Protective immunity against malaria liver stage after vaccination with live parasites

Despite nearly 100 years of research and control efforts, malaria remains one of the most important infectious diseases. An efficient vaccine would be a powerful to tool to reduce mortality and morbidity. Experimentally, induction of sterile immunity in humans after vaccination with attenuated sporozoites has been obtained. This observation has spurred the search for subunit vaccines that aim to reproduce this protection. As yet none of the current candidate subunit vaccines achieved complete protection reproducibly. This failure coupled to the recent advent of genetically modified Plasmodium parasites has led to a renewed interest in the use of live parasites for vaccination against malaria pre-erythrocytic stages. In this article, we review and discuss the recent developments in this field.     

Protective immunity to pre-erythrocytic stage malaria

The development of a vaccine against malaria is a major research priority given the burden of disease, death and economic loss inflicted upon the tropical world by this parasite. Despite decades of effort, however, a vaccine remains elusive. The best candidate is a subunit vaccine termed RTS,S but this provides only partial protection against clinical disease. This review examines what is known about protective immunity against pre-erythrocytic stage malaria by considering the humoral and T cell-mediated immune responses that are induced by attenuated sporozoites and by the RTS,S vaccine. On the basis of these observations a set of research priorities are defined that are crucial for the development of a vaccine capable of inducing long-lasting and high-grade protection against malaria.     

Protective CD8+ T lymphocytes in primates immunized with malaria sporozoites

Live attenuated malaria vaccines are more potent than the recombinant protein, bacterial or viral platform vaccines that have been tested, and an attenuated sporozoite vaccine against falciparum malaria is being developed for humans. In mice, attenuated malaria sporozoite vaccines induce CD8(+) T cells that kill parasites developing in the liver. We were curious to know if CD8(+) T cells were also important in protecting primates against malaria. We immunized 9 rhesus monkeys with radiation attenuated Plasmodium knowlesi sporozoites, and found that 5 did not develop blood stage infections after challenge with live sporozoites. We then injected 4 of these protected monkeys with cM-T807, a monoclonal antibody to the CD8 molecule which depletes T cells. The fifth monkey received equivalent doses of normal IgG. In 3 of the 4 monkeys receiving cM-T807 circulating CD8(+) T cells were profoundly depleted. When re-challenged with live sporozoites all 3 of these depleted animals developed blood stage malaria. The fourth monkey receiving cM-T807 retained many circulating CD8(+) T cells. This monkey, and the vaccinated monkey receiving normal IgG, did not develop blood stage malaria at re-challenge with live sporozoites. Animals were treated with antimalarial drugs and rested for 4 months. During this interval CD8(+) T cells re-appeared in the circulation of the depleted monkeys. When all vaccinated animals received a third challenge with live sporozoites, all 5 monkeys were once again protected and did not develop blood stage malaria infections. These data indicate that CD8(+) T cells are important effector cells protecting monkeys against malaria sporozoite infection. We believe that malaria vaccines which induce effector CD8+ T cells in humans will have the best chance of protecting against malaria.     

Protective CD8 T cell immunity triggered by CpG-protein conjugates competes with the efficacy of live vaccines

In contrast to infectious (live) vaccines are those based on subunit Ag that are notoriously poor in eliciting protective CD8 T cell responses, presumably because subunit Ags become insufficiently cross-presented by dendritic cells (DCs) and because the latter need to be activated to acquire competence for cross-priming. In this study, we show that CpG-Ag complexes overcome these limitations. OVA covalently linked to CpG-DNA (CpG-OVA complex), once it is efficiently internalized by DCs via DNA receptor-mediated endocytosis, is translocated to lysosomal-associated membrane protein 1 (LAMP-1)-positive endosomal-lysosomal compartments recently shown to display competence for cross-presentation. In parallel, CpG-OVA complex loaded DCs become activated and acquire characteristics of professional APCs. In vivo, a single s.c. dose of CpG-OVA complex (10 mug of protein) induces primary and secondary clonal expansion/contraction of Ag-specific CD8 T cells similar in kinetics to live vaccines; examples including Listeria monocytogenes genetically engineered to produce OVA (LM-OVA) and two viral vector-based OVA vaccines analyzed. Interestingly, CpG-OVA complex induced almost equal percentages of Ag-specific memory CD8 T cells as did infection with LM-OVA. A single dose vaccination with CpG-OVA complex protected mice against lethal doses of LM-OVA. These data underscore that the synergy imparted by CpG-OVA complex-mediated combined triggering of innate and specific immunity might be key to initiate CD8 T cell-based immunoprotection by synthetic vaccines based on subunit Ag.     

