Elevated production of Legionella-specific immunoglobulin A in A/J mice is accompanied by T-helper 1-type polarization

Legionella pneumophila (Lpn) is a Gram-negative bacterium and an intracellular parasite that causes Legionnaires' disease. Secretion of immunoglobulin A (IgA) against Lpn on the mucosal surface of the upper respiratory tract may be important as a self-defense mechanism. A/J mice have been demonstrated to be permissive as to Lpn replication in macrophages due to a natural mutation in neuronal apoptosis inhibitory protein 5. We compared A/J and BALB/c mice as to IgA production after repeated intranasal immunization using a heat-killed Lpn in the presence of cholera toxin as a mucosal adjuvant. A/J mice secreted more Lpn-specific IgA in nasal washes than BALB/c mice. The Lpn-specific serum IgA level was also higher in A/J than BALB/c mice. Because both BALB/c and A/J mice are known to exhibit T-helper 2 (Th2)-biased immune responses, we examined whether the Lpn-specific IgA production is related to the stronger Th2 bias. There was no difference in IgG1 (Th2-controlled) while A/J mice produced more IgG2a (Th1-controlled), suggesting that the elevated IgA response was rather correlated with Th1-controlled isotype switching. Our results also suggest that A/J mice will be useful hosts for Lpn-specific IgA production such as for the preparation of IgA monoclonal antibodies.     

Immunoglobulin class-specific antibody response in serum, spleen, lungs, and bronchoalveolar washings after primary and secondary sendai virus infection of germfree mice.

Immunoglobulin class-specific antibodies were measured by a solid-phase radioimmunoassay in serum, bronchoalveolar washings (BAW), lung cell lysates, and spleen cell lysates in germfree mice after intranasal (i.n.) and intraperitoneal (i.p.) primary and secondary 10(5), 10(4), and 10(3) mean tissue culture infective doses (TCID(50)) of live parainfluenza 1 (Sendai) virus. The earliest antibody detected in lungs after i.n. virus challenge was immunoglobulin G (IgG), followed by IgM and, lastly, IgA. The local IgA response after both primary and secondary i.n. virus challenge was lowest after the severest infection. It is suggested that the delayed appearance of IgA antibody and the lower response after severe lung damage may be related to a temporary local secretory component-producing cell deficiency. The lungs were a major source of serum IgG antibody after both primary and secondary i.n. virus challenge. Only IgG and IgM antibodies were detectable in lung cell lysates after the i.n. 10(3) TCID(50) secondary response. A secondary response was detected in IgG, IgA, and IgM after secondary i.n. challenge with the other two doses. The lung response to all of primary and secondary i.p. doses of virus was exclusively IgG and IgM. Calculation of radioimmunoassay antibody per microgram of IgG, IgA, and IgM in serum and BAW after both i.n. and i.p. virus challenges showed that, when BAW antibody was present, the ratio in BAW was always higher than that in serum. This finding in the i.n. mice, together with the presence of IgA antibody-containing cells in the lungs, strongly indicates local manufacture and secretion of IgA antibodies in these animals and suggests that the same conclusion could apply to local IgG and IgM antibodies after both i.n. and i.p. challenges.     

1141154113Expression of natural cytotoxicity receptors in peripheral blood NK cell subsets of women with recurrent spontaneous abortions (RSA) or implantation failures

