A synthetic peptide from Trypanosoma cruzi mucin-like associated surface protein as candidate for a vaccine against Chagas disease

Chagas disease, caused by Trypanosoma cruzi, is responsible for producing significant morbidity and mortality throughout Latin America. The disease has recently become a public health concern to nonendemic regions like the U.S. and Europe. Currently there are no fully effective drugs or vaccine available to treat the disease. The mucin-associated surface proteins (MASPs) are glycosylphosphatidylinositol (GPI)-anchored glycoproteins encoded by a multigene family with hundreds of members. MASPs are among the most abundant antigens found on the surface of the infective trypomastigote stage of T. cruzi, thus representing an attractive target for vaccine development. Here we used immunoinformatics to select a 20-mer peptide with several predicted overlapping B-cell, MHC-I, and MHC-II epitopes, from a MASP family member expressed on mammal-dwelling stages of T. cruzi. The synthetic MASP peptide conjugated to keyhole limpet hemocyanin (MASPpep-KLH) was tested in presence or not of an adjuvant (alum, Al) as a vaccine candidate in the C3H/HeNsd murine model of T. cruzi infection. In considerable contrast to the control groups receiving placebo, Al, or KLH alone or the group immunized with MASPpep-KLH/Al, the group immunized with MASPpep-KLH showed 86% survival rate after challenge with a highly lethal dose of trypomastigotes. As evaluated by quantitative real-time polymerase chain reaction, MASPpep-KLH-immunized animals had much lower parasite load in the heart, liver, and spleen than control animals. Moreover, protected animals produced trypanolytic, protective antibodies, and a cytokine profile conducive to resistance against parasite infection. Finally, in vivo depletion of either CD4⁺ or CD8⁺ T cells indicated that the latter are critical for protection in mice immunized with MASPpep-KLH. In summary, this new peptide-based vaccine with overlapping B- and T-cell epitopes is able to control T. cruzi infection in mice by priming both humoral and cellular immunity.     

Efficient protective immunity against Trypanosoma cruzi infection after nasal vaccination with recombinant Sendai virus vector expressing amastigote surface protein-2

Chagas’ disease, caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), is intractable showing a high mortality rate, and the development of effective vaccines is much desired. To examine the efficacy of a new mode of recombinant viral vaccine, we constructed two non-transmissible Sendai viruses (rSeV/dF) encoding the full-length parasite antigen amastigote surface protein-2 (ASP2) or ASP2 fused with a mono-ubiquitin on its N-terminus (UASP2). C57BL/6 mice immunized intranasally with rSeV/dF expressing either ASP2 or UASP2 showed significantly suppressed parasitemia and could be protected from lethal T. cruzi challenge. Depletion of CD8⁺ T cells around the time of infection with T. cruzi completely abolished this protection, confirming that acquired immunity against the infection of T. cruzi is dependent on CD8⁺ T cells. We also demonstrated that the protective immunity correlated with higher secretion of interferon-γ (IFN-γ) by spleen cells on in vitro-specific or non-specific stimulation. Increased CTL activity was also confirmed by degranulation or CTL assays. Interestingly, the control virus, rSeV/dF-GFP, induced even a higher IFN-γ production from spleen cells following non-specific but not specific stimulation in vitro, suggesting that SeV may also be a good adjuvant when used as a vaccine vehicle. Taking together, the current findings indicate that recombinant Sendai virus expressing the ASP2 or UASP2 antigens of T. cruzi are interesting candidates for the development of a new mode of recombinant viral vaccine against Chagas’ disease.     

Dendritic cells devoid of IL-10 induce protective immunity against the protozoan parasite Trypanosoma cruzi

In diverse models of microbial infections, protection is improved by immunization with dendritic cells (DC) loaded with whole pathogen lysate. However, pathogens that modulate DC function as a way to evade immunity may represent a challenge for these vaccination strategies. Thus, DC must be instructed in a particular manner to circumvent this issue and drive an effective immune response. Trypanosoma cruzi or its molecules alter DC function and, as we demonstrated, this phenomenon is associated with the parasite-driven stimulation of IL-10 production by DC. Here, we show that DC from IL-10-deficient mice pulsed in vitro with trypomastigote lysate secreted increased amounts of Th1-related cytokines and stimulated higher allogeneic and antigen-specific lymphocyte responses than their wild-type counterparts. In a model of DC-based immunization, these antigen-pulsed IL-10-deficient DC conferred protection against T. cruzi infection to recipient mice. Efficient immunity was associated with enhanced antigen-specific IFN-gamma production and endogenous DC activation. We illustrate for the first time a DC-based vaccination against T. cruzi and evidence the key role of IL-10 produced by sensitizing DC in inhibiting the induction of protection. These results support the rationale for vaccination strategies that timely suppress the effect of specific cytokines secreted by antigen presenting DC.     

