DNA-hydrolyzing IgG antibodies from the blood of patients with acquired immune deficiency syndrome

DNase activity was analyzed in 110 IgG preparations from the blood of AIDS patients. The relative activity of the preparations varied markedly among patients, being reliably detectable in 96% of the preparations. It was shown with several rigid criteria that DNAase activity is an intrinsic property of antibodies (Abs) from AIDS patients. Not only intact IgG, but also isolated light chains of polyclonal Abs were shown to possess catalytic activity. The abzymes efficiently catalyzed DNA hydrolysis in a wide range of pH (5.0–9.5). The K _M and V _max values were evaluated for Ab-dependent hydrolysis of DNA.     

Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses

Linear carbohydrate-peptide constructs based on the 13 amino acid nonnatural pan DR epitope (PADRE) and carbohydrate B cell epitopes are demonstrated to be potent immunogens. These data support our belief that PADRE should be considered as an alternative to more complex carriers for use in prophylaxis and therapeutic vaccines. Two model carbohydrate-PADRE glycoconjugates were used to demonstrate that PADRE could effectively provide T cell help for carbohydrate-specific Ab responses. Conjugates of PADRE covalently linked to the human milk oligosaccharide, lacto-N-fucopentose II or a dodecasaccharide derived from Salmonella typhimurium O-Ag induced high titer IgG Ab responses in mice, which were comparable to glycoconjugates employing human serum albumin (HSA) as the carrier protein. Different adjuvants, in combination with PADRE conjugates, allowed for the modulation of the isotype profile with alum supporting an IgG1 profile; QS-21 an IgG2a, 2b profile, while an alum/QS-21 mixture generated a balanced IgG1/IgG2b isotype profile. As defined by binding to synthetic glycoconjugates, dodecasaccharide-specific Abs exhibited fine specificity similar to protective polyclonal Ab responses previously reported for dodecasaccharide-protein conjugates. The same Abs bound to intact S. typhimurium cells, suggesting that biologically relevant specificities were produced. The affinity of the dodecasaccharide-specific Abs was further shown to be comparable to that of a well-characterized, high affinity monoclonal anti-carbohydrate Ab recognizing the same epitope.     

DNA-hydrolyzing IgG antibodies from the blood of patients with acquired immune deficiency syndrome

DNAase activity of 110 samples of IgG from the blood of AIDS patients was analyzed. It was shown that the relative activity of preparations varies very much from patient to patient, but 96% preparations show detectable level of DNAase activity. Several rigid criteria were applied and it was shown that DNAase activity is an intrinsic property of antibodies from AIDS patients. It was shown that catalytic activity could posses not only intact IgG, but also separated light chains of polyclonal antibodies. The abzymes catalyze DNA hydrolysis effectively in a wild range of pH (5.0-9.5). K(M) and V(MaKC) values of antibody-dependent hydrolysis of DNA was estimated.     

Reduced susceptibility of recombinant polyclonal antibodies to inhibitory anti-variable domain antibody responses

The immunogenicity of therapeutic Abs is a concern as anti-drug Abs may impact negatively on the pharmacodynamics and safety profile of Ab drugs. The factors governing induction of anti-drug Abs are not fully understood. In this study, we describe a model based on mouse-human chimeric Abs for the study of Ab immunogenicity in vivo. Six chimeric Abs containing human V regions and mouse C regions were generated from six human anti-Rhesus D Abs and the Ag-binding characteristics of the parental human Abs were retained. Analysis of the immune response toward the individual chimeric Abs revealed the induction of anti-variable domain Abs including anti-idiotypic Abs against some of these, thereby demonstrating the applicability of the model for studying anti-drug Ab responses in vivo. Immunization of BALB/c, C57, and outbred NMRI mice with a polyclonal composition consisting of all six chimeric Abs demonstrated that the immunogenicity of the individual Abs was haplotype dependent. Chimeric Abs, which were nonimmunogenic when administered individually, did not become immunogenic as part of the polyclonal composition, implying the absence of epitope spreading. Ex vivo Ab-binding studies established a clear correlation between the level of immunogenicity of the Abs comprised in the composition and the impact on the pharmacology of the Abs. These analyses demonstrate that under these conditions this polyclonal Ab composition was generally less susceptible to blocking Abs than the respective mAbs.     

