TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells

Whole cell pertussis vaccines (Pw) induce Th1 responses and protect against Bordetella pertussis infection, whereas pertussis acellular vaccines (Pa) induce Ab and Th2-biased responses and also protect against severe disease. In this study, we show that Pw failed to generate protective immunity in TLR4-defective C3H/HeJ mice. In contrast, protection induced with Pa was compromised, but not completely abrogated, in C3H/HeJ mice. Immunization with Pw, but not Pa, induced a population of IL-17-producing T cells (Th-17), as well as Th1 cells. Ag-specific IL-17 and IFN-gamma production was significantly lower in Pw-immunized TLR4-defective mice. Furthermore, treatment with neutralizing anti-IL-17 Ab immediately before and after B. pertussis challenge significantly reduced the protective efficacy of Pw. Stimulation of dendritic cells (DC) with Pw promoted IL-23, IL-12, IL-1beta, and TNF-alpha production, which was impaired in DC from TLR4-defective mice. B. pertussis LPS, which is present in high concentrations in Pw, induced IL-23 production by DC, which enhanced IL-17 secretion by T cells, but the induction of Th-17 cells was also dependent on IL-1. In addition, we identified a new effector function for IL-17, activating macrophage killing of B. pertussis, and this bactericidal activity was less efficient in macrophages from TLR4-defective mice. These data provide the first definitive evidence of a role for TLRs in protective immunity induced by a human vaccine. Our findings also demonstrate that activation of innate immune cells through TLR4 helps to direct the induction of Th1 and Th-17 cells, which mediate protective cellular immunity to B. pertussis.     

Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice

The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-alpha and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2(-/-)) or TLR1 (TLR1(-/-)) produced low titers of antibodies against OspA. Notably, macrophages from TLR2(-/-) mice were unresponsive to OspA and PGN, whereas those from TLR1(-/-) mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.     

Bordetella pertussis fimbriae are effective carrier proteins in Neisseria meningitidis serogroup C conjugate vaccines

Serogroup C meningococcal conjugate vaccines generally use diphtheria or tetanus toxoids as the protein carriers. The use of alternative carrier proteins may allow multivalent conjugate vaccines to be formulated into a single injection and circumvent potential problems of immune suppression in primed individuals. Bordetella pertussis fimbriae were assessed as carrier proteins for Neisseria meningitidis serogroup C polysaccharide. Fimbriae were conjugated to the polysaccharide using modifications of published methods and characterised by size exclusion chromatography; co-elution of protein and polysaccharide moieties confirmed conjugation. The conjugates elicited boostable IgG responses to fimbriae and serogroup C polysaccharide in mice, and IgG:IgM ratios indicated that the responses were thymus-dependent. High bactericidal antibody titres against a serogroup C strain of N. meningitidis were also observed. In a mouse infection model, the conjugate vaccine protected against lethal infection with N. meningitidis. Therefore, B. pertussis fimbriae are effective carrier proteins for meningococcal serogroup C polysaccharide and could produce a vaccine to protect against meningococcal disease and to augment protection against pertussis.     

Acellular pertussis booster in adolescents induces Th1 and memory CD8+ T cell immune response

In a number of countries, whole cell pertussis vaccines (wcP) were replaced by acellular vaccines (aP) due to an improved reactogenicity profile. Pertussis immunization leads to specific antibody production with the help of CD4(+) T cells. In earlier studies in infants and young children, wcP vaccines selectively induced a Th1 dominated immune response, whereas aP vaccines led to a Th2 biased response. To obtain data on Th1 or Th2 dominance of the immune response in adolescents receiving an aP booster immunization after a wcP or aP primary immunization, we analyzed the concentration of Th1 (IL-2, TNF-α, INF-γ) and Th2 (IL-4, IL-5, IL-10) cytokines in supernatants of lymphocyte cultures specifically stimulated with pertussis antigens. We also investigated the presence of cytotoxic T cell responses against the facultative intracellular bacterium Bordetella pertussis by quantifying pertussis-specific CD8(+) T cell activation following the aP booster immunization. Here we show that the adolescent aP booster vaccination predominantly leads to a Th1 immune response based on IFNgamma secretion upon stimulation with pertussis antigen, irrespective of a prior whole cell or acellular primary vaccination. The vaccination also induces an increase in peripheral CD8(+)CD69(+) activated pertussis-specific memory T cells four weeks after vaccination. The Th1 bias of this immune response could play a role for the decreased local reactogenicity of this adolescent aP booster immunization when compared to the preceding childhood acellular pertussis booster. Pertussis-specific CD8(+) memory T cells may contribute to protection against clinical pertussis.     

