Dendritic cells stimulated with outer membrane protein A (OmpA) of Salmonella typhimurium generate effective anti-tumor immunity

Gram-negative bacterial outer membrane proteins (Omps) have an important role in pathogenesis and signal reception. We previously reported that Acinetobacter OmpA (AbOmpA) induced maturation of bone marrow-derived dendritic cells (BMDCs) and that AbOmpA-primed DCs produced IL-12 which generated Th1 CD4⁺ T-cells. We analyzed the effects of Salmonella typhimurium OmpA (OmpA-Sal) on dendritic cell (DC) maturation in the present study, and determined that tumor antigen-pulsed DCs stimulated with OmpA-Sal induced anti-tumor responses in a mouse model. OmpA-Sal activated BMDCs by augmenting expression of MHC class II and of the co-stimulatory molecules CD80 and CD86. RT-PCR revealed that IL-12(p40) gene expression is highly augmented in OmpA-Sal-stimulated BMDCs. DNA (CRT/E7) vaccination combined with OmpA-Sal stimulation generated more antigen-specific CD8⁺ T-cells in the present study. Certain antigen-pulsed BMDCs stimulated with OmpA-Sal induced strong PADRE-specific CD4⁺ and E7-specific CD8⁺ T-cell responses. In addition, BMDCs stimulated with OmpA-Sal (OmpA-Sal-BMDCs) and pulsed with both E7 and PADRE peptide generated greater numbers of E7-specific CD8⁺ effector and memory T-cells than those pulsed with E7 peptide alone. E7- and PADRE-expressing OmpA-Sal-BMDC vaccines resulted in significant long-term protective anti-tumor effects in vaccinated mice. Our data suggested that E7- and PADRE-expressing BMDCs that were matured in the presence of OmpA-Sal might enhance anti-tumor immunity and support the therapeutic use of OmpA-Sal in DC-based immunotherapy.     

Evaluation of yellow fever virus 17D strain as a new vector for HIV-1 vaccine development

The failure to develop an effective vaccine against HIV-1 infection has led the research community to seek new ways of raising qualitatively different antibody and cellular immune responses. Towards this goal, we investigated the yellow fever 17D vaccine strain (YF17D), one of the most effective vaccines ever made, as a platform for HIV-1 vaccine development. A test antigen, HIV-1 p24 (clade B consensus), was inserted near the 5′ end of YF17D, in frame and upstream of the polyprotein (YF-5′/p24), or between the envelope and the first non-structural protein (YF-E/p24/NS1). In vitro characterization of these recombinants indicated that the gene insert was more stable in the context of YF-E/p24/NS1. This was confirmed in immunogenicity studies in mice. CD8⁺ IFN-γ T-cell responses against p24 were elicited by the YF17D recombinants, as were specific CD4⁺ T cells expressing IFN-γ and IL-2. A balanced CD4⁺ and CD8⁺ T-cell response was notable, as was the polyfunctionality of the responding cells. Finally, the protective efficacy of the YF17D recombinants, particularly YF-E/p24/NS1, in mice challenged with a vaccinia expressing HIV-1 Gag was demonstrated. These results suggest that YF17D warrants serious consideration as a live-attenuated vector for HIV-1 vaccine development.     

Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs

The diversity of contemporary swine influenza virus (SIV) strains impedes effective immunization of swine herds. Mucosally delivered, attenuated virus vaccines are one approach with potential to provide broad cross-protection. Reverse genetics-derived H3N2 SIV virus with truncated NS1 (NS1Δ126 TX98) is attenuated and immunogenic when delivered intranasally in young pigs. We analyzed T-cell priming and cross-protective efficacy in weanling piglets after intranasal inoculation with NS1Δ126 TX98 versus wild type TX98. In vivo replication of the truncation mutant was minimal compared to the wild type virus. T-cell responses were greater in magnitude in pigs infected with the wild type virus in in vitro restimulation assays. According to the expression of activation marker CD25, peripheral T cell recall responses in NS1Δ126 TX98 infected pigs were minimal. However, intracellular IFN-γ data indicate that the attenuated virus induced virus-specific CD4⁺CD8^-, CD4⁺CD8⁺, CD4^-CD8⁺, and γδ T cells within 28 days. The IFN-γ response appeared to contract, as responses were reduced at later time points prior to challenge. CD4⁺CD8⁺ cells isolated 5 days after heterosubtypic H1N1 challenge (day 70 overall) showed an elevated CD25 response to virus restimulation. Pigs previously infected with wild type TX98 were protected from replication of the H1N1 challenge virus. Vaccination with NS1Δ126 TX98 was associated with significantly lower levels of Th1-associated cytokines in infected lungs but provided partial cross-protection against the H1N1 challenge. These results demonstrate that NS1Δ SIV vaccines can elicit cell-mediated cross-protection against antigenically divergent strains.     

