Age-dependent immune responses and immune protection after avian coronavirus vaccination.

Infectious bronchitis virus (IBV) is an endemic disease of chickens and a major contributor to economic losses for the poultry industry despite vaccination. Recent observations indicated that chicks may have an immature immune system immediately after hatching when vaccinated for IBV. Therefore we hypothesized that early IBV vaccination will generate an immature, poorly protective IBV-specific immune response contributing to immune escape and persistence of IBV. To test this hypothesis the IBV-specific immune response and immune protection were measured in chicks vaccinated at different ages. This demonstrated a delayed production of IgG and IgA plasma antibodies in the 1, 7 and 14-day-old vaccination groups and also lower IgA antibody levels were observed in plasma of the 1-day-old group. Similar observations were made for antibodies in tears. In addition, IgG antibodies from the 1-day-old group had lower avidity indices than day 28 vaccinated birds. The delayed and/or lower antibody response combined with lower IgG avidity indices coincided with increased tracheal inflammation and depletion of tracheal epithelia cells and goblet cells upon IBV field strain challenge. The lack of vaccine-mediated protection was most pronounced in the 1-day-old vaccination group and to a lesser extent the 7-day-old group, while the 14-day-old and older chickens were protected. These data strongly support IBV vaccination after day 7 post hatch.

Immunoglobulin A as an early humoral responder after mucosal avian coronavirus vaccination.

Infectious bronchitis virus (IBV) is a highly contagious coronavirus prevalent in all countries with an extensive poultry industry and continues to cause economic losses. IBV strains of the Ark serotype are highly prevalent in the Southeastern United States despite extensive vaccination. One explanation for this observation is the high genetic variability of IBV. In addition, IBV Ark-type vaccines may induce suboptimal mucosal immune responses, contributing to the prevalence and persistence of the Ark serotype. To test this hypothesis, chickens were ocularly vaccinated with a commercially available live attenuated IBV Ark-Delmarva Poultry Industry vaccine strain and both mucosal and systemic antibody responses were measured. The highest immunoglobulin A (IgA) spot-forming cell (SFC) response was observed in the Harderian glands (HG) and to a lesser extent in the spleen and conjunctiva-associated lymphoid tissues, while a limited IgG SFC response was observed in either the mucosal or systemic immune compartment. Interestingly, the peak IgA SFC response occurred 2 days earlier in spleen than in the head-associated lymphoid tissues despite ocular vaccination. Furthermore, IgA IBV-specific antibody levels significantly increased over controls 3 days earlier in tears and 4 days earlier in plasma than did IgG antibodies. IgA antibody levels were higher than IgG antibody levels throughout the primary response in tears and were similar in magnitude in plasma. In addition, a very early increase in IgA antibodies on day 3 postvaccination was observed in tears; such a response was not observed in plasma. This early increase is consistent with a mucosal T-independent IgA response to IBV. In the secondary response the IBV antibody levels significantly increased over controls starting on day 1 after boosting, and the IgG antibody levels were higher than the IgA antibody levels in both tears and plasma. In summary, ocular vaccination induced higher IgA antibodies in the primary IBV response, while the memory response is dominated by IgG antibodies. Thus, lower mucosal IgA antibody levels are observed upon secondary exposure to IBV, which may contribute to vulnerability of host epithelial cells to infection by IBV and persistence of the Ark serotype.

Cell-mediated immune responses in the head-associated lymphoid tissues induced to a live attenuated avian coronavirus vaccine.

Humoral immunity is important for controlling viral diseases of poultry, but recent studies have indicated that cytotoxic T cells also play an important role in the immune response to infectious bronchitis virus (IBV). To better understand the cell mediated immune responses to IBV in the mucosal and systemic immune compartments chickens were ocularly vaccinated with IBV. This induced a lymphocyte expansion in head-associated lymphoid tissues (HALT) and to a lesser extent in the spleen, followed by a rapid decline, probably due to homing of lymphocytes out of these organs and contraction of the lymphocyte population. This interpretation was supported by observations that changes in mononuclear cells were mirrored by that in CD3(+)CD44(+) T cell abundance, which presumably represent T effector cells. Increased interferon gamma (IFN-γ) expression was observed in the mucosal immune compartment, i.e., HALT, after primary vaccination, but shifted to the systemic immune compartment after boosting. In contrast, the expression of cytotoxicity-associated genes, i.e., granzyme A (GZMA) and perforin mRNA, remained associated with the HALT after boosting. Thus, an Ark-type IBV ocular vaccine induces a central memory IFN-γ response in the spleen while the cytotoxic effector memory response, as measured by GZMA and perforin mRNA expression, remains associated with CALT after boosting.