Activity and safety of CTLA-4 blockade combined with vaccines in cynomolgus macaques

The immune modulatory molecule CTLA-4 (CD152), through interactions with the B7 costimulatory molecules, has been shown to be a negative regulator of T cell activation in various murine model systems. Abs that block CTLA-4 function can enhance immune responses that mediate potent antitumor activity. However, CTLA-4 blockade can also exacerbate autoimmune disease. The safety and activity of anti-CTLA-4 Abs in primates has not been addressed. To that end, we generated human Abs against CTLA-4 using transgenic mice expressing human Ig genes. A high affinity Ab (10D1) that blocked the binding of CTLA-4 to the B7-1 and B7-2 ligands and had cross-reactivity with macaque CTLA-4 was chosen for further development. Administration of 10D1 to cynomolgus macaques significantly enhanced Ab responses to hepatitis surface Ag and a human melanoma cell vaccine. Anti-self Ab responses as measured by immunoassays using lysate from melanocyte-rich tissues were elicited in those animals receiving the melanoma cell vaccine and anti-CTLA-4 Ab. Remarkably, chronic administration of 10D1 did not result in measurable polyclonal T cell activation, significant alteration of the lymphocyte subsets, or induce clinically observable autoimmunity. Repeated dosing of the 10D1 did not elicit monkey anti-human Ab responses in the monkeys. These observations support the development of CTLA-4 blockade for human immunotherapy.     

HIV-1 integrase-hydrolyzing antibodies from sera of HIV-infected patients

Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether Abs from patients with viral diseases can hydrolyze viral proteins. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of AIDS patients by chromatography on several affinity sorbents. We present evidence showing that 89.5% IgGs purified from the sera of HIV-infected patients using several affinity resins including Sepharose with immobilized integrase specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Several rigid criteria have been applied to show that the IN-hydrolyzing activity is an intrinsic property of AIDS IgGs but not from healthy donors. Similar to autoimmune proteolytic abzymes, IN-hydrolyzing IgGs from some patients were inhibited by specific inhibitors of serine and metal-dependent proteases but a significant inhibition of the activity by specific inhibitors of acidic- and thiol-like proteases was observed for the first time. Although HIV infection leads to formation of Abs to many viral and human antigens, no possible biological role for most of them is known. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for estimation of the immune status in AIDS patients.     

Immunization of rabbits with DNase I produces polyclonal antibodies with DNase and RNase activities

Immunization of rabbits with DNase I leads to the production of antiidiotypic Abs with DNase activity. It is not known at present whether antiidiotypic Abs against DNA-hydrolyzing enzymes can possess RNase activity. Here we show that immunization of healthy rabbits with bovine DNase I produces IgGs with intrinsic DNase and RNase activities. Electrophoretically and immunologically homogeneous polyclonal IgGs were obtained by sequential chromatography of the immune sera on Protein A-Sepharose and gel filtration. Affinity chromatography on DNA cellulose using elution of Abs with different concentrations of NaCl and an acidic buffer separated catalytic IgGs into four Ab subfractions, three of which demonstrated only DNase activity while one subfraction hydrolyzed RNA faster than DNA. The serum of patients with many different autoimmune (AI) diseases contains small fractions of antibodies (Abs) interacting with immobilized DNA, which possess both DNase and RNase activities. Our data suggest that a fraction of abzymes from AI patients hydrolyzing both DNA and RNA can contain a subfraction of Abs against DNase I.     