A bivalent form of nanoparticle-based dengue vaccine stimulated responses that potently eliminate both DENV-2 particles and DENV-2-infected cells

Dengue is the most prevalent mosquito-borne viral disease and continues to be a global public health concern. Although a licensed dengue vaccine is available, its efficacy and safety profile are not satisfactory. Hence, there remains a need for a safe and effective dengue vaccine. We are currently developing a bivalent dengue vaccine candidate. This vaccine candidate is composed of a C-terminus truncated non-structural protein 1 (NS1_1-279) and envelope domain III (EDIII) of DENV-2 encapsidated in the nanocarriers, N, N, N-trimethyl chitosan nanoparticles (TMC NPs). The immunogenicity of this bivalent vaccine candidate was investigated in the present study using BALB/c mice. In this work, we demonstrate that NS1 + EDIII TMC NP-immunized mice strongly elicited antigen-specific antibody responses (anti-NS1 and anti-EDIII IgG) and T-cell responses (NS1- and EDIII-specific-CD4⁺ and CD8⁺ T cells). Importantly, the antibody response induced by NS1 + EDIII TMC NPs provided antiviral activities against DENV-2, including serotype-specific neutralization and antibody-mediated complement-dependent cytotoxicity. Moreover, the significant upregulation of Th1- and Th2-associated cytokines, as well as the increased levels of antigen-specific IgG2a and IgG1, indicated a balanced Th1/Th2 response. Collectively, our findings suggest that NS1 + EDIII TMC NPs induced protective responses that can not only neutralize infectious DENV-2 but also eliminate DENV-2-infected cells.     

Safety and immunogenicity of an adjuvanted protein therapeutic HIV-1 vaccine in subjects with HIV-1 infection: A randomised placebo-controlled study

The human immunodeficiency virus type-1 (HIV-1) vaccine candidate F4/AS01 has previously been shown to induce potent and persistent polyfunctional CD4⁺ T-cell responses in HIV-1-seronegative volunteers. This placebo-controlled study evaluated two doses of F4/AS01 1-month apart in antiretroviral treatment (ART)-experienced and ART-naïve HIV-1-infected subjects (1:1 randomisation in each cohort). Safety, HIV-1-specific CD4⁺ and CD8⁺ T-cell responses, absolute CD4⁺ T-cell counts and HIV-1 viral load were monitored for 12 months post-vaccination. Reactogenicity was clinically acceptable and no vaccine-related serious adverse events were reported. The frequency of HIV-1-specific CD4⁺ T-cells 2 weeks post-dose 2 was significantly higher in the vaccine group than in the placebo group in both cohorts (p < 0.05). Vaccine-induced HIV-1-specific CD4⁺ T-cells exhibited a polyfunctional phenotype, expressing at least CD40L and IL-2. No increase in HIV-1-specific CD8⁺ T-cells or change in CD8⁺ T-cell activation marker expression profile was detected. Absolute CD4⁺ T-cell counts were variable over time in both cohorts. Viral load remained suppressed in ART-experienced subjects. In ART-naïve subjects, a transient reduction in viral load from baseline was observed 2 weeks after the second F4/AS01 dose, which was concurrent with a higher frequency of HIV-1-specific CD4⁺ T-cells expressing at least IL-2 in this cohort. In conclusion, F4/AS01 showed a clinically acceptable reactogenicity and safety profile, and induced polyfunctional HIV-1-specific CD4⁺ T-cell responses in ART-experienced and ART-naïve subjects. These findings support further clinical investigation of F4/AS01 as a potential HIV-1 vaccine for therapeutic use in individuals with HIV-1 infection.     