Infectious bronchitis virus subpopulations in vaccinated chickens after challenge.

Infectious bronchitis coronavirus (IBV) shows extensive genotypic and phenotypic variability. The evolutionary process involves generation of genetic diversity by mutations and recombination followed by replication of those phenotypes favored by selection. In the current study, we examined changes occurring in a wild Arkansas (Ark) challenge strain in chickens that were vaccinated either ocularly with commercially available attenuated ArkDPI-derived vaccines or in ovo with a replication-defective recombinant adenovirus expressing a codon-optimized IBV Ark S1 gene (AdArkIBV.S1(ck)). Commercial IBV Ark vaccines A, B, and C provided slightly differing levels of protection against homologous challenge. Most importantly for the current study, chickens vaccinated with the different vaccines displayed significant differences in specific B-lymphocyte responses in the Harderian gland (i.e., the challenge virus encountered differing immune selective pressure during invasion among host groups). Based on S1 sequences, five predominant populations were found in different individual vaccinated/challenged chickens. Chickens with the strongest immune response (vaccine A) were able to successfully impede replication of the challenge virus in most chickens, and only the population predominant in the challenge strain was detected in a few IBV-positive birds. In contrast, in chickens showing less than optimal specific immune responses (vaccines B and C) IBV was detected in most chickens, and populations different from the predominant one in the challenge strain were selected and became predominant. These results provide scientific evidence for the assumption that poor vaccination contributes to the emergence of new IBV strains via mutation and/or selection. In ovo vaccination with a low dose of AdArkIBV.S1(ck) resulted in a mild increase of systemic antibody and reduced viral shedding but no protection against IBV signs and lesions. Under these conditions we detected only virus populations identical to the challenge virus. Possible explanations are discussed. From a broad perspective, these results indicate that selection is an important force driving IBV evolution.

The proportion of specific viral subpopulations in attenuated Arkansas Delmarva poultry industry infectious bronchitis vaccines influences vaccination outcome.

We investigated the significance of differing proportions of specific subpopulations among commercial Arkansas (Ark) Delmarva poultry industry (DPI) vaccines with regard to vaccination outcome. Two ArkDPI-derived vaccines that contain a higher proportion of viruses with S1 genes that become selected during replication in chickens exhibited more rapid establishment of those selected subpopulations in chickens, produced significantly higher viral loads in tears, and induced higher antibody responses compared with two other ArkDPI vaccines with lower proportions of viruses that become selected in chickens. The presence of higher proportions of selected subpopulations was also associated with a significantly higher incidence of respiratory signs early after vaccination and in some cases more severe tracheal lesions. However, one of the ArkDPI-derived vaccines with a lower proportion of selected subpopulations, despite producing a lower viral load in tears, also induced a higher incidence of respiratory signs later after vaccination and more severe tracheal lesions. Furthermore, one of the ArkDPI-derived vaccines with a higher proportion of selected subpopulations, despite producing a higher viral loads in tears, resulted in less severe tracheal damage. These discrepancies suggest that infectious bronchitis virus (IBV) load in tears may not always predict degree of tracheal damage and that phenotypic characteristics other than S1 may also be involved in severity of vaccine reactions following ArkDPI vaccine administration. We observed lower antibody responses to the vaccines that produced lower viral loads, which might contribute to the persistence of Ark serotype IBV vaccines observed in commercial flocks.

Avian influenza mucosal vaccination in chickens with replication-defective recombinant adenovirus vaccine.

