutralization epitope, and GP13, binding to the CD4-binding domain, neutralized wildtype virus better than mutant virus. Altogether the data suggest that the three conserved N-linked glycans, despite their location in immediate association with the CD4-binding domain, which is an important neutralization epitope, are not essential for virus replication in cell culture and they are not engaged in shielding neutralization epitopes of gp120 from neutralizing antibodies. However, the glycans evidently influence the three-dimensional conformation of gp120, since their presence increases the availability of the neutralization epitope of 2G12.     

Broad cross-neutralizing activity in serum is associated with slow progression and low risk of transmission in primate lentivirus infections

Sera from human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2)-infected humans were tested with autologous (from the same individual) and heterologous (from other individuals) virus isolates in a neutralization assay. Similarly, sera from experimentally simian immunodeficiency virus (SIVsm from sooty mangabey) or HIV-2SBL6669-infected cynomolgus macaques were tested for neutralizing activity against autologous and heterologous reisolates. In the neutralization assay, the virus dose ranged between 10-75 50% infectious dose (ID50), sera were used in five 2- or 4-fold dilutions, beginning with 1:20, and human peripheral blood mononuclear cells (PBMCs) served as target cells. The readout of the 7-day assay was a HIV-1 or HIV-2 antigen enzyme-linked immunosorbent assay (ELISA). Our results show that SIVsm-inoculated monkeys who develop early immunodeficiency lack serum neutralizing activity or develop a neutralizing antibody response with narrow specificity. Long survival is associated with the ability to neutralize several autologous and heterologous SIVsm reisolates. Infection of macaques with HIV-2SBL6669 did not cause disease within the 5 years observation time and elicited a broadly cross-reactive neutralizing antibody response, including neutralization of other, independently obtained, HIV-2 isolates. In HIV-1-infected humans, neutralizing antibodies can only be detected in up to 50% of cases. Neutralizing activity, whenever present, may show a broad specificity, that is, neutralization may occur across genetic subtypes. Presence of broadly cross-reactive neutralizing antibodies is associated with a lower risk of HIV-1 (subtype B) transmission both from mother to child and sexually from male to female. Unlike HIV-1 infection, serum neutralizing activity is regularly present in HIV-2 infection. In view of the differences between HIV-1 and HIV-2 pathogenesis, we suggest that an effective neutralizing antibody response may contribute to a delay in disease progression and to a decrease in risk of transmission.     

Assessment using ELISA of the herd immunity levels induced in cattle by foot-and-mouth disease oil vaccines

The development of a liquid-phase blocking sandwich ELISA (LPBE) to measure antibodies (Ab) produced in cattle with the O, A and C foot-and-mouth disease virus (FMDV) types of commercial vaccines used in Argentina is described. The test was specific: 99% of na?ve cattle sera (n = 130) gave titres below log10 = 1.2, and none had a titre above log10 = 1.5. Comparative studies with serum neutralization test (SNT) using sera from cattle which received one or more vaccine doses is reported. The overall rank correlation coefficient (Spearman's rho, rs) between SNT and LPBE were highly significant (rs > 0.67, P < 0.0001) for all vaccine strains. LBPE Ab titres on sera collected 90 days post vaccination were compared with results of cattle protection tests by applying a logistic regression. The minimum Ab titres at which 85% and 75% of the cattle were protected for each FMDV type were determined in order to interpret field Ab data in terms of protection. Application of this method allows large scale serological examinations to monitor antibody levels in vaccinated animals as an indirect indicator of the FMD control program status in the field. Its use in the evaluation of commercial batches of FMD vaccine is discussed.     

