RNA interference-mediated prevention and therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and is on the increase worldwide. Hepatocellular carcinoma results from chronic liver disease and cirrhosis most commonly associated with chronic hepatitis B (HBV) or hepatitis C (HCV) infection. The highest incidences of HCC are found in China and Africa, where chronic HBV infection is the major risk component. In the United States, Europe and Japan, the significant increase in HCC and HCC-related deaths within the last three decades is mainly attributed to the rise in the number of HCV-infected individuals; smaller increases of HCC are associated with HBV. Given that HCV and HBV infection account for the majority of HCCs, therapeutic and prophylactic approaches to control or eliminate virus infection may prove effective in reducing the occurrence of HCC. Although anti-viral therapies exist for both HBV and HCV infections, they are ineffective for a significant number of patients. In addition, some treatments such as interferon therapy are dose limiting owing to toxic side effects. Clearly, new approaches are needed. RNA interference (RNAi)-based approaches may meet this need and have already shown promising preclinical results in cell culture and animal models. Although this paper focuses on the potential of RNAi as a prophylactic for HCC development, the potential use of RNAi-mediated approaches for HCC therapy will also be discussed.

Engineering of DNA vaccines using molecular adjuvant plasmids.

These studies support the view that additional goals of enhancing DNA vaccine technology will probably be at several levels. The ability to deliver antigens more efficiently to professional APCs is likely to have important implications for our studies of basic principles of immunology. Furthermore, there are simple practical approaches to vaccine enhancement that can be tested with the present group of DNA vaccines. These studies should include the use of cytokine molecular adjuvants as well as possible co-stimulatory molecules. It is expected that the delivery of these "adjuvanted" DNA vaccines will require additional safety evaluation; however, it is clear that studies can be easily designed to address the important safety issues associated with these novel vaccine adjuvants. Overall, the results indicate that further more precise quantitative studies and combination studies examining these additional promising adjuvant candidates are warranted.

DNA vaccines--challenges in delivery.

DNA vaccines are typically comprised of plasmid DNA molecules that encode an antigen(s) derived from a pathogen or tumor cell. Following introduction into a vaccine, cells take up the DNA, where expression and immune presentation of the encoded antigen(s) takes place. DNA can be introduced by viral or bacterial vectors or through uptake of 'naked' or complexed DNA. Vaccination with DNA is a recent technology possessing distinct advantages over traditional vaccines (killed or attenuated pathogens) and the more recently developed subunit vaccines. Unlike most subunit vaccines, DNA vaccines induce both the humoral and cellular arms of the immune response. The stimulation of both arms of the immune system is important not only for the prevention of many diseases including AIDS, but also allows the use of a vaccine for therapeutic purposes. While the traditional attenuated pathogen vaccines are also able to elicit both cellular and humoral immune responses, there is a risk of reversion from the attenuated state to the virulent state. This risk does not exist with DNA vaccines. DNA vaccines can be manufactured and formulated by generic processes. DNA vaccine technology, however, is still in its infancy and much research needs to be done to improve the efficiency with which these vaccines work in humans. While continued efforts toward improving both DNA expression and DNA delivery are equally important for increasing the utility of DNA vaccines, this review will focus both on non-viral delivery of plasmid DNA and delivery methods for the encoded antigen.

IL-12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Th1-type CD4+ T cell-mediated protective immunity against herpes simplex virus-2 challenge.

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-gamma) and chemokines (RANTES and macrophage inflammatory protein-1alpha) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.

Cytokine molecular adjuvants modulate immune responses induced by DNA vaccine constructs for HIV-1 and SIV.