Antibodies against RNA hydrolyze RNA and DNA

Immunization of animals with DNA leads to the production of anti-DNA antibodies (Abs) demonstrating both DNase and RNase activities. It is currently not known whether anti-RNA Abs can possess nuclease activities. In an attempt to address this question, we have shown that immunization of three rabbits with complex of RNA with methylated BSA (mBSA) stimulates production of IgGs with RNase and DNase activities belonging to IgGs, while polyclonal Abs from three non-immunized rabbits and three animals immunized with mBSA are catalytically inactive. Affinity chromatography of IgGs from the sera of autoimmune (AI) patients on DNA-cellulose usually demonstrates a number of fractions, all of which effectively hydrolyze both DNA and RNA, while rabbit catalytic IgGs were separated into Ab subfractions, some of which demonstrated only DNase activity, while others hydrolyzed RNA faster than DNA. The enzymic properties of the RNase and DNase IgGs from rabbits immunized with RNA distinguish them from all known canonical RNases and DNases and DNA- and RNA-hydrolyzing abzymes (Abzs) from patients with different AI diseases. In contrast to RNases and AI RNA-hydrolyzing Abs, rabbit RNase IgGs catalyze only the first step of the hydrolysis reaction but cannot hydrolyze the formed terminal 2',3'-cyclophosphate. The data indicate that Abzs of AI patients hydrolyzing nucleic acids in part may be Abs against RNA and its complexes with proteins. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Antibody responses to mycobacterial and self heat shock protein 65 in autoimmune arthritis: epitope specificity and implication in pathogenesis

Many autoimmune diseases are believed to involve primarily T cell-mediated effector mechanisms. There is increasing realization, however, that Abs may also play a vital role in the propagation of T cell-driven disorders. In this study, on the rat adjuvant-induced arthritis (AA) model of human rheumatoid arthritis, we examined the characteristics of serum Ab response to mycobacterial heat shock protein (hsp) 65 (Bhsp65), self (rat) hsp65 (Rhsp65), and linear peptides spanning these two molecules. The AA-resistant WKY (RT.1(l)) rat responded to the heat-killed Mycobacterium tuberculosis immunization with a rapid burst of Abs to both Bhsp65 and Rhsp65. These Abs reacted with numerous peptide epitopes; however, this response was reduced to a few epitopes with time. On the contrary, the susceptible Lewis (RT.1(l)) rat developed a relatively lower Ab response to Bhsp65, and Abs to Rhsp65 did not appear until the recovery from the disease. The Ab response in Lewis rats diversified with progression of AA, and there was an intriguing overlap between the repertoire of Bhsp65-reactive B and T cells during the recovery phase of AA. Nonetheless, subsets of the repertoire of the late Abs in both rat strains became focused on the same epitope regions of Bhsp65 and Rhsp65. The functional relevance of these Abs was evident from the results showing that sera from recovery phase Lewis or WKY rats, but not that of naive rats, afforded protection against subsequent AA. These results are of significance in further understanding of the role of humoral immunity in the pathogenesis of autoimmune arthritis.     

Molecular recognition patterns of serum anti-DNA topoisomerase I antibody in systemic sclerosis

Autoreactive anti-DNA topoisomerase I (anti-Topo I) Abs are commonly detected in sera of systemic sclerosis (SSc) patients. Our studies have established a positive correlation between the levels of serum anti-Topo I Abs and both disease severity and activity of SSc. The molecular targets of anti-Topo I Ab on Topo I domains remain to be further defined. In this report, we studied the molecular recognition pattern of serum anti-Topo I Ab in 52 SSc patients. The highest reactivity of serum anti-Topo I Abs was against the core subdomains I and II (aa 207-441) and, to a lesser extent, against the core subdomain III (aa 433-636) of Topo I. The linker domain (aa 636-712) and the C-terminal domain (aa 713-765) had much less reactivity than the core domain (aa 207-636). Strikingly, very little reactivity was directed against the N-terminal domain (aa 1-213) by serum anti-Topo I Ab. This molecular recognition pattern was consistent among all SSc serum samples studied. Results from patients with serial serum samples indicated that this pattern remained unchanged over time. Interestingly, some naive B cells from healthy controls, upon transformation by EBV, produced IgM Abs against Topo I. These Abs had low affinity for Topo I and reacted equally to all domains of Topo I. The molecular recognition pattern of serum anti-Topo I Ab in SSc suggests the presence of a unique antigenic stimulation in vivo in this disease.     