Diffusing capacity of the lung for carbon monoxide

A phage-display technology was used to produce a single-chain Fv antibody fragment (scFv) from the 30AA5 hybridoma secreting anti-glycoprotein monoclonal antibody (MAb) that neutralizes rabies virus. ScFv was constructed and then cloned for expression as a protein fusion with the g3p minor coat protein of filamentous phage. The display of antibody fragment on the phage surface allows its selection by affinity using an enzyme-linked immunosorbent assay (ELISA); the selected scFv fragment was produced in a soluble form secreted by E. coli. The DNA fragment was sequenced to define the germline gene family and the amino-acid subgroups of the heavy (VH) and light (VL) chain variable regions. The specificity characteristics and neutralization capacity of phage-displayed and soluble scFv fragments were found to be identical to those of the parental 30AA5 MAb directed against antigenic site II of rabies glycoprotein. Phage-display technology allows the production of new antibody molecule forms able to neutralize the rabies virus specifically. The next step could be to engineer and produce multivalent and multispecific neutralizing antibody fragments. A cocktail of multispecific neutralizing antibodies could contain monovalent, bivalent or tetravalent scFv fragments, for passive immunoglobulin therapy.     

Schizophrenic prelingual deaf and hearing patients. A comparison of premorbid and current data after several years of progression

The aim of the present study was to investigate the seroprevalence to Flavivirus, in young people living in risk areas. We analyzed 189 human sera from 3 towns in the Province of Formosa. This area corresponds to the border that limits Brasil and Paraguay and the aim was to search for a possible introduction of Dengue and Yellow Fever from these countries. Serological tests such as haemagglutination inhibition (HI), complement fixation (CF) and neutralization (NT) were performed using St. Louis encephalitis (SLE), Bussuquara, Ilheus, Yellow fever and dengue 1 and 2 viruses. No definite evidence for HI antibodies to dengue and Ilheus was obtained. One serum cross-reacted only with yellow fever and two sera only for Bussuquara by the HI test. Only one serum was confirmed to be positive for Bussuquara by NT test. A total of 22 sera from 189 were positive for SLE by the HI test and 40 were also reactive by the NT test. The seroprevalence measured by HI and NT antibodies was similar in the three departments studied. These results show that SLE virus is present in the North of Argentina with an important value of prevalence so that this agent could play an important role in the febrile infections not virologically confirmed.     

Preferential selection of receptor-binding variants of influenza virus hemagglutinin by the neutralizing antibody repertoire of transgenic mice expressing a human immunoglobulin mu minigene

An analysis was made of the neutralizing antibody repertoire, for influenza virus hemagglutinin (HA) of transgenic mice expressing a human immunoglobulin mu (IgH) minigene, by monoclonal antibody (MAb) selection and sequencing of the HA genes of X31 (H3N2 subtype) laboratory variants. Whereas previously reported laboratory variants, selected in ovo with high-affinity murine MAbs of the IgG class, differed from wild-type virus by a single amino acid residue change in one of the major antigenic sites, neutralizing MAbs from transgenic donors selected novel variant viruses with altered receptor-binding specificity and contained residue changes in both the receptor-binding pocket (HA1 225 or HA1 226) and an antigenic site (HA1 135, HA1 145, or HA1 158). Changes in receptor-binding specificities of the variant viruses were confirmed by their resistance to inhibition by horse serum glycoproteins and altered binding to neoglycoproteins. The residue changes in variant virus V-21.2 (HA1 135 G-->R, 225 G-->D) abrogated neutralization by each of the MAbs; nevertheless V-21.2 was recognized by its own selecting MAb in enzyme-linked immunosorbent assay and therefore qualified as an adsorptive mutant rather than an antigenic variant. We consider that a low-affinity neutralizing antibody response may preferentially select for receptor-binding variants of influenza virus HA.     

