Guinea Pig Transferrin Receptor 1 Mediates Cellular Entry of Junín Virus and Other Pathogenic New World Arenaviruses.

Several clade B New World arenaviruses (NWAs) can cause severe and often fatal hemorrhagic fever, for which preventive and therapeutic measures are severely limited. These NWAs use human transferrin receptor 1 (hTfR1) as a host cell receptor for virus entry. The most prevalent of the pathogenic NWAs is Junín virus (JUNV), the etiological agent of Argentine hemorrhagic fever. Small animal models of JUNV infection are limited because most laboratory rodent species are refractory to disease. Only guinea pigs are known to develop disease following JUNV infection, but the underlying mechanisms are not well characterized. In the present study, we demonstrate marked susceptibility of Hartley guinea pigs to uniformly lethal disease when challenged with as few as 4 PFU of the Romero strain of JUNV. In vitro, we show that infection of primary guinea pig macrophages results in greater JUNV replication compared to infection of hamster or mouse macrophages. We provide evidence that the guinea pig TfR1 (gpTfR1) is the principal receptor for JUNV, while hamster and mouse orthologs fail to support viral entry/infection of pseudotyped murine leukemia viruses expressing pathogenic NWA glycoproteins or JUNV. Together, our results indicate that gpTfR1 serves as the primary receptor for pathogenic NWAs, enhancing viral infection in guinea pigs.IMPORTANCE JUNV is one of five known NWAs that cause viral hemorrhagic fever in humans. Countermeasures against JUNV infection are limited to immunization with the Candid#1 vaccine and immune plasma, which are available only in Argentina. The gold standard small animal model for JUNV infection is the guinea pig. Here, we demonstrate high sensitivity of this species to severe JUNV infection and identify gpTfR1 as the primary receptor. Use of hTfR1 for host cell entry is a feature shared by pathogenic NWAs. Our results show that expression of gpTfR1 or hTfR1 comparably enhances JUNV virus entry/infectivity. Our findings shed light on JUNV infection in guinea pigs as a model for human disease and suggest that similar pathophysiological mechanisms related to iron sequestration during infection and regulation of TfR1 expression may be shared between humans and guinea pigs. A better understanding of the underlying disease process will guide development of new therapeutic interventions.

Antigenic Pressure on H3N2 Influenza Virus Drift Strains Imposes Constraints on Binding to Sialylated Receptors but Not Phosphorylated Glycans.

H3N2 strains of influenza A virus emerged in humans in 1968 and have continued to circulate, evolving in response to human immune pressure. During this process of "antigenic drift," viruses have progressively lost the ability to agglutinate erythrocytes of various species and to replicate efficiently under the established conditions for amplifying clinical isolates and generating vaccine candidates. We have determined the glycome profiles of chicken and guinea pig erythrocytes to gain insights into reduced agglutination properties displayed by drifted strains and show that both chicken and guinea pig erythrocytes contain complex sialylated N-glycans but that they differ with respect to the extent of branching, core fucosylation, and the abundance of poly-N-acetyllactosamine (PL) [-3Galß1-4GlcNAcß1-]n structures. We also examined binding of the H3N2 viruses using three different glycan microarrays: the synthetic Consortium for Functional Glycomics array; the defined N-glycan array designed to reveal contributions to binding based on sialic acid linkage type, branched structures, and core modifications; and the human lung shotgun glycan microarray. The results demonstrate that H3N2 viruses have progressively lost their capacity to bind nearly all canonical sialylated receptors other than a selection of biantennary structures and PL structures with or without sialic acid. Significantly, all viruses displayed robust binding to nonsialylated high-mannose phosphorylated glycans, even as the recognition of sialylated structures is decreased through antigenic drift.IMPORTANCE Influenza subtype H3N2 viruses have circulated in humans for over 50 years, continuing to cause annual epidemics. Such viruses have undergone antigenic drift in response to immune pressure, reducing the protective effects of preexisting immunity to previously circulating H3N2 strains. The changes in hemagglutinin (HA) affiliated with drift have implications for the receptor binding properties of these viruses, affecting virus replication in the culture systems commonly used to generate and amplify vaccine strains. Therefore, the antigenic properties of the vaccines may not directly reflect those of the circulating strains from which they were derived, compromising vaccine efficacy. In order to reproducibly provide effective vaccines, it will be critical to understand the interrelationships between binding, antigenicity, and replication properties in different growth substrates.