We evaluated protection conferred by mucosal vaccination with replication-competent adenovirus-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdTW68.H5ck). Commercial, layer-type chicken groups were either singly vaccinated ocularly at 5 days of age, singly vaccinated via spray at 5 days of age, or ocularly primed at 5 days and ocularly boosted at 15 days of age. Only chickens primed and boosted via the ocular route developed AI systemic antibodies with maximum hemagglutination inhibition mean titers of 3.9 log2 at 32 days of age. In contrast, single vaccination via the ocular or spray routes maintained an antibody status similar to unvaccinated controls. All chickens (16/16) subjected to ocular priming and boosting with AdTW68.H5ck survived challenge with highly pathogenic AI virus A/chicken/Queretaro/14588-19/95 (H5N2). Single ocular vaccination resulted in 63% (10/16) of birds surviving the challenge followed by a 44% (7/16) survival of single-sprayed vaccinated birds. Birds vaccinated twice via the ocular route also showed significantly lower (P < 0.05) AI virus RNA concentrations in oropharyngeal swabs compared to unvaccinated-challenged controls.

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine.

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell-tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c(+) expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.

Avian influenza vaccination in chickens and pigs with replication-competent adenovirus-free human recombinant adenovirus 5.

Protective immunity to avian influenza (AI) virus can be elicited in chickens by in ovo or intramuscular vaccination with replication-competent adenovirus (RCA)-free human recombinant adenovirus serotype 5 (Ad5) encoding AI virus H5 (AdTW68.H5) or H7 (AdCN94.H7) hemagglutinins. We evaluated bivalent in ovo vaccination with AdTW68.H5 and AdCN94.H7 and determined that vaccinated chickens developed robust hemagglutination inhibition (HI) antibody levels to both H5 and H7 AI strains. Additionally, we evaluated immune responses of 1-day-old chickens vaccinated via spray with AdCN94.H7. These birds showed increased immunoglobulin A responses in lachrymal fluids and increased interleukin-6 expression in Harderian gland-derived lymphocytes. However, specific HI antibodies were not detected in the sera of these birds. Because pigs might play a role as a "mixing vessel" for the generation of pandemic influenza viruses we explored the use of RCA-free adenovirus technology to immunize pigs against AI virus. Weanling piglets vaccinated intramuscularly with a single dose of RCA-free AdTW68.H5 developed strong systemic antibody responses 3 wk postvaccination. Intranasal application of AdTW68.H5 in piglets resulted in reduced vaccine coverage, i.e., 33% of pigs (2/6) developed an antibody response, but serum antibody levels in those successfully immunized animals were similar to intramuscularly vaccinated animals.

Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy.

Preclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)-mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification-mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years.

Neural stem/progenitor cells modulate immune responses by suppressing T lymphocytes with nitric oxide and prostaglandin E2.

We and others have reported that neural stem/progenitor cells (NSCs) may exert direct anti-inflammatory activity. This action has been attributed, in part, to T-cell suppression. However, how T-cells become suppressed by NSCs remains unresolved. In this study, we explored one of these mechanisms and challenged some previously advanced hypotheses regarding underlying NSC-mediated T-cell suppression. We employed an easily observable and manipulatable system in which activated and non-activated T-cells were co-cultured with a stable well-characterized clone of lacZ-expressing murine NSCs. As in previous reports, NSCs were found to inhibit T-cell proliferation. However, this inhibition by NSCs was not due to suppression of T cell activation or induction of apoptosis of T cells during the early activation stage. High levels of nitric oxide (NO) and prostaglandin E2 (PGE2) were induced in the T cells when co-cultured with NSCs. In addition, inducible NOS (iNOS) and microsomal type 1 PGES (mPGES-1) were readily detected in NSCs in co-culture with T-cells, but not at all in NSCs cultured alone or in activated T cells cultured with or without NSCs. This finding suggested that activated T cells induced NO and PGE2 production in the NSCs. Furthermore, T-cell proliferation inhibited by co-culture with the NSCs was significantly restored by inhibitors of NO and PGE2 production. Therefore, NSCs appear to suppress T-cells, at least in part, by NO and PGE2 production which, in turn, would account for the well-documented reduction of central nervous system immunopathology by transplanted NSCs.

Induction of mucosal immunity in the avian Harderian gland with a replication-deficient Ad5 vector expressing avian influenza H5 hemagglutinin.

