Protective immunity against the protozoan Leishmania chagasi is induced by subclinical cutaneous infection with virulent but not avirulent organisms

Protective immunity against Leishmania major is provided by s.c. immunization with a low dose of L. major promastigotes or with dihydrofolate-thymidylate synthase gene locus (DHFR-TS) gene knockout L. major organisms. Whether these vaccine strategies will protect against infection with other Leishmania species that elicit distinct immune responses and clinical syndromes is not known. Therefore, we investigated protective immunity to Leishmania chagasi, a cause of visceral leishmaniasis. In contrast to L. major, a high dose s.c. inoculum of L. chagasi promastigotes was required to elicit protective immunity. Splenocytes from mice immunized with a high dose produced significantly greater amounts of IFN-gamma and lower TGF-beta than mice immunized with a low dose of promastigotes. The development of protective immunity did not require the presence of NK cells. Protection was not afforded by s.c. immunization with either attenuated L. chagasi or with L. major promastigotes, and s.c. L. chagasi did not protect against infection with L. major. Subcutaneous immunization with DHFR-TS gene knockouts derived from L. chagasi, L. donovani, or L. major did not protect against L. chagasi infection. We conclude that s.c. inoculation of high doses of live L. chagasi causes a subclinical infection that elicits protective immune responses in susceptible mice. However, L. chagasi that have been attenuated either by long-term passage or during the raising of recombinant gene knockout organisms do not elicit protective immunity, either because they fail to establish a subclinical infection or because they no longer express critical antigenic epitopes.     

Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections

Several antigens have been tested as vaccine candidates against Leishmania infections but controversial results have been reported when different antigens are co-administered in combined vaccination protocols. Immunization with A2 or nucleoside hydrolase (NH) antigens was previously shown to induce Th1 immune responses and protection in BALB/c mice against Leishmania donovani and L. amazonensis (A2) or L. donovani and L. mexicana (NH) infections. In this work, we investigated the protective efficacy of A2 and NH DNA vaccines, in BALB/c mice, against L. amazonensis or L. chagasi challenge infection. Immunization with either A2 (A2-pCDNA3) or NH (NH-VR1012) DNA induced an elevated IFN-gamma production before infection; however, only A2 DNA immunized mice were protected against both Leishmania species and displayed a sustained IFN-gamma production and very low IL-4 and IL-10 levels, after challenge. Mice immunized with NH/A2 DNA produced higher levels of IFN-gamma in response to both specific recombinant proteins (rNH or rA2), but displayed higher IL-4 and IL-10 levels and increased edema and parasite loads after L. amazonensis infection, as compared to A2 DNA immunized animals. These data extend the characterization of the immune responses induced by NH and A2 antigens as potential candidates to compose a defined vaccine and indicate that a highly polarized type 1 immune response is required for improvement of protective levels of combined vaccines against both L. amazonensis and L. chagasi infections.     

Development of BCG as a live recombinant vector system: potential use as an HIV vaccine.

Bacille Calmette-Guèrin (BCG), a live attenuated tubercle bacillus, is currently the most widely used vaccine in the world. Because of its unique characteristics, including low toxicity, adjuvant potential, and long-lasting immunity, BCG represents a novel vaccine vehicle with which to deliver protective antigens of multiple pathogens. We have developed episomal and integrative expression vectors employing regulatory sequences of major BCG heat shock proteins for stable maintenance and expression of foreign antigens in BCG vaccine strains (22). Shuttle plasmids capable of autonomous replication in Escherichia coli and BCG were constructed with a DNA cassette containing a minimal replicon derived from the Mycobacterium fortuitum plasmid pAL5000. Efficient and stable chromosomal integration of recombinant plasmids into BCG was achieved using a DNA segment containing the mycobacteriophage L5 attachment site and integrase coding sequence. Using the BCG hsp60 and hsp70 stress gene promoters, we were able to express Escherchia coli beta-galactosidase to levels in excess of 10% of total cell protein. The major antigens of HIV-1 gag, pol, and env were also stably expressed using our vector systems. The recombinant BCG elicited long-lasting humoral and cellular immune responses to these antigens in mice. Antibody responses to beta-galactosidase using as few as 200 colony-forming units were detected 6 weeks after immunization, and titers (1:30,000) were sustained for more than 10 weeks. Cellular immune responses, of both cytotoxic T cell (CTL) and helper T lymphocytes, were detected to beta-galactosidase. CTL responses were also induced to the HIV-1 envelope protein. Thus, we have demonstrated stable recombinant antigen expression, processing, and presentation using our recombinant BCG vector system. This live recombinant vector system shows promise as a universally applicable and safe vaccine vehicle for protection against various infectious diseases.     