Rough brucella strain RM57 is attenuated and confers protection against Brucella melitensis.

Brucella melitensis (B. melitensis) is a facultative intracellular pathogen, which is the main epidemic strain in China. To overcome disadvantages of traditional live attenuated vaccines, in this study a rough mutant RM57 was induced from a B. melitensis isolate M1981. In order to uncover the reason of changes in the LPS of RM57, the nucleotide sequences and transcription levels of all known genes related to LPS synthesis were detected. As LPS plays an important role in outer membrane integrity, the sensitivities of RM57 to SDS and polymyxin B were examined. The results showed that the expression level of LPS genes of RM57 was not significantly changed, and RM57 was sensitive to polymyxin B, compared to its parent strain. In further study, the virulence and protective efficacy of RM57 in mice and guinea pigs were determined, and our data indicated that RM57 was attenuated and had good protection effects, especially in guinea pigs model. Overall, these results demonstrated that the artificially induced rough mutant strain RM57 was an efficacious vaccine candidate against the challenge of virulent B. melitensis. Our data presented here provided additional insight into the mechanism of LPS synthesis of Brucella spp.

Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

Antigenic differences between vaccine and circulating wild-type mumps viruses decreases neutralization capacity of vaccine-induced antibodies.

A recent resurgence of mumps in doubly vaccinated cohorts has been observed, identifying genotype G as the current predominant genotype. In this study, the neutralization efficacy of guinea pig sera immunized with three vaccine viruses: L-Zagreb, Urabe AM9 and JL5, was tested against seven mumps viruses: three vaccine strains and four wild-type strains (two of genotype G, one of genotype C, one of genotype D) isolated during 1998-2011. All sera neutralized all viruses although at different levels. The neutralization efficiency of sera decreases several fold by temporal order of virus isolation. Therefore, we concluded that gradual evolution of mumps viruses, rather than belonging to a certain genotype, results in an antigenic divergence from the vaccine strains that decrease the neutralization capacity of vaccine-induced antibodies. Moreover, the amino-acid sequence alignment revealed three new potentially relevant regions for escape from neutralization, i.e. 113-130, 375-403 and 440-443.

Evaluation of two new recombinant guinea-pig lipocalins, Cav p 2 and Cav p 3, in the diagnosis of guinea-pig allergy.

BACKGROUND: Guinea-pigs, classical laboratory animals now often kept as pets, regularly elicit important allergic reactions. Two guinea-pig allergens have been described previously to some extent; however, biomolecular and immunological data are lacking. OBJECTIVE: To identify major guinea-pig allergens, produce them as recombinant proteins and define their allergenic potential and clinical importance in allergic patients. METHODS: Protein extracts were prepared from various guinea-pig tissues and allergens were purified by ion exchange chromatography. The N-termini of two immunoglobulin E (IgE)-reactive proteins were determined and degenerate primers were designed for cDNA amplification by RT-PCR. Allergenic properties of recombinant proteins were assayed by immunoblotting, ELISA and mediator release assays. Specific IgE were quantified in the sera of 26 guinea-pig-allergic patients. RESULTS: Major IgE-reactive proteins were detected in submaxillary and harderian gland extracts. Two proteins were identified and the cDNAs were cloned. The 17 kDa protein expressed in the harderian gland corresponds to the previously described Cav p 2. The 19 kDa protein, Cav p 3, is a new allergen expressed in the submaxillary gland. Recombinant Cav p 2 and Cav p 3 were recognized by IgE antibodies from 65% and 54% of guinea-pig-allergic patients, respectively. Both proteins demonstrated equivalent allergenic activity in the mediator release assays. CONCLUSION AND CLINICAL RELEVANCE: Two major guinea-pig allergens, Cav p 2 and Cav p 3, have been characterized and produced as recombinant proteins. Combined to guinea-pig serum albumin, the new allergens proved to be valuable in the component-resolved diagnosis of guinea-pig allergy.