DNA or nucleic acid immunization has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. Moreover, immune responses induced by DNA immunization can be enhanced and modulated by the use of molecular adjuvants. To further engineer the immune response in vivo, we investigated the induction and regulation of immune responses from the codelivery of Thl cytokines (interleukin-2 [IL-2] and IL-12), Th2 cytokines (IL-4 and IL-10), and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes along with a DNA vaccine construct encoding for simian immunodeficiency virus (SIV) gag/pol proteins. We observed that coinjection with IL-2, IL-4, IL-10, and GM-CSF resulted in increased levels of antigen-specific antibodies. In addition, we found that coinjection with cytokine genes drove the immune responses toward a more Thl or Th2 phenotype. We also observed that coadministration of IL-2, IL-12, and GM-CSF genes resulted in a dramatic enhancement of Th proliferation responses. Moreover, coimmunization with IL-12 genes resulted in a dramatic enhancement of antigen-specific cytotoxic T lymphocyte (CTL) responses. These results support the potential utility of molecular adjuvants in DNA vaccine regimens.

Biochemical and genetic characterization of an extracellular protease from Pseudomonas fluorescens CY091.

Pseudomonas fluorescens CY091 cultures produce an extracellular protease with an estimated molecular mass of 50 kDa. Production of this enzyme (designated AprX) was observed in media containing CaCl2 or SrCl2 but not in media containing ZnCl2, MgCl2, or MnCl2. The requirement of Ca2+ (or Sr2+) for enzyme production was concentration dependent, and the optimal concentration for production was determined to be 0.35 mM. Following ammonium sulfate precipitation and ion-exchange chromatography, the AprX in the culture supernatant was purified to near electrophoretic homogeneity. Over 20% of the enzyme activity was retained in the AprX sample which had been heated in boiling water for 10 min, indicating that the enzyme is highly resistant to heat inactivation. The enzyme activity was almost completely inhibited in the presence of 1 mM 1,10-phenanthroline, but only 30% of the activity was inhibited in the presence of 1 mM EGTA. The gene encoding AprX was cloned from the genome of P. fluorescens CY091 by isolating cosmid clones capable of restoring the protease production in a nonproteolytic mutant of strain CY091. The genomic region of strain CY091 containing the aprX gene was located within a 7.3-kb DNA fragment. Analysis of the complete nucleotide sequence of this 7.3-kb fragment revealed the presence of a cluster of genes required for the production of extracellular AprX in P. fluorescens and Escherichia coli. The AprX protein showed 50 to 60% identity in amino acid sequence to the related proteases produced by Pseudomonas aeruginosa and Erwinia chrysanthemi. Two conserved sequence domains possibly associated with Ca2+ and Zn2+ binding were identified. Immediately adjacent to the aprX structural gene, a gene (inh) encoding a putative protease inhibitor and three genes (aprD, aprE, and aprF), possibly required for the transport of AprX, were also identified. The organization of the gene cluster involved in the synthesis and secretion of AprX in P. fluorescens CY091 appears to be somewhat different from that previously demonstrated in P. aeruginosa and E. chrysanthemi.

Identification of gene loci controlling pectate lyase production and soft-rot pathogenicity in Pseudomonas marginalis.

Pseudomonas marginalis is an important postharvest pathogen capable of causing soft rot in a wide variety of harvested fruits and vegetables. Following transposon mutagenesis, we isolated two groups of P. marginalis CY091 mutants deficient in production of pectate lyase (Pel) and soft-rot pathogenicity in plants. The first group, designated Pel-, was caused by the insertion of Tn5 into a pel structural gene, and the second group, designated LemA-, was caused by the insertion of Tn5 into a regulatory locus corresponding to the lemA gene previously identified in other Gram-negative bacteria. The LemA- mutants also exhibited alteration in colony morphology and showed deficiency in production of protease (Prt). A cosmid clone pCIC carrying the P. marginalis lemA gene was isolated and characterized. pCIC was capable of restoring Pel production and soft-rot pathogenicity in LemA- mutants of P. marginalis and Pseudomonas viridiflava, indicating that the function of lemA gene in these two pseudomonads was similar and interchangeable. Using MudI-mediated mutagenesis, we isolated a third group of P. marginalis mutants deficient in production of Pel, Prt, and soft-rot pathogenicity. Mutants in this group (designated GacA-1) contained an insertion of MudI in a locus corresponding to the gacA gene of P. viridiflava. Like LemA- mutants, GacA- mutants also exhibited alteration in colony morphology and showed deficiency in production of Pel and Prt. However, GacA- mutants produced much lower levels of levan and fluorescent pyoverdine siderophore than the wild type and LemA- mutants. These results provide the first genetic evidence that P. marginalis produces a single alkaline Pel for maceration of plant tissue and demonstrate that production of Pel, Prt, levan, and pyoverdin by this bacterium is mediated by the two-component lemA/gacA gene system.

Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination.

Novel approaches for the generation of more effective vaccines for HIV-1 are of significant importance. In this report we analyze the immunogenicity and efficacy of an HIV-1 DNA vaccine encoding env, rev and gag/pol in a chimpanzee model system. The immunized animals developed specific cellular and humoral immune responses. Animals were challenged with a heterologous chimpanzee titered stock of HIV-1 SF2 virus and followed for 48 weeks after challenge. Polymerase chain reaction coupled with reverse transcription (RT-PCR) results indicated infection in the control animal, whereas those animals vaccinated with the DNA constructs were protected from the establishment of infection. These studies serve as an important benchmark for the use of DNA vaccine technology for the production of protective immune responses.

The repB gene required for production of extracellular enzymes and fluorescent siderophores in Pseudomonas viridiflava is an analog of the gacA gene of Pseudomonas syringae.

Two genes, designated repA and repB, are involved in the regulation of the synthesis of extracellular pectate lyase, protease, and alginate in Pseudomonas viridiflava. The repA gene has been shown to encode a protein highly homologous to several bacterial sensors in the two-component regulator family including the LemA of Pseudomonas syringae. In this study, the repB locus, initially identified in a 6.3-kb EcoRI genomic fragment of P. viridiflava, was further characterized. Results obtained from restriction mapping, deletion subclonings, and mini-Mu-LacZ fusions indicated that the repB gene was contained within a 0.8-kb HindIII-PstI region. Sequence analysis of this repB region revealed the presence of an open reading frame, which was predicted to encode a protein similar or identical to the gacA response regulator found in P. syringae and Pseudomonas fluorescens. The repB gene of P. viridiflava also regulated the production of fluorescent siderophores, in addition to the aforementioned extracellular enzymes and alginate. The repB or gacA homologs were detected in the genomes of nine other strains of P. viridiflava, P. fluorescens, and P. syringae included in the study. The data presented here and earlier indicate that the repA/repB gene regulatory system of P. viridiflava is analogous to the lemA/gacA system of P. syringae and P. fluorescens.

DNA inoculation induces cross clade anti-HIV-1 responses.

Nucleic acid or DNA immunization represents a novel approach to vaccine and immune therapeutic development. The direct injection of expression cassettes into a living host results in in vivo gene expression and immune activation. In the case of HIV-1 it has been shown by our laboratory that facilitated injection mimicks aspects of live attenuated vaccines and that both humoral and cellular responses can be induced upon injection of a nucleic acid sequence directly into a host target tissue. Antisera from HIV-1 plasmid expression cassette-immunized animals contain anti-HIV envelope glycoprotein immune responses. The antiserum neutralizes HIV-1 infection and inhibits cell to cell infection in vitro. Cellular immune responses have also been evaluated. We observed both T cell proliferation and isotype switching consistent with the production of relevant T helper immune responses in immunized animals. Furthermore it was demonstrated that CTL lysis of relevant env-expressing targets was similarly induced. These studies further define the importance of evaluating this new technology for vaccine and immune therapeutic development for HIV-1 as well as for other human viral pathogens.

