Humoral immunity induced in the lower respiratory tract by local immunization with a temperature-sensitive mutant ofPseudomonas aeruginosa

The nature of the humoral immune mechanisms involved in the protection induced after local immunization with a temperature-sensitive (ts) mutant ofPseudomonas aeruginosa was investigated. We had previously shown that intranasal (i.n.) immunization of granulocytopenic mice protected the animals from lethal pulmonary challenge withP. aeruginosa, whereas mice immunized intraperitoneally were unprotected. Intranasal immunization induced high levels of anti-P. aeruginosa IgG and IgA in the lower respiratory tract, whereas only modest levels of IgG (and no IgA) could be detected in lung lavage fluids from mice immunized by the intraperitoneal (i.p.) route with ts mutant E/9/9. Plasma anti-P. aeruginosa IgG levels after i.n. immunization were lower than those observed after i.p. immunization with similar doses of the ts mutant. The main contribution to the protection induced when mice are immunized intranasally appears to be from IgA in the pulmonary secretions, although other immune mechanisms cannot be discounted.     

Evaluation of different temperature-sensitive mutant phenotypes ofSalmonella enteritidis as vaccine potentials

Temperature-sensitive (ts) mutants ofSalmonella enteritidis were isolated after mutagenesis with UV light and enrichment with antibiotic. Mutants were characterized according to their growth profile at the permissive (28°C) and the nonpermissive (37°C) temperatures, persistence of surface antigens, reversion frequencies, and potentials for inducing humoral immunity and protection against challenge with the parental wild-type (wt) in mice. We obtained 32 strains ofS. enteritidis able to grow well at 28°C, but capable of only limited or no replication at 37°C. The ts mutants were positive for factor 9 in an agglutination assay and were susceptible to infection with phage P22. Three mutants of different phenotypes were selected for protection studies. A single intraperitoneal (i.p.) immunization with any of the mutants studied induced significant protection from i.p. challenge with 100 LD₅₀ of the wt strain.     

Temperature-sensitive mutants ofStaphylococcus aureus: Isolation and preliminary characterization

Temperature-sensitive (ts) mutants ofStaphylococcus aureus were isolated after mutagenesis with nitrosoguanidine and two cycles of enrichment with Penicillin G and D-Cycloserine. The mutants expressed tight, coasting, and leaky phenotypes on solid media. In broth, however, most exhibited coasting for a limited number of generations. The reversion frequency of selected ts mutants was less than 10^–6. Intraperitoneal (i.p.) immunization with ts mutant G/1/2 conferred significant protection (0 dead/6 total vs. 7/7, immunized vs. control; p=0.0006) from lethal i.p. challenge with the parental wild-type (wt)S. aureus suspended in 5% porcine mucin, performed 28 days after i.p. administration of 10⁸ colony-forming units. Protection induced by mutants of coasting phenotype was higher and lasted longer than that induced by mutants of the tight phenotype. The results of this study demonstrate that ts mutants ofS. aureus can be obtained and that ts mutants are able to induce protective immunity from subsequent challenge with the parental wt strain.     

Intranasal administration of a detoxified endotoxin vaccine protects mice against heterologous Gram-negative bacterial pneumonia

When given passively or elicited actively, antibodies induced by a detoxified Escherichia coli J5 mutant lipopolysaccharide (J5dLPS)-group B meningococcal outer membrane protein (-OMP) vaccine previously protected animals from lethal sepsis. To assess the use of this vaccine for the treatment of Gram-negative bacillary pneumonia, we vaccinated mice, with or without the adjuvant CpG, by intranasal (i.n.) or intraperitoneal (i.p.) routes of administration. Local and systemic IgG levels were 2-3 logs higher following i.p. immunization compared to i.n. However, i.n. immunization elicited both local and systemic IgA, unlike i.p. administration. The addition of CpG to the vaccine, by either route of administration, elicited greater levels of antibody. Intranasal immunization protected mice against lethal heterologous Gram-negative bacillary pneumonia and post-immunization serum and broncho-alveolar lavage fluid mediated enhanced bacterial killing with peritoneal and alveolar macrophages in vitro. We conclude that further studies on the use of J5dLPS-OMP for the prevention of nosocomial pneumonia are warranted.     

