S1 of distinct IBV population expressed from recombinant adenovirus confers protection against challenge.

Protective properties of three distinct infectious bronchitis virus (IBV) Ark Delmarva poultry industry (ArkDPI) S1 proteins encoded from replication-defective recombinant adenovirus vectors were investigated. Using a suboptimal dose of each recombinant virus, we demonstrated that IBV S1 amino acid sequences showing > or = 95.8% amino acid identity to the S1 of the challenge strain differed in their ability at conferring protection. Indeed, the S1 sequence of the IBV population previously designated C4 (AdIBVS1.C4), which protected the most poorly, differs from the S1 sequence of population C2 (AdIBVS1.C2), which provided the highest protection, only at amino acid position 56. The fact that a change in one amino acid in this region significantly altered the induction of a protective immune response against this protein provides evidence that the first portion of S1 displays relevant immunoprotective epitopes. Use of an optimal dose of AdIBVS1.C2 effectively protected chickens from clinical signs and significantly reduced viral load after IBV Ark virulent challenge. Moreover, increased numbers of both IgA and IgG IBV-specific antibody secreting lymphocytes were detected in the spleen after challenge. The increased response detected for both IgA and IgG lymphocytes after challenge might be explained by vaccine-induced B memory cells. The fact that a single vaccination with Ad/IBVS1.C2 provides protection against IBV challenge is promising, because Ad-vectored vaccines can be mass delivered by in ovo inoculation using automated in ovo injectors.

Conjunctiva-associated lymphoid tissue in avian mucosal immunity.

Conjunctiva-associated lymphoid tissue's (CALT) role in generating avian mucosal adaptive immunity was measured by analyzing cellular composition, expression of the polymeric immunoglobulin receptor (pIgR), and production of cytokines and antibodies in chickens ocular exposed to a replication-deficient adenovirus of serotype 5 (Ad5). These studies demonstrate that CALT contains B cells, γδ T cells, T helper, and cytotoxic T cells, and a T lymphocyte composition, which more resembles Harderian glands than spleen. CALT-derived lymphocytes contain antigen-specific, IgA-secreting plasma cells and cytokine-producing lymphocytes after ocular Ad5 vaccination. The expression of the pIgR in the CALT's lymphoepithelium emphasizes the importance of mucosal immune protection by paraocular lymphoid tissues. The CALT immune response after ocular Ad5 boosting was influenced by prior high dose in ovo Ad5 priming. Thus, both mucosal and systemic immunization influenced Ad5-induced IFN-γ responses in CALT.

Effects of phytase supplementation in broiler diets on a natural Eimeria challenge in naive and vaccinated birds.

Our study was conducted to determine the effects of dietary phytase on a natural Eimeria challenge in naive and vaccinated broilers. Prior to the experiment the litter was seeded with Eimeria by orally infecting 10-d-old chicks with a cocktail containing 100,000 and 5,000 sporulated Eimeria acervulina and Eimeria tenella oocysts, respectively. Straight-run broiler chicks were placed across 48 floor pens on fresh or seeded litter. Eight treatment combinations were created to include 2 dietary Ca-nonphytate P (npP) levels [0.9% Ca, 0.45% npP; 0.7% Ca, 0.35% npP, 500 phytase units of Optiphos phytase (JBS United, Sheridan, IN)], unchallenged versus challenged, and unvaccinated versus vaccinated groups of chicks. Body weights and feed consumption (FC) were recorded on d 10, 18, and 21. A total of 10 birds/treatment were killed on d 10 and 18 to obtain tissue samples from the duodena and ceca for lesion scoring and cytokine response measurement. At 21 d of age, the left tibia was removed from 18 birds/treatment to assess bone strength. Body weight, FC, and bone strength were unaffected (P > 0.05) by diet or vaccination. By d 21, birds exposed to coccidia had lower FC (P < 0.01), higher feed conversion (P < 0.001), and decreased bone strength (P < 0.01) compared with those not challenged. Regardless of treatment, gross and microscopic scoring of the intestines showed few differences (P > 0.05). Expression of interferon-γ did not differ (P > 0.05) in the duodena or ceca at either time point. The IL-17 gene expression was increased (P < 0.05) in phytase-supplemented, vaccinated, or challenged birds by 18 d of age, with significant interactions (P < 0.05) occurring between birds challenged and fed the marginal diet or vaccinated. Phytase supplementation was unable to provide additional benefits to performance or P utilization in birds vaccinated, subjected to a coccidiosis infection, or both. Based on cytokine production in the intestinal tract on d 10 and 18 postchallenge, the response to the Eimeria challenge was characterized by a T-helper type (Th) 17-like immune response and to a lesser extent a Th1-like immune response, whereas no Th2 cytokine was detected.