An engineered human IgG1 antibody with longer serum half-life

The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). By binding to FcRn in endosomes, IgG Abs are salvaged from lysosomal degradation and recycled to the circulation. Several studies have demonstrated a correlation between the binding affinity of IgG Abs to FcRn and their serum half-lives in mice, including engineered Ab fragments with longer serum half-lives. Our recent study extended this correlation to human IgG2 Ab variants in primates. In the current study, several human IgG1 mutants with increased binding affinity to human FcRn at pH 6.0 were generated that retained pH-dependent release. A pharmacokinetics study in rhesus monkeys of one of the IgG1 variants indicated that its serum half-life was approximately 2.5-fold longer than the wild-type Ab. Ag binding was unaffected by the Fc mutations, while several effector functions appeared to be minimally altered. These properties suggest that engineered Abs with longer serum half-lives may prove to be effective therapeutics in humans.     

Toward selective covalent inactivation of pathogenic antibodies: a phosphate diester analog of vasoactive intestinal peptide that inactivates catalytic autoantibodies

We report the selective inactivation of proteolytic antibodies (Abs) to an autoantigen, the neuropeptide vasoactive intestinal peptide (VIP), by a covalently reactive analog (CRA) of VIP containing an electrophilic phosphonate diester at the Lys(20) residue. The VIP-CRA was bound irreversibly by a monoclonal Ab that catalyzes the hydrolysis of VIP. The reaction with the VIP-CRA proceeded more rapidly than with a hapten CRA devoid of the VIP sequence. The covalent binding occurred preferentially at the light chain subunit of the Ab. Covalent VIP-CRA binding was inhibited by VIP devoid of the phosphonate diester group. These results indicate the importance of noncovalent VIP recognition in guiding Ab nucleophilic attack on the phosphonate group. Consistent with the covalent binding data, the VIP-CRA inhibited catalysis by the recombinant light chain of this Ab with potency greater than the hapten-CRA. Catalytic hydrolysis of VIP by a polyclonal VIPase autoantibody preparation that cleaves multiple peptide bonds located between residues 7 and 22 essentially was inhibited completely by the VIP-CRA, suggesting that the electrophilic phosphonate at Lys(20) enjoys sufficient conformational freedom to react covalently with Abs that cleave different peptide bonds in VIP. These results suggest a novel route to antigen-specific covalent targeting of pathogenic Abs.     

Antibody reactivity to HIV-1 Vpu in HIV-1/AIDS patients on highly active antiretroviral therapy

Human immunodeficiency virus type 1 (HIV-1) Vpu protein promotes both extracellular release of viral particles and degradation of CD4 in the endoplasmic reticulum. The correlation of anti-Vpu antibody (Ab) reactivity to Vpu and AIDS disease progression was studied in 162 HIV-1/AIDS patients after they had received highly active antiretroviral therapy (HAART) for 1 year. Anti-Vpu Ab reactivity was analyzed by Western blot using a recombinant Vpu protein. Results showed that at baseline (prior to initiation of HAART), 31.5% of patients (51/162) had anti-Vpu Ab. The proportion of anti-Vpu Ab in patients with CD4 counts > or =500, 200-500 and <200/mm(3) were 40.6, 34.7 and 14.3%, respectively (chi(2) test, p < 0.05). In addition, decreasing levels of anti-Vpu Ab reactivity were significantly correlated with increasing levels of HIV-1 viral load. After receiving HAART for 1 year, 7 of 111 anti-Vpu Ab-negative patients (6.3%) seroconverted (- --> + group) and 8 of 51 anti-Vpu Ab-positive (15.7%) patients became negative (+ --> - group). Among 104 anti-Vpu Ab-negative patients, 40 were selected for analysis of the VPU gene. All of them had an intact VPU gene. Patients were further divided into four groups according to their anti-Vpu Ab serostatus and anti-HIV-1 Ab was measured. The results showed that only the anti-Vpu Ab seroconverted group (- --> +) had increased serum levels of anti-HIV-1 Abs after 1 year of HAART, while the other three groups (+ --> +, - --> - and + --> -) had decreased serum levels of anti-HIV-1 Abs after 1 year of HAART (p < 0.05). In conclusion, the presence of anti-Vpu Ab is associated with improved prognosis following HIV-1 infection, and seroconversion of anti-Vpu Ab in patients on HAART indicates significant recovery of immunity.     