Effect of a combination DNA vaccine for the prevention and therapy of Trypanosoma cruzi infection in mice: Role of CD4 and CD8 T cells

Chagas disease is a major public health problem, with about 10 million infected people, and DNA vaccines are a promising alternative for the control of Trypanosoma cruzi, the causing agent of the disease. We tested here a new DNA vaccine encoding a combination of two leading parasite antigens, TSA-1 and Tc24, for the prevention and therapy of T. cruzi infection. Immunized Balb/c mice challenged by T. cruzi presented a significantly lower parasitemia and inflammatory cell density in the heart compared to control mice. Similarly, the therapeutic administration of the DNA vaccine was able to significantly reduce the parasitemia and inflammatory reaction in acutely infected Balb/c and C57BL/6 mice, and reduced cardiac tissue inflammation in chronically infected ICR mice. Therapeutic vaccination induced a marked increase in parasite-specific IFNγ producing CD4⁺ and CD8⁺ T cells in the spleen as well as an increase in CD4⁺ and CD8⁺ T cells in the infected cardiac tissue. In addition, some effect of the DNA vaccine could still be observed in CD4-knockout C57BL/6 mice, which presented a lower parasitemia and inflammatory cell density, but not in CD8-deficient mice, in which the vaccine had no effect. These results indicate that the activation of CD8⁺ T cells plays a major role in the control of the infection by the therapeutic DNA vaccine, and to a somewhat lesser extent CD4⁺ T cells. This observation opens interesting perspectives for the potentiation of this DNA vaccine candidate by including additional CD8⁺ T cell antigens/epitopes in future vaccine formulations.     

CD8 T cell immunity to Plasmodium permits generation of protective antibodies after repeated sporozoite challenge

Individuals living in malaria endemic areas are subject to repeated infections yet fail to develop sterilizing immunity, however, immunization of mice with attenuated sporozoites or subunit vaccines has shown the ability to protect mice against a sporozoite challenge. We recently reported that mice primed with dendritic cells coated with the dominant circumsporozoite CD8 T cell epitope from Plasmodium berghei followed by a boost with recombinant Listeria monocytogenes expressing the same epitope exhibited sterile immunity against a sporozoite challenge for more than one year. In this report we show those mice do not contain protective antibodies and that depletion of CD4 T cells in the immunized mice did not affect sterile immunity. In contrast, CD8 T cell depletion eliminated protection. Thus, protective immunity generated by this immunization approach is entirely memory CD8 T cell-dependent. We also show here that mice initially protected by circumsporozoite-specific memory CD8 T cells develop sterilizing sporozoite-specific antibodies after repeated asymptomatic challenges with physiologic numbers of viable sporozoites. Therefore, initial protection by a CD8 T cell-targeted liver stage subunit vaccine allows the generation of enhanced sterilizing immune responses from repeated exposure to Plasmodium parasites.     

CD8 T cell independent immunity after single dose infection-treatment-vaccination (ITV) against Plasmodium yoelii

Sporozoite vaccination of both humans and rodents elicits potent anti-malarial immunity, but the dose of sporozoites and the number of immunizations required varies with vaccination approach. Here we examine the immunological basis for superior protection afforded from single-dose vaccination with virulent sporozoites administered under prophylatic chloroquine-cover, referred to as infection-treatment-vaccination (ITV), compared to the well-studied approach of administering radiation-attenuated Plasmodium sporozoites (RAS). Earlier rodent studies utilizing ITV and RAS vaccination suggested a major role of CD8 T cells in reducing liver parasite burden after sporozoite challenge in a BALB/c mouse model. Consistent with this, we find that in C57Bl/6 mice ITV elicits substantially higher parasite-specific CD8 T cell responses than RAS vaccination and enhances immunity against P. yoelii infection. However, we show ITV-induced CD8 T cells are not necessary for protection following liver-stage sporozoite or blood-stage parasite challenge. Mechanistically, we found protection afforded from single-dose ITV is associated with low grade, transient parasitemia shortly following cessation of chloroquine treatment and generation of potent antibody responses to blood-stage parasites. Collectively, our data show the mechanistic basis for enhanced protective immunity against P. yoelli elicited by ITV in highly susceptible C57Bl/6 mice is independent of CD8 T cells. These studies may be relevant in understanding the potent immunity observed with ITV in humans.     