Avian influenza adenovirus-vectored in ovo vaccination: target embryo tissues and combination with Marek's disease vaccine.

We investigated embryo tissues targeted by replication competent adenovirus (Ad)-free recombinant Ad expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdH5) when injected into 18-day embryonated eggs. We also evaluated the effects of concurrent in ovo vaccination with the experimental AdH5 vaccine and commercially available Marek's disease virus (MDV) vaccine combinations Rispens/turkey herpesvirus (HVT) or HVT/SB-1. Computed tomography indicates that in ovo injection on day 18 of incubation places the solution in the amnion cavity, allantoic cavity, or both. Ad DNA was consistently detected in the chorioallantoic membranes as well as in the embryonic bursa of Fabricius, esophagus, and thymus 3 days postinoculation. H5 expression in these tissues also was detected by immunofluorescence assay. These results indicate possible swallowing of vaccine virus contained in the amnion. In contrast, vaccine localization in the allantoic fluid would have allowed bursal exposure through the cloaca. When the AdH5 vaccine was used in combination with MDV, chickens responding to the AdH5 vaccine had similar AI antibody levels compared with AdH5-only-vaccinated birds. However, combined vaccinated groups showed reduced vaccine coverage to AI, suggesting some level of interference. The combination of AdH5 with MDV Rispens/HVT affected the vaccine coverage to AI more severely. This result suggests that the replication rate of the more aggressive Rispens strain of serotype 1 may have interfered with the Ad-vectored vaccine. Increasing the Ad concentration produced similar AI antibody titers and AI vaccine coverage when applied alone or in combination with the HVT/SB-1 vaccine. Ad DNA was detected in hatched chickens 2 days after hatch but was undetectable on day 9 after hatch. MDV DNA was detected in feather follicles of all vaccinated birds at 12 days of age. Thus, Ad-vector vaccination does not interfere with the efficacy of MDV vaccination by using any of the commonly used vaccine strains.

Pathogenicity of infectious bursal disease virus variant AL2 in young chickens.

Limited information is available on the effects of the recently emerged infectious bursal disease virus (IBDV) variant AL2. In this study, the effects of inoculation of 4-day-old chickens with increasing doses of IBDV AL2 were characterized. IBDV AL2 induced neither overt clinical signs nor mortality. Infected chickens showed reduced bursa indices (BI) and bursa lymphocytic depletion, as determined by histomorphometry. However, histomorphometry and BI values differed during the early stages of the infection. Because data from bursa histomorphometry were consistent with viral RNA detection, such values seem to be more appropriate for the assessment of AL2 viral infectivity in chickens. Both the histomorphometry and BI data indicated a dose-effect pattern. However, with time, even low doses of the virus ultimately resulted in significant damage to the bursa. Samples of spleen were used to assess B- (IgM+) and T- (CD4+ and CD8+) cell populations by flow cytometry. Infected chickens showed a significant increase of splenic IgM+ cells at 5 and 8 days postinoculation (PI). On day 8 PI, the number of total IgM+ cells in the spleen was inversely related to the virus concentration. Others have shown that cell-mediated immunity is essential for protection against IBDV. Our results indicate a significant increase (P < 0.05) of total spleen CD4+ cell counts on day 8 PI in birds that received higher virus concentrations, indicating a role for these cells in protective immunity, while CD8 cell counts remained unchanged. We speculate that the changes in splenic CD4+ and IgM+ cell populations are associated with protective immune responses against IBDV in the host.