The chicken Harderian gland (HG) plays an important role in adaptive immune responses upon ocular exposure to avian pathogens such as avian influenza (AI). To determine the role of HGs in generating immunity, chickens were immunized ocularly with an adenovirus (Ad5) vector expressing the AI hemagglutinin H5 gene. The Ad5-H5 vector induced H5 transgene expression and induced H5- and Ad5-specific IgA and IgG spot-forming cells (SFCs) in the HGs. The IgA and IgG SFC peaked on day 9 forAd5 and day 11 for the H5 protein. In addition, Ad5- and H5-specific antibodies were induced in serum. IgA in chicken tears was predominantly dimeric, while in serum monomeric IgA was most abundant. Analysis of HG mRNA confirmed expression of the polymeric immunoglobulin receptor (plgR). These data demonstrated the importance of HGs to generate mucosal and systemic immunity to AI following ocular Ad5-H5 administration to chickens.

Temporal delay of peak T-cell immunity determines Chlamydia pneumoniae pulmonary disease in mice.

Severe chlamydial disease typically occurs after previous infections and results from a hypersensitivity response that is also required for chlamydial elimination. Here, we quantitatively dissected the immune and disease responses to repeated Chlamydia pneumoniae lung infection by multivariate modeling with four dichotomous effects: mouse strain (A/J or C57BL/6), dietary protein content (14% protein and 0.3% L-cysteine-0.9% L-arginine, or 24% protein and 0.5% L-cysteine-2.0% L-arginine), dietary antioxidant content (90 IU alpha-tocopherol/kg body weight versus 450 IU alpha-tocopherol/kg and 0.1% g L-ascorbate), and time course (3 or 10 days postinfection). Following intranasal C. pneumoniae challenge, C57BL/6 mice on a low-protein/low-antioxidant diet, but not C57BL/6 mice on other diets or A/J mice, exhibited profoundly suppressed early lung inflammatory and pan-T-cell (CD3delta(+)) and helper T-cell (CD45) responses on day 3 but later strongly exacerbated disease on day 10. Contrast analyses characterized severe C. pneumoniae disease as being a delayed-type hypersensitivity (DTH) response with increased lung macrophage and Th1 cell marker transcripts, increased Th1:Th2 ratios, and Th1 cytokine-driven inflammation. Results from functional analyses by DTH, enzyme-linked immunospot, and immunohistofluorescence assays were consistent with the results obtained by transcript analysis. Thus, chlamydial disease after secondary infection is a temporal dysregulation of the T-cell response characterized by a profoundly delayed T-helper cell response that results in a failure to eliminate the pathogen and provokes later pathological Th1 inflammation. This delayed T-cell response is under host genetic control and nutritional influence. The mechanism that temporally and quantitatively regulates the host T-cell population is the critical determinant in chlamydial pathogenesis.

Immunity to a self-derived, channel-forming peptide in the respiratory tract.

The channel-forming peptide NC-1130 was generated based on the amino acid sequence of the M2 segment of the spinal cord alpha-subunit of the glycine receptor and has been proposed as a therapeutic agent for anion channelopathies such as cystic fibrosis. Lysine adduction and amino acid substitutions at positions T19R and S22W of the peptide improved its performance as an ion channel. However, these modifications generated an altered self, potentially making this NC-1130 peptide immunogenic, which could preclude the repeated use of NC-1130 as a therapeutic agent. To measure the ability of NC-1130 to induce an immune response, it was administered nasally with or without cholera toxin (CT). The NC-1130 peptide, when given alone without adjuvant, induced very little peptide-specific immunity based on analyses of peptide-specific antibodies by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay, induction of cytokine production, and delayed-type hypersensitivity (DTH) responses. The administration of NC-1130 with the mucosal adjuvant CT induced peptide-specific immunoglobulin G (IgG) antibodies and DTH responses and a Th2-dominant cytokine response. The coadministration of the strong mucosal adjuvant CT induced a systemic NC-1130-specific IgG response but not a mucosal peptide-specific antibody response. The lack of peptide-specific immunity and specifically mucosal immunity should allow repeated NC-1130 peptide applications to epithelial surfaces to correct anion channelopathies.