Intranasal Immunization with Yersinia enterocolitica O:8 Cellular Extract Protects against Local Challenge Infection

Yersinia enterocolitica is enteropathogenic for humans and rodents. Immune protection from oral and respiratory pathogens may be most effectively elicited following intranasal (i.n.) vaccination. An experimental murine intranasal challenge model was used to evaluate the immunogenicity of a Y. enterocolitica O:8 cellular extract (CE) in mucosa. This antigenic preparation has demonstrated to induce protection by subcutaneous immunization. Mice were immunized intranasally with two doses of CE. Immunized and nonimmunized animals were challenged with 5×10⁶ colony-forming units (CFU) by nasal infection. Antibodies in serum and bronchoalveolar lavage (b.a.l.) fluid were assessed before and 48 hr after challenge. The CFU were determined by analysis of lung homogenate samples. The CE immunization induced significant b.a.l.-specific IgA and IgG, and serum-specific IgG, IgA and IgM. Histopathological studies 24 and 48 hr postchallenge demonstrated that immunization protected against progressive lesions resulting from Y. enterocolitica invasion of the pulmonary mucosa. The CFU in the lungs showed that CE immunization led to significant clearance as compared to the bacterial level in nonimmunized controls. From the results obtained, it can be concluded that CE can induce local and systemic immunity and protect against nasal infection.     

Local and systemic immunity against Streptococcus pneumoniae: Humoral responses against a non-capsulated temperature-sensitive mutant

Abstract Temperature-sensitive mutants of Streptococcus pneumoniae were isolated after chemical mutagenesis. Intranasal immunization with temperature-sensitive mutant J/3 induced higher levels of circulating antibody than those obtained after immunization with the heat-killed parental wild type. Moreover, local immunization with mutant J/3 induced high levels of anti- S. pneumoniae IgG and IgA in the lower respiratory tract, whereas only moderate IgG (and no IgA) antibodies were detected in lung lavage fluids from mice immunized intranasally with the heat-killed strain.     

Lack of development of phagocytic inhibitory activity in serum of cats immunized withEnterobacter cloacae lipopolysaccharide

Sequentially collected sera from cats immunized withEnterobacter cloacae lipopolysaccharide (LPS) were assessed for their effect on phagocytosis by incubating alveolar marcophage monolayers in the presence of³H-labeled bacteria and 5% serum from control or immunized animals. Unlike serum fromPseudomonas aeruginosa LPS-immunized orP. aeruginosa-infected cats in previous studies, which contained phagocytic inhibitory activity specific forP. aeruginosa, serum from 12 of 13 cats immunized withE. cloacae LPS did not contain phagocytic inhibitory activity forE. cloacae orP. aeruginosa. An enzyme-linked immunosorbent assay (elisa) demonstrated a significant rise inE. cloacae LPS specific IgG (peak titer 1:10,240) by week 13 after immunization. This study suggests that long-term immunization withE. cloacae LPS does not result in the induction of macrophage phagocytic inhibitory activity as previously demonstrated withP. aeruginosa LPS immunization.     