Infectious bronchitis virus in the chicken Harderian gland and lachrymal fluid: viral load, infectivity, immune cell responses, and effects of viral immunodeficiency.

We compared detection of infectious bronchitis virus (IBV) by quantitative RT-PCR (qRT-PCR) in tears and trachea of IBV-infected chickens and found that quantitative detection of IBV RNA in tears is more sensitive than in tracheal homogenates. Furthermore, we demonstrated that IBV contained in chicken lachrymal fluid is infectious and that tears of IBV-infected chickens can be used to infect naive chickens. We compared the immune responses to IBV in the Harderian gland and cecal tonsils of immunocompetent chickens and chickens infected with chicken anemia virus (CAV) and/or infectious bursal disease virus (IBDV). Flow cytometry analyses of lymphocytes in Harderian glands and cecal tonsils indicated that the relative abundance of IgM+ B cells in the Harderian glands and cecal tonsils following exposure to IBV in combination with immunosuppressive viruses was reduced compared to chickens infected with IBV alone. CAV, but not IBDV, reduced the CD4+/CD8+ T cell ratios compared to chickens infected with IBV alone. Enzyme-linked immuno-spot forming assays on cells in the Harderian glands and cecal tonsils of IBV-infected chickens indicated that maximum IBV-specific IgA-secreting cell responses were reduced in chickens infected with CAV. IBDV co-infected chickens displayed a delayed IgA response to IBV. Thus immunosuppressive viruses reduced B cells and T helper cells in the Harderian glands and cecal tonsils in response to IBV, and slowed the kinetics and/or reduced the magnitude of the mucosal immune response against IBV. We have shown for the first time that CAV affects pathogen-specific B cell responses in a mucosal effector site.

Pathogenesis of infectious bronchitis virus in vaccinated chickens of two different major histocompatibility B complex genotypes.

Infectious bronchitis (IB) disease progression in vaccinated chickens after challenge was evaluated in a single commercial line of layer chickens presenting two different major histocompatibility complex (MHC) B complex genotypes. MHC B genotypes were determined by DNA sequence-based typing of BF2 alleles. In total, 33 B2/B15 and 47 B2/B21 chickens were vaccinated with an Ark-type IB virus (IBV) attenuated vaccine and challenged with Ark-type IBV field isolate AL/4614/98 14 days later. Additional chickens of both genotypes served as unvaccinated/challenged and unvaccinated/nonchallenged controls. Clinical signs, histopathologic analysis, detection of IBV genomes in tears, and IBV-specific immunoglobulin A (IgA) in tears were used to evaluate disease progression and immune response. The incidence of IBV respiratory signs was significantly higher in B2/21 than in B2/B15 MHC genotype birds. However, neither the severity and duration of respiratory signs nor the severity and incidence of histologic lesions differed significantly with MHC genotype. The levels of IBV-specific IgA in tears of vaccinated and challenged chickens did not differ significantly between MHC genotypes. IBV genomes were present in the tears of vaccinated and challenged birds, and the incidence of detectable IBV genomes did not vary significantly with MHC B genotype. From an applied perspective, these results indicate that vaccinated commercial outbred chickens with these MHC genotypes are equally resistant to IBV.

A revisit of mucosal IgA immunity and oral tolerance.

Induction of mucosal immunity by oral immunization with protein antigen alone is difficult: potent mucosal adjuvants, vectors, or other special delivery systems are required. Cholera toxin (CT) has been shown to be an effective adjuvant for the development of mucosal vaccines and, when given with vaccine, induces both mucosal and systemic immune responses via a Th2 cell-dependent pathway. However, and in addition to potential type-I hypersensitivity, a major concern for use of mucosal adjuvants such as CT is that this molecule is not suitable for use in humans because of its inherent toxicity. When we examined the potential toxicity of CT for the central nervous system, both CT and CT-B accumulated in the olfactory nerves/epithelium and olfactory bulbs of mice when given by the nasal route. The development of effective mucosal vaccines for the elderly is also an important issue; however, only limited information is available. When mucosal adjuvanticity of CT was evaluated in aged mice, an early immune dysregulation was evident in the mucosal immune system. The present review discusses these potential problems for effective mucosal vaccine development. Tolerance represents the most common and important response of the host to environmental antigens, including food and commensal bacterial components, for the maintenance of an appropriate immunological homeostasis. We have examined whether Peyer patches could play a more important role for the maintenance of oral tolerance. Using Peyer patch-null mice, we found that mice lacking this gut-associated lymphoid tissue retained their capability to produce secretory IgA antibodies but did not develop normal oral tolerance to protein antigens.