Specific immune response genes of the guinea pig. 3. Linkage of the GA and GT immune response genes to histocompatibility genotypes in inbred guinea pigs.

The ability of guinea pigs to form immune responses specific for each of the random copolymers, L-glutamic acid and L-alanine (GA) and L-glutamic acid and L-tyrosine (GT), is under the control of distinct autosomal dominant genes. By testing for the ability to respond to these copolymers among the progeny from the reciprocal backcross mating of responder (2 x 13)F(1) animals with the appropriate nonresponder parental strain, we have demonstrated that different unigenic autosomal dominant traits control the ability to respond to GA and GT respectively. The data further shows that the GA gene is linked to the poly-L-lysine (PLL) gene and to the locus determining the major strain 2 histocompatibility specificities and that the GT gene is linked to the locus controlling the expression of major strain 13 histocompatibility specificities. Analysis of the inheritance of the GT and PLL genes among the offspring from a mating of responder (2 x 13)F(1) guinea pigs with random-bred guinea pigs unable to respond to GT or PLL demonstrate that these genes segregate away from each other. Thus, the PLL gene and the genes to which it is linked, the GA gene and the major strain 2 histocompatibility locus, behave as alleles or pseudoalleles to the GT gene and the major strain 13 histocompatibility locus.

Histocompatibility specificity.

The lymph node cells from all L-glutamic acid and L-tyrosine (GT) responder random-bred guinea pigs were susceptible to lysis by strain 2 anti-strain 13 isoantisera in the presence of complement. These same antisera were cytolytic for lymph node cells of only some of the GT nonresponder animals. However, after absorption with cells, from a nonresponder guinea pig, susceptible to lysis, the anti-strain 13 antisera were no longer able to lyse cells from any GT nonresponder guinea pigs while retaining a large measure of their cytolytic activity for cells of all GT responder guinea pigs. Thus, at least two major strain 13 histocompatibility specificities are expressed on the cells of random-bred guinea pigs. The genetic locus controlling the expression of only one of those strain 13 histocompatibility specificities is linked to the GT immune response gene.

The guinea pig I region. II. Functional analysis.

We have examined whether an association exists between specific Ia antigen genes and Ir genes which are encoded within the same haplotype. Functionally monospecific sera to the Ia antigens of the guinea pig MHC were selective in their ability to inhibit antigen-specific T-cell proliferation and we were thus able to demonstrate an association between individual Ia specificities and specific Ir genes. The results of these studies in inbred animals were confirmed by examining the association of Ir genes and Ia antigens in the outbred guinea pig population. Of great interest was the observation that antisera made against cross-reactive Ia antigens of strains lacking specific Ir genes would still inhibit immune responses of strains possessing the Ir gene, if the Ir gene was associated with that Ia antigen in the responder strain.

The guinea pig I region. I. A structural and genetic analysis.

The Ia antigens of the guinea pig have been shown to play a central role in the regulation of the immune response. We have previously partially characterized the chemical structure of these antigens. In this communication, we further characterize the structure of the five Ia antigens already described, as well as two new Ia antigens. Evidence is presented which shows that these seven Ia antigens can be organized into three distinct groups, each with a characteristic structure. The Ia.2 determinant of strain 2 and the Ia.3 and Ia.5 determinants of strain 13 animals are found on molecules composed of a 25,000 dalton chain and a 33,000 dalton chain in noncovalent association, or else are individually expressed on nonlinked 33,000 and 25,000 dalton molecules. The Ia.4 and Ia.5 determinants of strain 2 and the Ia.7 determinant of strain 13 are borne on 58,000 dalton molecules in which two chains are linked by disulfide bonds. The Ia.1 and Ia.6 determinants of strain 13 are found on a molecule of 26,000-27,000 daltons. Ia.6 of strain 2 has yet to be definitively assigned. Furthermore, in strain 13 animals the Ia.3 and Ia.5 determinants are borne on the same molecule, as are the Ia.1 and Ia.6 determinants. In strain 2 animals, the Ia.4 and Ia.5 determinants are found on the same molecule. On the basis of chemical structure, we have divided the guinea pig I region into three subregions. The accompanying paper presents evidence of associations between particular Ia genes and Ir genes.