A novel role for defensins in intestinal homeostasis: regulation of IL-1beta secretion.

Impaired expression of alpha-defensin antimicrobial peptides and overproduction of the proinflammatory cytokine IL-1beta have been associated with inflammatory bowel disease. In this study, we examine the interactions between alpha-defensins and IL-1beta and the role of defensin deficiency in the pathogenesis of inflammatory bowel disease. It was found that matrix metalloproteinase-7-deficient (MMP-7(-/-)) mice, which produce procryptdins but not mature cryptdins (alpha-defensins) in the intestine, were more susceptible to dextran sulfate sodium-induced colitis. Furthermore, both baseline and dextran sulfate sodium-induced IL-1beta production in the intestine were significantly up-regulated in MMP-7(-/-) mice compared with that in control C57BL/6 mice. To elucidate the molecular mechanism for the increased IL-1beta production in defensin deficiency in vivo, we evaluated the effect of defensins on IL-1beta posttranslational processing and release. It was found that alpha-defensins, including mouse Paneth cell defensins cryptdin-3 and cryptdin-4, human neutrophil defensin HNP-1, and human Paneth cell defensin HD-5, blocked the release of IL-1beta from LPS-activated monocytes, whereas TNF-alpha expression and release were not affected. Unlike alpha-defensins, human beta-defensins and mouse procryptdins do not have any effect on IL-1beta processing and release. Thus, alpha-defensins may play an important role in intestinal homeostasis by controlling the production of IL-1beta.

Enterotoxin-based mucosal adjuvants alter antigen trafficking and induce inflammatory responses in the nasal tract.

The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (OB). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1beta or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.

Nasal colonization with Streptococcus pneumoniae includes subpopulations of surface and invasive pneumococci.

We demonstrated that during colonization with Streptococcus pneumoniae the nasal mucosal tissues of mice support two populations of pneumococci. Transparent-phase pneumococci can be readily washed from the outer surface, while a second population composed of primarily opaque-phase pneumococci is released only by homogenization of the nasal tissue. The fact that the opaque phase has previously been associated with invasion and the fact that opaque-phase pneumococci were released by homogenization of previously washed nasal tissue suggest that the opaque-phase pneumococci may have invaded the nasal tissue. Consistent with this hypothesis was our observation that there was inflammation in portions of the nasal mucosa of the colonized mice but not in the mucosa of noncolonized mice, but this observation did not prove the hypothesis. If the opaque-phase pneumococci released from the nasal tissue were from within the tissue and/or if resistance of the opaque-phase subpopulation to antibody, complement, and phagocytes is essential for long-term carriage, it seems likely that the virulence factors of S. pneumoniae that are necessary for killing humans exist to facilitate carriage. Although this speculation is unproven, the observation that there are separate populations of pneumococci during colonization may help guide future attempts to understand the biology of nasal colonization by this pathogen.

A novel adjuvant for mucosal immunity to HIV-1 gp120 in nonhuman primates.

The development of a safe and effective mucosal adjuvant is a crucial step toward a mucosal HIV/AIDS vaccine. This study seeks to determine the promise of a nontoxic mutant of cholera toxin (mCT; E112K) as a mucosal adjuvant in nonhuman primates. HIV-1 gp120 was nasally administered together with mCT E112K or native CT (nCT) as adjuvant on five to six occasions over a 6- to 8-wk period to groups of four rhesus macaques and alone to two monkeys that acted as controls. Macaques given nasal gp120 with either mCT E112K or nCT showed elevated gp120-specific IgG and IgA Ab responses with virus-neutralizing activity in both their plasma and mucosal external secretions, as well as higher numbers of gp120-specific IgA Ab-forming cells in their mucosal and peripheral lymphoid tissues and of IL-4-producing Th2-type CD4-positive (CD4(+)) T cells than did controls. Even though significant mucosal adjuvanticity was seen with both mCT E112K and nCT, neuronal damage was observed only in the nCT-treated, but not in the control or mCT E112K-treated groups. These results clearly show that mCT E112K is an effective and safe mucosal adjuvant for the development of a nasal HIV/AIDS vaccine.

Pneumococcal carriage results in ganglioside-mediated olfactory tissue infection.