Protective immunity of Nile tilapia against Ichthyophthirius multifiliis post-immunization with live theronts and sonicated trophonts

Two immunization trials were conducted to evaluate host protection of Nile tilapia, Oreochromis niloticus against Ichthyophthirius multifiliis (Ich). Immunizations were done with live theronts or sonicated trophonts by bath immersion and intraperitoneal (IP) injection. The immunized fish were challenged with theronts 21 days post-immunization in trial I and 180 days post-immunization in trial II. The serum anti-Ich antibody and cumulative mortalities of tilapia were determined after theront challenge. Serum anti-Ich antibody was significantly higher (P<0.05) in tilapia immunized with live theronts by immersion or IP injection or with sonicated trophonts administered by IP injection than tilapia immunized with sonicated trophonts by immersion, with bovine serum albumin by IP injection, or non-immunized controls. Host protection was acquired in fish immunized with live theronts by immersion or IP injection. Tilapia immunized with sonicated trophonts by IP injection were partially protected with a 57-77% survival in both trials. At 180 days post-immunization, serum antibody titers had declined in immunized fish yet they were still able to survive challenge. The protection appears not to be solely depending on serum antibody response against Ich.     

Protective immunity induced by vaccination with SAG1 gene-transfected cells against Toxoplasma gondii-infection in mice

To develop a vaccine by augmenting the protective cellular immunity against Toxoplasma gondii (T. gondii)-infection, T gondii SAG1 gene-transfectants were established by using RMA.S (H-2b), a murine transporter associated with the antigen processing (TAP) molecule-deficient lymphoma line, as a host antigen-presenting cell (APC). Immunization of C57BL/6 mice with the SAG1-transfected RMA.S induced CD8+ cytotoxic T lymphocytes (CTL) specific for not only SAG1-transfected RMA.S but also T gondii-infected RMA.S, and elicited protective responses to infection with a virulent T. gondii strain, RH.     

Superior antimalarial immunity after vaccination with late liver stage-arresting genetically attenuated parasites

While subunit vaccines have shown partial efficacy in clinical trials, radiation-attenuated sporozoites (RAS) remain the "gold standard" for sterilizing protection against Plasmodium infection in human vaccinees. The variability in immunogenicity and replication introduced by the extensive, random DNA damage necessary to generate RAS could be overcome by genetically attenuated parasites (GAP) designed via gene deletion to arrest at defined points during liver-stage development. Here, we demonstrate the principle that late liver stage-arresting GAP induce larger and broader CD8 T cell responses that provide superior protection in inbred and outbred mice compared to RAS or early-arresting GAP immunizations. Late liver stage-arresting GAP also engender high levels of cross-stage and cross-species protection and complete protection when administered by translationally relevant intradermal or subcutaneous routes. Collectively, our results underscore the potential utility of late liver stage-arresting GAP as broadly protective next-generation live-attenuated malaria vaccines and support their potential as a powerful model for identifying antigens to generate cross-stage protection.     