Evaluation of recombinant adenovirus vectors and adjuvanted protein as a heterologous prime-boost strategy using HER2 as a model antigen

Induction of strong antigen-specific cell-mediated and humoral responses are critical to developing a successful therapeutic vaccine. Herein, using HER2 as a model antigen, we aim to evaluate a therapeutic vaccine protocol that elicits anti-tumor antibody and cytotoxic T cells to HER2/neu antigen. Replication-competent (ΔPS AdV) and non-replicating recombinant adenoviral vectors (AdV) expressing a rat HER2/neu (ErbB2) oncogene, were generated and compared for four different doses and over four time points for their ability to induce antigen-specific T and B cell responses in mice. Although ΔPS AdV:Her2 vector was shown to induce more durable antigen-specific CD8⁺ T cell responses, overall, the AdV:Her2 vector induced broader T and B cell responses. Hence the AdV:Her2 vector was used to evaluate a heterologous prime-boost vaccination regimen using rat HER2 protein encapsulated in archaeosomes composed of a semi-synthetic glycolipid (sulfated S-lactosylarchaeol, SLA; and lactosylarchaeol, LA) (SLA/LA:HER2enc) or admixed with archaeosomes composed of SLA alone (SLA:HER2adm). We first tested AdV:Her2 using a prime-boost approach with SLA/LA:HER2enc, and thereafter evaluated a sub-optimal AdV:Her2 dose in a heterologous prime-boost approach with SLA:HER2adm. A single administration of AdV:Her2 alone induced strong cell-mediated immune responses, whereas SLA/LA:HER2enc alone induced strong antigen-specific IgG titers. In mice primed with a suboptimal dose of AdV:Her2, strong CD8⁺ T-cell responses were observed after a single dose which were not further augmented by protein boost. AdV:Her2 induced CD4⁺ specific T-cell responses were augmented by SLA:HER2adm. Homologous vaccination using SLA:HER2adm induced strong antigen-specific antibody responses. However, the overall magnitude of the responses was similar with three doses of SLA:HER2adm or Ad:HER2 prime followed by two doses of SLA:HER2adm.We demonstrate that AdV:Her2 is capable of inducing strong antigen-specific CD8⁺ T cell responses, even at a low dose, and that these responses can be broadened to include antigen-specific antibody responses by boosting with SLA adjuvanted proteins without compromising CD8 T cell responses elicited by AdV priming.     

DNA/Ad5 vaccination with SIV epitopes induced epitope-specific CD4⁺ T cells, but few subdominant epitope-specific CD8⁺ T cells

The goals of a T cell-based vaccine for HIV are to reduce viral peak and setpoint and prevent transmission. While it has been relatively straightforward to induce CD8⁺ T cell responses against immunodominant T cell epitopes, it has been more difficult to broaden the vaccine-induced CD8⁺ T cell response against subdominant T cell epitopes. Additionally, vaccine regimens to induce CD4⁺ T cell responses have been studied only in limited settings. In this study, we sought to elicit CD8⁺ T cells against subdominant epitopes and CD4⁺ T cells using various novel and well-established vaccine strategies. We vaccinated three Mamu-A*01⁺ animals with five Mamu-A*01-restricted subdominant SIV-specific CD8⁺ T cell epitopes. All three vaccinated animals made high frequency responses against the Mamu-A*01-restricted Env TL9 epitope with one animal making a low frequency CD8⁺ T cell response against the Pol LV10 epitope. We also induced SIV-specific CD4⁺ T cells against several MHC class II DRBw*606-restricted epitopes. Electroporated DNA with pIL-12 followed by a rAd5 boost was the most immunogenic vaccine strategy. We induced responses against all three Mamu-DRB*w606-restricted CD4 epitopes in the vaccine after the DNA prime. Ad5 vaccination further boosted these responses. Although we successfully elicited several robust epitope-specific CD4⁺ T cell responses, vaccination with subdominant MHC class I epitopes elicited few detectable CD8⁺ T cell responses. Broadening the CD8⁺ T cell response against subdominant MHC class I epitopes was, therefore, more difficult than we initially anticipated.     

Active hexose correlated compound potentiates the antitumor effects of low-dose 5-fluorouracil through modulation of immune function in hepatoma 22 tumor-bearing mice

BACKGROUND/OBJECTIVES

A variety of immunomodulators can improve the efficacy of low-dose chemotherapeutics. Active hexose correlated compound (AHCC), a mushroom mycelia extract, has been shown to be a strong immunomodulator. Whether AHCC could enhance the antitumor effect of low-dose 5-fluorouracil (5-FU) via regulation of host immunity is unknown.

MATERIALS/METHODS

In the current study Hepatoma 22 (H22) tumor-bearing mice were treated with PBS, 5-FU (10 mg·kg^-1·d^-1, i.p), or AHCC (360 mg·kg^-1·d^-1, i.g) plus 5-FU, respectively, for 5 d. CD3⁺, CD4⁺, CD8⁺, and NK in peripheral blood were detected by flow cytometry. ALT, AST, BUN, and Cr levels were measured by biochemical assay. IL-2 and TNFα in serum were measured using the RIA kit and apoptosis of tumor was detected by TUNEL staining. Bax, Bcl-2, and TS protein levels were measured by immunohistochemical staining and mRNA level was evaluated by RT-PCR.