An IL-15 adjuvant enhances the efficacy of a combined DNA vaccine against Brucella by increasing the CD8 cytotoxic T cell response

We investigated whether a combined DNA vaccine delivered together with the IL-15 gene (DNA-IL-15(+)) enhanced the immune response against Brucella abortus in mice. Mice vaccinated with DNA-IL-15(+) developed a robust humoral response; Brucella-specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. Splenocytes from DNA-IL-15(+)-vaccinated mice induced significantly higher levels of IFN-γ (P < 0.01) and CD8⁺ T cell response (P < 0.01), suggesting induction of a T-helper-1-dominated immune response. In a specific cytotoxic-T-lymphocyte activity assay, DNA-IL-15(+) immunization elicited mainly CD8⁺ T cells, which mediate cytotoxicity, but also CD4⁺ T cells. In vivo depletion of T cell subsets showed that the DNA-IL-15(+)-induced protection against Brucella infection is mediated predominantly by CD8⁺ T cells, although CD4⁺ T cells also contribute. These data indicate that plasmid-delivered IL-15 increases the efficacy of the Brucella DNA vaccine.     

Recombinant pro-apoptotic Mycobacterium tuberculosis generates CD8 T cell responses against human immunodeficiency virus type 1 Env and M. tuberculosis in neonatal mice

Mycobacterium bovis BCG is an attractive vaccine vector against breast milk HIV transmission because it elicits Th1-type responses in newborns. However, BCG causes disease in HIV-infected infants. Genetically attenuated Mycobacterium tuberculosis (Mtb) mutants represent a safer alternative for immunocompromised populations. In the current study, we compared the immunogenicity in mice of three different recombinant attenuated Mtb strains expressing an HIV envelope (Env) antigen construct. Two of these strains (ΔlysA ΔpanCD Mtb and ΔRD1 ΔpanCD Mtb) failed to induce significant levels of HIV Env-specific CD8⁺ T cell responses. In striking contrast, an HIV-1 Env-expressing attenuated ΔlysA Mtb containing a deletion in secA2, which encodes a virulence-related secretion system involved in evading adaptive immunity, generated consistently measurable Env-specific CD8⁺ T cell responses that were significantly greater than those observed after immunization with BCG expressing HIV Env. Similarly, another strain of ΔlysA ΔsecA2 Mtb expressing SIV Gag induced Gag- and Mtb-specific CD8⁺ T cells producing perforin or IFNγ, and Gag-specific CD4⁺ T cells producing IFNγ within 3 weeks after immunization in adult mice; in addition, IFNγ-producing Gag-specific CD8⁺ T cells and Mtb-specific CD4⁺ T cells were observed in neonatal mice within 1 week of immunization. We conclude that ΔlysA ΔsecA2 Mtb is a promising vaccine platform to construct a safe combination HIV-TB vaccine for use in neonates.     

Oral vaccination with Ts87 DNA vaccine delivered by attenuated Salmonella typhimurium elicits a protective immune response against Trichinella spiralis larval challenge

We have previously reported that Ts87 is an immunodominant antigen that induces protective immunity against Trichinella spiralis larval challenge. In this study, the Ts87 gene was cloned into an expression plasmid, pVAX1, and the recombinant Ts87 DNA was transformed into attenuated Salmonella typhimurium strain SL7207. Oral immunization of mice with Ts87 DNA delivered in S. typhimurium elicited a significant local mucosal IgA response and a systemic Th1/Th2 immune response. Cytokine profiling also showed a significant increase in the Th1 (IFN-γ) and Th2 (IL-5, 6, 10) responses in splenocytes of immunized mice upon stimulation with Ts87 antigen. An immunofluorescence assay performed with antisera revealed that the recombinant Ts87 protein was expressed in mesenteric lymph nodes of immunized mice. The mice immunized with Salmonella-delivered Ts87 DNA displayed a statistically significant 29.8% reduction in adult worm burden and a 34.2% reduction in muscle larvae following challenge with T. spiralis larvae, compared with mice immunized with empty Salmonella or a PBS control. Our results demonstrate that Ts87 DNA delivered by attenuated live S. typhimurium elicits a local IgA response and a balanced Th1/Th2 immune response and produces partial protection against T. spiralis infection in mice.     