T cell response of asymptomatic Leishmania chagasi infected subjects to recombinant leishmania antigens.

In areas of Leishmania chagasi transmission the ability to control leishmania infection is associated with IFN-gamma production. In visceral leishmaniasis down-regulation of T cell responses is mediated by interleukin-10 (IL-10). In this study we evaluated the lymphoproliferative response, IFN-gamma and IL-10 production on lymphocyte cultures stimulated with recombinant leishmania antigens in subjects with asymptomatic L. chagasi infection. There was a statistically significant difference in the lymphoproliferative response of the subjects with asymptomatic infection as compared to patients with visceral leishmaniasis and healthy subjects with respect to crude antigens (p<0.01), gp-63 (p<0.05) and hsp-70 (p<0. 01), as well as between asymptomatic L. chagasi infected subjects and patients with visceral leishmaniasis with respect to the response to all antigens tested. The IFN-gamma production observed in the group with asymptomatic infection with all the three recombinant antigens tested was higher (p<0.01) than that observed in patients with visceral leishmaniasis and in healthy subjects. Furthermore, in individuals with asymptomatic infection, IL-10 levels in cultures stimulated with recombinant antigens were very low. This study shows that lymphocytes from individuals with asymptomatic L. chagasi infection are able to recognize recombinant leishmania antigens with production of a cytokine that is associated with leishmania killing.     

Immunological properties of recombinant Mycobacterium bovis bacillus Calmette-Guérin strain expressing fusion protein IL-2-ESAT-6

The live vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) provides variable efficacy against adult pulmonary tuberculosis (TB). Recombinant BCG, expressing either immunodominant antigens or Th1 cytokines, is a promising strategy for developing a new TB vaccine. However, not much is known about whether the introduction of cytokine and specific antigen genes concurrently into the BCG strain could improve the immunogenicity of BCG. In this study, a recombinant BCG strain (rBCG) expressing the fusion protein human interleukin (IL)-2 and ESAT-6 (early secreted antigenic target-6 kDa) antigen of Mycobacterium tuberculosis was constructed. Six weeks after BALB/c mice (H-2d) were immunized with 106 colony forming units (CFUs) BCG or rBCG, splenocyte proliferation was determined with MTT [3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay, IL-4 and interferon (IFN)-gamma produced by splenocytes were tested by enzyme linked immunosorbent assay (ELISA,) and the cytotoxicity of splenocytes from immunized mice to P815 cells (H-2d) expressing ESAT-6 protein was measured using CytoTox 96 Non-Radioactive Cytotoxicity Assay. Compared with native BCG-vaccinated mice, rBCG induced stronger Th1 responses that were confirmed by high lymphoproliferative responses and IFN-gamma production to culture filtrate protein (CFP) or ESAT-6 protein. Moreover, rBCG induced significant enhanced CTL responses against P815-ESAT-6 cells. Results from rBCG-immunized mice demonstrated that introducing the il-2 and esat-6 genes into BCG could enhance Th1 type immune responses to ESAT-6. Further investigation is needed by introducing other Th1 cytokines and antigens into BCG to optimize the protective efficacy against TB.     

Development of Novel Prime-Boost Strategies Based on a Tri-Gene Fusion Recombinant L. tarentolae Vaccine against Experimental Murine Visceral Leishmaniasis

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB^-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB^-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB^-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL.     