Aim:  To determine if IgA is required for protection against Chlamydia infection in the male reproductive tract (MRT).
Materials and Methods:  Male polyimmunoglobulin receptor knockout mice (PIgR^-/-) and wild-type C57BL/6 (WT) mice were immunised intranasally with chlamydial major outer membrane protein (MOMP) and cholera toxin (CT). MOMP-specific IgG and IgA in serum and prostatic fluids were measured by ELISA. Serum and PF were also assayed for inhibition of in vitro chlamydial infection. Immunized WT and PIgR^-/- mice were challenged by direct inoculation of C. muridarum into the meatus urethra. Four weeks post challenge Chlamydia levels in the penile urethra, epididymis and testis were determined by PCR.
Results:  Equivalent levels of IgG were found in the serum of both WT and PIgR^-/- mice however IgA in serum of PIgR^-/- mice was 19- to 20-fold higher than in WT animals consistent with the lack of the PIgR IgA transport molecule. IgA levels were significantly lower in PIgR^-/- PF compared to WT PF after both immunization and infection. Only PF from WT but not PIgR^-/- animals was able to inhibit in vitro chlamydial infection. Following challenge significantly higher levels of Chlamydia were recovered from the MRT of PIgR^-/- mice compared to WT animals.
Conclusions:  Male mice lacking a functional PIgR were unable to clear a genital tract Chlamydia infection despite high levels of serum IgA. These data show that mucosal IgA plays a major role in preventing chlamydial infection of the male genital tract and suggest that immunization strategies to protect males should target a strong mucosal IgA response.     

The relative susceptibility of mouse strains to pulmonary Cryptococcus neoformans infection is associated with pleiotropic differences in the immune response

CBA/J mice were highly susceptible to intratracheal (i.t.) Cryptococcus neoformans infection relative to BALB/c mice, while both strains were equally susceptible to intravenous (i.v.) infection. Increased susceptibility in i.t. infection was associated with higher brain CFU, lower serum immunoglobulin M (IgM) and IgG responses to glucuronoxylomannan (GXM), lack of IgE regulation during infection, and alveolar macrophage permissiveness to intracellular replication in vitro. In contrast, for BALB/c mice, relative resistance was associated with increased interleukin-12 (IL-12) and decreased IL-10 pulmonary levels. In CBA/J mice, relative susceptibility was associated with a decreased proportion of CD4+ and CD8+ T cells and an increase in macrophage percentage in pulmonary infiltrates. In contrast, no significant differences in these cytokines or cell recruitment were observed in the i.v. model, consistent with no differences in the survival rate. Passive antibody (Ab) protection experiments revealed a prozone effect in the BALB/c mice with i.v. infection, such that Ab efficacy decreased at higher doses. In the i.t. model using CBA/J mice, low Ab doses were disease enhancing and protection was observed only at high doses. Our results show (i) that differences in mouse strain susceptibility are a function of the infection model, (ii) that susceptibility to pulmonary infection was associated with macrophage permissiveness for intracellular replication, and (iii) that the efficacy of passive Ab in pulmonary infection is a function of dose and mouse strain. The results highlight significant differences in the pathogenesis of cryptococcal infection among inbred mice and associate their relative susceptibility with differences in numerous components of the innate and adaptive immune responses.     

Induction of antibody responses in the common mucosal immune system by respiratory syncytical virus immunostimulating complexes

Immunostimulating complexes (ISCOMs) containing envelope proteins of respiratory syncytial virus (RSV) were explored as a mucosal delivery system for the capacity of inducing a common mucosal antibody response. Two intranasal (i.n.) administrations of BALB/c mice with ISCOMs induced potent serum IgG, and strong IgA responses to RSV locally in the lungs and the upper respiratory, and remotely in the genital and the intestinal tracts. Virtually no measurable IgA response was found in these mucosal organs after two subcutaneous (s.c.) immunizations. Virus neutralizing (VN) antibodies were detected in serum and in all of the mucosal organ extracts after both s.c. and i.n. immunizations indicating that the neutralizing epitopes were preserved after both mucosal and parenteral modes of administration. While the mucosal IgA response appears to be of mucosal origin, the IgG antibodies to RSV detected in the mucosal organs were likely of serum origin. However, the mucosal VN antibodies correlated with the IgG rather than the IgA levels. An enhanced IgA response to gp120 in various mucosal organs was recorded after i.n. immunization with gp120 incorporated in RSV ISCOMs, indicating a role of RSV envelope proteins in enhancing and targeting mucosal responses to passenger antigens. Received: 23 October 1998     