Kinetic, affinity, and diversity limits of human polyclonal antibody responses against tetanus toxoid

Due to technical limitations, little knowledge exists on the composition of Ag-specific polyclonal Ab responses. Hence, we here present a molecular analysis of two representative human Ab repertoires isolated by using a novel single-cell cloning approach. The observed genetic diversity among tetanus toxoid-specific plasma cells indicate that human polyclonal repertoires are limited to the order of 100 B cell clones and hypermutated variants thereof. Affinity and kinetic binding constants are log-normally distributed, and median values are close to the proposed affinity ceilings for positive selection. Abs varied a million-fold in affinity but were restricted in their off-rates with an upper limit of 2 x 10(-3) s(-1). Identification of Abs of high affinity without hypermutations in combination with a modest effect of hypermutations on observed affinity increases indicate that Abs selected from the naive repertoire are not only of low affinity but cover a relatively large span in affinity, reaching into the subnanomolar range.     

Lacto-N-fucopentaose III found on Schistosoma mansoni egg antigens functions as adjuvant for proteins by inducing Th2-type response.

We have recently demonstrated that induction of Th2 responses by Schistosoma mansoni egg Ag is largely due to carbohydrates on the Ag functioning as adjuvants. Lacto-N-fucopentaose III (LNFPIII), a polylactosamine sugar, is the predominant carbohydrate found in S. mansoni egg Ag. Therefore, using neoglycoprotein, we investigated whether LNFPIII induces in vivo Th2 response and functions as an adjuvant. Following intranasal immunization with LNFPIII linked to human serum albumin (HSA) (HSA-LNFPIII), BALB/c mice mounted a strong Th2 response and produced significantly higher levels of total IgE as well as HSA-specific IgG, IgG1, and IgE. HSA-LNFPIII was over 1000-fold more potent in inducing Ab production as compared with HSA alone. Although LNFPIII itself did not function as an epitope for either IgG or IgE, its conjugation with protein was essential for the adjuvant activity. Moreover, fucose residue on LNFPIII was crucial for induction of Ab production. Nasal lymphocytes from mice immunized with HSA-LNFPIII produced IL-4, IL-5, and IL-10, but not IFN-gamma following in vitro stimulation with HSA or HSA-LNFPIII. In addition, these activated nasal lymphocytes also showed a significant increase of B7-2 expression on B220-positive cells. Furthermore, not only intranasal but also both i.p. and s.c. immunization with HSA-LNFPIII induced significant production of HSA-specific Abs compared with the immunization with HSA alone, suggesting that the activity of LNFPIII was not restricted on particular route of immunization. These results demonstrate that Lewis type carbohydrate LNFPIII can function as an adjuvant by their ability to induce a Th2 response.     

Influence of the antibody purification method on immunoassay performance: hapten-antibody binding in accordance with the structure of the affinity column ligand.

The effects of ligands for immunoaffinity chromatography on the immunoassay were investigated with three goat anti-methamphetamine (anti-MA) antibodies (Abs). An N-4-aminobutyl derivative of methamphetamine (4-ABMA) was conjugated with proteins and used as immunogens. All the antisera produced were purified by affinity chromatography with various ligands of 4-ABMA-proteins and of haptens as well as protein G: 4-ABMA-bovine serum albumin (4-ABMA-BSA), 4-ABMA-keyhole limpet hemocyanine (4-ABMA-KLH), 4-ABMA-ovalbumin (4-ABMA-OVA), MA, 4-ABMA, and amphetamine were used as ligands. Enzyme-linked immunosorbent assay (ELISA) was conducted to examine characteristics of the purified Abs with the 4-ABMA-OVA competitor coated. The results obtained revealed that characters of the purified Abs were closely related with chemical structures of ligands used. The Abs from the MA and the amphetamine columns showed better sensitivities than those from the others in each antiserum. Particularly, the Ab from the amphetamine column gave the best results in terms of sensitivity and specificity. The recognition or the affinity of the Ab selected was considered to be affected by the structure of the ligand concerned. These results suggest that the Ab purification method should be considered as an important parameter which has great influence on the performance of immunoassays with polyclonal Abs.     