Recombinant antibodies in bioactive peptide design.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important in many immune and inflammatory processes. GM-CSF binds to specific cellular receptors which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design, and such design depends on a molecular understanding of ligand-receptor interactions. One approach to dissecting out critical intermolecular interactions is to develop analogs of specific interaction sites of potential importance. Monoclonal antibodies have been employed for these purposes in prior studies. Here we present application of recombinant antibody technology to the development of analogs of a site on GM-CSF bound by a neutralizing anti-GM-CSF monoclonal antibody. Polyclonal antisera with high titer neutralizing activity against human GM-CSF were developed in BALB/c mice. Purified immunoglobulins were prepared and used to immunize syngeneic mice. Anti-anti-GM-CSF was developed which demonstrated biological antagonist activity against GM-CSF-dependent cellular proliferation. RNA was extracted from spleen cells of mice with biologically active anti-anti-GM-CSF, cDNA synthesized, and polymerase chain reaction performed with primers specific for murine kappa light chain V regions. Polymerase chain reaction products were cloned into the pDABL vector and an expression library developed. This was screened with anti-GM-CSF neutralizing mAb 126.213, and several binding clones isolated. One clone (23.2) which inhibited 126.213 binding to GM-CSF was sequenced revealing a murine kappa light chain of subgroup III. Comparison of the 23.2 sequence with the human GM-CSF sequence revealed only weak sequence similarity of specific complementarity determining regions (CDRs) with human GM-CSF. Structural analysis revealed potential mimicry of specific amino acids in the CDR I, CDR II and FR3 regions of 23.2 with residues on the B and C helices of GM-CSF. A synthetic peptide analog of the CDR I was bound by 126.213, specifically antagonized GM-CSF binding to cells and blocked GM-CSF bioactivity. These studies indicate the feasibility of using recombinant antibody libraries as sources of interaction site analogs.

Facilitated DNA inoculation induces anti-HIV-1 immunity in vivo.

Vaccine design against HIV-1 is complicated both by the latent aspects of lentiviral infection and the diversity of the virus. The type of vaccine approach used is therefore likely to be critically important. In general, vaccination strategies have relied on the use of live attenuated material or inactivated/subunit preparations as specific immunogens. Each of these methodologies has advantages and disadvantages in terms of the elicitation of broad cellular and humoral immune responses. Although most success has been achieved with live attenuated vaccines, there is a conceptual safety concern associated with the use of these vaccines for the prevention of human infections. In contrast, subunit or killed vaccine preparations enjoy advantages in preparation and conceptual safety; however, their ability to elicit broad immunity is more limited. In theory, inoculation of a plasmid DNA that supports in vivo expression of proteins, and therefore presentation of the processed protein antigen to the immune system, could be used to combine the features of a subunit vaccine and a live attenuated vaccine. We have designed a strategy for intramuscular DNA inoculation to elicit humoral and cellular immune responses against expressed HIV antigens. Uptake and expression are significantly enhanced if DNA is administered in conjunction with the facilitating agent bupivacaine-HCl. Using this technique we have demonstrated functional cellular and humoral immune responses against the majority of HIV-1 encoded antigens in both rodents and non-human primates.

Isolated VH4 heavy chain variable regions bind DNA characterization of a recombinant antibody heavy chain library derived from patient(s) with active SLE.

In many autoimmune diseases autoantibodies are intimately involved in disease manifestations. Molecular characterization of these autoantibodies should provide insights into the pathogenesis of these diseases, as well as suggest novel avenues for development of therapeutics. While some prior studies suggest that DNA binding may be a characteristic of individual heavy chain variable regions, the ability of these V regions to bind DNA in isolation has not been investigated. We have utilized a bacterial vector for cloning and expressing isolated antibody heavy chain variable regions. RNA was extracted from peripheral blood mononuclear cells of patients with active SLE, cDNA synthesized and heavy chain V regions amplified with VH specific oligonucleotide primers. The VH fragments were cloned into a bacterial expression plasmid including the pelB leader peptide to direct appropriate expression. Recombinant antibodies were screened for binding to 32P-labeled double-stranded plasmid DNA and later also characterized for binding to single-stranded DNA. Binding was confirmed by standard ELISA methodology. Sequence analysis of seven DNA binding VH fragments revealed that they utilized the VH gene family previously described to be associated with autoimmune responses, with a JH6 segment. On VH sequence analysis only one residue substitution in the consensus sequence is needed to form a VH4 germline gene. Potential contact residues with DNA were delineated by three-dimensional structure analysis. We concluded that the DNA binding characteristics of VH regions can be examined in the absence of light chain. DNA binding specificity appears to be a property of the germline VH4 gene. Analysis of such V regions can aid in the identification of hypervariable region contact residues important for DNA binding.