Secretory IgA antibodies provide cross-protection against infection with different strains of influenza B virus

This study examined whether secretory IgA (S-IgA) antibodies (Abs) could confer cross-protective immunity against infection with influenza B viruses of antigenically distinct lineages. Wild-type or polymeric Ig receptor (pIgR)-knockout (KO) mice were immunized by infection with different B viruses or by intranasal (i.n.) administration with different inactivated vaccines. Four weeks later mice were challenged with either the B/Ibaraki/2/85 virus, representative of the B/Victoria/2/87 (B/Victoria)-lineage, or B/Yamagata/16/88 virus, representative of the B/Yamagata-lineage. Three days after challenge, nasal wash and serum specimens were assayed for IgA and IgG Abs specific for challenge viral antigens and for protection against challenge viruses. In wild-type mice, B/Ibaraki (or B/Yamagata) cross-reactive IgA Abs were detected at higher levels when infected or immunized with homologous-lineage viruses and at lower levels when infected or immunized with heterologous-lineage viruses. There was a correlation between the amount of nasal cross-reactive IgA Ab and the efficacy of cross-protection with a homologous-lineage virus. In mice lacking the pIgR, nasal cross-protective IgA Abs were only marginally detected in vaccinated mice and an accumulation of IgA in the serum was observed. This reduction of nasal IgA was accompanied by inefficient cross-protection against the B/Ibaraki (or B/Yamagata) virus infection. These results suggest that challenge viral-antigen cross-reactive S-IgA in nasal secretions induced by i.n. infection or vaccination is involved in providing cross-protection against challenge infection with virus within either the B/Victoria- or B/Yamagata-lineage.     

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific IgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49, VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice; these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG.     

Efficient intranasal immunization of newborn mice with recombinant adenovirus expressing rotavirus protein VP4 against oral rotavirus infection

Efficacy of passive protection of newborn mice against rotavirus infection by the rotavirus VP4 protein expressed by an adenoviral vector in mice was studied. The VP4 gene was inserted into the E1 region of adenoviral vector pJM17. Recombinant adenovirus Ad5N/VP4 was grown in 293 cells. Intramuscular (i.m.), oral or intranasal (i.n.) immunization of newborn mice with Ad5/VP4 resulted in appearance of VP4-specific antibodies. Specific IgG antibodies were detected in the serum and intestine specimens of i.m. vaccinated mice. Oral immunization elicited serum IgG antibodies and intestinal IgG and IgA antibodies. Compared with i.m. and oral applications, i.n. immunization led to higher levels of serum IgG and intestinal IgG and IgA antibodies. Pups were challenged twice with simian rotavirus SA11 strain orally at the days 7 and 8 after birth. Pups born to i.n. immunized dams achieved 100% protection from rotavirus-induced diarrhea after both challenges. The protection of pups born to orally immunized dams was 80%, while only 30% of pups born to i.m. immunized dams were protected after both challenges. I.n. immunization was most efficient in inducing rotavirus VP4-specific serum, intestinal and milk IgG or IgA in mice that protected newborn mice completely.     

Intranasal immunization with pneumococcal conjugate vaccines with LT-K63, a nontoxic mutant of heat-Labile enterotoxin, as adjuvant rapidly induces protective immunity against lethal pneumococcal infections in neonatal mice

