Molecular epidemiology of Shigella flexneri in a diarrhoea-endemic area of Lima, Peru.

A year-long community-based study of diarrhoeal diseases was conducted in Canto Grande, a periurban community in Lima, Peru. In 109 (34%) houses out of 323 that were visited, at least one individual was detected with shigellosis. The frequency of the 161 shigella isolates obtained was as follows: 117 S. flexneri (73%), 21 S. boydii (13%), 15 S. dysenteriae (9%), and 8 S. sonnei (5%). Using a non-radioactive ipaH gene probe as a molecular epidemiological tool, a total of 41 S. flexneri strains were shown to be distributed in 25 intra-family comparisons by pairs (icp). Further subdivision, based on a comparison of the serotype, plasmid profile, antibiotic resistances and ipaH hybridization patterns indicated that Group I, with 11 icp (44%), had strains that were identical. Group II with 8 icp (32%), had strains that were different and Group III with 6 icp (24%), had strains with the same serotype and identical ipaH profiles but with differences in other markers. This data indicates that a diversity of shigella clones circulated in this community resulting from both clonal spread and horizontal transfer of genetic elements. Furthermore, ipaH profiling of isolates can be used not only to differentiate between closely related shigella strains but also with other parameters, help to understand the dynamics of the generation of new clones of pathogenic bacteria.

Effects of buprenorphine on immunogenicity and protective efficacy in the guinea pig keratoconjunctivitis model (Sereny test).

Shigellosis is a disease of global proportions, with an estimated 164.7 million episodes annually throughout the world as well as an estimated 1.1 million associated mortalities in developing countries. Due to increasing incidence, and continued emergence of multi-drug resistant strains, Shigella vaccine development is considered a top public health priority. The guinea pig keratoconjunctivitis model, the basis for the Sereny test, remains the most reliable in vivo indicator of virulence of Shigella strains and immunogenicity and protective efficacy of Shigella vaccine candidates. The model is effective in evaluating the ability of Shigella strains to invade the corneal epithelia of guinea pigs and spread to contiguous cells, with the more virulent strains causing ulcerative keratoconjunctivitis. However, analgesia is not routinely used to relieve this painful condition because of potential immunomodulation and confounding of experimental results. The objective of the study reported here was to evaluate use of buprenorphine hydrochloride as an analgesic during the Sereny test. Local and systemic immune responses were measured in guinea pigs given buprenorphine versus those responses in controls. Results of this study suggest that buprenorphine, administered at an analgesic dose of 0.05 mg/kg of body weight twice daily, can be successfully used with the model without significantly affecting immunologic evaluation of Shigella vaccine candidates. However, in buprenorphine-treated animals, there was a significant increase in the amount of mucopurulent ocular discharge, requiring frequent cleaning of the affected eyes. Additionally, animals treated with buprenorphine had significant reduction in body weight, in comparison with saline controls.

Isolation and characterization of a Shigella flexneri invasin complex subunit vaccine.

The invasiveness and virulence of Shigella spp. are largely due to the expression of plasmid-encoded virulence factors, among which are the invasion plasmid antigens (Ipa proteins). After infection, the host immune response is directed primarily against lipopolysaccharide (LPS) and the virulence proteins (IpaB, IpaC, and IpaD). Recent observations have indicated that the Ipa proteins (IpaB, IpaC, and possibly IpaD) form a multiprotein complex capable of inducing the phagocytic event which internalizes the bacterium. We have isolated a complex of invasins and LPS from water-extractable antigens of virulent shigellae by ion-exchange chromatography. Western blot analysis of the complex indicates that all of the major virulence antigens of Shigella, including IpaB, IpaC, and IpaD, and LPS are components of this macromolecular complex. Mice or guinea pigs immunized intranasally with purified invasin complex (invaplex), without any additional adjuvant, mounted a significant immunoglobulin G (IgG) and IgA antibody response against the Shigella virulence antigens and LPS. The virulence-specific response was very similar to that previously noted in primates infected with shigellae. Guinea pigs (keratoconjunctivitis model) or mice (lethal lung model) immunized intranasally on days 0, 14, and 28 and challenged 3 weeks later with virulent shigellae were protected from disease (P<0.01 for both animal models).

Shigella flexneri IpaH(7.8) facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages.