CD4+ and CD8+ T cell- and IL-17-mediated protection against Entamoeba histolytica induced by a recombinant vaccine

Amebiasis in the murine model can be prevented by vaccination with the Gal/GalNAc lectin through a T cell-dependent mechanism. In this work we further decipher the mechanism of this protection. Mice vaccinated with the recombinant “LecA” fragment of the Gal/GalNAc lectin with alum were capable of transferring protection to naïve recipients by both CD4+ T cells and surprisingly CD8+ T cells. We then examined the cytokine profile of these cells. CD4+ T cells from PBMC of LecA-alum vaccinated mice were observed to be a major source of IFN-γ, known to be a protective cytokine with this vaccine. In contrast, CD8+ T cells produced relatively little IFN-γ but more IL-17 than the CD4 compartment. We thus examined the role of IL-17 in vaccine mediated protection and found through neutralization experiments that this cytokine contributed to LecA-alum vaccine protection. In addition we examined whether these cells exhibited direct amebicidal activity in vitro and found that both populations had amebicidal activity at high concentrations (1000:1) but CD8+ T cells appeared more potent, capable of cytotoxicity at a 100:1 ratio. In conclusion, both CD4 and CD8 T cells exert protection with this amebiasis vaccine. The mechanism of CD8 T cell-mediated protection may include direct amebicidal activity and/or IL-17 production. Both IL-17 and IFN-γ are useful surrogates for immune protection.     

Circulating regulatory T cells (CD4CD25FOXP3) decrease in breast cancer patients after vaccination with a modified MHC class II HER2/neu (AE37) peptide

Regulatory T cells (T_Reg), CD4⁺CD25⁺FOXP3⁺, are implicated in suppressing tumor immune responses. We analyzed peripheral blood lymphocytes (PBL) from breast cancer patients receiving a modified HLA class II HER2/neu peptide (AE37) vaccine for T_Reg cells and correlated their levels with vaccine-specific immune responses. The mean CD4⁺CD25⁺FOXP3⁺ T_Reg cells decreased in patients with vaccination with no significant difference in serum TGF-β levels. IFN-γ ELISPOT and DTH increased after vaccination with a good correlation between T_Reg cell reduction and size of DTH to AE37. The T_Reg cell reduction and associated immune response suggest that AE37 may be clinically useful.     

Phenotype of the anti-Rickettsia CD8 T cell response suggests cellular correlates of protection for the assessment of novel antigens

The obligately intracellular bacteria Rickettsia infect endothelial cells and cause systemic febrile diseases that are potentially lethal. No vaccines are currently available and current knowledge of the effective immune response is limited. Natural and experimental rickettsial infections provide strong and cross-protective cellular immunity if the infected individual survives the acute infection. Although resistance to rickettsial infections is attributed to the induction of antigen-specific T cells, particularly CD8⁺ T cells, the identification and validation of correlates of protective cellular immunity against rickettsial infections, an important step toward vaccine validation, remains a gap in this field. Here, we show that after a primary challenge with Rickettsia typhi in the C3H mouse model, the peak of anti-Rickettsia CD8⁺ T cell-mediated responses occurs 7 days post-infection (dpi), which coincides with the beginning of rickettsial clearance. At this time point, both effector-type and memory-type CD8⁺ T cells are present, suggesting that 7 dpi is a valid time point for the assessment of CD8⁺ T cell responses of mice previously immunized with protective antigens. Based on our results, we suggest four correlates of cellular protection for the assessment of protective rickettsial antigens: (1) production of IFN-γ by antigen-experienced CD3⁺CD8⁺CD44^high cells, (2) production of Granzyme B by CD27^lowCD43^low antigen-experienced CD8⁺ T cells, (3) generation of memory-type CD8⁺ T cells [Memory Precursor Effector Cells (MPECs), as well as CD127^highCD43^low, and CD27^highCD43^low CD8⁺ T cells], and (4) generation of effector-like memory CD8⁺ T cells (CD27^lowCD43^low). We propose that these correlates could be useful for the general assessment of the quality of the CD8⁺ T cell immune response induced by novel antigens with potential use in a vaccine against Rickettsia.     