Neutralization of HIV-1 primary isolates by polyclonal and monoclonal human antibodies

To examine antibody-mediated neutralization of HIV-1 primary isolates in vitro, we tested sera and plasma from infected individuals against four clade B primary isolates. These isolates were analyzed further for neutralization by a panel of several human anti-HIV-1 mAb in order to identify the neutralizing epitopes of these viruses. Each of the HIV-1+ serum and plasma specimens tested had neutralizing activities against one or more of the four primary isolates. Of the three individual sera, one (FDA-2) neutralized all of the four isolates, while the other two sera were effective against only one virus. The pooled plasma and serum samples reacted broadly with these isolates. Based on the neutralizing activities of the mAb panel, each virus isolate exhibited a distinct pattern of reactivity, suggesting antigenic diversity among clade B viruses. Neutralizing epitopes were found in the V3 loop and CD4-binding domain of gp120, as well as near the transmembrane region (cluster II epitope) of gp41. A mAb directed to the cluster I epitope of gp41 near the immunodominant disulfide loop weakly neutralized one primary isolate. None of the mAb in the panel affected one primary isolate, US4, although this virus was sensitive to neutralization by some of the polyclonal antibody specimens. This isolate was also resistant to neutralization by a cocktail of 10 mAb, most of which individually inhibited at least one of the other three viruses tested. These results suggest that neutralizing activity for this latter virus is present in certain HIV-1+ sera/plasma, but is not exhibited by the mAb in the panel. Thus, effective neutralizing antibodies against primary isolates can be generated by humans upon exposure to HIV-1, but not all of these antigenic specificities are represented in a large panel of human anti-HIV-1 mAb.     

Using distilled water for the extraction of mucosal antibodies and the subsequent application in RSV neutralization test

Virus neutralization (VN) is an important functional test for evaluating RSV vaccines, also encompassing in mucosal secretion of the respiratory tract considering the infection route. In our previous study, an immunoglobin extraction method described by Bergquist et al. was adopted for RSV ELISA, but it was not suitable for virus neutralization test due to the cell toxicity of the 2% saponin solution used for the antibody extraction. In order to overcome this problem, several solvents including distilled water were tested in the present study for the capacity to extract immunogloblins. Antibodies in the extracts were evaluated and compared by ELISA. Distilled water was as efficient as the 2% saponin solution for extraction of total IgA, RSV specific IgA and IgG. More importantly, the organ extracts obtained subsequently could be used for virus neutralization test without causing adverse effect on the cell culture. Therefore, distilled water was finally chosen as the solvent for immunoglobulin extraction from mucosal organs when both ELISA and virus neutralization test are required.     

Serologic responses of young colostrum fed dairy calves to antigens of Pasteurella haemolytica A1

The potential and limitations of early calfhood vaccination to induce active immunity to Pasteurella haemolytica A1 in conventional colostrum fed calves were investigated. Holstein dairy calves (n = 29) were vaccinated at 2 and 4 weeks of age, or at 6 and 8 weeks of age with a commercial culture supernatant vaccine (Presponse, Langford Inc., Guelph, Ont., Canada), or remained unvaccinated as controls. Serum antibody titres were measured using an indirect bacterial agglutination assay, a leukotoxin neutralization assay, and enzyme immunoassays for antibodies of the IgM, IgG1, and IgG2 isotypes binding purified capsular polysaccharide of P. haemolytica A1. Seroconversion (fourfold or greater increase in serum antibody titre) rates were compared using Fisher's exact test. The effects of passive antibody titres and age on response to vaccination were assessed by linear modelling. Vaccination at 2 and 4 weeks of age was associated with 40%, and 0% of calves seroconverting on the basis of agglutinating antibody titres, and leukotoxin neutralizing titres respectively, and 50%, 0%, and 0% seroconverting on the basis of IgM, IgG1 and IgG2 antibodies to capsular polysaccharide, respectively. Agglutinating antibody responses were not related to prevaccination antibody titres, or to age at vaccination. Higher responses (p = 0.08) to leukotoxin were observed in older calves (after taking differences in prevaccination titres into account). Statistical analyses of responses to capsular polysaccharide among calves with comparable prevaccination IgG1 antibody titres revealed significantly higher IgM, IgG1 and IgG2 responses in older calves. Rising titres of IgM antibodies in nonvaccinated calves after 5 weeks of age suggest natural exposure to P. haemolytica A1 or antigens which result in serologic cross-reactions as a means of priming immune responses.     

Transgenic mice secreting coronavirus neutralizing antibodies into the milk

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.