The behavior of Shigella flexneri ipaH mutants was studied in human monocyte-derived macrophages (HMDM), in 1-day-old human monocytes, and in J774 mouse macrophage cell line. In HMDM, strain pWR700, an ipaH(7.8) deletion mutant of S. flexneri 2a strain 2457T, behaved like the wild-type strain 2457T. This strain caused rapid host cell death by oncosis, and few bacterial CFU were recovered after incubation in the presence of gentamicin as previously described for 2457T-infected HMDM. However, analysis of bacterial compartmentalization within endocytic vacuoles with gentamicin and chloroquine indicated that more pWR700 than 2457T was present within the endocytic vacuoles of HMDM, suggesting that ipaH(7.8) deletion mutant transited more slowly from the vacuoles to the cytoplasm. In contrast to findings with HMDM, CFU recovered from pWR700-infected mouse J774 cells were 2 to 3 logs higher than CFU from 2457T-infected J774 cells. These values exceeded CFU recovered after infection of J774 cells with plasmid-cured avirulent strain M4243A1. Incubation with gentamicin and chloroquine clearly showed that pWR700 within J774 cells was mostly present within the endocytic vacuoles. This distribution pattern was similar to that seen with M4243A1 and contrasted with the pattern seen with 2457T. Complementation of pWR700 with a recombinant clone expressing ipaH(7. 8) restored the intracellular distribution of bacteria to that seen with the wild-type strain. Strains with deletions in ipaH(4.5) or ipaH(9.8), however, behaved like 2457T in both HMDM and J774 cells. The distribution profile of pWR700 in 1-day-old monocytes was similar to that seen in J774 cells. Like infected J774 cells, 1-day-old human monocytes demonstrated apoptosis upon infection with virulent Shigella. These results suggest that a role of the ipaH(7. 8) gene product is to facilitate the escape of the virulent bacteria from the phagocytic vacuole of monocytes and macrophages.

Native and mutant forms of cholera toxin and heat-labile enterotoxin effectively enhance protective efficacy of live attenuated and heat-killed Shigella vaccines.

Both native and mutant forms of cholera toxin (CT) and heat-labile enterotoxin (LT) are effective adjuvants for antigens and killed whole-cell preparations. To determine whether these toxin molecules could also boost the immunogenicity and efficacy of live attenuated vaccines directed against shigellosis, the guinea pig keratoconjunctivitis model was used to evaluate the adjuvant effect of these toxin molecules on EcSf2a-3, a DeltavirG DeltaaroD Escherichia coli-Shigella flexneri 2a hybrid vaccine strain that was previously found to be less protective than its parent strain in the guinea pig model. Experiments using native and mutant toxin molecules showed that both CT and LT and mutant derivatives were effective as an adjuvant for EcSf2a-3 and that the mutant toxin molecules, which were developed to retain adjuvanticity without the toxicity associated with the native molecules, were as effective as the native toxin molecules as adjuvants. Protective efficacy was enhanced for both the oral and intranasal routes of immunization. Serum antibody response to the S. flexneri 2a O antigen, the primary antigen for protective immunity, was not dependent on the addition of an adjuvant. However, enumeration of the O-antigen-specific immunoglobulin G (IgG) and IgA antibody-secreting cells in the spleen and draining lymph nodes following intranasal immunization suggested that enhancement of the local immune response by the toxin molecules may contribute to the observed increase in protective efficacy. The efficacy of heat-killed S. flexneri 2a was enhanced only by mutant LT molecules. These results suggest that the best candidates for enhancing the efficacy of both live attenuated and heat-killed Shigella vaccines with minimal reactogenicity are the mutant toxin molecules.

Vaccination against shigellosis with attenuated Shigella flexneri 2a strain SC602.

The Shigella flexneri 2a SC602 vaccine candidate carries deletions of the plasmid-borne virulence gene icsA (mediating intra- and intercellular spread) and the chromosomal locus iuc (encoding aerobactin) (S. Barzu, A. Fontaine, P. J. Sansonetti, and A. Phalipon, Infect. Immun. 64:1190-1196, 1996). Dose selection studies showed that SC602 causes shigellosis in a majority of volunteers when 3 x 10(8) or 2 x 10(6) CFU are ingested. In contrast, a dose of 10(4) CFU was associated with transient fever or mild diarrhea in 2 of 15 volunteers. All volunteers receiving single doses of >/=10(4) CFU excreted S. flexneri 2a, and this colonization induced significant antibody-secreting cell and enzyme-linked immunosorbent assay responses against S. flexneri 2a lipopolysaccharide in two-thirds of the vaccinees. Seven volunteers who had been vaccinated 8 weeks earlier with a single dose of 10(4) CFU and 7 control subjects were challenged with 2 x 10(3) CFU of virulent S. flexneri 2a organisms. Six of the control volunteers developed shigellosis with fever and severe diarrhea or dysentery, while none of the vaccinees had fever, dysentery, or severe symptoms (P = 0. 005). Three vaccinees experienced mild diarrhea, and these subjects had lower antibody titers than did the fully protected volunteers. Although the apparent window of safety is narrow, SC602 is the first example of an attenuated S. flexneri 2a candidate vaccine that provides protection against shigellosis in a stringent, human challenge model.