Pseudomonas aeruginosa outer membrane protein F produced inEscherichia coli retains vaccine efficacy

Pseudomonas aeruginosa outer membrane protein F was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Antisera directed against protein F purified fromP. aeruginosa PAO1 reacted with thisE. coli strain by immunofluorescence assay and immunoblotting, whereas these antisera were nonreactive withE. coli strains lacking thePseudomonas protein F gene. The protein F purified from thisE. coli strain was used to immunize mice by intramuscular injection of 10 µg of protein F preparation on days 1 and 14, followed by burn and challenge of the mice on day 28. As compared with control mice immunized withE. coli K-12 lipopolysaccharide, immunization with theE. coli-derived protein F afforded significant protection against subsequent challenge with heterologous Fisher-Devlin immunotype 5 and 6 strains ofP. aeruginosa. Antisera from mice immunized with theE. coli-derived protein F reacted at bands corresponding to protein F and 2-mercaptoethanol-modified protein F upon immunoblotting against cell envelope proteins of the PAO1, immunotype 5, and immunotype 6 strains ofP. aeruginosa and theE. coli strain containing the cloned F gene, but failed to react at these sites in anE. coli strain lacking the F gene. These data demonstrate thatP. aeruginosa protein F produced inE. coli through genetic engineering techniques retains its vaccine efficacy in the complete absence of anyP. aeruginosa lipopolysaccharide.     

Intranasal Immunization of Mice Against Naegleria fowleri1

ABSTRACT. The purpose of this research was to determine whether mice could be protected from lethal challenge with Naegleria fowleri by prior intranasal exposure to pathogenic and nonpathogenic Naegleria. Mortality ranged from 0 to 100% for mice inoculated intranasally (i.n.) with 5 × 10³ amebae of 13 human isolates of N. fowleri. Mice were immunized and challenged i.n. using live amebae of strains of low, medium, and high virulence. The greatest protection against lethal challenge was afforded by three immunizing doses of 10³ amebae per dose of the strain of medium virulence. Nonpathogenic N. gruberi also was used to immunize mice i.n. against lethal challenge with N. fowleri. Protection was greater following immunization with N. gruberi than it was after immunization with N. fowleri, suggesting that nonpathogenic N. gruberi may be a better immunogen in protecting mice against lethal naeglerial challenge.     

Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.     

Intranasal Immunization of Baculovirus Displayed Hemagglutinin Confers Complete Protection against Mouse Adapted Highly Pathogenic H7N7 Reassortant Influenza Virus

Background

Avian influenza A H7N7 virus poses a pandemic threat to human health because of its ability for direct transmission from domestic poultry to humans and from human to human. The wide zoonotic potential of H7N7 combined with an antiviral immunity inhibition similar to pandemic 1918 H1N1 and 2009 H1N1 influenza viruses is disconcerting and increases the risk of a putative H7N7 pandemic in the future, underlining the urgent need for vaccine development against this virus.

Methodology/Principal Findings

In this study, we developed a recombinant vaccine by expressing the H7N7-HA protein on the surface of baculovirus (Bac-HA). The protective efficacy of the live Bac-HA vaccine construct was evaluated in a mouse model by challenging mice immunized intranasally (i.n.) or subcutaneously (s.c.) with high pathogenic mouse adapted H7N7 reassorted strain. Although s.c. injection of live Bac-HA induced higher specific IgG than i.n. immunization, the later resulted in an elevated neutralization titer. Interestingly, 100% protection from the lethal viral challenge was only observed for the mice immunized intranasally with live Bac-HA, whereas no protection was achieved in any other s.c. or i.n. immunized mice groups. In addition, we also observed higher mucosal IgA as well as increased IFN-γ and IL-4 responses in the splenocytes of the surviving mice coupled with a reduced viral titer and diminished histopathological signs in the lungs.

Conclusion

Our results indicated that protection from high pathogenic H7N7 (NL/219/03) virus requires both mucosal and systemic immune responses in mice. The balance between Th1 and Th2 cytokines is also required for the protection against the H7N7 pathogen. Intranasal administration of live Bac-HA induced all these immune responses and protected the mice from lethal viral challenge. Therefore, live Bac-HA is an effective vaccine candidate against H7N7 viral infections.     

