Flt3 ligand pretreatment promotes protective immunity to Listeria monocytogenes

Flt3 ligand (Flt3L) plays a critical role in the proliferation, differentiation and survival of haematopoietic progenitor cells. Its potential use in a clinical setting has been suggested. Here, we report that mice administered Flt3L displayed a nine-fold increase in size of their hepatic non-parenchymal cell population and an approximate 365-fold increase in number of mature dendritic cells within their livers. Such mice exhibited an elevated resistance to secondary infections with Listeria monocytogenes, an intracellular bacterial pathogen. More than 2.0 log(10)fewer listeriae were recovered in the livers of Flt3L-treated, than untreated, mice on day 2 following secondary challenge. Importantly, Flt3L-pretreated mice immunized with an avirulent (listeriolysin O-negative) strain of Listeria harbored significantly fewer ( approximately 1.5 log(10)) organisms in their spleens and livers than did control mice immunized with listeriolysin O-negative listeriae and challenged with a lethal dose of bacteria. The latter finding supports a potential role for Flt3L in strategies to develop vaccines to intracellular pathogens.     

Nasal Flt3 ligand cDNA elicits CD11c+CD8+ dendritic cells for enhanced mucosal immunity

The embryo expresses paternal Ags foreign to the mother and therefore has been viewed as an allograft. It has been shown that anergic T cells generated by blocking of the CD28/B7 costimulatory pathway with anti B7-1 and anti B7-2 mAbs can be transferred as suppresser cells to prevent allograft rejection. Little is known, however, about the in vivo function of anti-B7-treated T cells after their transfer into abortion-prone mice in the maintenance of materno-fetal tolerance. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered anti-B7-1 and anti-B7-2 mAbs on day 4 of gestation (murine implantation window). The anti-B7-treated T cells subsequently were adoptively transferred into abortion-prone CBA/J mice. We demonstrated that costimulation blockade with anti-B7 mAbs at the time of implantation resulted in altered allogeneic T cell response and overcame increased maternal rejection to the fetus in the CBA/JxDBA/2 system. The transferred anti-B7-treated T cells appeared to be regulatory, decreasing responsiveness and generating clonal deviation in maternal recipient T cells. The transferred CFSE-labeled T cells were found to reside in the spleen and uterine draining lymph nodes, and a few were localized to the materno-fetal interface of the maternal recipient. Our findings suggest that the anti-B7-treated T cells not only function as potent suppresser cells, but also exert an immunoregulatory effect on the maternal recipient T cells, which cosuppresses maternal rejection to the fetus. This procedure might be considered potentially useful for fetal survival when used as an immunotherapy for human recurrent spontaneous abortion.     

Dendritic cell‐targeting DNA‐based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae

To develop safe vaccines for inducing mucosal immunity to major pulmonary bacterial infections, appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants must be considered. Such vaccine constructs can induce Ag‐specific immune responses that protect against mucosal infections. In particular, it has been shown that simply mixing the adjuvant with the bacterial Ag is a relatively easy means of constructing adjuvant‐based mucosal vaccine preparations; the resulting vaccines can elicit protective immunity. DNA‐based nasal adjuvants targeting mucosal DCs have been studied in order to induce Ag‐specific mucosal and systemic immune responses that provide essential protection against microbial pathogens that invade mucosal surfaces. In this review, initially a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule that is a growth factor for DCs, as an effective adjuvant for mucosal immunity to pneumococcal infections, is introduced. Next, the potential of adding unmethylated CpG oligodeoxynucleotide and pFL together with a pneumococcal Ag to induce protection from pneumococcal infections is discussed. Pneumococcal surface protein A has been used as vaccine for restoring mucosal immunity in older persons. Further, our nasal pFL adjuvant system with phosphorylcholine‐keyhole limpet hemocyanin (PC‐KLH) has also been used in pneumococcal vaccine development to induce complete protection from nasal carriage by Streptococcus pneumoniae . Finally, the possibility that anti‐PC antibodies induced by nasal delivery of pFL plus PC‐KLH may play a protective role in prevention of atherogenesis and thus block subsequent development of cardiovascular disease is discussed.