Construction of a stable attenuated Shigella sonnei DeltavirG vaccine strain, WRSS1, and protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model.

Construction of a stable Shigella sonnei vaccine has been complicated by the instability of the virulence phenotype caused by the spontaneous loss of the invasion plasmid. To select a suitable candidate for vaccine construction, 16 S. sonnei strains were screened for stability of the virulence phenotype. A stable strain, S. sonnei Mosely, was selected for further work. pDeltavirG2, a deletion derivative of the virG gene in the sacB suicide vector pCVD442, was used to generate an S. sonnei virG deletion strain, WRSS1, which was invasive in HeLa cells but negative in the Sereny test. WRSS1 was found to be both immunogenic and protective in the guinea pig keratoconjunctivitis model.

Identification and molecular characterization of a 27 kDa Shigella flexneri invasion plasmid antigen, IpaJ.

Shigella species and enteroinvasive Escherichia coli contain a core set of virulence genes whose coordinated expression results in the invasion of host colonic epithelial cells and the dysenteric syndrome. A number of virulence determinants are carried by the 230 kb invasion plasmid found in all virulent strains of Shigellae. Many of these invasion plasmid genes encode immunogens that are recognized by convalescent serum, including proteins that mediate the invasion (IpaB, IpaC, IpaD) and cell spreading (VirG or IcsA and IcsB) phenotypes. In this report, we describe the molecular characterization of a novel invasion plasmid antigen from Shigella flexneri, designated IpaJ. The ipaJ gene encodes a 780 bp open reading frame (ORF), separated from the ipaR (virB) stop codon by 944 bp. The predicted amino acid sequence for IpaJ revealed a consensus signal peptide for protein export. TnphoA mutagenesis of the ipaJ ORF confirmed the presence of export signal sequences in IpaJ. Unlike ipaBCDA genes, transcription analysis of ipaJ indicated that the gene is not expressed in a temperature-dependent fashion. The IpaJ protein was expressed and purified as a His6-tagged fusion protein that reacted with convalescent sera in Western blot analyses, confirming its identification as a Shigella immunogen. Construction and phenotypic characterization of ipaJ mutants in two serotypes of S. flexneri showed that the mutants were not compromised in their ability to invade cultured epithelial cells or to form plaques on BHK cell monolayers. In addition, the ipaJ mutants were Sereny positive indicating a capacity for intercellular dissemination; however, in the limited number of guinea-pigs tested, the keratoconjunctivitis reaction appeared attenuated.

Outer membrane protein of Neisseria meningitidis as a mucosal adjuvant for lipopolysaccharide of Brucella melitensis in mouse and guinea pig intranasal immunization models.

A mucosal vaccine against brucellosis consisting of the lipopolysaccharide (LPS) of Brucella melitensis complexed with the outer membrane protein (GBOMP) of group B Neisseria meningitidis was tested in small-animal models of intranasal immunization. Mice given two doses of the vaccine developed high levels of immunoglobulin G (IgG) and IgA antibodies specific for B. melitensis LPS in lung lavages and specific IgG and IgA antibody-secreting cells in the lungs and spleen. Similarly, in guinea pigs immunized twice intranasally, IgG and IgA LPS-specific antibodies were detected in lung lavages, and specific antibody-secreting cells were isolated from the spleen and cervical nodes. In mice immunized with LPS only, pulmonary responses consisted mostly of IgM antibodies, while guinea pigs given LPS alone developed local antibody of all three isotypes, but at lower levels compared to animals given the complex vaccine. Both mice and guinea pigs also developed high levels of serum IgG and moderate levels of IgA as a result of intranasal immunization with the complex vaccine. The serum antibodies in both cases were found to cross-react with the LPS of B. abortus, which shares an immunogenic epitope with B. melitensis LPS. In mice given the complex vaccine, there was a prominent serum IgG1 response that was absent in the mice given LPS alone. In conclusion, the N. meningitidis GBOMP was an effective mucosal adjuvant for secretory IgA and IgG responses in the lungs of both mice and guinea pigs. The IgG1 subclass response in mice suggests that GBOMP may have favored a Th2 type of response to the LPS. A vaccine capable of stimulating high levels of antibody at local sites has the potential to protect against brucellae, since these pathogens gain entry to the host via mucosal routes.