Murine cytokine responses following multiple oral immunizations using lipid-formulated mycobacterial antigens

Oral vaccination of mice with live Mycobacterium bovis BCG in lipid-formulation induces a gamma-interferon response that can be measured systemically, and confers protection against aerosolized mycobacterial challenge. Here, we have investigated cytokine responses following the vaccination, drawing comparisons between mice that received single or multiple oral immunizations and between mice receiving formulations containing live BCG or non-replicating mycobacterial antigens. Single oral immunization with lipid-formulated live BCG invoked secreted and cellular IFN-gamma responses in mice 8 weeks post-vaccination, the magnitudes of which were significantly elevated in mice receiving multiple immunizations over the 8-week period. Single oral immunization with live BCG also invoked an interleukin-2 response (but not TNF-alpha or IL-4), although the magnitude was not elevated by multiple immunizations. Multiple immunizations with lipid-formulated soluble or particulate non-replicating mycobacterial antigens failed to invoke cytokine responses, except for a low-level IFN-gamma response in mice multiple immunized with lipid-formulated heat-killed BCG. These results are discussed in contrast to the known patterns of cytokine induction following parenteral-route immunization with live or non-replicating mycobacterial antigens and with practical reference to the development of oral-delivery vaccines against tuberculosis.     

Manipulation of immune responses to Mycobacterium bovis by vaccination with IL-2- and IL-18-secreting recombinant bacillus Calmette Guerin

Bacillus Calmette Guerin (BCG) has been reported to show variable efficacy as a vaccine against tuberculosis. We demonstrated that the secretion of biologically active IL-2 (rBCG/IL-2),but not IL-18 (rBCG/IL-18), by BCG improves its ability to induce and maintain a strong type 1 immune response in BALB/c mice. rBCG/IL-2 induced significantly higher Ag-specific proliferative responses, high IFN-gamma production and serum titres of IgG2a 16 weeks after vaccination. This immune profile was correlated to an increased rate of clearance of non-pathogenic mycobacteria (live BCG delivered intranasally). Surprisingly, however,this strong type 1 immune profile induced no greater protective immunity against aerosol challenge with virulent Mycobacterium bovis than that induced by normal BCG (nBCG). By comparison,vaccination with rBCG/IL-18 was found to induce significantly less IFN-gamma production in splenic lymphocytes than nBCG.This impaired induction of IFN-gamma was correlated to a significantly lower protective efficacy against M. bovis challenge, as compared to nBCG. The data suggest that manipulation of the immune response to tuberculosis and tuberculosis vaccines will require a more complete understanding of the factors that are important in generating a protective immune response.     

Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes

BALB/c mice can be protected against a fatal Leishmania major infection by immunization with whole radio-attenuated promastigotes; however, neither the antigens responsible for protection nor the protective immunologic mechanisms have been defined. In this study, the ability of promastigote fractions to elicit similar immunity to that obtained with whole organisms, and the immune responses associated with such protection were analyzed. Intraperitoneal immunization with a soluble, membrane-free parasite extract was found to induce protection against L. major challenge equal to that obtained with whole organisms. Induction of immunity (89% protection in seven experiments) was most effective with 100 micrograms of the soluble leishmanial antigen (SLA) and required concomitant injection of the bacterial adjuvant, Corynebacterium parvum (CP), followed by an i.p. boost of SLA alone 1 wk later. Vaccinated animals exhibited Leishmania-specific cell-mediated immunity, as assessed both by lymphocyte transformation and the production of macrophage-activating factors (MAF). In addition, although SLA + CP-immunized mice failed to exhibit delayed-type hypersensitivity (DTH) before challenge, splenic lymphocytes from these mice could transfer a local DTH reaction to naive recipients. Immunization also induced the production of antibodies against two major metabolically labeled proteins of m.w. 30,000 and 53,000, but failed to stimulate a detectable humoral response against promastigote surface antigens. Thus, these experiments demonstrate that vaccine-induced immunity against cutaneous leishmaniasis is strongly associated with the induction of cell-mediated immunity, but does not require the development of an antibody response to promastigote surface antigens. In addition, these studies establish the feasibility of employing soluble, nonmembrane-derived parasite material as a source of protective immunogens.     