     

A CpG oligodeoxynucleotide enhances the immune response to rabies vaccination in mice

Background

Rabies is a fatal disease that is preventable when post exposure prophylaxis (PEP) is administered in a timely fashion. CpG oligodeoxynucleotides (ODNs) can trigger cells that express Toll-like receptor 9, and their immunopotentiation activity in an inactivated aluminum-adjuvanted rabies vaccine for dogs has been identified using mouse and dog models.

Methods

A human diploid cell rabies vaccine (HDCV) of humans and a CpG ODNs with cross-immunostimulatory activity in humans and mice were used to evaluate the immunogenicity and protective efficacy of CpG ODN in a mouse model that simulates human PEP.

Results

HDCV combined with CpG ODN (HDCV–CpG) stimulated mice to produce rabies virus-specific neutralizing antibody (RVNA) earlier and increased the seroconversion rate. Compared with HDCV alone, either HDCV–1.25 μg CpG or HDCV–5 μg CpG increased the levels of RVNA. In particular, 5 μg CpG ODN per mouse significantly boosted the levels of RVNA compared with HDCV alone. IFN-γ producing splenocytes generated in the HDCV-5 μg CpG group were significantly increased compared to the group treated with HDCV alone. When the immunization regimen was reduced to three injections or the dose was reduced to half of the recommended HDCV combined with CpG ODN, the RVNA titers were still higher than those induced by HDCV alone. After viral challenge, 50% of mice immunized with a half-dose HDCV–CpG survived, while the survival rate of mice immunized with HDCV alone was 30%.

Conclusions

The immunopotentiation activity of CpG ODNs for a commercially available human rabies vaccine was first evaluated in a mouse model on the basis of the Essen regimen. Our results suggest that the CpG ODN used in this study is a potential adjuvant to rabies vaccines for human use.

     

A plasmid encoding Japanese encephalitis virus PrM and E proteins elicits protective immunity in suckling mice

A plasmid encoding Japanese encephalitis virus (JEV) prM and E proteins was constructed, and its efficacy as a candidate vaccine against JEV was evaluated in suckling mice. Groups of 10 BALB/c mice (5-7 days old) were immunized twice via muscular injection with this DNA vaccine, an empty vector or PBS at an interval of 3 weeks, and were challenged with a lethal dose of JEV 3 weeks after the second inoculation. Both cellular and humoral immune responses were examined before the challenge. Two animals from each group were sacrificed to detect the JEV-specific cytotoxic T lymphocyte activity. JEV-specific lactate dehydrogenase release in the DNA vaccine, empty vector and PBS groups was 37.5%, 18% and 8.5% respectively. JEV-specific antibody was detected in 8 of 10 animals in DNA vaccine group with a geometrical mean titer of 1: 28.3. The pooled serum from the same group also showed a neutralizing activity. Six of 8 mice in the DNA vaccine group survived the challenge, with a protection rate of 75%, but all the mice died in the two control groups. These results show that this JEV prM and E DNA vaccine is immunogenic and protective against JEV infection in the mouse model.     

FMS-like tyrosine kinase 3 ligand aggravates the lung inflammatory response to Streptococcus pneumoniae infection in mice: role of dendritic cells

Pretreatment of mice with the hemopoietic growth factor, FMS-like tyrosine kinase 3 ligand (Flt3L), has been shown to increase monocyte-derived myeloid dendritic cells (DC) in lung parenchymal tissue, with possible implications for protective immunity to lung bacterial infections. However, whether Flt3L treatment improves lung innate immunity of mice to challenge with Streptococcus pneumoniae has not been investigated previously. Mice pretreated with Flt3L exhibited a peripheral monocytosis and a strongly expanded lung myeloid DC pool, but responded with a similar proinflammatory cytokine release (TNF-alpha, IL-6, keratinocyte derived cytokine, MIP-2, CCL2) and neutrophilic alveolitis upon infection with S. pneumoniae as did control mice with a normal lung DC pool. Unexpectedly, however, Flt3L-pretreated mice, but not control mice, infected with S. pneumoniae developed vasculitis and increased lung permeability by days 2-3 postinfection, and florid pneumonia accompanied by sustained increased bacterial loads by days 3-4 postinfection. This was associated with an overall increased mortality of approximately 35% by day 4 after pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block inflammatory monocyte-dependent lung mononuclear phagocyte mobilization significantly reduced the lung leakage, but not vasculitis in Flt3L-pretreated mice infected with S. pneumoniae, without affecting the intra-alveolar cytokine liberation or the concomitantly developing neutrophilic alveolitis. Together, the data demonstrate that previous Flt3L-induced lung DC accumulation is not protective in lung innate immunity to challenge with S. pneumoniae, and support the concept that CCR2-dependent mononuclear phagocyte as opposed to neutrophil recruitment contributes to increased lung leakage in Flt3L-pretreated mice challenged with S. pneumoniae.     