Specificity of monoclonal antibodies elicited by mucosal infection of BALB/c mice with virulent Shigella flexneri 2a.

Protective immunity against shigellosis is thought to be determined by the O-antigen side chains of the lipopolysaccharide (LPS) molecule. To study possible common protective epitopes, monoclonal antibodies reacting with Shigella flexneri 2a LPS were generated from BALB/c mice infected ocularly with the virulent serotype 2a strain S. flexneri 2457T and tested against a panel of S. flexneri LPSs by enzyme-linked immunosorbent and immunoblot assays. Four monoclonal antibodies were identified, all of which showed restricted specificity patterns. Three different patterns of reactivity to LPS possessing the 3,4 group antigen were seen: (i) 2a only, (ii) 2a and 5a, and (iii) 2a, 4a, 5a, and Y. These results have implications for designing a Shigella vaccine that will be protective against related serotypes. Electron microscopy studies showed that the monoclonal antibodies bind to the bacterial surface in a patchy pattern, suggesting their potential use for examining the LPS distribution on the surface of the bacteria.

Construction and characterization of virG (icsA)-deleted Escherichia coli K12-Shigella flexneri hybrid vaccine strains.

Human challenge studies with EcSf2a-2, an aroD deletion-attenuated Escherichia coli K12-Shigella flexneri hybrid vaccine expressing S. flexneri 2a somatic antigen and the invasive phenotype indicated that, at doses of 2 x 10(9) bacteria, EcSf2a-2 was immunogenic but also reactogenic and therefore not sufficiently attenuated. Two factors that may contribute to the residual reactogenicity are the spontaneous appearance of plaque-positive variants in the E. coli K12 recipient and the presence of the arg locus encoding enterotoxin or cytotoxin, transferred from S. flexneri 2a into the E. coli recipient. EcSf2a-3 was derived from EcSf2a-2 by introducing a deletion in the virG gene, whose expression is required for plaque formation and keratoconjunctivitis in guinea pigs. EcSf2a-5 contains the same deletion in the E. coli-S. flexneri hybrid strain, 7921, but does not contain the arg locus. Lack of virG expression in these hybrid strains did not affect the immune response to LPS or the development of protective immunity in the guinea pig model.

Cross-reactivity of Shigella flexneri serotype 2a O antigen antibodies following immunization or infection.

To study the cross-reactivity pattern of Shigella flexneri 2a O-antigen antibodies, sera from humans and monkeys challenged with S. flexneri 2a, and from humans and guinea pigs immunized with a recombinant vaccine expressing serotype 2a O-antigen, were tested against a panel of lipopolysaccharide extracted from heterologous S. flexneri. Sera from the two groups of humans, who were volunteers in either a clinical challenge or vaccination study, showed similar patterns: cross-reactivity was more often seen with IgA antibodies, and these were mostly cross-reactive with serotype 2b, which shares the type II antigen, and serotypes 1a, 5a, and Y, which share 4 or 3, 4 group antigen, with 2a. The majority of sera from immunized guinea pigs showed both IgG and IgA cross-reactivity with 1a, 5a, and Y, but not 2b. The majority of sera from challenged monkeys showed cross-reactivity with almost all flexneri serotypes tested, with 1a, 2b, and Y being recognized most often, and the cross-reactive antibodies were more often IgG than IgA. These results show that either immunization or challenge with the 2a serotype induces cross-reactive antibodies which recognize similar subsets of heterologous serotypes, and suggest that it may be possible to design multivalent vaccines against S. flexneri.

Genetic polymorphism of the ipaH multicopy antigen gene in Shigella spps. and enteroinvasive Escherichia coli.