A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis.

Immunologic reactivity to lipid-DNA conjugates has traditionally been viewed as less of an issue than with viral vectors. We performed a dose escalation safety trial of aerosolized cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lower airways of eight adult cystic fibrosis patients, and monitored expression by RT-PCR. The cDNA was complexed to a cationic lipid amphiphile (GL-67) consisting of a cholesterol anchor linked to a spermine head group. CFTR transgene was detected in three patients at 2-7 days after gene administration. Four of the eight patients developed a pronounced clinical syndrome of fever (maximum of 103.3EF), myalgias, and arthralgia beginning within 6 hr of gene administration. Serum IL-6 but not levels of IL-8, IL-1, TNF-alpha, or IFN-gamma became elevated within 1-3 hr of gene administration. No antibodies to the cationic liposome or plasmid DNA were detected. We found that plasmid DNA by itself elicited minimal proliferation of peripheral blood mononuclear cells taken from study patients, but led to brisk immune cell proliferation when complexed to a cationic lipid. Lipid and DNA were synergistic in causing this response. Cellular proliferation was also seen with eukaryotic DNA, suggesting that at least part of the immunologic response to lipid-DNA conjugates is independent of unmethylated (E. coli-derived) CpG sequences that have previously been associated with innate inflammatory changes in the lung.

Oral QS-21 requires early IL-4 help for induction of mucosal and systemic immunity.

The highly purified saponin derivative, QS-21, from the Quillaja saponaria Molina tree has been proved to be safe for parenteral administration and represents a potential alternative to bacterial enterotoxin derivatives as a mucosal adjuvant. Here we report that p.o. administration of QS-21 with the vaccine protein tetanus toxoid elicited strong serum IgM and IgG Ab responses, which were only slightly enhanced by further oral immunization. The IgG Ab subclass responses were predominantly IgG1 followed by IgG2b for the 50-microg p.o. dose of QS-21, whereas the 250-microg p.o. dose also induced IgG2a and IgG3 Abs. Low oral QS-21 doses induced transient IgE Ab responses 7 days after the primary immunization, whereas no IgE Ab responses were seen in mice given the higher QS-21 dose. Further, low but not high p.o. QS-21 doses triggered Ag-specific secretory IgA (S-IgA) Ab responses. Th cell responses showed higher IFN-gamma (Th1-type) and lower IL-5, IL-6, and IL-10 (Th2-type) secretion after the high QS-21 p.o. dose than after low doses. Interestingly, the mucosal adjuvant activity of low oral QS-21 doses was diminished in IL-4(-/-) mice, suggesting a role for this cytokine in the initiation of mucosal immunity by oral QS-21. In summary, our results show that oral QS-21 enhances immunity to coadministered Ag and that different doses of QS-21 lead to distinct patterns of cytokine and serum Ab responses. We also show that an early IL-4 response is required for the induction of mucosal immunity by oral QS-21 as adjuvant.

Cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues.

We tested the notion that the mucosal adjuvant cholera toxin (CT) could target, in addition to nasal-associated lymphoreticular tissues, the olfactory nerves/epithelium (ON/E) and olfactory bulbs (OBs) when given intranasally. Radiolabeled CT ((125)I-CT) or CT-B subunit ((125)I-CT-B), when given intranasally to mice, entered the ON/E and OB and persisted for 6 days; however, neither molecule was present in nasal-associated lymphoreticular tissues beyond 24 h. This uptake into olfactory regions was monosialoganglioside (GM1) dependent. Intranasal vaccination with (125)I-tetanus toxoid together with unlabeled CT as adjuvant resulted in uptake into the ON/E but not the OB, whereas (125)I-tetanus toxoid alone did not penetrate into the CNS. We conclude that GM1-binding molecules like CT target the ON/E and are retrograde transported to the OB and may promote uptake of vaccine proteins into olfactory neurons. This raises concerns about the role of GM1-binding molecules that target neuronal tissues in mucosal immunity.

Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component.

The acute-phase response (APR) is regulated by TNF-alpha, IL-1beta, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1beta and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1beta gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1beta to promote up-regulation of the CRP and SAP genes.

Gamma interferon is not required for mucosal cytotoxic T-lymphocyte responses or heterosubtypic immunity to influenza A virus infection in mice.

Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-gamma) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-gamma, we have investigated the mechanism of HSI by analyzing the role of IFN-gamma for HSI in IFN-gamma gene-deleted (IFN-gamma(-/-)) mice. It has been reported that IFN-gamma is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-gamma is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-gamma(-/-) mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-gamma(-/-) mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-gamma does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.

Vaccines for mucosal immunity to combat emerging infectious diseases.

The mucosal immune system consists of molecules, cells, and organized lymphoid structures intended to provide immunity to pathogens that impinge upon mucosal surfaces. Mucosal infection by intracellular pathogens results in the induction of cell- mediated immunity, as manifested by CD4-positive (CD4 + ) T helper-type 1 cells, as well as CD8 + cytotoxic T-lymphocytes. These responses are normally accompanied by the synthesis of secretory immunoglobulin A (S-IgA) antibodies, which provide an important first line of defense against invasion of deeper tissues by these pathogens. New-generation live, attenuated viral vaccines, such as the cold-adapted, recombinant nasal influenza and oral rotavirus vaccines, optimize this form of mucosal immune protection. Despite these advances, new and reemerging infectious diseases are tipping the balance in favor of the parasite; continued mucosal vaccine development will be needed to effectively combat these new threats.

Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response.

Protective immunity to enterotoxigenic Escherichia coli (ETEC) is antibody (Ab) dependent; however, oral immunization with purified ETEC fimbriae fails to elicit protective immunity as a consequence of antigenic alteration by the gastrointestinal (GI) tract. Unless unaltered ETEC fimbriae can reach the inductive lymphoid tissues of the GI tract, immunity to ETEC cannot be induced. To produce immunity, live vectors, such as Salmonella typhimurium, can effectively target passenger antigens to the inductive lymphoid tissues of the GI tract. By convention, oral immunizations with Salmonella vectors induce CD4(+) T helper (Th) cell responses by gamma interferon (IFN-gamma)-dominated pathways both to the vector and passenger antigen, resulting in serum immunoglobulin G2a (IgG2a) and modest mucosal IgA Ab responses. In the present study, mice orally immunized with a Salmonella vector engineered to stably express ETEC colonization factor antigen I (CFA/I) showed initially elevated serum IgG1 and mucosal IgA anti-CFA/I Ab responses. As expected, mice orally immunized with an E. coli-CFA/I construct elicited poor anti-CFA/I Ab responses. In fact, the addition of cholera toxin during oral E. coli-CFA/I immunization failed to greatly enhance mucosal IgA Ab responses. Seven days after immunization with the Salmonella-CFA/I construct, cytokine-specific ELISPOT showed induction of predominant Th2-type responses in both mucosal and systemic immune compartments supporting the early IgG1 and IgA anti-CFA/I Abs. By 4 weeks, the Th cell response became Th1 cell dominant from the earlier Th2-type responses, as evidenced by increased mucosal and systemic IFN-gamma-producing T cells and a concomitant elevation of serum IgG2a Ab responses. This biphasic response offers an alternative strategy for directing Salmonella vector-induced host immunity along a Th2 cell-dependent pathway, allowing for early promotion of mucosal and systemic Abs.

Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.

Strategies for mucosal vaccine development.

Vaccines able to induce both secretory IgA for protection of mucosal surfaces and systemic immunity to pathogens invading the host are of great interest in the war against infectious diseases. Mucosal vaccines trigger immune cells in mucosal inductive sites and thus can induce immunity in both the mucosal and systemic compartments. This review presents a critical survey of adjuvants and delivery systems currently being tested for mucosal immunization. A better understanding of cellular and molecular factors involved in the regulation of mucosal immunity will help in the design of safer mucosal vaccines to elicit the appropriate protective immune response to a given pathogen.