A protective role of locally administered immunostimulatory CpG oligodeoxynucleotide in a mouse model of genital herpes infection

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODNs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. We therefore investigated whether such a CpG-containing ODN (CpG ODN) given mucosally in the female genital tract could enhance innate immunity and protect against genital herpes infection. Groups of C57BL/6 mice were treated intravaginally with either CpG ODN or a non-CpG ODN control in the absence of any antigen either 2 days before or 4 h after an intravaginal challenge with a normally lethal dose of herpes simplex virus type 2 (HSV-2). Mice treated with CpG ODN exhibited significantly decreased titers of HSV-2 in their vaginal fluids compared with non-CpG ODN-treated mice. Furthermore, CpG ODN pretreatment significantly protected against development of disease and death compared to non-CpG ODN pretreatment. Most strikingly, CpG ODN conferred protection against disease and death even when given after the viral challenge. The CpG ODN-induced protection was associated with a rapid production of gamma interferon (IFN-gamma), interleukin-12 (IL-12), IL-18, and RANTES in the genital tract mucosa following CpG ODN treatment. The observed protection appeared to be dependent on IFN-gamma, IL-12, IL-18, and T cells, as CpG ODN pretreatment did not confer any significant protection in mice deficient in IFN-gamma, IL-12, IL-18, or T cells. Further, a complete protective immunity to reinfection was elicited in CpG ODN-treated, HSV-2-challenged mice, suggesting a role for mucosally administered CpG ODN in inducing the development of an acquired immune response in addition to its potent stimulation of innate immunity.     

A bivalent CMV vaccine formulated with human compatible TLR9 agonist CpG1018 elicits potent cellular and humoral immunity in HLA expressing mice

There is now convincing evidence that the successful development of an effective CMV vaccine will require improved formulation and adjuvant selection that is capable of inducing both humoral and cellular immune responses. Here, we have designed a novel bivalent subunit vaccine formulation based on CMV-encoded oligomeric glycoprotein B (gB) and polyepitope protein in combination with human compatible TLR9 agonist CpG1018. The polyepitope protein includes multiple minimal HLA class I-restricted CD8⁺ T cell epitopes from different antigens of CMV. This subunit vaccine generated durable anti-viral antibodies, CMV-specific CD4⁺ and CD8⁺ T cell responses in multiple HLA expressing mice. Antibody responses included broad T_H1 isotypes (IgG2a, IgG2b and IgG3) and potently neutralized CMV infection in fibroblasts and epithelial cells. Furthermore, polyfunctional antigen-specific T cell immunity and antiviral antibody responses showed long-term memory maintenance. These observations argue that this novel vaccine strategy, if applied to humans, could facilitate the generation of robust humoral and cellular immune responses which may be more effective in preventing CMV-associated complications in various clinical settings.     