The ipaH loci comprise a multicopy antigen gene family unique to Shigella species and enteroinvasive Escherichia coli (EIEC). DNA probes derived from the Shigella flexneri serotype 5 ipaH7.8 gene were used to compare the molecular arrangement of ipaH alleles in a variety of Shigella and EIEC strains. Multiple copies of ipaH-homologous sequences were detected in all invasion plasmids examined. Oligonucleotide probes covering discrete 24 bp segments of the ipaH7.8 gene and sequences flanking the ipaH4.5 (probe H25) and ipaH2.5 (probe H24) loci were used to define the extent of homology among invasion plasmid copies of ipaH in S. flexneri serotypes 1, 2 and 5 and in S. sonnei. IpaH alleles carried by these invasion plasmids were not structurally equivalent and showed sequence divergence at their amino- and carboxy-terminal ends. The H25 probe was shown to correspond to an IS629 sequence genetically linked to the ipaH alleles, while the H24 probe defined a DNA sequence found only in Shigella invasion plasmids. Chromosomal DNA from invasion plasmid-cured S. flexneri and S. sonnei strains hybridized a core ipaH7.8 gene segment, indicating that portions of the ipaH7.8 structural gene were reiterated and contained within the shigellae chromosomes. Based on the specificity of the ipaH7.8 core probe and the detection of ipaH sequences on the invasion plasmids and chromosomes of Shigella strains, three polymorphic groups within a collection of forty S. dysenteriae 1 isolates received by the United States Centers for Disease Control in 1988 were identified using this probe. These results suggest that ipaH restriction fragment length polymorphisms may be useful in genetic lineage and epidemiologic studies of virulent shigellae.

Local immune response and protection in the guinea pig keratoconjunctivitis model following immunization with Shigella vaccines.

This study used the guinea pig keratoconjunctivitis model to examine the importance of route of administration (mucosal versus parenteral), frequency and timing of immunization (primary versus boosting immunization), and form of antigen given (live attenuated vaccine strain versus O-antigen-protein conjugate) on the production of protective immunity against Shigella infection. Since local immune response to the lipopolysaccharide (LPS) O-antigen of Shigella spp. is thought to be important for protection against disease, O-antigen-specific antibody-secreting cells (ASC) in the spleen and regional lymph nodes of immunized animals were measured by using an ELISPOT assay. Results indicated that protective efficacy was associated with a strong O-antigen-specific ASC response, particularly in the superficial ventral cervical lymph nodes draining the conjunctivae. In naive animals, a strong ASC response in the cervical lymph nodes and protection against challenge were detected only in animals that received a mucosal immunization. Protection in these animals was increased by a boosting mucosal immunization. While parenteral immunization alone with an O-antigen-protein conjugate vaccine did not protect naive animals against challenge, a combined parenteral-mucosal regimen elicited enhanced protection without the addition of a boosting immunization. Although O-antigen-specific serum immunoglobulin A titers were significantly higher in animals receiving a mucosal immunization, there was no apparent correlation between levels of serum antibody and protection against disease.

Small-animal model to measure efficacy and immunogenicity of Shigella vaccine strains.

The development of a small-animal model to test the protective efficacy and immunogenicity of a vaccine strain against shigellosis would greatly facilitate the evaluation of potential vaccine candidates. In guinea pigs, the ability of shigellae to invade and multiply within the corneal epithelium, causing keratoconjunctivitis, closely mimics the invasion process in the intestinal epithelium (B. Sereny, Acta Microbiol. Acad. Sci. Hung. 4:367-376, 1957). The serum response of animals recovering from a Shigella keratoconjunctival infection was determined and found to be consistent with that shown by convalescent humans and primates. This model was used to test the efficacy of two vaccine candidates, and the immune response of the guinea pigs to the vaccine strains was examined. Both vaccine strains demonstrated significant protection against challenge by homologous virulent Shigella strains, and the results were comparable with results obtained in trials with monkeys. The guinea pig model also provides a rapid and inexpensive means of evaluating different immunization regimens as well as of testing other variables such as length of protection against disease.

Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG-like family of proteins.