Evaluation of a multisubunit recombinant polymorphic membrane protein and major outer membrane protein T cell vaccine against Chlamydia muridarum genital infection in three strains of mice

An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of Chlamydia muridarum genital infection, multifunctional mucosal CD4 T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4 T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH + MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population.     

Vaccination with a piggyBac plasmid with transgene integration potential leads to sustained antigen expression and CD8 T cell responses

DNA vaccination with plasmid has conventionally involved vectors designed for transient expression of antigens in injected tissues. Next generation plasmids are being developed for site-directed integration of transgenes into safe sites in host genomes and may provide an innovative approach for stable and sustained expression of antigens for vaccination. The goal of this study was to evaluate in vivo antigen expression and the generation of cell mediated immunity in mice injected with a non-integrating plasmid compared to a plasmid with integrating potential. Hyperactive piggyBac transposase-based integrating vectors (pmhyGENIE-3) contained a transgene encoding either eGFP (pmhyGENIE-3-eGFP) or luciferase (pmhyGENIE-3-GL3), and were compared to transposase-deficient plasmids with the same transgene and DNA backbone. Both non-integrating and integrating plasmids were equivalent at day 1 for protein expression at the site of injection. While protein expression from the non-integrating plasmid was lost by day 14, the pmhyGENIE-3 was found to exhibit sustained protein expression up to 28 days post-injection. Vaccination with pmhyGENIE-3-eGFP resulted in a robust CD8⁺ T cell response that was three-fold higher than that of non-integrating plasmid vaccinations. Additionally we observed in splenocyte restimulation experiments that only the vaccination with pmhyGENIE-3-eGFP was characterized by IFNγ producing CD8⁺ T cells. Overall, these findings suggest that plasmids designed to direct integration of transgenes into the host genome are a promising approach for designing DNA vaccines. Robust cell mediated CD8⁺ T cell responses generated using integrating plasmids may provide effective, sustained protection against intracellular pathogens or tumor antigens.     

Protective immunity induced by a DNA vaccine expressing eIF4A of Toxoplasma gondii against acute toxoplasmosis in mice

Toxoplasma gondii is an obligate intracellular protozoan parasite infecting humans, mammals and birds. Eukaryotic translation initiation factor (eIF4A) is a newly identified protein associated with tachyzoite virulence. To evaluate the protective efficacy of T. gondii eIF4A, a DNA vaccine (pVAX-eIF4A) encoding T. gondii eIF4A (Tg-eIF4A) gene was constructed. The expression ability of this recombinant DNA plasmid was examined in Marc145 cells by IFA. Then, Kunming mice were intramuscularly immunized with pVAX-eIF4A and followed by challenge infection with the highly virulent T. gondii RH strain. The results showed that vaccination with pVAX-eIF4A elicited specific humoral responses, with high IgG antibody titers and specific lymphocyte proliferative responses. The cellular immune response was associated with significant production of IFN-γ, IL-2 in Kunming mice, and a mixed IgG1/IgG2a response with predominance of IgG2a production, indicating that a Th1 type response was elicited after immunization with pVAX-eIF4A. In addition, the increase of the percentage of CD8+ T cells in lymphoid in mice suggested the activation of MHC class I restricted antigen presentation pathways. After lethal challenge, the mice vaccinated with the pVAX-eIF4A showed a significantly prolonged survival time (23.0 ± 5.5 days) compared with control mice which died within 7 days of challenge (P < 0.05). These results demonstrate that pVAX-eIF4A could elicit strong humoral, Th1-type cellular immune responses and increase survival time of immunized mice, suggesting that eIF4A is a promising vaccine candidate against acute T. gondii infection in mice.     