IFN-gamma modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis.

Resistance to Leishmania major in mice is associated with the generation of distinct CD4+ Th subsets, termed TH1 and TH2. To define the factors contributing to the genesis of these Th cells, we first investigated when these subsets developed following L. major infection. Lymph node (LN) cells collected 3 days after infection of BALB/c mice secreted IL-4 and IL-5 in vitro, but little IFN-gamma, whereas LN cells from a resistant strain, C3H/HeN, secreted IFN-gamma and no IL-4 or IL-5. Cytokine production was eliminated in both cases by in vivo or in vitro depletion of CD4+ cells, but not after depletion of CD8+ cells. Similar responses were observed after inoculation of killed promastigotes or a soluble leishmanial Ag preparation. These data indicate that the development of Th1- and Th2-like responses can precede lesion formation and does not require a live infection. We next investigated whether IFN-gamma was important in the differentiation of Th1 and Th2 cells. C3H/HeN mice have previously been shown to be susceptible to leishmanial infection after treatment with anti-IFN-gamma. We confirmed this observation and found that the abrogation of resistance was associated with enhanced production of IL-4 and IL-5, and decreased production of IFN-gamma by cells taken from these mice. Conversely, LN cells from BALB/c mice inoculated with parasites plus IFN-gamma produced significantly higher levels of IFN-gamma, and decreased levels of IL-4 and IL-5, than mice infected with parasites alone. Finally, we determined if IFN-gamma might augment vaccine induced immunity. We found that s.c. immunization with soluble leishmanial Ag, the bacterial adjuvant, Corynebacterium parvum and IFN-gamma could protect mice against L. major infection, and that this protection was associated with induction of Th1 responses. From these data we conclude that levels of IFN-gamma at the time of infection or immunization dramatically alters the type of response elicited: high levels of IFN-gamma favor Th1 type responses, whereas low levels promote a Th2 response.     

Leishmania donovani recombinant iron superoxide dismutase B1 protein in the presence of TLR-based adjuvants induces partial protection of BALB/c mice against Leishmania major infection

In this study, we tested the protective efficacy of recombinant Leishmania donovani iron superoxide dismutase B1 (SODB1) against Leishmania major infection in BALB/c mice. Mice were challenged with L. major 3weeks after the second boost immunization with rSODB1 alone or in the presence of adjuvants. Injection of BALB/c mice with rSODB1 alone elicited both humoral and cellular immune responses. Administration of rSODB1 with CpG ODN or GLA-SE (a synthetic toll-like receptor 4 agonist) adjuvant resulted in the induction of anti-SODB1 IgG1, and more importantly of significantly high levels of IgG2a isotype. Immunization of mice with rSODB1 alone or with adjuvant induced the production of IFN-γ by splenocytes in response to stimulation with L. major soluble leishmanial antigens (SLA). Moreover, immunization protocols involving rSODB1 resulted in a significant decrease in IL-10 as compared to controls. The presence of CpG ODN or GLA-SE adjuvant in the immunization protocols resulted in a relative increase in IFN-γ in response to stimulation with rSODB1 in comparison to immunization with rSODB1 alone. Mice immunized with rSODB1 plus CpG ODN or GLA-SE, were able to partially control their Leishmania infections, as indicated by the reduction in footpad swelling and parasite numbers, compared to controls. These results suggest that immunization with recombinant SODB1 protein together with CpG ODN or GLA-SE can be potential vaccine candidate against leishmaniasis.     

Recombinant Mycobacterium bovis BCG producing the circumsporozoite protein of Plasmodium falciparum FCC-1/HN strain induces strong immune responses in BALB/c mice

The current vaccine against tuberculosis, Mycobacterium bovis strain bacillus Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for expression and delivery of protective recombinant antigens. Malaria is one of the severest parasitic diseases in humans especially in the developing world. No efficacious vaccine is currently available. However, circumsporozoite protein (CSP) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the immune response to recombinant BCG (rBCG) vaccine expressing Plasmodium falciparum CSP (BCG-CSP) under the control of heat shock protein 70 promoter in BALB/c mice. The lymphocytes proliferative response to P. falciparum soluble antigen was significantly higher than those in the groups of BCG and normal saline, and the production of cytokines (IFN-gamma and IL-2) in response to malaria antigen was significantly higher in rBCG and BCG groups than control group of normal saline. A specific IgG antibody response against P. falciparum antigen of CSP was also characterized. The booster injection could enhance the production of cytokine, proliferation responses of spleen lymphocytes and the antibodies titer of BCG-CSP. The results in the study demonstrated that rBCG vaccine producing CSP is an appropriate vaccine for further evaluation in non-human primates.     