Specific HIV gp120-cleaving antibodies induced by covalently reactive analog of gp120

We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314-28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and Km for this reaction was approximately 200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization.     

Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-alpha inducer in systemic lupus erythematosus

Patients with systemic lupus erythematosus (SLE) have increased blood levels of IFN-alpha, which correlate to disease activity. We previously identified an IFN-alpha-inducing factor (IIF) in the blood of SLE patients that activated the natural IFN-alpha-producing cells in cultures of normal PBMC. The SLE-IIF contained DNA and IgG, possibly as small immune complexes. In our study, we demonstrated that SLE-IIF correlated to the presence of anti-dsDNA Abs in patients and contained anti-dsDNA Abs as an essential component. Purified anti-DNA Abs or SLE-IgG caused only a weak IFN-alpha production in cultures of normal PBMC in the presence of costimulatory IFN-alpha2b. However, they converted the plasmid pcDNA3, which itself induced no IFN-alpha production in PBMC, into an efficient IFN-alpha inducer. A human monoclonal anti-ss/dsDNA Ab had the same effect. This IFN-alpha-inducing activity of the plasmid was abolished by methylation, suggesting that unmethylated CpG DNA motifs were important. Like IIF in SLE serum, the combination of SLE-IgG and pcDNA3 appeared to stimulate IFN-alpha production in natural IFN-alpha-producing cells, a unique cell population resembling immature dendritic cells. The IFN-alpha production was greatly enhanced by IFN-alpha2b and IFN-beta, and for SLE-IIF it was also enhanced by GM-CSF but inhibited by IL-10. We have therefore identified a new function of DNA-anti-DNA Ab complexes, IFN-alpha induction, that might be important in the pathogenesis of SLE.     

IgY Antibodies Protect against Human Rotavirus Induced Diarrhea in the Neonatal Gnotobiotic Piglet Disease Model

Group A Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The aim of the present work was to evaluate protection against rotavirus (RV) diarrhea conferred by the prophylactic administration of specific IgY antibodies (Ab) to gnotobiotic piglets experimentally inoculated with virulent Wa G1P[8] human rotavirus (HRV). Chicken egg yolk IgY Ab generated from Wa HRV hyperimmunized hens specifically recognized (ELISA) and neutralized Wa HRV in vitro. Supplementation of the RV Ab free cow milk diet with Wa HRV-specific egg yolk IgY Ab at a final ELISA Ab titer of 4096 (virus neutralization –VN- titer = 256) for 9 days conferred full protection against Wa HRV associated diarrhea and significantly reduced virus shedding. This protection was dose-dependent. The oral administration of semi-purified passive IgY Abs from chickens did not affect the isotype profile of the pig Ab secreting cell (ASC) responses to Wa HRV infection, but it was associated with significantly fewer numbers of HRV–specific IgA ASC in the duodenum. We further analyzed the pigś immune responses to the passive IgY treatment. The oral administration of IgY Abs induced IgG Ab responses to chicken IgY in serum and local IgA and IgG Ab responses to IgY in the intestinal contents of neonatal piglets in a dose dependent manner. To our knowledge, this is the first study to show that IgY Abs administered orally as a milk supplement passively protect neonatal pigs against an enteric viral pathogen (HRV). Piglets are an animal model with a gastrointestinal physiology and an immune system that closely mimic human infants. This strategy can be scaled-up to inexpensively produce large amounts of polyclonal IgY Abs from egg yolks to be applied as a preventive and therapeutic passive Ab treatment to control RV diarrhea.