Molecular characterization of two gene loci required for production of the key pathogenicity factor pectate lyase in Pseudomonas viridiflava.

Four pleiotropic mutants of Pseudomonas viridiflava strain PJ-08-6A that were deficient in production of both pectate lyase (Pel) and protease (Prt) were isolated following transposon mutagenesis. Unlike secretion-defective (Out-) mutants, these four showed no accumulation of enzymes within the cells. Southern hybridization analysis revealed that each mutant had Tn5 inserted in one of two EcoRI genomic fragments. These EcoRI fragments (5.2- and 6.3-kb) appeared to contain two distinct gene loci, designated repA and repB, which were required for production of extracellular enzymes in this bacterium. Cosmid clones carrying the functional repA and repB DNA fragments were identified in a genomic library of strain PJ-08-6A. After analysis of repA+ plasmids by restriction mapping and marker-exchange mutagenesis, the repA gene was located in a joint region between the 1.8-kb EcoRI-HindIII and 2.8-kb EcoRI fragments cloned. Nucleotide sequence analysis of the repA region revealed the presence of an open reading frame consisting of 2,790 bases. The RepA protein predicted from the DNA sequence showed 93% similarity in amino acid sequence to the LemA protein of P. syringae pv. syringae, which was previously identified as a member of a two-component global regulatory system. A plasmid carrying the lemA gene of P. syringae pv. syringae was capable of complementing the RepA- mutation in P. viridiflava. The functions of the repA and lemA genes thus appear to be similar and interchangeable. Mutants of P. viridiflava strain SF312A deficient in production of Pel, Prt, and the exopolysaccharide alginate also were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular diagnosis of Borrelia burgdorferi infection (Lyme disease).

In spite of significant advances in immunologically based testing, accurate diagnosis of Lyme borreliosis remains problematic. To address this issue, a DNA amplification-based diagnostic test was developed utilizing the polymerase chain reaction (PCR) and oligonucleotide primers specific for the OspA and OspB genes of Borrelia burgdorferi. In this approach, a relatively large DNA fragment is amplified with an outer set of primers, and a "nested" internal sequence of the PCR product subsequently reamplified with an inner set of primers. This nested approach coupled with simple differential centrifugation allowed specific detection of as few as four B. burgdorferi organisms mixed in 2 ml of blood. This methodology was utilized on patients' samples, and it allowed detection of B. burgdorferi in the peripheral blood and urine of several individuals with clinical evidence of Lyme borreliosis. PCR became negative and symptoms improved following antibiotic therapy of treated individuals. These studies suggest that direct detection of Borrelia in infected individuals can aid in diagnosis and evaluation of therapy for Lyme borreliosis.

Construction of a recombinant bacterial human CD4 expression system producing a bioactive CD4 molecule.

The CD4 protein expressed on helper T lymphocytes is a restriction element for major histocompatibility class II immune responses. This molecule is also used by the human immunodeficiency virus as its specific cellular receptor facilitating binding of virus to cells. As soluble forms of CD4 inhibit HIV infection in tissue culture, attention has focused on this molecule. Bacterially produced CD4 would facilitate studies of the biology of the CD4 molecule. However, bacterially expressed CD4 must be refolded for assumption of its interaction with conformationally dependent anti-CD4 monoclonal antibodies as well as the HIV-1 envelope protein gp120. We report here the engineering of an external domain construct of the CD4 gene into a novel expression vector containing the nucleotide sequence encoding the pelB leader peptide of Erwinia carotovara (pDABL), to facilitate correct folding of CD4 in bacteria. Monoclonal antibodies specific for important conformational epitopes of the CD4 molecule were able to bind bacterial colonies containing the pDABL/CD4 vector but not colonies with vector alone. Importantly, recombinant gp120 produced in baculovirus bound specifically to bacterial colonies expressing the CD4 recombinant molecule. This system presents a simple screening mechanism for molecules that bind to the external domain of the CD4 glycoprotein. Vectors such as pDABL will also facilitate the production of large amounts of biologically active proteins in bacteria.