Streptococcus pneumoniae cause considerable morbidity and mortality, with persistent neurological sequelae, particularly in young children and the elderly. It is widely assumed that carriage occurs through direct mucosal colonization from the environment whereas meningitis results from invasion from the blood. However, the results of published studies can be interpreted that pneumococci may enter the brain directly from the nasal cavity by axonal transport through olfactory nerves. This hypothesis is based on findings that (i) teichoic acid of the pneumococcal cell wall interact with gangliosides (GLS), (ii) the interaction of GLS with cholera toxin leads to axonal transport through the olfactory nerves into the brain, and (iii) viruses enter the brain through axonal transport into olfactory nerves. After nasal inoculation, we observe high numbers of pneumococci in nasal washes and the olfactory nerves and epithelium. Significant numbers of pneumococci also infected the olfactory bulbs, brain, and the trigeminal ganglia. The absence of bacteremia in this model makes it unlikely that the bacteria entered the brain from the blood stream. Recovery of colony-forming units from the brain, lungs, olfactory nerves, and epithelium and nasal washes was inhibited by incubating pneumococci with GLS before nasal inoculation. These findings, confirmed by PCR and immunohistochemistry, support a GLS-mediated process of infection and are consistent with pneumococci reaching the brain through retrograde axonal transport.

Immunizations with pneumococcal surface protein A and pneumolysin are protective against pneumonia in a murine model of pulmonary infection with Streptococcus pneumoniae.

Intranasal infection of mice with certain strains of capsular group 19 Streptococcus pneumoniae can result in focal pneumonia in the absence of bacteremia. Using this model of murine pneumonia, we demonstrated that immunization with recombinant forms of either pneumococcal surface protein A (PspA) or PdB (a genetically detoxified derivative of pneumolysin) elicited significant protection against focal pulmonary infection. This may be the first demonstration that a proposed vaccine antigen can protect against pneumococcal pneumonia. The best protection was obtained by immunizing mice with a mixture of PspA and PdB, indicating that the protection elicited by these antigens can complement each other. This result is in agreement with previous studies that used pneumococcal sepsis and nasal colonization models and demonstrate that the best protein vaccines for prevention of infection may be those that include more than one protection-eliciting pneumococcal protein.

Delayed lupus onset in (NZB x NZW)F1 mice expressing a human C-reactive protein transgene.

OBJECTIVE: Human C-reactive protein (CRP) binds apoptotic cells and alters blood clearance of injected chromatin in mice. To test whether CRP participates in the pathogenesis of systemic lupus erythematosus (SLE), we examined disease development in lupus-prone (NZB x NZW)F(1) (NZB/NZW) mice expressing a human CRP transgene (hCRPtg/BW). METHODS: Mortality was monitored, proteinuria was determined by dipstick, and serum levels of human CRP and anti-double-stranded DNA (anti-dsDNA) were determined by enzyme-linked immunosorbent assay in NZB/NZW and hCRPtg/BW mice. Thin sections of kidneys were analyzed by immunofluorescence microscopy to compare deposition of IgG, IgM, C3, and human CRP, and electron microscopy was used to reveal differences in ultrastructure. In situ hybridization was performed to detect human CRP messenger RNA expression. RESULTS: The hCRPtg/BW mice had less proteinuria and longer survival than NZB/NZW mice. They also had lower IgM and higher IgG anti-dsDNA titers than NZB/NZW mice, although the differences were transient and small. In hCRPtg/BW mice, accumulation of IgM and IgG in the renal glomeruli was delayed, reduced, and more mesangial than in NZB/NZW mice, while end-stage accumulation of IgG, IgM, and C3 in the renal cortex was prevented. There was less glomerular podocyte fusion, basement membrane thickening, mesangial cell proliferation, and occlusion of capillary lumens in hCRPtg/BW mice, but dense deposits in the mesangium were increased. With disease progression in hCRPtg/BW mice, there was little rise in the plasma CRP level, but CRP in the kidneys became increasingly apparent due to local, disease-independent, age-related expression of the transgene. CONCLUSION: In hCRPtg/BW mice, CRP protects against SLE by increasing blood and mesangial clearance of immune complexes and by preventing their accumulation in the renal cortex.