Structural characteristics of the alloantigens determined by the major histocompatibility complex of the guinea pig.

The GPLA B and Ia (I region-associated) antigens are the products of genes found in the guinea pig major histocompatibility complex. Because of their importance in immune response phenomena, a structural study of these antigens was undertaken. [3H]leucine and [3H]fucose were internally incorporated into guinea pig lymph node cells. The GPLA B and Ia antigens were solubilized by the nonionic detergent Nonidet P-40, purified by affinity chromatography using an adsorbent column of lentil lectin, isolated by immunoprecipitation, and examined by discontinuous polyacrylamide-sodium dodecyl sulfate gel electrophoresis. The GPLA antigens B.1, B.2, B.3, and B.4, were shown to be glycoproteins of mol wt 40,000 daltons and to be noncovalently associated with a 12,000 dalton protein. The molecules bearing B.2 and B.3 in a B.2/B.3 heterozygote are shown to be separable, suggesting the antigenic determinant is a primary gene product. In addition, a new GPLA determinant, S, which resembles the B antigen in that it is found on a molecule of approximately 40,000 daltons, was studied. In a B.2/B.3 S+ animal the molecule bearing antigen S was shown to be independent of those bearing B.2 and B.3, providing evidence that the genes determining B and S are at separate loci. The Ia-bearing molecules identified by anti-Ia.2,4 are glycoproteins of mol wt 58,000 daltons which are composed of two subunits of 33,000 and 25,000 daltons, respectively, linked by disulfide bonds. The Ia-bearing molecules are independent of GPLA-bearing molecules, indicating different loci determining these antigens. By all criteria, the guinea pig GPLA B antigens appear homologous to the murine H-2D and H-2K antigens, while the guinea pig Ia antigens appear homologous to the Ia antigens of the mouse.

Regulation of delayed hypersensitivity. Failure to transfer delayed hypersensitivity to desensitized guinea pigs.

Two potential mechanisms for terminating delayed hypersensitivity (DH) reactions have been examined in desensitized guinea pigs. Lack of macrophage responsiveness to lymphokines was sought as an explanation for the reduced ability of these animals to express delayed hypersensitivity. Skin-reactive factor was injected into the skin of desensitized guinea pigs and a control group of similarly immunized animals. The resulting inflammatory reactions were similar in size and intensity in both groups indicating normal macrophage responsiveness in the desensitized state. Passive cellular transfer of DH responses to desensitized animals was markedly less successful than transfer to normal animals. However, cells from desensitized guinea pigs did transfer DH responsiveness to normal animals. These data support the concept of a humoral suppressant of cellular immunity, perhaps acting as a feedback inhibitor, produced when guinea pigs are desensitized.

Idiotype expression and the inheritance of mouse antibody clones.

The inheritance of idiotypes was investigated using idiotypic antisera against two monoclonal antibodies to streptococcal carbohydrates derived from A/J mice. Each of the two idiotypes was characterized by a special frequency of expression. One of the idiotypes was expressed in more than 80% of A/J mice, the other in less than 20%. The idiotypes of both antibodies were strain specific and were transmitted to (A/J x BALB/cJ)F(1) hybrid mice. Furthermore, both idiotypes remained associated with the A/J heavy chain C region allotype in (A/J x BALB/cJ)F(1) hybrid mice. The results suggest that idiotypes are specified by allelic V genes, and that the heavy chain idiotype locus is linked to the heavy chain allotype locus.