Comparison of the functional properties of murine dendritic cells generated in vivo with Flt3 ligand, GM-CSF and Flt3 ligand plus GM-SCF

Flt3 ligand (FL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are important growth factors for dendritic cells (DC). Substantial numbers of DC can be generated in vivo following the administration of either factor. We sought to extend our knowledge of the functional properties of these cells including their ability to prime na?ve CD8(+) T cells. In addition, we compared the nature of the DC generated in vivo with the single cytokines to those generated with the combination of FL+polyethylene glycol-modified GM-CSF (pGM-CSF). Treatment with FL+pGM-CSF yielded greater numbers of both CD11b(low) and CD11b(high) DC than with either cytokine alone, and these DC were more efficient at antigen (Ag) capture. The FL+pGM-CSF-generated CD11b(low) DC lacked expression of CD8alpha. Following treatment with LPS in vivo, all DC subsets upregulated CD40, CD80, CD86, and MHC class II expression, but surprisingly Ag capture was not downregulated and some DC subsets retained expression of intracellular MHC class II vesicles. Thus, even after activation in vivo with LPS, DC retained Ag capture properties of immature DC, and Ag presentation/costimulation properties of mature DC. Though all DC subsets stimulated CD4(+) T cell proliferation equivalently, FL-generated DC were more efficient at priming Ag-specific CD8(+) cytolytic T cells than DC generated with either pGM-CSF alone or FL+pGM-CSF, and CD11b(high) DC were more efficient at priming CD8(+) T cells than CD11b(low) DC.     

Mucosal administration of CpG oligodeoxynucleotide elicits strong CC and CXC chemokine responses in the vagina and serves as a potent Th1-tilting adjuvant for recombinant gD2 protein vaccination against genital herpes

Although sexually transmitted pathogens are capable of inducing pathogen-specific immune responses, vaginal administration of nonreplicating antigens elicits only weak, nondisseminating immune responses. The present study was undertaken to examine the potential of CpG-containing oligodeoxynucleotide (CpG ODN) for induction of chemokine responses in the genital tract mucosa and also as a vaginal adjuvant in combination with glycoprotein D of herpes simplex virus type 2 (HSV-2) for induction of antigen-specific immune responses. We found that a single intravaginal administration of CpG ODN in mice stimulates a rapid and potent response of CC chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES as well as of CXC chemokines MIP-2 and IP-10 in the vagina and/or the genital lymph nodes. Importantly, intravaginal vaccination with recombinant gD2 in combination with CpG ODN gave rise to a strong antigen-specific Th1-like immune response in the genital lymph nodes as well as the spleens of the vaccinated mice. Further, such an immunization scheme conferred both systemic and mucosal immunoglobulin G antibody responses as well as protection against an otherwise lethal vaginal challenge with HSV-2. These results illustrate the potential of CpG ODN for induction of potent chemokine responses in the genital tract and also as a vaginal adjuvant for generation of Th1-type mucosal and systemic immune responses towards a nonreplicating antigen derived from a sexually transmitted pathogen. These data have implications for the development of a mucosal vaccine against genital herpes and possibly other sexually transmitted diseases.     

Stimulation of protective and cross-protective immunity against influenza B virus after adjuvant mucosal immunization of mice

Intratracheal immunization of mice with inactivated influenza B virus and delipidated Bacillus firmus as adjuvant increases protection of mice against infection with the homologous virus strain and induces cross-protection: mice immunized by influenza virus B/Yamanashi 166/98 were protected even against phylogenetically distant virus drift variant B/Lee/40 lethal for mice.     

Withdrawn: Immunization with pseudotype baculovirus expressing envelope protein of Japanese encephalitis virus elicits protective immunity in mice

This paper appeared in the February issue, occupying pp. 150‐159. It was a duplicate of JGM 1271, which appears on pp. 57‐65 of the January issue. It has therefore been withdrawn from Wiley InterScience. The publisher apologies to the authors for the error. Copyright © 2009 John Wiley & Sons, Ltd.     

Contributions of edema factor and protective antigen to the induction of protective immunity by Bacillus anthracis edema toxin as an intranasal adjuvant