TRIF is a critical negative regulator of TLR agonist mediated activation of dendritic cells in vivo

Despite recent advances in developing and licensing adjuvants, there is a great need for more potent formulations to enhance immunogenicity of vaccines. An Eimeria tenella derived antigen (rEA) augments immune responses against several pathogens in animal models and recently was confirmed to be safe for human use. In this study, we have analyzed the molecular mechanisms underlying rEA activity in mice, and confirmed that rEA activates multiple immune cell types, including DCs, macrophages, NK, B, and T cells. The rEA adjuvant also elicits the induction of pleiotropic pro-inflammatory cytokines, responses that completely depend upon the presence of the TLR adaptor protein MyD88. Surprisingly, we also found that the TRIF adaptor protein acts as a potent negative regulator of TLR agonist-triggered immune responses. For example, IL12 production and the induction of co-stimulatory molecule expression by DCs and IFNγ production by NK cells in vivo were significantly increased in rEA-treated TRIF-KO mice. Importantly, however, TRIF suppressive effects were not restricted to rEA-mediated responses, but were apparent in LPS- or ODN2006-activated DCs as well. Taken together, our findings confirm that rEA is a potent adjuvant, triggering robust activation of the innate immune system, in a manner that is augmented by MyD88 and inhibited by TRIF; thereby unveiling the potential complexities of modulating TLR activity to augment vaccine efficacy.     

Skin immune responses to peptide and protein antigen are TLR4 independent

Little is known about the innate immune mechanisms regulating adaptive immune responses elicited through the skin. Tissue injury is postulated to liberate Toll like receptor 4 (TLR4) ligands. In this study, we determined whether TLR4 signaling modulates the response to epidermal injury induced by tape stripping (TS) and whether it alters humoral and cellular immune responses generated through epicutaneous immunization with peptide+cholera toxin (CT). The combined use of cholera toxin and TS with antigen promoted optimal antigen-specific CD4(+) and CD8(+) T cell proliferation in Balb/c and C57BL/6 mice, respectively. TLR4 mutant mice had similar T cell responses to wild type mice. Further, OVA-protein specific IgG, IgG(1), IgG(2a), and IgE titers were similar in wild type and TLR4 mutant mice. Thus, TLR4 signaling was not required for the generation of epicutaneous T cell or antibody mediated immune responses and did not alter the quality of the immune responses elicited.     

TLR7 imidazoquinoline ligand 3M-019 is a potent adjuvant for pure protein prototype vaccines

Cancer vaccines, while theoretically attractive, present difficult challenges that must be overcome to be effective. Cancer vaccines are often poorly immunogenic and may require augmentation of immunogenicity through the use of adjuvants and/or immune response modifiers. Toll-like receptor (TLR) ligands are a relatively new class of immune response modifiers that may have great potential in inducing and augmenting both cellular and humoral immunity to vaccines. TLR7 ligands produce strong cellular responses and specific IgG2a and IgG2b antibody responses to protein immunogens. This study shows that a new TLR7 ligand, 3M-019, in combination with liposomes produces very strong immune responses to a pure protein prototype vaccine in mice. Female C57BL/6 mice were immunized subcutaneously with ovalbumin (OVA, 0.1 mg/dose) weekly 4x. Some groups were immunized to OVA plus 3M-019 or to OVA plus 3M-019 encapsulated in liposomes. Both antibody and cellular immune responses against OVA were measured after either two or four immunizations. Anti-OVA IgG antibody responses were significantly increased after two immunizations and were substantially higher after four immunizations in mice immunized with OVA combined with 3M-019. Encapsulation in liposomes further augmented antibody responses. IgM responses, on the other hand, were lowered by 3M-019. OVA-specific IgG2a levels were increased 625-fold by 3M-019 in liposomes compared to OVA alone, while anti-OVA IgG2b levels were over 3,000 times higher. In both cases encapsulation of 3M-019 in liposomes was stronger than either liposomes alone or 3M-019 without liposomes. Cellular immune responses were likewise increased by 3M-019 but further enhanced when it was encapsulated in liposomes. The lack of toxicity also indicates that this combination may by safe, effective method to boost immune response to cancer vaccines.     

Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4(+) and CD8(+) T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8(+) T cell responses against even low doses of protein Ags, and this effect was independent of CD4(+) T cell help. Ag-specific CD8(+) T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.     

Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T cell responses

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-alpha, and increased expression of CD80, CD86, and CD40 on CD11c(+) dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8(+) T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8(+) T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8(+) CD62(low) cells, approximately 50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.     

TLR2, but not TLR4, is required for effective host defence against Chlamydia respiratory tract infection in early life

Chlamydia pneumoniae commonly causes respiratory tract infections in children, and epidemiological investigations strongly link infection to the pathogenesis of asthma. The immune system in early life is immature and may not respond appropriately to pathogens. Toll-like receptor (TLR)2 and 4 are regarded as the primary pattern recognition receptors that sense bacteria, however their contribution to innate and adaptive immunity in early life remains poorly defined. We investigated the role of TLR2 and 4 in the induction of immune responses to Chlamydia muridarum respiratory infection, in neonatal wild-type (Wt) or TLR2-deficient ((-/-)), 4(-/-) or 2/4(-/-) BALB/c mice. Wt mice had moderate disease and infection. TLR2(-/-) mice had more severe disease and more intense and prolonged infection compared to other groups. TLR4(-/-) mice were asymptomatic. TLR2/4(-/-) mice had severe early disease and persistent infection, which resolved thereafter consistent with the absence of symptoms in TLR4(-/-) mice. Wt mice mounted robust innate and adaptive responses with an influx of natural killer (NK) cells, neutrophils, myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, and activated CD4(+) and CD8(+) T-cells into the lungs. Wt mice also had effective production of interferon (IFN)γ in the lymph nodes and lung, and proliferation of lymph node T-cells. TLR2(-/-) mice had more intense and persistent innate (particularly neutrophil) and adaptive cell responses and IL-17 expression in the lung, however IFNγ responses and T-cell proliferation were reduced. TLR2/4(-/-) mice had reduced innate and adaptive responses. Most importantly, neutrophil phagocytosis was impaired in the absence of TLR2. Thus, TLR2 expression, particularly on neutrophils, is required for effective control of Chlamydia respiratory infection in early life. Loss of control of infection leads to enhanced but ineffective TLR4-mediated inflammatory responses that prolong disease symptoms. This indicates that TLR2 agonists may be beneficial in the treatment of early life Chlamydia infections and associated diseases.     

Mucosal and systemic immune responses of mice to tetanus toxoid coadministered nasally with AFCo1

Mucosal immune responses are an early and important line of defense against pathogens. The current understanding of the mucosal immune system allows us to consider the use of nasal immunization for induction of antigen-specific immune responses at the mucosal surface and the systemic compartment. Mucosal adjuvants are key for developing novel mucosal vaccines and represent 1 approach to improving mucosal and systemic immunity. However, few mucosal vaccine adjuvants are currently approved for human use. Neisseria meningitidis B proteoliposome-derived cochleate (AFCo1 - Adjuvant Finlay Cochleate 1) has been demonstrated to be a potent mucosal adjuvant. The present work demonstrates that intranasal immunization of 3 doses of tetanus toxoid (TT) coadministered with AFCo1 in mice promotes high systemic and mucosal responses. The anti-TT IgG serum titers and the mucosal anti-TT IgA in saliva and vaginal wash were significantly higher than TT alone. The analysis of antibody subclasses showed that intranasal administration of AFCo1 + TT induced not only IgG1 but also IgG2a anti-TT antibodies at levels comparable to those obtained with TT vaccine (vax-TET). These data support the fact that AFCo1 is a potent mucosal adjuvant in nasal immunization to a coadministered protein antigen.     