Oligonucleotide primers derived from the ipaH7.8 sequence have been used to determine the boundaries of DNA sequence homology among five ipaH genes on the invasion plasmid (pWR100) of Shigella flexneri 5, strain M9OT-W. The primary structure of IpaH4.5 has been established from DNA sequence analysis. The first 197 amino acids in IpaH7.8 were replaced in IpaH4.5 by a unique set of 251 amino acids, generating two related proteins with variable and conserved sequences. The amino-terminal region of IpaH4.5 displayed an internal repeat structure, also seen in IpaH7.8, characteristic of members of the leucine-rich glycoprotein (LRG) family. The DNA sequences of ipaH2.5 and ipaH1.4 indicate that these genes are truncated versions of ipaH7.8. Western blot analysis of a lambda gt11 ipaH recombinant (W7) subclone demonstrated that the antigenicity of IpaH7.8 resides outside the leucine-rich repetitive region.

Molecular analysis of variant plasmid forms of a bivalent Salmonella typhi-Shigella sonnei vaccine strain.

The Salmonella typhi-Shigella sonnei hybrid vaccine strain 5076-IC was constructed to express the S. sonnei form I antigen, which may play an important role in producing protective immunity. Three clonal variants which existed in preparations of the vaccine could be distinguished phenotypically by lactose utilization, S. sonnei form I antigen expression, and restriction enzyme analysis of large plasmid DNA. Since expression of the form I antigen was lost in two of the clonal variants, genetic instability of the 120-MDa vaccine plasmid appeared to be a potential problem. To examine the molecular basis of this genetic instability, we hybridized large plasmid DNA isolated from the clonal variants to a variety of DNA probes encoding virulence-associated antigens and to Escherichia coli lacZ DNA. Results indicated that DNA rearrangement accompanied by deletions of plasmid material occurred in the vaccine plasmid. In addition, the vaccine plasmid did not contain some S. sonnei genetic material encoding antigenic polypeptides necessary for virulence.

Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri.

A lambda gt11 expression library of Tn5-tagged invasion plasmid pWR110 (from Shigella flexneri serotype 5, strain M90T-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens (J. M. Buysse, C. K. Stover, E. V. Oaks, M. M. Venkatesan, and D. J. Kopecko, J. Bacteriol. 169:2561-2569, 1987). Southern blot analysis of wild-type S. flexneri serotype 5 invasion plasmid DNA (pWR100) digested with various combinations of five restriction enzymes and hybridized with defined ipaH probes showed complex hybridization patterns resulting from multiple copies of the ipaH gene on pWR100. DNA sequence analysis of a 2.9-kilobase (kb) EcoRI fragment directing IpaH antigen synthesis in plasmid recombinant pWR390 revealed an open reading frame coding for a 532-amino-acid protein (60.8 kilodaltons); this size matched well with the estimated size of IpaH determined by Western blot analysis of M90T-W cells and maxicell analysis of Escherichia coli HB101(pWR390) transformants. Examination of the amino acid sequence of IpaH revealed a hydrophilic protein with six evenly spaced 14-residue (L-X2-L-P-X-L-P-X2-L-X2-L) repeat motifs in the amino-terminal end of the molecule. Southern blot analysis of HindIII-digested pWR100 DNA probed with defined segments of the pWR390 2.9-kb insert demonstrated that the multiple band hybridization pattern resulted from repeats of a significant portion of the ipaH structural gene in five distinct HindIII fragments (9.8, 7.8, 4.5, 2.5, and 1.4 kb). Affinity-purified IpaH antibody, used to monitor the expression of the antigen in M90T-W cells grown at 30 and 37 degrees C, showed that IpaH synthesis was not regulated by growth temperature.

Direct quantitative in situ hybridization studies of Ig VH utilization. A comparison between unstimulated B cells from autoimmune and normal mice.

This study examines Ig VH utilization in murine lupus with emphasis on the relative contribution of 3' and 5' gene families. We used in situ hybridization with 35S-labeled ssRNA probes to detect VH expression in individual spleen cells. Cells were taken from unmanipulated animals, and were not stimulated in vitro. This approach allows analysis of VH usage among only those B cells which have undergone activation in vivo, while minimizing the potential for skewing in vitro. We compared usage of the 3' 7183 and Q52 families with the more 5' J558 family in adult NZB, MRL-lpr/lpr, and nonautoimmune NIH Swiss mice. VH utilization in the autoimmune strains was proportionate to VH family size, and was not biased toward the 3' families when compared with the Swiss repertoire. Moreover, 3' skewing did not develop in NZB mice with increasing age. Thus, systemic autoimmunity is not associated with impaired normalization of the adult repertoire away from the 3' bias of early ontogeny. Instead, our data support a stochastic model for VH gene usage in the activated B cells and plasma cells of adult lupus mice.