Immunization with pneumococcal polysaccharides (PPS) conjugated to tetanus toxoid (TT) (Pnc-TT) elicits protective immunity in an adult murine pneumococcal infection model. To assess immunogenicity and protective immunity in early life, neonatal (1 week old) and infant (3 weeks old) mice were immunized intranasally (i.n.) or subcutaneously (s.c.) with Pnc-TT of serotype 1 (Pnc1-TT). Anti-PPS-1 and anti-TT immunoglobulin G (IgG) and IgM antibodies were measured in serum and saliva, and vaccine-induced protection was evaluated by i.n. challenge with serotype 1 pneumococci. Pnc1-TT was immunogenic in neonatal and infant mice when administered s.c. without adjuvant: a majority of the young mice were protected from bacteremia and a reduction of pneumococcal density in the lungs was observed, although antibody responses and protective efficacy remained lower than in adults. The addition of LT-K63, a nontoxic mutant of heat-labile enterotoxin, as adjuvant significantly enhanced PPS-1-specific IgG responses and protective efficacy following either s.c. or i.n. Pnc1-TT immunization. Mucosal immunization was particularly efficient in neonates, as a single i.n. dose of Pnc1-TT and LT-K63 induced significantly higher PPS-1-specific IgG responses than s.c. immunization and was sufficient to protect neonatal mice against pneumococcal infections, whereas two s.c. doses were required to induce complete protection. In addition, i.n. immunization with Pnc1-TT and LT-K63 induced a vigorous salivary IgA response. This suggests that mucosal immunization with pneumococcal conjugate vaccines and LT-K63 may be able to circumvent some of the limitations of neonatal antibody responses, which are required for protective immunity in early life.     

Protection of mice against Mycoplasma pulmonis infection using purified mouse immunoglobulins: comparison between protective effect and biological properties of immunoglobulin classes.

Purified mouse immunoglobulins with specific activity against Mycoplasma pulmonis were examined for their ability to protect mice against intranasal (i.n.) infection with this organism. The survival of mycoplasmas in the respiratory tract of mice inoculated i.n. with M. pulmonis mixed with either IgG1, IgGa, IgG2b or IgA antibodies was less than that seen in control animals inoculated with mycoplasmas mixed with normal mouse serum. The ways in which the immunoglobulin fractions could have mediated resistance were examined in vitro. IgG1, IgG2a and IgG2b antibody fractions killed M. pulmonis in the presence of guinea-pig complement, and also promoted the phagocytosis of M. pulmonis by mouse lung macrophages. In contrast, IgA antibody did not appear to be either mycoplasmacidal or opsonic.     

Biliary Immune Response to Orally Presented Food Antigen, Ovomucoid, and its Potentiation by Cholera Toxin B Subunit

To clarify the biliary immune response against food antigen, we studied biliary antibody response to intravenous and oral primary immunization with ovomucoid (OM) and the effects of cholera toxin B subunit (CTB) on the oral response in mice. Specific antibodies against OM were induced in biliary and serum immunoglobulin (Ig) A, IgG and IgM by intravenous (i.v.) administration of the antigen. However, the antibodies were induced only in biliary Igs, but not in serum Igs, by oral intubation of the antigen. The higher levels of anti-OM in bile than in serum observed in the oral group may favour the assumption that antigen-stimulated lymphoid cells migrate to the liver, gall bladder or bile duct where they produce antibody. Both serum and biliary anti-OM responses to oral immunization were potentiated remarkably by oral administration of CTB with the antigen, the IgM anti-OM response being potentiated to the largest extent. In the CTB-treated mice, the IgA anti-OM level was higher in bile than in serum. Serum level of IgG anti-OM was much lower in the CTB-treated mice than in the i.v.-immunized mice, but the biliary level of IgG anti-OM in the CTB-treated mice was comparable to that in the i.v.-immunized mice. The relationship between serum and biliary IgA and IgG antibodies suggests that CTB potentiates biliary anti-OM responses not solely through increasing systemic levels of the antibodies but through modulating the processes specific to mucosal presentation of antigen.     