Toll-like receptor 4 (TLR4) is required for protective immunity to larval Strongyloides stercoralis in mice

TLR4 is important for immunity to various unicellular organisms and has been implicated in the immune responses to helminth parasites. The immune response against helminths is generally Th2-mediated and studies have shown that TLR4 is required for the development of a Th2 response against allergens and helminth antigens in mice. C3H/HeJ mice, which have a point mutation in the Tlr4 gene, were used in this study to determine the role of TLR4 in protective immunity to the nematode Strongyloides stercoralis. It was demonstrated that TLR4 was not required for killing larval S. stercoralis during the innate immune response, but was required for killing the parasites during the adaptive immune response. No differences were seen in the IL-5 and IFN-gamma responses, antibody responses or cell recruitment between wild type and C3H/HeJ mice after immunization. Protective immunity was restored in immunized C3H/HeJ mice by the addition of wild type peritoneal exudate cells in the environment of the larvae. It was therefore concluded that the inability of TLR4-mutant mice to kill larval S. stercoralis during the adaptive immune response is due to a defect in the effector cells recruited to the microenvironment of the larvae.     

Neisserial porin-induced dendritic cell activation is MyD88 and TLR2 dependent

Neisserial porins have been shown to act as B cell mitogens and immune adjuvants. PorA and PorB are the major outer membrane porin proteins of the human pathogen Neisseria meningitidis. We have shown that the mechanism of the immunopotentiating capability of porin involves up-regulation of the T cell costimulatory ligand, CD86. Due to neisserial porin's ability to activate B cells and potentiate immune responses, we hypothesized that porin also employs the potent immune stimulatory function of dendritic cells (DC). We examined the ability of purified N. meningitidis PorB to induce maturation of murine splenic and bone marrow-derived DC. PorB treatment induced DC maturation, as demonstrated by increased expression of CD86 and class I and II MHC molecules. In addition, PorB not only enhanced the allostimulatory activity of DC, but also augmented the ability of DC to stimulate T cells in an Ag-specific manner. PorB-matured DC secreted the inflammatory cytokine IL-6, which may have implications for the adjuvant property of porin. Induction of IL-6 by PorB is also significant because IL-6 is one of a number of cytokines produced during infection with N. meningitidis and may be involved in the inflammatory process observed during infection and disease. We previously demonstrated the requirement of MyD88 and TLR2 for PorB-induced B cell activation. In the present study, MyD88 and TLR2 were also essential for PorB-induced DC activation. This work is significant for elucidating the mechanism(s) of neisserial porin's immune stimulatory activity.     

A New Adenovirus Based Vaccine Vector Expressing an Eimeria tenella Derived TLR Agonist Improves Cellular Immune Responses to an Antigenic Target

Background

Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals.

Methodology/Principal Findings

In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8⁺ and CD8⁻ T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions.

Conclusion/Significance

The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.     

Multiple innate immune pathways contribute to the immunogenicity of recombinant adenovirus vaccine vectors