Protective immunity against lethal anaphylactic reaction in Toxoplasma gondii-infected mice by DNA vaccination with T. gondii-derived heat shock protein 70 gene

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) was proven to induce lethal anaphylactic reaction in T. gondii-infected mice through platelet-activating factor (PAF)-mediated, but not classical IgE-dependent, pathway via TLR4/MyD88 signal pathway. The effector cells generating PAF and causing T.g.HSP70-induced anaphylactic reaction were CD11b⁺ and CD11c⁺ cells, although the reaction was enhanced by marked IFN-γ production by CD11b⁺, CD11c⁺, CD4⁺ and CD8⁺ splenocytes. In the present study, the effects of T.g.HSP70 gene vaccine targeting peripheral dendritic cells were evaluated against T.g.HSP70-induced anaphylactic reaction in T. gondii-infected mice. C57BL/6 mice receiving T.g.HSP70 gene vaccine showed prolonged survival. Platelets of peripheral blood, which completely disappeared during the T.g.HSP70-induced anaphylactic reaction, were partially restored with the T.g.HSP70 gene vaccination. The T.g.HSP70-induced marked production of PAF and IFN-γ from splenocytes of infected mice during the T.g.HSP70-induced anaphylactic reaction was shown to decrease after the T.g.HSP70 gene vaccination. Thus, T.g.HSP70 gene vaccine induced protective immunity against T.g.HSP70-induced PAF-mediated lethal anaphylactic reaction in T. gondii-infected mice.     

Protective CD8+ T cell responses against the pre-erythrocytic stages of malaria parasites: an overview

CD8+ T cells have been implicated as critical effector cells in protection against the pre-erythrocytic stage of malaria in mice and humans following irradiated sporozoite immunization. Immunization experiments in animal models by several investigators have suggested different strategies for vaccination against malaria and many of the targets from liver stage malaria antigens have been shown to be immunogenic and to protect mice from the sporozoite challenge. Several prime/boost protocols with replicating vectors, such as vaccinia/influenza, with non-replicating vectors, such as recombinant particles derived from yeast transposon (Ty-particles) and modified vaccinia virus Ankara, and DNA, significantly enhanced CD8+ T cell immunogenicity and also the protective efficacy against the circumsporosoite protein of Plasmodium berghei and P. yeti. Based on these experimental results the development of a CD8+ T cell inducing vaccine has moved forward from epitope identification to planning stages of safety and immunogenicity trials of candidate vaccines.     

Protective immunity against Taenia crassiceps murine cysticercosis induced by DNA vaccination with a Taenia saginata tegument antigen

This study investigated the protective capacity of the recombinant Taenia saginata Tso18 antigen administered as a DNA vaccine in the Taenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from a T. saginata oncosphere cDNA library, has homologies with Taenia solium and Echinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice. Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3-81.4%, while lower levels of non-specific protection were induced in control mice injected with the plasmid pcDNA3.1 (18.8-33.1%) or a plasmid with irrelevant construct, pcDNA3.1/3D15 (33.4-38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid and pcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies. These antibodies were shown to recognize a 66 kDa antigen fraction of T. crassiceps and T. solium. Splenocytes enriched in both CD4+CD8- and CD4-CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from both T. solium and T. crassiceps cestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres of T. saginata and T. solium, in the adult tapeworm and in the tegument of T. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera of cestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.     

Optimal control analysis of a malaria disease transmission model that includes treatment and vaccination with waning immunity

We derive and analyse a deterministic model for the transmission of malaria disease with mass action form of infection. Firstly, we calculate the basic reproduction number, R(0), and investigate the existence and stability of equilibria. The system is found to exhibit backward bifurcation. The implication of this occurrence is that the classical epidemiological requirement for effective eradication of malaria, R(0)<1, is no longer sufficient, even though necessary. Secondly, by using optimal control theory we derive the conditions under which it is optimal to eradicate the disease and examine the impact of a possible combined vaccination and treatment strategy on the disease transmission. When eradication is impossible, we derive the necessary conditions for optimal control of the disease using Pontryagin's Maximum Principle. The results obtained from the numerical simulations of the model show that a possible vaccination combined with effective treatment regime would reduce the spread of the disease appreciably.     

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.     