Antibody content of rabbit milk and serum following inhalation or ingestion of respiratory syncytial virus and bovine serum albumin

The contribution of bronchus-associated or gut-associated lymphoid tissues to the development of specific immunologic reactivity in lactating mammary glands was studied by evaluating the effect of the nature of the antigen and the route of immunization on milk antibody content. Groups of pregnant rabbits were immunized with respiratory syncytial virus (RSV) and bovine serum albumin (BSA) administered i.v., per oral (p.o.) or transtracheal (i.t.) routes. The response to RSV was characterized by the regular appearance of IgA anti-RSV in the colostrum, milk, bronchial, and intestinal secretions following p.o. or i.t. immunization, but not after i.v. immunization. RSV-specific IgG appeared in the colostrum, milk, and serum regardless of the route of immunization. On the other hand, the response to BSA by all three routes of immunization was characterized by the appearance of anti-BSA in serum, colostrum and milk which was solely associated with IgG. The anti-BSA isotype did not change during the 30-day nursing period and was not affected by BSA ingestion before or during pregnancy or during nursing. If BSA was reintroduced after 20 days of nursing, a sharp rise in the Ab content of milk occurred in p.o. but not i.v. immunized dams. This increased anti-BSA was also of the IgG isotype. These observations suggest that the isotypes of specific Ab responses in the lactating mammary gland of the rabbit may be determined by the physical and chemical nature of the antigens contacted on respiratory or intestinal mucosal surfaces.     

Protection from Clostridium difficile Infection in CD4 T Cell- and Polymeric Immunoglobulin Receptor-Deficient Mice

Clostridium difficile rivals methicillin-resistant Staphylococcus aureus as the primary hospital-acquired infection. C. difficile infection (CDI) caused by toxins A and/or B can manifest as mild diarrhea to life-threatening pseudomembranous colitis. Although most patients recover fully from CDI, ∼20% undergo recurrent disease. Several studies have demonstrated a correlation between anti-toxin antibody (Ab) and decreased recurrence; however, the contributions of the systemic and mucosal Ab responses remain unclear. Our goal was to use the CDI mouse model to characterize the protective immune response to C. difficile. C57BL/6 mice infected with epidemic C. difficile strain BI17 developed protective immunity against CDI and did not develop CDI upon rechallenge; they generated systemic IgG and IgA as well as mucosal IgA Ab to toxin. To determine if protective immunity to C. difficile could be generated in immunodeficient individuals, we infected CD4^−/− mice and found that they generated both mucosal and serum IgA anti-toxin Abs and were protected from CDI upon rechallenge, with protection dependent on major histocompatibility complex class II (MHCII) expression; no IgG anti-toxin Ab was found. We found that protection was likely due to neutralizing mucosal IgA Ab. In contrast, pIgR^−/− mice, which lack the receptor to transcytose polymeric Ab across the epithelium, were also protected from CDI, suggesting that although mucosal anti-toxin Ab may contribute to protection, it is not required. We conclude that protection from CDI can occur by several mechanisms and that the mechanism of protection is determined by the state of immunocompetence of the host.     

Route of Infection That Induces a High Intensity of Gamma Interferon-Secreting T Cells in the Genital Tract Produces Optimal Protection against Chlamydia trachomatis Infection in Mice