Autogenous immunity to endogenous RNA tumor virus. Radioimmune precipitation assay of mouse serum antibody levels.

The radioimmune precipitation assay using (3)H-labeled AKR leukemia virus was applied to the detection and quantitation of natural serum antibodies directed against endogenous murine leukemia virus (MuLV) envelope antigens B6C3F(1) and BALB/c mice, which have low natural incidences of leukemia and lymphoma, and AKR mice, which have a high incidence, were used in this study. Sera from mice of various age groups were assayed. A marked difference in age-associated levels of the autogenous immune response to endogenous murine leukemia virus was detected, and the quantitative differences among these strains were inversely related to the incidence of lymphoma. The radioimmune precipitation test as applied was 500 times more sensitive than virus neutralization. That the reactions we have observed are specific is suggested by several lines of evidence, including the nonreactivity of normal hamster and absorbed rat serum, the positive reaction of absorbed rat anti-AKR serum, the inhibition of precipitation of labeled virus by purified unlabeled virus, and isopycnic gradient analysis of the reactive products.

Variation in susceptibility of a human lymphoid cell line to immune lysis during the cell cycle. Lack of correlation with antigen density and complement binding.

Cultured human lymphoid cells RPMI 8866 at different stages of their growth cycle vary in their susceptibility to lysis by rabbit, human, and guinea pig complement activated by HL-A antibodies or heterologous antibodies directed to membrane antigens; cells in G(1) phase are the least sensitive to lysis. To investigate the cause of differential susceptibility of cells RPMI 8866 to lysis, the expression of HL-A determinants and the ability of cells to react with complement were investigated. No change was detected in the density of HL-A antigens on RPMI 8866 cells in synchronous growth as determined by quantitative microabsorption assays, isotopic antiglobulin tests and yields of soluble HL-A antigens. Cells did not vary during the growth cycle in their ability to interact with complement components and in their capacity to activate the complement system through the classical or alternate pathway. These data suggest that variability in lytic susceptibility is due to changes in the structure of the cell membrane or in its ability to repair complement induced damage at certain intervals during the cell cycle. Therefore, this cell line constitutes a useful model to investigate the final steps of the cytolytic reaction.

Events after the binding of antigen to lymphocytes: removal and regeneration of the antigen receptor.

The behavior of the immunoglobulin antigen receptor on lymphocytes was studied using both fluorescent antiimmunoglobulin antibody to detect B cells and autoradiography with radiolabeled antigens to detect antigen-binding cells. It was shown that after binding of antiimmunoglobulin antibody to the lymphocyte there was a rapid loss of surface immunoglobulin and then a progressive reappearance over 18 h. This could be quantitated using an inhibition assay for surface immunoglobulin. Similarly, after binding various dinitrophenyl-conjugated proteins or keyhole limpet hemocyanin to their specific antigen-binding cells, there was a loss of the antigen receptor from the surface and then a progressive reappearance of the receptor. The reappearance of surface immunoglobulin and of the antigen receptor proceeded at about the same rate. Repeated exposure to antibody or prolonged exposure to antigen did not diminish the capacity of the lymphocyte to re-express its receptor. These events, which follow the interaction of antigen and its receptor, are of possible importance in understanding the mechanism of triggering of the immune response and of tolerance.

The immune response against myelin basic protein in two strains of rat with different genetic capacity to develop experimental allergic encephalomyelitis.