T cell immunity induced by live, necrotic, and apoptotic tumor cells

The rules that govern the engagement of antitumor immunity are not yet fully understood. Ags expressed by tumor cells are prone to induce T cell tolerance unless the innate immune system is activated. It is unclear to what extent tumors engage this second signal link by the innate immune system. Apoptotic and necrotic (tumor) cells are readily recognized and phagocytosed by the cells of the innate immune system. It is unknown how this affects the tumor's immunogenicity. Using a murine melanoma (B16m) and lymphoma (L5178Y-R) model, we studied the clonal sizes and cytokine signatures of the T cells induced by these tumors in syngeneic mice when injected as live, apoptotic, and necrotic cells. Both live tumors induced a type 2 CD4 cell response characterized by the prevalent production of IL-2, IL-4, and IL-5 over IFN-gamma. Live, apoptotic, and necrotic cells induced CD4 (but no CD8) T cells of comparable frequencies and cytokine profiles. Therefore, live tumors engaged the second signal link, and apoptotic or necrotic tumor cell death did not change the magnitude or quality of the antitumor response. A subclone of L5178Y-R, L5178Y-S cells, were found to induce a high-frequency type 1 response by CD4 and CD8 cells that conveyed immune protection. The data suggest that the immunogenicity of tumors, and their characteristics to induce type 1 or type 2, CD4 or CD8 cell immunity is not primarily governed by signals associated with apoptotic or necrotic cell death, but is an intrinsic feature of the tumor itself.     

Studies on the transfer of protective immunity with lymphoid cells from mice immune to malaria sporozoites.

In an effort to understand the mechanisms involved in the protective immunity to malarial sporozoites, an A/J mouse/Plasmodium berghei model was studied. Protective immunity could consistently be adoptively transferred only by using sublethal irradiation of recipients (500 R); a spleen equivalent (100 X 10(6))of donor cells from immune syngeneic mice; and a small booster immunization (1 X 10(4)) of recipients with irradiation-attenuated sporozoites. Recipient animals treated in this manner were protected from lethal challenge with 1 X 10(4) nonattenuated sporozoites. Immune and nonimmune serum and spleen cells from nonimmune animals did not protect recipient mice. Fewer immune spleen cells (50 X 10(6)) protected some recipients. In vitro treatment of immune spleen cells with anti-theta sera and complement abolished their ability to transfer protection. This preliminary study suggests that protective sporozoite immunity can be transferred with cells, and that it is T cell dependent.     

Regulatory T cell vaccination without autoantigen protects against experimental autoimmune encephalomyelitis

Regulatory T (T(reg)) cells show promise for treating autoimmune diseases, but their induction to elevated potency has been problematic when the most optimally derived cells are from diseased animals. To circumvent reliance on autoantigen-reactive T(reg) cells, stimulation to myelin-independent Ags may offer a viable alternative while maintaining potency to treat experimental autoimmune encephalomyelitis (EAE). The experimental Salmonella vaccine expressing colonization factor Ag I possesses anti-inflammatory properties and, when applied therapeutically, reduces further development of EAE in SJL mice. To ascertain T(reg) cell dependency, a kinetic analysis was performed showing increased levels of FoxP3(+)CD25(+)CD4(+) T cells. Inactivation of these T(reg) cells resulted in loss of protection. Adoptive transfer of the vaccine-induced T(reg) cells protected mice against EAE with greater potency than naive or Salmonella vector-induced T(reg) cells, and cytokine analysis revealed enhanced production of TGF-beta, not IL-10. The development of these T(reg) cells in conjunction with immune deviation by Th2 cells optimally induced protective T(reg) cells when compared those induced in the absence of Th2 cells. These data show that T(reg) cells can be induced to high potency to non-disease-inducing Ags using a bacterial vaccine.     