The innate immune pathways that contribute to the potent immunogenicity of recombinant adenovirus (rAd) vaccine vectors remain largely undefined. Previous studies assessing innate immunity triggered by vaccine vectors have largely focused on in vitro studies involving antigen-presenting cells and on early in vivo inflammatory responses. Here, we systematically explore the Toll-like receptor (TLR) signaling requirements for the generation of cellular immune responses by intramuscular immunization with common and alternative serotype rAd vectors in mice. Antigen-specific CD8(+) T-lymphocyte responses elicited by these rAd vectors were significantly diminished in MyD88(-/-) mice but not in TRIF(-/-) or TLR3(-/-) mice, suggesting the importance of MyD88-dependent TLR signaling. However, the absence of each individual TLR resulted in minimal to no effect on vaccine-elicited cellular immune responses. Moreover, responses were not diminished in IL-1R(-/-) or IL-18R(-/-) mice. These data suggest that rAd vectors engage multiple MyD88-dependent signaling pathways, none of which are individually critical; rather, they are integrated to contribute to the potent immunogenicity of rAd vectors. Stimulation of multiple innate immune mechanisms may prove a generalizable property of potent vaccines, and this strategy could be harnessed in the development of next-generation vaccine vectors and adjuvants.     

Evaluating vaccinia virus cytokine co-expression in TLR GKO mice

Using Toll-like receptor (TLR) and MyD88 gene knock-out (GKO) mice the effect of TLRs and MyD88 on virus replication, interferon (IFN)-β production, natural killer (NK) cell and CD8T cell responses were assessed following ectromelia virus (ECTV) and recombinant vaccinia virus (rVV) infection. The capacity for rVVs encoding cytokines to restore immune function in MyD88(-/-) mice was clearly demonstrated. Results showed that TLR2(-/-), TLR4(-/-)and TLR7(-/-) mice survived ECTV infection whereas MyD88(-/-) and TLR9(-/-)mice, in contrast, were highly susceptible. Next, following infection with rVV, MyD88(-/-) mice elicited reduced serum IFN-β, NK cell and CD8T cell responses compared with wild-type mice, whereas TLR9(-/-) mice showed elevated CD8T cell responses. When MyD88(-/-)mice were infected with rVV co-expressing IFN-β these mice were able to restore IFN-β levels and CD8T cell responses but not NK cell activation. Interestingly, even though rVV co-expressing interleukin (IL)-2 enhanced NK cell activation in MyD88(-/-) mice, this was not associated with an antiviral effect, as observed in normal mice. Surprisingly, co-infection with rVV IL-2/rVV IL-12, but not rVV IL-2/rVV IFN-β, restored the attenuated phenotype of rVV IL-2 in MyD88(-/-) mice indicating that the IL-2/IL-12 combination promotes antiviral responses. Our results clearly show that the CD8T cell defect observed in MyD88(-/-) mice to vaccinia virus infection can be restored by rVV-encoding IFN-β demonstrating the critical role of this cytokine in T cell mediated immunity and illustrates that the model can provide an effective platform for the elucidation of cytokine immunobiology.     

Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis

TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the live vaccine strain were used to investigate the contribution of mast cell/TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHC class II and lysosomal-associated membrane protein 2. Infected TLR2(-/-) mast cells, in contrast to wild-type and TLR4(-/-) cells, lacked detectable IL-4 and displayed increased cell death with a 2-3 log increase of F. tularensis replication, but could be rescued with rIL-4 treatment. Importantly, MHC class II and lysosomal-associated membrane protein 2 localization with labeled F. tularensis in the lungs was greater in wild-type than in TLR2(-/-) mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses.     

An oral Salmonella vaccine promotes the down-regulation of cell surface Toll-like receptor 4 (TLR4) and TLR2 expression in mice

A single oral immunization with the Lon-protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) induced protective immunity in mice against a subcutaneous challenge with virulent Listeria monocytogenes as well as virulent Salmonella serovar Typhimurium. The populations of cell surface Toll-like receptor 4 (TLR4) and TLR2 on peritoneal macrophages decreased at week 6 after immunization. This population decrease was not reversed after a challenge with either Salmonella or Listeria. These results suggest that oral immunization with CS2022 induced immune tolerance correlated with the down-regulation of cell surface TLR expression. This down-regulation may in part account for the development of cross-protection against a Listeria challenge by immunization with CS2022.     

A Whole Virus Pandemic Influenza H1N1 Vaccine Is Highly Immunogenic and Protective in Active Immunization and Passive Protection Mouse Models

The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.