Identification of a 30 kDa antigen from leishmania (L.) chagasi amastigotes implicated in protective cellular reponses in a murine model

An antigen of apparent molecular mass of 30 kDa, termed p30, was purified from Leishmania (L.) chagasi amastigotes after separation of parasite extracts by sodium dodecyl sulfate-polyacrylamide gel eletroctrophoresis followed by electroelution. The use of the purified antigen in lymphocyte cultures from BALB/c mice previously immunised with L. (L.) chagasi amastigotes led to high levels of proliferation. Animal immunisation with p30 plus complete Freund's adjuvant either by subcutaneous or intraperitoneal route led to comparable antigenic stimulation. Similar stimulation indices induced by p30 were also obtained when animals were immunised with Corynebacterium parvum as adjuvant by the intraperitoneal route. Detection of IL-2 and IFN-gamma in the supernatants from lymphocytes stimulated by p30 and inhibition of the production of these lymphokines in the presence of anti-CD4 strongly indicated the involvement of the Th1 subset in the responses elicited by p30 antigen. Immunisation of BALB/c mice with p30 provided partial protection against challenge with L. (L.) chagasi amastigotes, indicating a protective role for p30 and that Th1 can be related to accquired resistance to visceral leishmaniasis in a murine model. Further characterisation studies were performed by the use of a monoclonal antibody directed to a cysteine proteinase of 30 kDa from L. (L.) amazonensis amastigotes. Despite the cross-reactivity presented by p30 from both Leishmania species, the p30 from L. (L.) chagasi amastigotes lacks proteolytic activity.     

Protection against Leishmania major infection by oligomannose-coated liposomes

Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidylethanolamine (Man5-DPPE) have been shown to induce cellular immunity against antigens encapsulated in the liposomes. To assess whether these neoglycolipid-coated liposomes can elicit protective immune response against challenge infection, effects of immunization with soluble leishmanial antigens encapsulated in the liposomes were evaluated using Leishmania major infection in susceptible BALB/c mice. Intraperitoneal immunization of mice with leishmanial antigens in the Man5-DPPE-coated liposomes significantly suppressed footpad swelling in comparison to the control, non-immunized mice, while progression of the disease was observed in mice administered antigens in uncoated liposomes and those administered soluble antigens alone, as seen with control mice. Similarly, the number of parasites decreased substantially in local lymph nodes of mice immunized with the antigen in the Man5-DPPE-coated liposomes. Protection against L. major infection in the immunized mice also coincided with an elevated ratio of antigen-specific IgG2a/IgG1 antibodies, which is a profile of T helper-type 1-like immune response. Taken together, these results indicate the possibility that Man5-DPPE-coated liposome-encapsulated antigens could serve as a vaccine that triggers protection against infectious disease.     

沙眼衣原体分泌性蛋白Pgp3滴鼻免疫增强小鼠对衣原体生殖道感染的保护作用

【目的】探讨不同免疫途径沙眼衣原体(Chlamydia trachomatis,Ct)分泌性蛋白Pgp3的免疫保护效果,分析其可能的保护机制,以确定Pgp3蛋白疫苗在Ct疫苗研制中的应用价值。【方法】分泌性蛋白Pgp3经滴鼻或肌注途径免疫雌性Balb/c小鼠,免疫60 d后,阴道接种鼠沙眼衣原体(Chlamydia muridarum,Cm)建立生殖道感染动物模型,在该模型中评价Pgp3蛋白疫苗抗Cm感染的保护效果,并探讨其机制。【结果】滴鼻或肌注免疫后,小鼠血清及生殖道中检测到了特异性抗体;小鼠脾淋巴细胞产生IFN-γ、IL-17及IL-5水平均明显高于对照组,且滴鼻免疫组IFN-γ水平升高较肌注组更显著(P<0.05);Pgp3蛋白滴鼻免疫组小鼠经Cm生殖道感染后,阴道带菌时间明显缩短,输卵管病理改变轻而肌注免疫组其保护作用不明显。【结论】Pgp3蛋白经滴鼻免疫可有效诱导小鼠产生明显的抗Cm保护效应。其可能的免疫保护机制与诱导Th1型为主的细胞免疫应答及高效价的特异性抗体有关,提示Pgp3蛋白疫苗具有潜在的疫苗研究与开发价值。     

Lymphatic tracing and T cell responses following oral vaccination with live Mycobacterium bovis (BCG)

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis bacille Calmette-Guérin (BCG) expands a subset of interferon-gamma (IFN-gamma)-secreting T cells and mediates protection against aerosol mycobacterial challenge. We have traced the movement of the live vaccine through the regional lymphatics of mice and monitored the resultant immune response. Six hours after oral vaccination BCG was detected in low numbers systemically and in draining lymphatic tissue. However, after 48 h, BCG was predominantly associated with alimentary tract lymphatic tissues, such as the cervical and mesenteric lymph nodes and Peyer's patches. Lymphocytes that produced IFN-gamma in response to PPD-B or BCG-pulsed dendritic cells predominated in the spleen and were almost exclusively CD4(+), CD44(+) and CD62L(-), thus resembling an effector memory T cell population. Despite the fact that an oral route was used for immunization, splenic IFN-gamma-secreting T cells in vaccinated mice did not express the mucosal homing antigens alpha(4)beta(7) integrin or alphaIEL (CD103). However, a proportion of BCG-specific CD4(+) T cells expressed the CD29 integrin (beta(1)) chain, potentially involved in lung homing function. Thus, oral priming with M. bovis BCG appears to induce a subset of spleen-resident CD4(+) T cells with the potential to provide protective immunity in the lung.     

Comparison of adjuvant efficacy of herpes simplex virus type 1 recombinant viruses expressing TH1 and TH2 cytokine genes

The adjuvant effects of cytokines in humoral and cell-mediated immunity to herpes simplex virus type 1 (HSV-1) have been examined in mice using HSV-1 recombinant viruses expressing murine interleukin-2 (IL-2), IL-4, or gamma interferon (IFN-gamma) gene. Groups of naive BALB/c mice were immunized intraperitoneally with one or three doses of the HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-gamma or with parental control virus. Despite similar replication kinetics, these three recombinant viruses elicited different immune responses to HSV-1 on immunization. Immunization with the recombinant virus expressing IL-4 elicited a humoral response of greater magnitude than immunization with the recombinant viruses expressing IL-2 or IFN-gamma or with parental virus. In contrast, immunization with recombinant virus expressing IL-2 elicited a higher cytotoxic T-cell response than immunization with viruses expressing IL-4 or IFN-gamma. Stimulation in vitro of splenocytes obtained from the mice immunized with UV-inactivated HSV-1 McKrae resulted in a T(H)1 pattern of cytokine expression irrespective of the recombinant virus used in the immunization. As observed for the parental virus, both CD4(+) and CD8(+) T cells contributed equally to the production of IL-2 by the splenocytes of mice immunized with any of the three recombinant viruses. However, the pattern of IFN-gamma production by CD4(+) and CD8(+) T cells differed according to the recombinant virus used. After lethal ocular challenge, all immunized mice were protected completely against death and manifestations of eye disease caused by HSV-1, which are typical responses in unimmunized mice. Mice immunized with IL-4-expressing virus cleared the virus from their eyes more rapidly than mice immunized with IL-2- or IFN-gamma-expressing virus. Taken together, our results suggest that, in contrast to IFN-gamma which did not exhibit an adjuvant effect, both IL-4 and IL-2 act as adjuvants in immunization with HSV, with IL-4 showing greater efficacy.     

Induction of cellular immune responses to simian immunodeficiency virus gag by two recombinant negative-strand RNA virus vectors

A recombinant Newcastle disease virus (rNDV) expressing simian immunodeficiency virus (SIV) Gag protein (rNDV/SIVgag) was generated. The rNDV/SIVgag virus induced Gag-specific cellular immune responses in mice, leading to a specific anti-Gag antiviral immunity. This was evidenced by the inhibition of growth of recombinant vaccinia virus expressing an identical Gag antigen (rVac/SIVgag) but not of wild-type vaccinia virus in rNDV/SIVgag-immunized mice. Among intravenous, intraperitoneal, or intranasal immunization routes, intranasal administration induced the strongest protective response against challenge with rVac/SIVgag. We further demonstrated that these immune responses were greatly enhanced after booster immunization with recombinant influenza viruses expressing immunogenic portions of SIV Gag. The magnitude of the protective immune response correlated with the levels of cellular immune responses to Gag, which were still evident 9 weeks after immunization. These results suggest that rNDV and influenza virus vectors are suitable candidate vaccines against AIDS as well as against other infectious diseases.     

Development of protective immunity in a murine model of melioidosis is influenced by the source of Burkholderia pseudomallei antigens

Melioidosis is a potentially fatal disease caused by the bacterium, Burkholderia pseudomallei. The current study was carried out to determine the mechanisms involved in the development of protective immunity in a murine model of melioidosis. Following intravenous infection with B. pseudomallei, both C57BL/6 and BALB/c mice demonstrated delayed-type hypersensitivity responses and lymphocyte proliferation towards B. pseudomallei antigens, indicating the generation of B. pseudomallei-specific lymphocytes. Adoptive transfer of these lymphocytes to na?ve C57BL/6 mice was demonstrated by a delayed-type hypersensitivity response. Mice were not protected from a subsequent lethal challenge with a highly virulent strain of B. pseudomallei, suggesting that a single intravenous dose of the bacterium is insufficient to induce a protective adaptive immune response. Attempts to induce resistance in susceptible BALB/c mice used repetitive low-dose exposure to live B. pseudomallei. Immune responses and resistance following subcutaneous immunization with live B. pseudomallei were compared with exposure to heat-killed, culture filtrate and sonicated B. pseudomallei antigens. Compared to heat-killed B. pseudomallei, significant protection was generated in BALB/c mice following immunization with live bacteria. Our studies also demonstrate that the type of immune response generated in vivo is influenced by the antigenic preparation of B. pseudomallei used for immunization.     

Differential antigen requirements for protection against systemic and intranasal vaccinia virus challenges in mice

The development of a subunit vaccine for smallpox represents a potential strategy to avoid the safety concerns associated with replication-competent vaccinia virus. Preclinical studies to date with subunit smallpox vaccine candidates, however, have been limited by incomplete information regarding protective antigens and the requirement for multiple boost immunizations to afford protective immunity. Here we explore the protective efficacy of replication-incompetent, recombinant adenovirus serotype 35 (rAd35) vectors expressing the vaccinia virus intracellular mature virion (IMV) antigens A27L and L1R and extracellular enveloped virion (EEV) antigens A33R and B5R in a murine vaccinia virus challenge model. A single immunization with the rAd35-L1R vector effectively protected mice against a lethal systemic vaccinia virus challenge. The rAd35-L1R vector also proved more efficacious than the combination of four rAd35 vectors expressing A27L, L1R, A33R, and B5R. Moreover, serum containing L1R-specific neutralizing antibodies afforded postexposure prophylaxis after systemic vaccinia virus infection. In contrast, the combination of rAd35-L1R and rAd35-B5R vectors was required to protect mice against a lethal intranasal vaccinia virus challenge, suggesting that both IMV- and EEV-specific immune responses are important following intranasal infection. Taken together, these data demonstrate that different protective antigens are required based on the route of vaccinia virus challenge. These studies also suggest that rAd vectors warrant further assessment as candidate subunit smallpox vaccines.