Novel vaccination protocol with two live mucosal vectors elicits strong cell-mediated immunity in the vagina and protects against vaginal virus challenge

Most HIV infections result from heterosexual transmission to women. Because cellular immunity plays a key role in the control of the infection, we sought to strengthen cellular immune responses in vaginal tissue. We explored a novel prime-boost protocol that used two live mucosal agents that trigger different pathways of innate immunity and induce strong cellular immunity. Adenovirus serotype 5 (Ad5) has frequently been used as a boost for DNA vaccines. In this study we used attenuated, recombinant L. monocytogenes-gag (rLm-gag) to prime mice by various mucosal routes-oral, intrarectal, and intravaginally (ivag)-followed by a systemic or mucosal boost with replication-defective rAd5-gag. Mice primed with a single administration of rLm-gag by any route and then boosted with rAd5-gag intramuscularly exhibited abundant Gag-specific CD8 T cells in spleen and vaginal lamina propria. Conversely, when boosted with rAd5-gag ivag, the immune response was reoriented toward the vagina with strikingly higher CD8 T cell responses in that tissue, particularly after ivag immunization by both vectors (ivag/ivag). Five weeks to 5 mo later, ivag/ivag-immunized mice continued to show high levels of effector memory CD8 T cells in vagina, while the pool of memory T cells in spleen assumed a progressively more central memory T cell phenotype. The memory mice showed high in vivo CTL activity in vagina, a strong recall response, and robust protection after ivag vaccinia-gag challenge, suggesting that this prime-boost strategy can induce strong cellular immunity, especially in vaginal tissues, and might be able to block the heterosexual transmission of HIV-1 at the vaginal mucosa.     

IgA-driven T cell-mediated anti-bacterial immunity in man after live oral Ty 21a vaccine

Cellular immunity against Salmonella typhi was observed by using a direct anti-bacterial in vitro assay in volunteers orally vaccinated with the live S. typhi mutant strain Ty 21a. With this experimental approach, it was demonstrated that Ty 21a vaccine also induces cellular immunity against S. paratyphi A and B. Interestingly, the mechanism involved in cellular immunity against bacteria seems to be of an antibody-dependent cellular cytotoxicity (ADCC) type, with IgA acting as the humoral arm and CD4+ T lymphocytes as the cellular one. In accordance with the increase in IgA-driven ADCC against S. typhi, a major rise in IgA against O and H antigens was observed in the serum of vaccinees in parallel to an increase in IgG of identical specificity. Furthermore, a Ty 21 vaccine induced cellular activity against flagellar antigens. These results indicate that IgA-ADCC by T lymphocytes against bacteria can originate from local stimulation of the gut mucosal immune system. This cellular defense mechanism might be at the origin of the protection induced by Ty 21a vaccine.     

T cell immunity to lymphoma following treatment with anti-CD40 monoclonal antibody

In this study we demonstrate that treatment with anti-CD40 mAb eradicates a range of mouse lymphomas (BCL(1), A31, A20, and EL4), but only when used against i.v. tumor doses in excess of 10(7) cells. Only partial protection was seen against smaller tumor loads. We saw no evidence that anti-CD40 mAb changed the phenotype of the lymphomas or inhibited their growth in the initial period following treatment, but it did result in a rapid expansion of cytotoxic CD8(+) cells that was able to clear the neoplastic disease and provide long-term protection against tumor rechallenge. The CTL responses were blocked by mAb against a range of coreceptors and cytokines, including CD8, B7-1, B7-2, LFA-1, and IFN-gamma, but not CD4 or CTLA-4, indicating the presence of a conventional cellular Th1 response. Furthermore, we found evidence of cross-recognition between lymphomas (BCL(1) and A20) as measured by cytotoxicity and IFN-gamma responses in vitro and using tumor rechallenge experiments, suggesting common target Ags. Finally, although anti-CD40 was shown to stimulate NK cell killing, we could find no role for these cells in controlling tumor growth. These data underline the ability of anti-CD40 mAb to potentiate CTL responses and the potency of cellular immunity in eradicating large quantities of syngeneic tumor.