We have shown that intranasal coapplication of Bacillus anthracis protective Ag (PA) together with a B. anthracis edema factor (EF) mutant having reduced adenylate cyclase activity (i.e., EF-S414N) enhances anti-PA Ab responses, but also acts as a mucosal adjuvant for coadministered unrelated Ags. To elucidate the role of edema toxin (EdTx) components in its adjuvanticity, we examined how a PA mutant lacking the ability to bind EF (PA-U7) or another mutant that allows the cellular uptake of EF, but fails to efficiently mediate its translocation into the cytosol (PA-dFF), would affect EdTx-induced adaptive immunity. Native EdTx promotes costimulatory molecule expression by macrophages and B lymphocytes, and a broad spectrum of cytokine responses by cervical lymph node cells in vitro. These effects were reduced or abrogated when cells were treated with EF plus PA-dFF, or PA-U7 instead of PA. We also intranasally immunized groups of mice with a recombinant fusion protein of Yersinia pestis F1 and LcrV Ags (F1-V) together with EdTx variants consisting of wild-type or mutants PA and EF. Analysis of serum and mucosal Ab responses against F1-V or EdTx components (i.e., PA and EF) revealed no adjuvant activity in mice that received PA-U7 instead of PA. In contrast, coimmunization with PA-dFF enhanced serum Ab responses. Finally, immunization with native PA and an EF mutant lacking adenylate cyclase activity (EF-K346R) failed to enhance Ab responses. In summary, a fully functional PA and a minimum of adenylate cyclase activity are needed for EdTx to act as a mucosal adjuvant.     

Live virulent Rhodococcus equi, rather than killed or avirulent, elicits protective immunity to R. equi infection in mice

Mice inoculated intravenously with a sublethal dose of live virulent Rhodococcus equi ATCC 33701 that contained an 85-kb virulence plasmid were immune to a lethal intravenous challenge of ATCC 33701. This immunity depended upon the dose of immunization and developed rapidly: mice primed with 10(5) live ATCC 33701 eliminated the challenged bacteria more rapidly than mice primed with doses ranging from 10(2) to 10(4) bacteria, and mice given 10(5) live ATCC 33701 intravenously withstood the lethal challenge as early as 5 days after the initial inoculation. However, this protective immunity did not develop in mice immunized with doses of heat-killed ATCC 33701 ranging from 10(6) to 10(8), or in mice immunized with doses of live ATCC 33701P-, a plasmid-cured derivative (avirulent), in doses ranging from 10(5) to 10(7). These mice had positive antibody titers against R. equi at the challenge (14 days after priming). Adoptive transfer of resistance to virulent R equi was obtained with spleen cells from mice immunized with live ATCC 33701, but not monoclonal antibody to 15- to 17-kDa virulence-associated antigens. These results revealed that live ATCC 33701P-, a plasmid-cured derivative of virulent R equi, could not elicit protective immunity, and are consistent with previous observations that protective immunity was induced by live virulent, but not killed organisms.     

PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans

Streptococcus pneumoniae (pneumococci) adhere to human nasopharyngeal (NP) epithelial cells as a first step in colonization and adherence of pneumococci to lung epithelia may be required to establish pneumonia. We sought to determine if PcpA can serve as an adhesin to human NP (D562) and lung (A549) epithelial cells and whether PcpA mediated adherence can be inhibited by human anti-PcpA antibodies. A PcpA isogenic mutant was constructed in a pneumococcal TIGR4 background. When the mutant and wild type strains were compared for their adherence to D562 and A549 cell lines, a reduction in adherence by the mutant was observed (p = 0.0001 for both cell types). PcpA was ectopically expressed on the surface of minimally-adherent heterologous host Escherichia coli resulting in augmented adherence to D562 (p = 0.002) and A549 (p = 0.015) cells. Total IgG was purified from a pool of 6 human sera having high IgG titers of anti-pneumococcal proteins. The purified IgG reduced TIGR4 adherence to D562 cells but we determined that this effect was largely due to bacterial cell aggregation as determined by flow cytometry and confocal microscopy. Fab fragments were prepared from pooled IgG sera. Inhibition of TIGR4 adherence to D562 cells was observed using the Fab fragments without causing bacterial aggregation (p = 0.0001). Depletion of PcpA-specific Fab fragments resulted in an increase in adherence of TIGR4 to D562 cells (p = 0.028). We conclude that PcpA can mediate adherence of pneumococci to human NP and lung epithelial cells and PcpA mediated adherence can be inhibited by human anti-PcpA antibodies.     