Further analysis of protection induced by the MIC3 DNA vaccine against T. gondii: CD4 and CD8 T cells are the major effectors of the MIC3 DNA vaccine-induced protection,both Lectin-like and EGF-like domains of MIC3 conferred protection

The present study was conducted mainly to evaluate the contribution of the cellular and the humoral responses in protection conferred by the MIC3 DNA vaccine (pMIC3i) that was proved as a potent vaccine against toxoplasmosis. We performed the adoptive transfer of CD4⁺ and CD8⁺ T lymphocytes from pMIC3i immunized mice to naive ones and the role of humoral immunity was evaluated by in vitro invasion assays. We also constructed plasmids encoding the EGF-like domains and the Lectin-like domain of MIC3, to define which domains of MIC3 are involved in the protection. Furthermore, the adjuvant effect of the GM-CSF-expressing vector (granulocyte-macrophage colony-stimulating factor) required the precise temporal and spatial codelivery of GM-CSF with antigen, thus, we constructed a bicistronic plasmid expressing MIC3 and GM-CSF. In conclusion, the protection induced by pMIC3i was mainly mediated by CD4⁺ and CD8⁺ T lymphocytes and both EGF and Lectin domains of MIC3 conferred protection. Furthermore, the codelivery of GM-CSF by a bicistronic plasmid appeared to be a most effective way for enhancing the adjuvant properties of GM-CSF.     

Lack of cross-protection against invasive pneumonia caused by heterologous strains following murine Streptococcus pneumoniae nasopharyngeal colonisation despite whole cell ELISAs showing significant cross-reactive IgG

Prior exposure to intact Streptococcus pneumoniae can induce a protective antibody response to proteins antigens, which prevents subsequent invasive disease. This may be achieved either by colonisation with live bacteria or by immunisation with killed cells. Such approaches could provide novel vaccine strategies that overcome the serotype restriction of conjugate vaccines, and would aim to prevent disease caused by all strains of S. pneumoniae. Serum antibody is required to prevent invasive disease, but which in vitro measure of antibody response correlates best with protective immunity has not been established for protein antigens.     

Protection promoted by pGP3 or pGP4 against Chlamydia muridarum is mediated by CD4 cells in C57BL/6N mice

Urogenital tract infection with Chlamydia trachomatis is a leading cause of sexually transmitted infections. There is currently no commercially available vaccine against C. trachomatis. The highly conserved plasmid of chlamydiae has been considered to be a virulence factor and the plasmid proteins have important roles in the Chlamydia-specific immune response. This study was designed to evaluate the efficacy of vaccination with plasmid proteins in the prevention of C. muridarum lung infection in a mouse model. C57BL/6N mice were immunised 3 times subcutaneously with recombinant pGP3 or pGP4 and infected with C. muridarum. Immunisation of the mice with recombinant pGP3 or pGP4 protein caused a significantly lower chlamydial burden in the lungs of the infected mice; the lower IFN-γ level indicated a reduced extent of inflammation. In vitro or in vivo neutralisation of C. muridarum with sera obtained from immunised mice did not reduce the number of viable C. muridarum in the lungs of mice. However, adoptive transfer of the CD4⁺ spleen cells isolated from the immunised mice resulted in a significantly reduced bacterial burden. Our results indicate that it is not the pGP3- and pGP4-specific antibodies, but the CD4⁺ cells that are responsible for the protective effect of the immune response to plasmid proteins.     

Effects of Signal 3 during CD8 T cell priming: Bystander production of IL-12 enhances effector T cell expansion but promotes terminal differentiation

Adjuvants are commonly used in vaccines to augment immune response, but how the inflammatory cytokines elicited by adjuvants directly influence effector and memory CD8 T cell differentiation remains poorly characterized. Here, we used a peptide-pulsed dendritic cell (DC) vaccination model to examine the role of primary cytokines, IL-12 and IFNγ, elicited by CpG-B adjuvant on CD8 T cell priming and memory CD8 T cell development. During DC vaccination, simultaneous exposure to antigen and a heterologous Listeria infection, CpG-B or IL-12 enhanced a portion of the effector CD8 T cells to expand and differentiate to a larger extent. Simultaneously, this also decreased their ability to become long-lived memory CD8 T cells. However, development of memory CD8 T cells and their precursors was largely unaffected by the additional inflammatory cytokines. Moreover, IL-12 production by the antigen-presenting cell (APC) was not required during DC + CpG vaccination or Listeria infection, but rather ‘bystander’ macrophages and DCs appeared to be the physiologically relevant cellular sources of this cytokine. Furthermore, IFNγ induced by CpG was required in vivo for optimal production of IL-12, which in turn, influenced effector CD8 T cell longevity. Together, these findings demonstrate the importance of an interconnected multicellular network between APCs, naïve T cells and bystander cells of the innate immune system that regulate effector and memory CD8 T cell development during vaccination.