The induction of local T helper type 1 (Th1)-mediated cellular immunity is crucial for resistance of mice to genital infection by the obligate intracellular bacterium Chlamydia trachomatis. We tested the hypothesis that the route of immunization that elicits relatively high numbers of chlamydia-specific, gamma interferon (IFN-γ)-secreting T lymphocytes (ISTLs) in the genital tract would induce optimal protective immunity against reinfection. Female BALB/c mice were infected intravaginally (i.v.), intranasally (i.n.), orally (p.o.), or subcutaneously (s.c.) with C. trachomatis. At days 7, 14, 21, and 28 postinfection, T cells isolated from the genital tract tissues were restimulated with chlamydial antigen in vitro, and the amounts of IFN-γ induced were measured by a sandwiched enzyme-linked immunosorbent assay method. At day 7 postinfection, i.n.- and i.v.-immunized mice had high levels of chlamydia-specific ISTLs in their genital tracts (203.58 ± 68.1 and 225.5 ± 12.1 pg/ml, respectively). However, there were no detectable ISTLs in the genital tracts of p.o.- or s.c.-infected mice. When preinfected mice were challenged i.v. 70 days later, animals preexposed by the i.n. route were highly resistant to reinfection, with greatly reduced chlamydial burden, and suffered an attenuated infection that resolved by day 6 postchallenge. Animals preexposed by the i.v. route were modestly protected, whereas p.o. and s.c. groups were indistinguishable in this regard from control mice. The resistance of i.n.-immunized mice (and to some extent the i.v.-exposed mice) to reinfection was associated with early appearance (within 24 h) of high levels of genital ISTLs compared with mice preinfected by other routes. Furthermore, although i.n. and i.v.-immunized mice had comparable levels of chlamydia-specific immunoglobulin A (IgA) antibodies in their vaginal washes, the levels of IgG2a were four- sixfold higher in i.n.-immunized mice than in any of the other groups. The results suggested that immunization routes that foster rapid induction of vigorous genital mucosal cell-mediated immune (CMI) effectors (e.g., IFN-γ), the CMI-associated humoral effector, IgG2a, and to some extent secretory IgA produce protective immunity against chlamydial genital infection. Therefore, i.n. immunization is a potential delivery route of choice in the development of a vaccine against Chlamydia.     

A mutant pertussis toxin molecule that lacks ADP-ribosyltransferase activity, PT-9K/129G, is an effective mucosal adjuvant for intranasally delivered proteins.

We examined the capacity of a genetically detoxified derivative of pertussis toxin (PTX), PT-9K/129G, to act as a mucosal adjuvant for an intranasally (i.n.) administered tetanus vaccine. Groups of mice were immunized i.n. with the nontoxic C-terminal 50-kDa portion of tetanus toxin (fragment C [Frg C]) either alone or mixed with PT-9K/129G, PTX, or cholera toxin (CT) or were immunized subcutaneously (s.c.) with an equivalent amount of Frg C adsorbed to alhydrogel. In response to a single immunization, mice receiving Frg C plus PT-9K/129G or CT i.n. and parenterally immunized mice developed high-titer (> 20,000) anti-Frg C antibodies, whereas mice immunized i.n. with Frg C plus PTX or with Frg C alone seroconverted only after being boosted. The serum anti-Frg C response was dominated by immunoglobulin G1 (IgG1) in mice immunized with Frg C plus PT-9K/129G, with Frg C plus PTX, or s.c. In contrast, IgG1, IgG2a, and IgG2b contributed almost equally to the Frg C response when CT was the adjuvant. Anti-Frg C IgE was detected only in the sera of mice immunized i.n. with Frg C plus PTX and immunized s.c. with Frg C plus alhydrogel. High levels of IgA antibodies were present in nasal lavage fluid from mice immunized i.n. with Frg C plus PT-9K/129G, PTX, or CT but not in that from mice given Frg C alone i.n. or parenterally. The mucosal adjuvanticity of PT-9K/129G was manifested in inbred as well as outbred mice. A single i.n. dose of Frg C plus either PT-9K/129G or PTX (with high specific activity) was sufficient to protect all immunized mice from tetanus toxin challenge, in contrast to the case for mice that received Frg C alone i.n. We conclude that the pertussis toxin analog PT-9K/129G, which is devoid of ADP-ribosyltransferase activity, is a potent mucosal adjuvant for vaccines delivered via the respiratory tract.     

Further characterization of delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for delivering antigens of enterotoxigenic Escherichia coli.

The use of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a live vector for enterotoxigenic Escherichia coli (ETEC) antigens is reported. CVD 1203 alone or expressing colonization factor antigen fimbriae and CS3 fibrillae of ETEC was given to guinea pigs and mice, orogastrically (o.g.) or intranasally (i.n.). CVD 1203 given i.n. elicited high titers of antilipopolysaccharide (anti-LPS) immunoglobulin A (IgA) and was protective in guinea pigs against a homologous conjunctival challenge. Whereas a strong IgA response against colonization factor antigen CS3, and Shigella LPS was detected in tears and serum of guinea pigs after o.g. or i.n. immunization, the i.n. route elicited significantly higher antibody titers. A strong serum IgG response was also observed against the ETEC antigens, although no serum anti-LPS IgG response was detected. The immune response in mice followed a pattern similar to that in guinea pigs, and the difference between the responses after o.g. and i.n. administration was even more remarkable.     