Protective immunity to pseudomonas aeruginosa induced with a capsid-modified adenovirus expressing P. aeruginosa OprF

This study focuses on the development of a new clinical vaccine candidate (AdOprF.RGD.Epi8) against Pseudomonas aeruginosa using an E1⁻ E3⁻ adenovirus (Ad) vector expressing OprF (AdOprF.RGD.Epi8) and modifications of the Ad genome providing two capsid changes: (i) modification of the Ad hexon gene to incorporate an immune-dominant OprF epitope (Epi8) into loop 1 of the hexon, enabling repeat administration to boost the anti-OprF immune response, and (ii) modification of the fiber gene to incorporate an integrin-binding RGD sequence to enhance gene delivery to antigen-presenting cells. Western analysis confirmed that AdOprF.RGD.Epi8 expresses OprF, contains Epi8 in the hexon protein, and enhances gene transfer to dendritic cells compared to AdOprF, a comparable Ad vector expressing OprF with an unmodified capsid. Intramuscular immunization of C57BL/6 mice with AdOprF.RGD.Epi8 resulted in the generation of anti-OprF antibodies at comparable levels to those induced following immunization with AdOprF, but immunization with AdOprF.RGD.Epi8 was associated with increased CD4 and CD8 gamma interferon T-cell responses against OprF as well as increased survival against lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeat administration of AdOprF.RGD.Epi8 resulted in boosting of the humoral anti-OprF response as well as increased protection, whereas no boosting could be achieved with repeat administration of AdOprF. This suggests that the capsid-modified AdOprF.RGD.Epi8 vector is a more effective immunogen compared to a comparable wild-type Ad capsid, making it a good candidate for an anti-P. aeruginosa vaccine.     

Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon

In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF^ts1. The mutations that resulted in a temperature-sensitive phenotype in mSF^ts1 were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42 °C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 h of growth. In order to facilitate its utilization, the mSF^ts1 was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF^ts1 for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.     

Comparative study of immunobiological activities ofPseudomonas aeruginosa andBrucella melitensis lipopolysaccharides

The toxic activity ofBrucella melitensis andPseudomonas aeruginosa lipopolysaccharides as well as their behavior as immunogens, mitogens, and interferon inducers have been studied. Although their toxicities were very similar, the former molecule was incapable of eliciting a primary immune response in mice. Rabbit hyperimmunization gave titers half of those obtained withP. aeruginosa lipopolysaccharide. Optimal mitogenic responses of spleen cell cultures were obtained using 10–50 μg/ml and 50–100 μg/ml ofPseudomonas andBrucella lipopolysaccharide, respectively, giving the latter a lower stimulation of³H-thymidine uptake. Interferon titers induced in chickens byBrucella lipopolysaccharide were three times lower than those obtained withPseudomonas lipopolysaccharide.     

Salmonella Typhimurium strain expressing OprF‐OprI protects mice against fatal infection by Pseudomonas aeruginosa

Pseudomonas aeruginosa poses a major threat to human health and to the mink industry. Thus, development of vaccines that elicit robust humoral and cellular immunity against P. aeruginosa is greatly needed. In this study, a recombinant attenuated Salmonella vaccine (RASV) that expresses the outer membrane proteins fusion OprF_190–342‐OprI_21–83 (F1I2) from P. aeruginosa was constructed and the potency of this vaccine candidate assessed by measuring F1I2‐specific humoral immune responses upon vaccination through s.c. or oral routes. S.C. administration achieved higher serum IgG titers and IgA titers in the intestine and induced stronger F1I2‐specific IgG and IgA titers in lung homogenate than did oral administration, which resulted in low IgG titers and no local IgA production. High titers of IFN‐γ, IL‐4, and T‐lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized s.c., indicating elicitation of cellular immunity. Importantly, when immunized mice were challenged with P. aeruginosa by the intranasal route 30 days after the initial immunization, s.c. vaccination achieved 77.78% protection, in contrast to 41.18% via oral administration and 66.67% via Escherichia coli‐expressed F1I2 (His‐F1I2) vaccination. These results indicate that s.c. vaccination provides a better protective response against P. aeruginosa infection than do oral administration and the His‐F1I2 vaccine.     