Ocular tissue involvement in HIV infection: immunological and pathological aspects.

The CNS afflictions in AIDS are myriad and suggest a tropism of HIV to neural tissue. Ocular involvement is a frequent manifestation of the HIV infection, resulting in a high incidence of blindness within this patient population. Ocular lesions include cotton wool spots, presumably from HIV-induced microvasculopathy, retinal hemorrhage in cytomegalovirus retinitis and conjunctival Kaposi's sarcoma. These manifestations have been noted in up to 71% of AIDS patients. In fact, ocular disease is often the presenting symptom in an HIV-infected individual. Despite the high incidence of ocular involvement in AIDS patients, the etiology and pathogenesis of these manifestations are not well understood. The immunosuppressive action of HIV is the most likely primary cause for the development of ocular complications in AIDS. Here we review some of the important immunological and pathological features of AIDS affliction in the eye.

Development and evaluation of an enzyme-linked immunosorbent assay for endotoxin in milk.

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of endotoxin in milk samples. Bovine and rabbit antisera raised in response to vaccination with the J5 mutant of Escherichia coli 0111:B4 were used. Antiserum to this mutant has been shown to be cross-reactive with endotoxin from other gram-negative organisms. Known quantities of endotoxin were added to milk samples to generate a standard curve. Acid treatment of whole milk enhanced the detection of endotoxin as compared to untreated whole milk, skim milk and chloroform-treated milk. Milk samples from experimentally induced mastitic cows were then assayed for endotoxin content. Recovery of endotoxin, as measured by ELISA, positively correlated with the amount of endotoxin infused and the time post-infusion of sampling. However, when endotoxin from these samples was quantitated using the Limulus Amebocyte Lysate (LAL) assay, readings tended to increase, suggesting false-positive reactions with the LAL assay. Milk samples from cases of clinical mastitis were assayed by ELISA with 64% of these showing measurable levels of endotoxin. While further studies of this assay are needed, refinements may produce an assay important for clinical applications.

Antibody specific for Escherichia coli J5 cross-reacts to various degrees with an Escherichia coli clinical isolate grown for different lengths of time.

Rabbit antiserum raised against the rough mutant of Escherichia coli O111:B4, designated J5, was examined for cross-reactivity to an E. coli clinical isolate (A2385). In whole-cell enzyme-linked immunosorbent assays, J5 antiserum reacted to a greater extent with A2385 grown for 5 h than with the same bacteria grown for 19 h, while the homologous antiserum reacted similarly with bacteria grown for different lengths of time. J5 antiserum reacted to the greatest extent with lipopolysaccharide (LPS) from A2385 grown for up to 10 h, and reactivity greatly diminished thereafter; homologous antiserum showed no difference in reaction over time. LPS from smooth bacteria grown for 19 h showed no reaction with J5 antiserum in immunoblots, while LPS from A2385 grown for 5 or 10 h showed a positive reaction. Little or no difference among the three LPS samples could be seen when homologous antiserum was used. Mice vaccinated with J5 LPS before lethal challenge with live A2385 were protected from this challenge, whereas most nonimmunized mice died. Toxicity tests in mice showed LPS from A2385 grown for 19 h to be twice as lethal as LPS from A2385 grown for 3 h. Mice vaccinated with J5 LPS were protected to a greater extent when challenged with a lethal dose of LPS from A2385 grown for 3 h than when challenged with LPS from A2385 grown for 19 h. The results reported here may explain the means by which J5 vaccination (active or passive) sometimes protects against heterologous challenge.