Mechanisms of monophosphoryl lipid A augmentation of host responses to recombinant HagB from Porphyromonas gingivalis

Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Our laboratory has shown the induction of protection to P. gingivalis infection after subcutaneous immunization with recombinant hemagglutinin B (rHagB). The purpose of this study was to determine if humoral antibody responses are induced after intranasal (i.n.) immunization of rHagB and if monophosphoryl lipid A (MPL), a nontoxic derivative of the lipid A region of lipopolysaccharide, acts as a mucosal adjuvant and potentiates responses to rHagB. Further, the effects of MPL on the nature of the response to HagB and on the costimulatory molecules B7-1 and B7-2 on different antigen-presenting cells (APC) were evaluated. Groups of BALB/c mice were immunized three times (2-week intervals) by the i.n. route with HagB (20 microg) alone or with MPL (25 microg). A group of nonimmunized mice served as control. Serum and saliva samples were collected prior to immunization and at approximately 2-week intervals and evaluated for serum immunoglobulin G (IgG) and IgG subclass and for salivary IgA antibody activity by enzyme-linked immunosorbent assay. Mice immunized with rHagB plus MPL had significantly higher salivary IgA (P < 0.05) and serum IgG (P < 0.05) anti-HagB responses than mice immunized with rHagB alone. The IgG1 and IgG2a subclass responses seen in mice immunized with rHagB plus MPL were significantly higher (P < 0.05) than those seen in mice immunized with rHagB only. Further, the IgG2a/IgG1 ratio in the latter group was approximately 1, whereas in mice immunized with rHagB plus MPL the ratio was <1. These results provide evidence for the participation of T helper (Th) 1 and Th2 cells in responses to rHagB and that MPL potentiates a type 2 response to HagB. MPL was also shown to preferentially up-regulate B7-2 expression on B cells, whereas a preferential increase in B7-1 costimulatory molecule was seen on macrophages and dendritic cells. These results provide evidence that MPL exerts a differential regulation in the expression of costimulatory molecules on APC.     

Intranasal Immunization with Pneumococcal Polysaccharide Conjugate Vaccines with Nontoxic Mutants of Escherichia coli Heat-Labile Enterotoxins as Adjuvants Protects Mice against Invasive Pneumococcal Infections

Host defenses against Streptococcus pneumoniae depend largely on phagocytosis following opsonization by polysaccharide-specific immunoglobulin G (IgG) antibodies and complement. Since colonization of the respiratory mucosa is the first step in pneumococcal pathogenesis, mucosal immune responses may play a significant role. In addition to inducing systemic immune responses, mucosal vaccination with an effective adjuvant has the advantage of inducing mucosal IgA antibodies. The heat-labile enterotoxin (LT) of Escherichia coli is a well-studied mucosal adjuvant, and adjuvant activity of nontoxic LT mutants has been demonstrated for several protein antigens. We investigated the immunogenicity of pneumococcal polysaccharide conjugate vaccines (PNC) of serotypes 1 and 3 in mice after intranasal (i.n.) immunization by using as an adjuvant the nontoxic LT mutant LT-K63 or LT-R72, which has minimal residual toxicity. Pneumococcal serotype-specific antibodies were measured in serum (IgM, IgG, and IgA) and saliva (IgA), and vaccine-induced protection was evaluated by i.n. challenge with virulent pneumococci of the homologous serotype. When administered with LT mutants, i.n. immunization with both conjugates induced systemic and mucosal immune responses, and serum IgG antibody levels were significantly higher than after subcutaneous immunization. All mice immunized i.n. with PNC-1 and LT mutants were protected against bacteremia and cleared the pneumococci from the lung 24 h after i.n. challenge; pneumococcal density correlated significantly with serum IgG antibody levels. Similarly, the survival of mice immunized i.n. with PNC-3 and LT mutants was significantly prolonged. These results demonstrate that i.n. vaccination with PNC and potent adjuvants can protect mice against invasive and lethal pneumococcal infections, indicating that mucosal vaccination with PNC may be an alternative vaccination strategy for humans.     