Analysis of Cellular Response and Gamma Interferon Synthesis in Bronchoalveolar Lavage Fluid and Lung Homogenate of Mice Infected with Pneumocystis carinii

The cellular and cytokine responses in the lungs of mice infected with Pneumocystis carinii were examined on both lung homogenates and bronchoalveolar lavage (BAL) fluids. In the lungs of infected mice, the number of P. carinii cysts rapidly decreased by day 7, then started to increase with a peak on day 14, and thereafter decreased gradually. When the presence of P. carinii was examined at the DNA level by dot blot hybridization, a similar clearance curve was obtained, and the organisms were shown to be completely eliminated on day 28. In the late phase of infection, leukocytes, mainly lymphocytes, increased in number when analyzed on lung homogenates, while no significant increase of inflammatory cells was observed in BAL fluids. An accumulation of both CD4⁺ and CD8⁺ T cells and an increase of activated T cells expressing IL-2Rα were observed in lung homogenates of the infected mice. In addition, a considerable amount of IFN-γ was detected in lung homogenates, but not in BAL fluids. These data indicate that lung homogenates are more suitable than BAL fluids for the analysis of cellular and cytokine responses in the lungs of mice infected with P. carinii. To define the involvement of IFN-γ in host defense against P. carinii, the effect of this cytokine on the killing activity of macrophages against P. carinii was examined in vitro. IFN-γ was found to augment this activity by increasing nitric oxide synthesis of the macrophages. Thus, it is suggested that IFN-γ plays an important role in the protection of mice from P. carinii infection.     

Vaccination with M2e-based multiple antigenic peptides: characterization of the B cell response and protection efficacy in inbred and outbred mice

Background

The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains.

Methodology/Principal Findings

Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs.

Conclusion/Significance

Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population.     

Protective immunity induced by systemic and mucosal delivery of DNA vaccine expressing glycoprotein B of pseudorabies virus

A murine model immunized by systemic and mucosal delivery of plasmid DNA vaccine expressing glycoprotein B (pCIgB) of pseudorabies virus (PrV) was used to evaluate both the nature of the induced immunity and protection against a virulent virus. With regard to systemic delivery, the intramuscular (i.m.) immunization with pCIgB induced strong PrV-specific IgG responses in serum but was inefficient in generating a mucosal IgA response. Mucosal delivery through intranasal (i.n.) immunization of pCIgB induced both systemic and mucosal immunity at the distal mucosal site. However, the levels of systemic immunity induced by i.n. immunization were less than those induced by i.m. immunization. Moreover, i.n. genetic transfer of pCIgB appeared to induce Th2-biased immunity compared with systemic delivery, as judged by the ratio of PrV-specific IgG isotypes and Th1- and Th2-type cytokines produced by stimulated T cells. Moreover, the immunity induced by i.n. immunization did not provide effective protection against i.n. challenge of a virulent PrV strain, whereas i.m. immunization produced resistance to viral infection. Therefore, although i.n. immunization was a useful route for inducing mucosal immunity at the virus entry site, i.n. immunization did not provide effective protection against the lethal infection of PrV.     

Increased susceptibility of gentamincin-resistantPseudomonas aeruginosa to human sera

A total of 40 clinical strains ofPseudomonas aeruginosa were tested for in vitro resistance to the bactericidal action of pooled normal human sera (PHS). Getamicin-resistantP. aeruginosa (GRPA) were found to be significantly more susceptible to the killing action of 6% PHS than their gentamincin-sensitiveP. aeruginosa (GSPA) counterparts. In vitro mutants of GRPA were also more susceptible to PHS than their progenitor GSPA strain. The increased susceptibility of GRPA to PHS may help explain their lack of dissemination to internal body sites.     

Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice

Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1–3), domains I and II (D1–2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1–3, D1–2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1–3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-γ T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins.