Specific immunization of mice against Leishmania mexicana amazonensis using solubilized promastigotes.

Successful immunization of highly susceptible BALB/c mice against progressive infection by Leishmania mexicana amazonensis, using whole solubilized promastigotes was achieved. The best immunization schedule consisted of three weekly injections of 5 X 10(7) parasite equivalents. Intravenous was superior to intraperitoneal or subcutaneous immunization. Protection persisted for up to 2 months after immunization, and beneficial effects could be observed in long-term follow-up (24 weeks after infection). Immunized mice exhibited marked reduction in primary lesion size, as well as reduction of the number of parasites in the spleen, and developed less metastases. High titres of specific anti-L. m. amazonensis IgG antibodies resulted from immunization, but titres did not correlate with protection. Groups with widely differing pre-infection antibody titres were equally protected, and similar antibody titres resulted in different levels of protection. Immunization alone did not induce significant serum interferon-gamma levels and specific delayed-type hypersensitivity (DTH) reactions, but resulted in the persistence of positive (DTH) reactions after infection, at a time when infected control animals had suppressed responses. Resistance to leishmaniasis appears to depend on cell mediated immune mechanisms, and the possibility of immunization with a solubilized antigen without adjuvant is intriguing and opens new perspectives in this area.     

Expression of hypoxia-inducible factor-1alpha in the cutaneous lesions of BALB/c mice infected with Leishmania amazonensis

The hypoxia-inducible factor-1alpha (HIF-1alpha) is expressed in response to hypoxia and has been recently demonstrated in a variety of cells such as tumor cells and tumor-associated macrophages. Several characteristics of leishmanial lesions in humans and in animal models, such as microcirculation impairment, metabolic demand for leukocyte infiltration into infected tissue, parasite proliferation, and secondary bacterial infection, are strong indications of a hypoxic microenvironment in the lesions. We evaluated HIF-1alpha expression in the cutaneous lesions of BALB/c mice during Leishmania amazonensis infection. Immunohistochemical analyses of the lesions demonstrated, only in the later stages of infection when the lesion size is maximal and parasite burden is enormous and massive numbers of recruited macrophages and ulcers are observed, positive HIF-1alpha-infected cells throughout the lesions. HIF-1alpha is expressed mainly in the cytoplasm and around parasites inside the parasitophorous vacuoles of macrophages. This is the first evidence that macrophages in the microenvironment of lesions caused by a parasite produce a hypoxia-inducible factor.     

Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1

The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.     

Oral immunization with pBsVP6-transgenic alfalfa protects mice against rotavirus infection

A critical factor in edible plant-derived vaccine development is adequate expression of the exogenous antigens in transgenic plants. We synthesized a codon-optimized gene (sVP6) encoding the VP6 protein of human group A rotavirus and inserted it into the alfalfa genome using agrobacterium-mediated transformation. As much as 0.28% of the total soluble protein of the pBsVP6-transgenic alfalfa was sVP6. Female BALB/c mice were gavaged weekly with 10 mg of transgenic alfalfa extract containing 24 microg of sVP6 protein and 10 microg of CpG-rich oligodeoxynucleotides as mucosal adjuvant. Immunized mice developed high titers of anti-VP6 serum IgG and mucosal IgA. Offspring of immunized dams developed less severe diarrhea after challenge with simian rotavirus SA-11, indicating that antibodies generated in the dams provided passive heterotypic protection to the pups. These results suggest that oral immunization with pBsVP6-transgenic alfalfa provides a potential means of protecting children and young animals from severe acute rotavirus-induced diarrhea.     

Leishmania major-infected murine langerhans cell-like dendritic cells from susceptible mice release IL-12 after infection and vaccinate against experimental cutaneous Leishmaniasis

Leishmania major-infected C57BL / 6 skin-dendritic cells (DC) are activated and release cytokines (including IL-12 p70), and likely initiate protective Th1 immunity in vivo (von Stebut, E. et al., J. Exp. Med. 188: 1547 - 1552). To characterize differences in DC function in mice that are genetically susceptible (BALB / c) and resistant (C57BL / 6) to cutaneous leishmaniasis, we analyzed the effects of L. major on Langerhans cell-like, fetal skin-derived DC (FSDDC) from both strains. BALB / c- and C57BL / 6-FSDDC ingested similar numbers of amastigotes, but did not ingest metacyclic promastigotes. Like C57BL / 6-FSDDC, infection of BALB / c-FSDDC led to up-regulation of MHC class I and II antigens, CD40, CD54, and CD86 within 18 h. L. major-induced BALB / c DC activation also led to the release of TNF-alpha, IL-6 and IL-12 p40 into 18-h supernatants. Infected BALB / c- and C57BL / 6-DC both released small amounts of IL-12 p70 within 72 h. Additional stimulation with IFN-gamma and / or anti-CD40 induced the release of more IL-12 p70 from infected BALB / c-DC than C57BL / 6-DC. Co-culture of control or infected BALB / c- and C57BL / 6-DC with naive syngeneic CD4(+) T cells and soluble anti-CD3 resulted in mixed, IFN-gamma-predominant responses after restimulation with immobilized anti-CD3. Finally, syngeneic L. major-infected DC effectively vaccinated BALB / c mice against cutaneous leishmaniasis. Genetic susceptibility to L. major that results from induction of Th2 predominant immune responses after infection does not appear to reflect failure of skin DC to internalize or respond to parasites, or the inability of BALB / c T cells to mount a Th1 response to DC-associated Leishmania antigens.     

DNA immunization of mice against the VP1 capsid protein of coxsackievirus B4

The protective activity of a DNA plasmid encoding the immunodominant capsid protein VP1 of coxsackievirus B4 (CBV-4) was studied in BALB/c mice. The plasmid pCI-B4-1-c - which gave the highest expression level of VP1 in cultured monkey and human cells - was chosen for immunization. Two injections of pCI-B4-1-c (1 month apart) into the regenerating mouse muscle tissue induced a specific antibody response to CBV-4, as shown by immunoenzyme and neutralization assays. Upon challenge with live CBV-4, the mortality rate of mice vaccinated with the recombinant plasmid was significantly reduced (21% versus >58%) as compared with that of mice that had been either nontreated or injected with a control plasmid devoid of the insert. The VP1-based vaccine, however, did not provide complete protection as - after virus challenge - moderate viraemia occurred together with modest plasma elevations of pathogenesis-related enzymes (amylase and creatine kinase). Yet, immunofluorescence of the small intestine and heart did confirm the protective effect of the VP1-encoding vaccine. In order to obtain a more complete protection against CBV-4, it may be beneficial to immunize mice with combinations of separate DNA plasmids encoding not only VP1 but also the VP2 and VP3 capsid proteins.     

DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus

The immune response to a naked plasmid DNA encoding the nucleoprotein (N protein) of porcine transmissible gastroenteritis virus (TGEV) was investigated in this study. A complementary DNA of the entire N gene was amplified by RT-PCR, and inserted into a mammalian expression vector (pcDNA3.1) to construct a recombinant plasmid (pcDNA/N). To evaluate the immunogenicity of the construct, BALB/c mice were intramuscularly immunized with different doses (50, 100 and 200 μg/mouse) of pcDNA/N twice at a 5-week interval. An optimal antibody response was achieved with 100 μg of pcDNA/N. The response lasted at least 11 weeks after primary immunization. By western blotting analysis, the antibodies specifically recognized a 47 kDa protein corresponding to the viral N protein, but they did not reveal neutralizing activity against infectious TGEV in vitro. Immunoglobulin G2a was predominant among these antibodies, which was indicative of Th1 type cell activation in pcDNA/N immunized mice. Moreover, spleen cells from these mice showed stronger immune responses than those from live vaccine or parental vector immunized mice. These results suggest that the construct can elicit both humoral and cell-mediated immune (CMI) responses against TGEV N protein in mice.     

N-acetyl-l-cysteine reduces the parasitism of BALB/c mice infected with Leishmania amazonensis

Leishmania amazonensis infection leads to progressive diseases in a majority of inbred strains of mice. Glutathione (GSH) participates in a large number of cellular phenomena and seems to be essential for several immune functions, including host defense during leishmaniasis. In this study, we evaluated the effects of N-acetyl-l-cysteine (NAC), as GSH supplement, on the course of L. amazonensis infection in susceptible BALB/c mice. The treatment with NAC (200 mg/kg daily) was effective in raising GSH levels in both lymph node and spleen cells. Although this treatment did not change the footpad swelling development in L. amazonensis-infected mice, it caused a significant decrease in the number of parasites recovered from the footpad lesion and draining popliteal lymph node. Our data suggest that intracellular Leishmania killing in vivo was improved by the augment of GSH levels through NAC administration.     

Induction of hyperthyroidism in mice by intradermal immunization with DNA encoding the thyrotropin receptor

Intramuscular injection with plasmid DNA encoding the human thyrotropin receptor (TSHR) has been known to elicit symptoms of Graves' disease (GD) in outbred but not inbred mice. In this study, we have examined, firstly, whether intradermal (i.d.) injection of TSHR DNA can induce hyperthyroidism in BALB/c mice and, secondly, whether coinjection of TSHR- and cytokine-producing plasmids can influence the outcome of disease. Animals were i.d. challenged at 0, 3 and 6 weeks with TSHR DNA and the immune response was assessed at the end of the 8th or 10th week. In two experiments, a total of 10 (67%) of 15 mice developed TSHR-specific antibodies as assessed by flow cytometry. Of these, 4 (27%) mice had elevated thyroxine (TT4) levels and goitrous thyroids with activated follicular epithelial cells but no evidence of lymphocytic infiltration. At 10 weeks, thyroid-stimulating antibodies (TSAb) were detected in two out of the four hyperthyroid animals. Interestingly, in mice that received a coinjection of TSHR- and IL-2- or IL-4-producing plasmids, there was no production of TSAbs and no evidence of hyperthyroidism. On the other hand, coinjection of DNA plasmids encoding TSHR and IL-12 did not significantly enhance GD development since two out of seven animals became thyrotoxic, but had no goitre. These results demonstrate that i.d. delivery of human TSHR DNA can break tolerance and elicit GD in inbred mice. The data do not support the notion that TSAb production is Th2-dependent in murine GD but they also suggest that codelivery of TSHR and Th1-promoting IL-12 genes may not be sufficient to enhance disease incidence and/or severity in this model.     

Mucosal immunization with attenuated Shigella flexneri harboring an influenza hemagglutinin DNA vaccine protects mice against a lethal influenza challenge

Mucosal surfaces are important for the induction of immunity against influenza virus. In a murine intranasal immunization model, we demonstrated that the attenuated Shigella flexneri Deltaasd strain 15D, carrying a DNA construct encoding the influenza virus hemagglutinin (HA), induces protective immunity against a lethal respiratory challenge with influenza A/WSN/33. Influenza virus-specific IFN-gamma T cells were detected among splenocytes, and anti-HA IgG was detected in serum post-immunization, albeit at low levels. Following influenza virus challenge, an accelerated anti-HA IgA antibody response was detected in bronchoalveolar lavage (BAL) washings from mice vaccinated with attenuated shigella containing the HA construct. These results suggest that S. flexneri Deltaasd strain 15D is a promising vector for mucosal DNA vaccine immunization against influenza virus and other mucosal pathogens.     

Chitosan-DNA基因免疫诱导粘膜特异性免疫应答及其抗CVB3感染作用

目的：构建新型粘膜基因疫苗，诱生CVB3VP1特异性粘膜免疫应答，探讨粘膜免疫抗CVB3感染的作用，为病毒性心肌炎的特异性防治奠定基础。方法：抽提CVB3 RNA，以RT-PCR扩增得VP1基因，插入真核表达载体pcDNA3中，构建质粒pcDNA3-VP1，以Chitosan多糖包裹形成Chitosan-DNA基因疫苗。将该质粒转染Hela细胞，观察其体外表达情况。以50辉DNA剂量的Chitosan-DNA疫苗滴鼻免疫BALB／C小鼠3次，检测CVB3特异性体液和细胞免疫应答；隔4周以5LD50活CVB3致死攻击小鼠，观察攻击后存活情况。结果：制备了直径为80-100nm的Chitosan-DNA复合物颗粒，体外转染证实Chitosan-DNA复合物中VPl的表达高于pcDNA3-VP1质粒-Lipofectamine的表达水平。该疫苗滴鼻3次免疫后，不仅诱生了高水平的IgG，而且诱生了高水平的粘膜IgA抗体，第6周抗体P/N值分别达3．5和3．2。特异性细胞免疫应答研究发现，该疫苗诱生了较强的VP1特异性CTL杀伤作用，并显著高于pcDNA3-VP1和pcDNA3组。CVB3攻击后，可保护33．3％小鼠长期存活，而pcDNA3和pcDNA3-VP1质粒滴鼻免疫对照组的平均存活天数分别为8．5和10．8天。病理学研究显示：Chitosan-DNA免疫小鼠心肌组织基本正常，而对照小鼠死亡前心肌显示大量的灶性坏死和炎性浸润。结论：Chitosan-DNA基因疫苗滴鼻免疫可诱生CVB3特异性粘膜IgA应答及CTL反应，具有一定的抗CVB感染的免疫保护力。     

Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague.

The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase-V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Recombinant V antigen was inoculated intraperitoneally into mice and shown to induce a protective immune response against a subcutaneous challenge with 3.74 x 10(6) CFU of virulent Y. pestis. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague.     

Identification and molecular characterization of a gene encoding a protective Leishmania amazonensis Trp-Asp (WD) protein

Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates the formation of protein complexes. The single-copy LAWD gene is transcribed as a approximately 3.1-kb mRNA in both promastigotes and amastigotes, with homologues being detected in several other Leishmania species. Immunoelectron microscopy revealed a predominant localization of the LAWD protein in the flagellar pocket. Analyses of sera from human patients with cutaneous and mucocutaneous leishmaniasis indicated that these individuals mounted significant humoral responses against LAWD. Given that recombinant LAWD protein elicited the production of high levels of gamma interferon, but no detectable levels of interleukin-10 (IL-10), in CD4(+) cells of L. amazonensis-infected mice, we further examined whether LAWD could elicit protective immunity. DNA vaccination with the LAWD and IL-12 genes significantly delayed lesion development, which correlated with a dramatic reduction in parasite burdens. Thus, we have successfully identified a promising vaccine candidate and antigenic vehicle to aid in the dissection of the complicated pathogenic immune response of L. amazonensis.     

Intranasal immunization with multivalent group A streptococcal vaccines protects mice against intranasal challenge infections

We have previously shown that a hexavalent group A streptococcal M protein-based vaccine evoked bactericidal antibodies after intramuscular injection. In the present study, we show that the hexavalent vaccine formulated with several different mucosal adjuvants and delivered intranasally induced serum and salivary antibodies that protected mice from intranasal challenge infections with virulent group A streptococci. The hexavalent vaccine was formulated with liposomes with or without monophosphorylated lipid A (MPL), cholera toxin B subunit with or without holotoxin, or proteosomes from Neisseria meningitidis outer membrane proteins complexed with lipopolysaccharide from Shigella flexneri. Intranasal immunization with the hexavalent vaccine mixed with these adjuvants resulted in significant levels of antibodies in serum 2 weeks after the final dose. Mean serum antibody titers were equivalent in all groups of mice except those that were immunized with hexavalent protein plus liposomes without MPL, which were significantly lower. Salivary antibodies were also detected in mice that received the vaccine formulated with the four strongest adjuvants. T-cell proliferative assays and cytokine assays using lymphocytes from cervical lymph nodes and spleens from mice immunized with the hexavalent vaccine formulated with proteosomes indicated the presence of hexavalent protein-specific T cells and a Th1-weighted mixed Th1-Th2 cytokine profile. Intranasal immunization with adjuvanted formulations of the hexavalent vaccine resulted in significant levels of protection (80 to 100%) following intranasal challenge infections with type 24 group A streptococci. Our results indicate that intranasal delivery of adjuvanted multivalent M protein vaccines induces protective antibody responses and may provide an alternative to parenteral vaccine formulations.     

Subcutaneous immunization with recombinant adenovirus expressing influenza A nucleoprotein protects mice against lethal viral challenge

Current influenza vaccines mainly induce strain-specific neutralizing antibodies and need to be updated each year, resulting in significant burdens on vaccine manufacturers and regulatory agencies. Genetic immunization strategies based on the highly conserved nucleoprotein (NP) of influenza have attracted great attention as NP could induce heterosubtypic immunity. It is unclear, however, whether different forms of vectors and/or vaccination regimens could have contributed to the previously reported discrepancies in the magnitude of protection of NP-based genetic vaccinations. Here, we evaluated a plasmid DNA vector (pNP) and a recombinant adenovirus vector (rAd-NP) containing the NP gene through various combinations of immunization regimens in mice. We found that pNP afforded only partial protection even after 4 injections, with full protection against lethal challenge achieved only with the fourth boost using rAd-NP. Alternatively, only two doses of rAd-NP delivered subcutaneously were needed to induce an enhanced immune response and completely protect the animals, a finding which, to our knowledge, has not been reported before.     

Intranasal immunization with SAG1 and nontoxic mutant heat-labile enterotoxins protects mice against Toxoplasma gondii

Effective protection against intestinal pathogens requires both mucosal and systemic immune responses. Intranasal administration of antigens induces these responses but generally fails to trigger a strong protective immunity. Mucosal adjuvants can significantly enhance the immunogenicities of intranasally administered antigens. Cholera toxin (CT) and heat-labile enterotoxin (LT) are strong mucosal adjuvants with a variety of antigens. Moreover, the toxicities of CT and LT do not permit their use in humans. Two nontoxic mutant LTs, LTR72 and LTK63, were tested with Toxoplasma gondii SAG1 protein in intranasal vaccination of CBA/J mice. Vaccination with SAG1 plus LTR72 or LTK63 induced strong systemic (immunoglobulin G [IgG]) and mucosal (IgA) humoral responses. Splenocytes and mesenteric lymph node cells from mice immunized with LTR72 plus SAG1, but not those from mice immunized with LTK63 plus SAG1, responded to restimulation with a T. gondii lysate antigen in vitro. Gamma interferon and interleukin 2 (IL-2) production by splenocytes and IL-2 production by mesenteric lymph node cells were observed in vitro after antigen restimulation, underlying a Th1-like response. High-level protection as assessed by the decreased load of cerebral cysts after a challenge with the 76K strain of T. gondii was obtained in the group immunized with LTR72 plus SAG1 and LTK63 plus SAG1. They were as well protected as the mice immunized with the antigen plus native toxins. This is the first report showing protection against a parasite by using combinations of nontoxic mutant LTs and SAG1 antigen. These nontoxic mutant LTs are now attractive candidates for the development of mucosally delivered vaccines.     

Intranasal vaccination against cutaneous leishmaniasis with a particulated leishmanial antigen or DNA encoding LACK

We have previously demonstrated that oral delivery of a disease-promoting particulated antigen of Leishmania amazonensis (LaAg) partially protects mice against cutaneous leishmaniasis. In the present work, we sought to optimize a mucosal vaccine by using the intranasal route for delivery of different antigen preparations, including (i) LaAg, (ii) soluble recombinant p36/LACK leishmanial antigen (LACK), and (iii) plasmid DNA encoding LACK (LACK DNA). BALB/c mice that received two intranasal doses of 10 microg of LaAg and were challenged 1 week postvaccination with L. amazonensis developed delayed but effective control of lesion growth. A diminished parasite burden was accompanied by enhancement of both gamma interferon (IFN-gamma) and interleukin-10 levels in the lesion-draining lymph nodes. The vaccine efficacy improved with time. At 4 months postvaccination, when a strong parasite-specific TH1-type response was present in vivo, the infection was controlled for at least 5 months after challenge. In contrast to the particulated LaAg, soluble LACK (10 microg/dose) had no effect. Interestingly, LACK DNA (30 microg/dose), but not empty DNA, promoted rapid and durable protective immunity. Parasite growth was effectively controlled, and at 5 months after challenge LACK-reactive cells in both the mucosal and lesion-draining lymph nodes produced high levels of IFN-gamma. These results demonstrate for the first time the feasibility of using the intranasal route for long-lived memory vaccination against cutaneous leishmaniasis with adjuvant-free crude antigens or DNA.     

Oral immunization with a recombinant malaria protein induces conformational antibodies and protects mice against lethal malaria

The increasing death toll from malaria, due to the decreasing effectiveness of current prophylactic and therapeutic regimens, has sparked a search for alternative methods of control, such as vaccines. Although several single proteins have shown some promise as subunit vaccines against sexual blood stages in experimental systems, it is clear that multicomponent vaccines are required. Many logistic difficulties make such an approach prohibitively expensive. In an effort to try to overcome some of these issues, we examined the possibility of oral immunization as a route for inducing host protective immunity. We report here that oral feeding of a malaria protein induced serum antibody levels similar to those induced by intraperitoneal immunization with Freund's adjuvant. Further, responses to conformational epitopes were induced. In the rodent challenge system, significant levels of protection to lethal challenge with malaria were induced in mice. The protective efficacy was highly correlated with antibody levels, which depended on the antigen dosage and required cholera toxin subunit B as an oral adjuvant. These findings offer new approaches to the development of a malaria vaccine and provide justification for the investigation of transgenic plants as a means of vaccine delivery.     

Immunization with a DNA vaccine cocktail protects mice lacking CD4 cells against an aerogenic infection with Mycobacterium tuberculosis

Tuberculosis (TB) is the most common opportunistic disease and a potentially fatal complication among immunocompromised individuals infected with human immunodeficiency virus (HIV). Effective vaccination against TB in persons with HIV has been considered unlikely because of the central role that CD4 cells play in controlling tuberculous infections. Here we show that the vaccination of CD8^−/− mice with a TB DNA vaccine cocktail did not significantly enhance protective responses to a Mycobacterium tuberculosis infection. In contrast, immunization with a DNA vaccine cocktail or with the current TB vaccine, Mycobacterium bovis BCG, induced considerable antituberculosis protective immunity in immune-deficient mice lacking CD4 cells. In vaccinated CD4^−/− animals, substantially reduced bacterial burdens in organs and much improved lung pathology were seen 1 month after an aerogenic M. tuberculosis challenge. Importantly, the postchallenge mean times to death of vaccinated CD4^−/− mice were significantly extended (mean with DNA cocktail, 172 ± 7 days; mean with BCG, 156 ± 22 days) compared to that of naïve CD4^−/− mice (33 ± 6 days). Furthermore, the treatment of DNA-vaccinated CD4^−/− mice with an anti-CD8 or anti-gamma interferon (IFN-γ) antibody significantly reduced the effect of immunization, and neither IFN-γ^−/− nor tumor necrosis factor receptor-deficient mice were protected by DNA immunization; therefore, the primary vaccine-induced protective mechanism in these immune-deficient mice likely involves the secretion of cytokines from activated CD8 cells. The substantial CD8-mediated protective immunity that was generated in the absence of CD4 cells suggests that it may be possible to develop effective TB vaccines for use in HIV-infected populations.Tuberculosis (TB) remains a significant global threat to public health, with two million people dying from Mycobacterium tuberculosis infections each year and eight million cases of TB developing annually (6). The increasing linkage of TB with the human immunodeficiency virus (HIV) pandemic has magnified this tragedy in the past decade. The World Health Organization estimates that at least six million people worldwide are coinfected with M. tuberculosis and HIV (18). The HIV-M. tuberculosis coinfection rates exceed 5% in eight African countries, and in South Africa alone two million adults are coinfected (9). In these developing countries, TB is the most prevalent cause of morbidity and mortality for HIV-positive adults. In contrast to immunocompetent individuals, who have a 10% lifetime risk of disease following TB infection, persons coinfected with M. tuberculosis and HIV have a nearly 10% annual risk of developing disease.A primary reason for the continued failure to curb the global TB epidemic is the absence of a highly effective vaccine. Although the current TB vaccine, Mycobacterium bovis BCG, has been widely used for decades, its efficacy in controlled clinical trials has been extremely variable and its value in protecting against the most prevalent form of the disease, adult pulmonary TB, is doubtful (7). Moreover, the effectiveness of BCG in preventing TB in HIV-infected individuals is uncertain. Since BCG is a live vaccine (attenuated but not avirulent) and since clinical cases of reactivated BCG have been reported for HIV-infected persons, BCG vaccination has not been indicated for immunocompromised individuals (33).The development of a new vaccine against TB for use in HIV-positive persons has been considered unlikely because of the presumed essential roles that CD4 cells play in controlling TB infections. Mice that lack CD4 cells or that are aberrant in major histocompatibility complex class II presentation are extremely sensitive to a TB challenge and cannot effectively control acute TB infections (3, 13). The greatly enhanced susceptibility of HIV patients to both primary and reactivated disease argues that CD4 cells also play a prominent role in protective immune responses against human TB. The primary antituberculosis effector function of CD4⁺ T cells involves the production and secretion of cytokines which activate macrophages to control or eliminate the intracellular bacilli (13). In addition to this more direct role of CD4 cells in limiting tuberculous infections, CD4 cells also assist in the development of primary CD8 T-cell responses (23). CD4 cells stimulate professional antigen-presenting cells (APCs) primarily via CD40-CD40 ligand interactions; these activated APCs efficiently costimulate antigen-specific naïve CD8 T cells. Additionally, cytokine production from CD4 cells enhances the proliferation of primed CD8 cells.Despite the critical immune functions of CD4 cells, recent studies have demonstrated that protective immunity to pathogens can be generated in the absence of CD4 cells. For instance, substantial CD8 T-cell responses to the influenza virus, lymphocytic choriomeningitis virus (LCMV), and pathogenic fungi can occur in mice lacking CD4 cells (17, 26, 34, 35). In a Listeria monocytogenes model, similar levels of activated CD8 T cells were detected in CD4^−/− and wild-type (WT) C57BL/6 mice after an infection. Importantly, the epitope-specific CD8 T cells that were generated established long-term memory in CD4^−/− mice and were capable of producing an effective recall response (21). Under circumstances in which CD4 help is not required for the generation of effective CD8 T-cell responses, alternate pathways for the activation of APCs exist. For influenza virus, a direct infection of dendritic cells results in the upregulation of costimulatory molecules. For bacteria, dendritic cells can be activated through recognition by Toll-like receptors (TLRs) of bacterial products such as peptidoglycan, glycolipids, and lipoproteins (2, 29, 30).Based on these findings in other immune-deficient disease models, we evaluated whether cell-mediated antituberculosis protective immunity could be induced in mice lacking CD4 cells by immunization with a DNA vaccine cocktail or with BCG. Previously, members of our laboratory generated a DNA cocktail that expressed mycobacterial proteins fused at the N terminus to ubiquitin (UB), a eukaryotic intracellular targeting sequence (10, 11). These UB-conjugated proteins were designed to enhance major histocompatibility complex class I presentation. In studies using WT C57BL/6 mice, it was shown that vaccination with this combination was more effective than immunization with the individual single components and that the sustained protective immunity induced by the plasmid mixture was equivalent to the level of protection elicited by the BCG vaccine (10, 11). We report here that substantial antituberculosis protective immunity can be induced in the absence of CD4 cells. Immunization of CD4^−/− mice with either the DNA vaccine cocktail or BCG leads to significantly decreased lung and spleen bacterial burdens and much improved lung pathology, relative to naïve controls, after an aerogenic M. tuberculosis infection. Most importantly, the survival of the vaccinated animals was substantially extended compared to nonimmunized CD4^−/− controls.     

Mycobacterium bovis BCG-induced protection against cutaneous and systemic Leishmania major infections of mice.

We examined the protective effects of Mycobacterium bovis bacillus Calmette-Guérin (BCG) administration on Leishmania major infections of BALB/c and P/J mice. There were two treatment protocols. In the first, the footpads of naive animals were inoculated with mixtures of L. major and BCG (viable or heat killed) or the soluble mycobacterial antigen, purified protein derivative. Viable BCG, but not heat-killed BCG or purified protein derivative, inoculated with L. major amastigotes into the footpads of naive BALB/c or P/J mice protected these animals from the metastatic spread of parasites to the viscera and from ensuing lethal systemic infection. This treatment also induced cures of the cutaneous lesions of P/J mice but not of BALB/c mice. In the second protocol, we induced an immune response to BCG before inoculation of L. major. BCG given intraperitoneally 10 days before infection of footpads with leishmania offered protection against the metastatic spread of amastigotes in both P/J and BALB/c mice, regardless of intralesional treatment, and modulated the severity of cutaneous infection by 30 to 50%. Inoculation of a mixture of viable BCG and L. major amastigotes into BCG-immune mice completely protected both BALB/c and P/J strains from cutaneous disease; we recovered no parasites from the inoculated footpads of these animals. Furthermore, each of the nonspecifically protected mice of both the BALB/c and P/J strains developed immunity to rechallenge with viable L. major. Injection of amastigotes at a site remote from the original lesion, the contralateral footpad, resulted in the complete clearance of parasites in the inoculum with no evidence of either cutaneous or systemic disease over an extended observation period.