Heterosubtypic immunity to lethal influenza A virus infection is associated with virus-specific CD8(+) cytotoxic T lymphocyte responses induced in mucosa-associated tissues.

Heterosubtypic immunity, defined as cross-reactive immune responses to influenza virus of a different serotype than the virus initially encountered, was investigated in association with virus-specific cytotoxic T lymphocyte (CTL) responses induced in systemic and mucosa-associated lymph nodes after immunization via different routes. Mice immunized by the pulmonary route with live nonpathogenic influenza virus, strain Udorn (H3N2), survived challenge with mouse-adapted pathogenic influenza virus, strain PR/8/34 (H1N1). These mice developed strong heterosubtypic CTL responses in spleen, cervical lymph nodes (CLN), and mediastinal lymph nodes (MLN). Alternately, only 20% of mice immunized intravenously, intraperitoneally, or intranasally survived the challenge; all of these developed CTL responses in spleen and CLN, but not in MLN. Direct correlation between short-term and long-term memory heterosubtypic CTL responses induced in MLN and host recovery after lethal infection indicates that these CTL responses may play an important role in heterosubtypic immunity. Furthermore, induction and maintenance of memory CTL in regional mucosa-associated lymphoid tissues are highly dependent on mucosal immunization. The results implicate the mechanism of heterosubtypic immunity and should be an important consideration in the development of protective mucosal vaccines against variant strains of influenza and HIV.

Measurement of HIV-1 Specific and Total Antibody Secreting Cells by ELISPOT.

B-cells play an important role in protection against pathogens, and they secrete specific antibodies in serum and mucosal secretions upon antigenic stimulation contributing to immune exclusion and clearance of pathogens. The frequency of antibody forming cells (AFC) in specific organs is often a reflection of the route of antigen exposure, i.e., systemic, oral, or intranasal, as well as of antigenic load. Enumeration of AFC was originally performed by plaque-forming cell assay measuring lysis of sheep red blood cells. The nature of this assay, that requires coupling of the antigen to sheep red blood cells (SRBC), made detection of various antigen-specific AFC rather cumbersome. However, the development of the enzyme linked immunodetection of AFCs (ELISPOT) combined with the development of standardized lymphocyte isolation techniques enables detection of AFC secreting antibodies specific for many different antigens and derived from various immunologic effector sites (1-6).

Testosterone and IL-6 requirements for human C-reactive protein gene expression in transgenic mice.

In vitro, IL-6 is the main inducer of the human C-reactive protein (CRP) gene, and IL-1 and steroids can enhance this effect. However, in mice, IL-6 is necessary but not sufficient for induction of the human CRP transgene, and testosterone is required for its constitutive expression by males. To examine the relative contributions of testosterone and IL-6 in the regulation of CRP gene expression, we produced CRP-transgenic (CRPtg), IL-6-deficient (IL-6-/-) mice. Male CRPtg/IL-6-/- mice expressed CRP constitutively, but CRP levels were not increased after injection of LPS. However, acute-phase CRP levels were attained after injection of IL-6. In contrast, female CRPtg/IL-6-/- mice did not express CRP constitutively or after administration of LPS, IL-6, IL-1, or IL-6 plus IL-1. Like males, testosterone-treated CRPtg/IL-6-/- females expressed CRP constitutively, and their transgene responded to injection of IL-6. The endogenous acute-phase protein serum amyloid P (SAP) was expressed constitutively equally by male and female IL-6-/- mice, responded minimally to LPS, and did not respond to either IL-6 or IL-1 alone. Acute-phase levels of SAP were induced in IL-6-/- mice by injection of IL-6 together with IL-1 or LPS. We conclude that in vivo, both constitutive and IL-6-dependent acute-phase expression of the CRP transgene require testosterone. In contrast, testosterone is not required for expression of the SAP gene, which requires IL-1 plus IL-6 for acute-phase induction.