Enhancement of Serum and Mucosal Immune Responses to a Haemophilus influenzae Type B Vaccine by Intranasal Delivery

Intranasal (i.n.) vaccination is potentially the most direct method for conveying upper respiratory and mucosal immunity to respiratory pathogens. However, for unclear reasons, vaccines introduced into the nasal sinuses often have lower efficacy than vaccines administered by the more frequently used parenteral routes. We examined i.n. vaccination in a mouse immune-response model with a commonly used Haemophilus influenzae type B vaccine (Hibv) composed of the polyribosylribitol phosphate (PRP) capsule antigen conjugated to tetanus toxoid. Intranasal vaccination with Hibv using a Toll-like receptor 4 (TLR4) agonist as an adjuvant significantly increased the levels of IgA specific for the PRP capsule antigen in blood serum, saliva, and mucosal secretion specimens. In contrast, control mice vaccinated transdermally (t.d.) with Hibv did not produce significant levels of PRP-specific IgA in the blood serum and saliva, and anti-PRP IgG was increased only in serum. The i.n. and t.d. vaccinations resulted in equivalent bactericidal antibody responses in blood serum, suggesting that vaccine-derived IgG is protective against infection. Elevated levels of IgG specific for the tetanus toxoid carrier protein were measured in nasal sinuses and vaginal secretions in mice vaccinated by either the t.d. or i.n. route. Tissue culture studies confirmed that the nasopharynx-associated lymphoid tissue (NALT) was at least one of the sources of PRP-specific IgA and carrier-specific IgG within the nasal sinuses. We conclude that i.n. vaccination aided by a TLR4 agonist results in robust immune responses to both the carrier protein and bacterial polysaccharide components of the Hibv.     

Carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens

We have evaluated the ability of two carbohydrate biopolymers, chitosan and gellan, to enhance antibody responses to subunit influenza virus vaccines delivered to the respiratory tracts of mice. Groups of mice were vaccinated three times intranasally (i.n.) with 10 microg of purified influenza B/Panama virus surface antigens (PSAs), which consist of hemagglutinin (HA) and neuraminidase (NA), either alone or admixed with chitosan or gellan solutions. Separate groups were vaccinated subcutaneously (s.c.) with PSAs adsorbed to Alhydrogel or chitosan or gellan alone i.n. Serum antibody responses were determined by enzyme-linked immunosorbent assay (ELISA) for influenza virus-specific immunoglobulin G (IgG) and by HA inhibition (HAI) and NA inhibition (NAI) assays. The local respiratory immune response was measured by assaying for influenza virus-specific IgA antibody in nasal secretions and by enumerating nasal and pulmonary lymphocytes secreting IgA, IgG, and IgM anti-influenza virus-specific antibodies by enzyme-linked immunospotting (ELISPOT). When administered alone i.n., B/Panama PSA was poorly immunogenic. Parenteral immunization with B/Panama PSA with Alhydrogel elicited high titers of anti-B/Panama antibodies in serum but a very poor respiratory anti-B/Panama IgA response. In contrast, i.n. immunization with PSA plus chitosan stimulated very strong local and systemic anti-B/Panama responses. Gellan also enhanced the local and serum antibody responses to i.n. PSA but not to the same extent as chitosan. The ability of chitosan to augment the immunogenicity of influenza vaccines given i.n. was confirmed using PSA prepared from an influenza A virus (A/Texas H1N1).