After challenge with guiena pig basic protein (GPBP) Lewis (Le) rats, which are homozygous for the immune response experimental allergic encephalomyelitis (Ir-EAE) gene, developed positive delayed skin tests against GPBP and the 43 residue encephalitogenic fragment (EF); in addition, Le rat lymph node cells (LNC) were stimulated and produced migration inhibitory factor (MIF) when incubated in vitro with these antigens. In contrast Brown Norway (BN) rats, which lack the Ir-EAE gene, did not develop delayed skin tests to EF and their LNC were not stimulated and did not produce MIF when incubated in vitro with EF. These observations indicate that the Ir-EAE gene controls a T-cell response against the EF. Le rats produced measurable anti-BP antibody by radioimmunoassay after primary challenge. Although no antibody was detectable in BN rats by radioimmunoassay, radioimmunoelectrophoresis indicated that a small amount of antibody was formed after primary immunization. After boosting intraperitoneally, both strains of rat exhibited a rise in anti-BP antibody; which was greater in Le rats. In both strains of rat the anti-BP antibody reacted with a portion of the molecule other than the EF. Since EF primarily evokes a T cell response, it is suggested that the EF portion of the BP molecule may contain a helper determinant in antibody production.

The glomerular mesangium. 3. Acute immune mesangial injury: a new model of glomerulonephritis.

A mechanism of immune glomerular injury is described based on the fixation of antibody (Ab) to an antigen (Ag) that has localized in the glomerular mesangium. Rabbits were given, intravenously (i.v.), aggregated human IgG (AHIgG) or albumin (AHSA) and 10 h later, when the Ag by immunofluorescent microscopy was present in the mesangium, a kidney was removed and transplanted into a normal rabbit. The recipient then received, i.v., rabbit anti-HIgG or anti-HSA. Within minutes of Ab infusion, glomeruli of the donor kidney had polymorphonuclear (PMN) infiltration that over the next few hours became marked and was associated with glomerular cell swelling. At 24 h a decrease in PMN's and early mesangial proliferation was seen. By 3 days there was marked mesangial hypercellularity and increased mesangial matrix. Within minutes after Ab administration rabbit IgG, C3, and fibrin were seen in the glomerular mesangium. There was a fall in complement titer by 1 min after Ab infusion that was due to complement consumption by the donor kidney. Complement then returned to normal levels by 48 h. Significant glomerular injury did not occur (a) in the recipient's own kidney, (b) from Ag administration and transplantation without recipient Ab administration, or (c) from transplantation and Ab administration without prior Ag administration. These studies demonstrated that Ag localized in the glomerular mesangium can react with circulating Ab and complement resulting in severe glomerular injury.

Antibody-induced movement of membrane components of Leishmania enriettii.

Incubation in vitro of viable Leishmania enriettii with antibodies from infected or immune guinea pigs and a fluorescein-labeled antiguinea pig Ig conjugate induced aggregation of surface antigens to form a "cap" over the anterior pole of the amastigote and over both the anterior and posterior poles of the promastigote form of the parasite. Cap formation occurred only with optimum quantities of guinea pig antibodies and was inhibited by low temperature and the metabolic inhibitors, sodium azide and iodoacetamide. The aggregated antigens were rapidly lost from the surface of the parasite but reappeared after 3 h of incubation at 23 degrees C. This phenomenon of ligand-induced membrane antigen movement is apparently similar to that described in mammalian cells, and may represent the first stage of the interaction between host antibodies and the surface membrane of protozoal parasites.

Interactions of C-reactive protein with the complement system. I. Protamine-induced consumption of complement in acute phase sera.

Protamine sulfate was found to consume large amounts of C selectively during preincubation with sera of individuals in the "acute phase". Marked depletion of C1, C4, and C2 with minimal, if any, depletion of C3-9, was observed. The consumption was time and temperature dependent, occurring most rapidly and extensively at 37 degrees C, 0.10 M relative salt concentration and pH 7.5-8.0; it required calcium ions. It was mediated by a heat-stable nondialyzable factor which separated with C-reactive protein (CRP) during fractionation and purification, correlated with serum CRP levels, and, like other known reactivities of CRP, was inhibited by phosphoryl choline. Preparations of CRP purified either from serum or ascites resulted in consumption of large amounts of C1, C4, and C2 when preincubated with normal serum and protamine. We conclude that CRP is a potent activator of the C system at the level of C1, and that polycations such as protamine sulfate are substrates of CRP which can bring about this activation. It seems not unlikely that one role of CRP in health and disease involves its ability to interact with the C system.