Immunogenicity and protective efficacy of GroEL (hsp60) of Streptococcus pneumoniae against lethal infection in mice

The present study was carried out to evaluate the immunogenicity and protective efficacy of GroEL (hsp60) of Streptococcus pneumoniae , by expressing full length GroEL in heterologous host Escherichia coli BL21(DE3). PCR-amplified groEL was ligated in pQE 30 expression vector and subsequently transformed in E. coli DH5α strains. Cloning of groEL was confirmed by double digestion, followed by DNA sequencing. The His-tag containing recombinant GroEL was purified by Ni-NTA affinity chromatography. To determine the immunogenicity of GroEL, the mice were immunized by injecting 40 μg GroEL protein per mouse intraperitoneally. The results showed a significant increase in antibody titre and lymphocyte proliferation in animals immunized with GroEL as compared with control. Further, there was an appreciable increase in interleukin-2 (IL-2) and IL-4 production in lymphocytes isolated from immunized mice as compared with control. To determine the efficacy of GroEL in eliciting protection, the mice were challenged with the lethal dose of S. pneumoniae A66 type 3 capsular strain intranasally after the seventh day of the last immunization. In the GroEL-immunized mice the onset of death was insignificantly delayed and all the mice died by the seventh day postinfection.     

A Powassan virus domain III nanoparticle immunogen elicits neutralizing and protective antibodies in mice

Powassan virus (POWV) is an emerging tick borne flavivirus (TBFV) that causes severe neuroinvasive disease. Currently, there are no approved treatments or vaccines to combat POWV infection. Here, we generated and characterized a nanoparticle immunogen displaying domain III (EDIII) of the POWV E glycoprotein. Immunization with POWV EDIII presented on nanoparticles resulted in significantly higher serum neutralizing titers against POWV than immunization with monomeric POWV EDIII. Furthermore, passive transfer of EDIII-reactive sera protected against POWV challenge in vivo. We isolated and characterized a panel of EDIII-specific monoclonal antibodies (mAbs) and identified several that potently inhibit POWV infection and engage distinct epitopes within the lateral ridge and C-C′ loop of the EDIII. By creating a subunit-based nanoparticle immunogen with vaccine potential that elicits antibodies with protective activity against POWV infection, our findings enhance our understanding of the molecular determinants of antibody-mediated neutralization of TBFVs.     

CD8+ cells enhance resistance to pulmonary serotype 3 Streptococcus pneumoniae infection in mice

Despite the success of the pneumococcal conjugate vaccine, pneumococcal pneumonia remains a significant clinical problem, and there is still much to learn about natural resistance and cellular immunity to pneumococcus. We investigated the role of T lymphocytes in resistance to serotype (ST) 3 Streptococcus pneumoniae in an intranasal infection model in C57BL/6 (wild-type [Wt]) and CD8(+) (CD8(-/-))- and CD4(+) (MHC class II(-/-))-deficient mice. CD8(-/-) mice exhibited significantly more bacterial dissemination and lung inflammation and a significantly more lethal phenotype than Wt mice. However, there was no difference in the bacterial dissemination, lung inflammation, or survival of Wt and MHC class II(-/-) mice. Perforin (Pfn)(-/-) and IFN-γ(-/-) mice, which were used to dissect the role of CD8(+) T cells in our model, also exhibited a more lethal survival phenotype than Wt mice. Comparison of lung chemokine/cytokine levels by Luminex and cellular recruitment by FACS in Wt mice and knockout strains revealed that CD8(-/-) and IFN-γ(-/-) mice, which had the most lethal survival phenotype, had more CD4(+)IL-17(+) T (Th17) cells, IL-17, neutrophil chemoattractants, and lung neutrophils, and fewer regulatory T cells than Wt mice. CD4(+) T cell depletion improved the survival of ST-infected CD8(-/-) mice, and survival studies in Th17-deficient mice revealed that the Th17 response was dispensable for ST3 resistance in our model. Taken together, these findings demonstrate that CD8(+) cells are required, but CD4(+) T cells are dispensable for resistance to ST3 pneumonia in mice and suggest a previously unsuspected role for CD8(+) cells in modulating the inflammatory response to ST3.