RESULTS

Diet consumption and body weight showed that AHCC had no apparent toxicity. AHCC could reverse liver injury and myelosuppression induced by 5-FU (P < 0.05). Compared to mice treated with 5-FU, mice treated with AHCC plus 5-FU had higher thymus index, percentages of CD3⁺, CD4⁺, and NK cells (P < 0.01), and ratio of CD4⁺/CD8⁺ (P < 0.01) in peripheral blood. Radioimmunoassay showed that mice treated with AHCC plus 5-FU had the highest serum levels of IL-2 and TNFα compared with the vehicle group and 5-FU group. More importantly, the combination of AHCC and 5-FU produced a more potent antitumor effect (P < 0.05) and caused more severe apoptosis in tumor tissue (P < 0.05) compared with the 5-FU group. In addition, the combination of AHCC and 5-FU further up-regulated the expression of Bcl-2 associated X protein (Bax) (P < 0.01), while it down-regulated the expression of B cell lymphoma 2 (Bcl-2) (P < 0.01).

CONCLUSIONS

These results support the claim that AHCC might be beneficial for cancer patients receiving chemotherapy.     

Heterosubtypic immunity to influenza mediated by liposome adjuvanted H5N1 recombinant protein vaccines

A non-egg, non-culture based influenza vaccine that intervenes large influenza outbreaks and protects against heterosubtypic infections is needed. Candidates of such vaccine are likely to be conserved influenza virus proteins or their coding DNA. The vaccine must be conveniently produced at reasonable cost, safe, highly immunogenic and should be able to recall rapidly the immunological memory upon the antigenic re-exposure. In this study vaccines made of full length recombinant NP and M2 of the H5N1 influenza A virus were entrapped either alone or together into liposome (L) made of phosphatidylcholine and cholesterol. The vaccines (L-NP, L-M2 or L-NP + M2) and mocks (L or PBS) were safe without causing any adverse reaction in the intramuscularly injected mice. They were readily immunogenic at a single dose and a recalled response could be detected within one day post booster. Cytokine and antibody data indicated that the vaccines induced a Th1 bias immune response. NP containing vaccines stimulated a marked increase of cytotoxic lymphocytes, i.e., CD8⁺, intracellular IFNγ⁺ cells, while M2 containing vaccines elicited good antibody response which neutralized infectivity of heterologous influenza viruses. Although the three vaccines elicited different immunological defense factors; nevertheless, they similarly and readily abrogated lung histopathology mediated by viruses belonging to different H5N1 clade/subclade and heterosubtypes including swine H1N1 and human H1N1/2009 viruses. They protected the vaccinated mice against lethal challenges with mouse adapted avian H5N1 virus. The liposome adjuvanted vaccines which demonstrated high protective efficacy in mice warrant testing further in a non-rodent model as well as in humans.     

Enhanced immune response by amphotericin B following NS1 protein prime-oral recombinant Salmonella vaccine boost vaccination protects mice from dengue virus challenge

A recombinant vaccine strain SL3261/pLT105 of attenuated aroA Salmonella enterica serovar Typhimurium SL3261 strain expressing a secreted dengue virus type 2 non-structural NS1 and Yersinia pestis F1 (Caf1) fusion protein, rNS1:Caf1, was generated. Immunological evaluation was performed by prime-boost vaccine regimen. Oral immunization of mice with 1 x 10(9)cfu of SL3261/pLT105 only induced low levels of NS1-specific antibody response and protective immunity following dengue virus challenge. The parenteral NS1 protein priming-oral Salmonella boosting protocol enhanced both NS1-specific serum IgG response and protective efficacy as compared to mice immunized with each type vaccine alone. Addition of an antifungal antibiotic amphotericin B (AmB) to Salmonella vaccine further enhanced the synergic effects of prime-boost vaccine regimen on the elicited NS1-specific serum IgG response and the protective efficacy. Together, the results demonstrated that the rNS1:Caf1 producing Salmonella SL3261/pLT105 strain fails to provide effective protection as an oral vaccine alone despite co-administration of AmB as an adjuvant capable of enhancing the immune responses, and moreover, the protein priming-oral Salmonella vaccine boosting approach in combination with AmB as an immunization regimen may have the potential to be further explored as an alternative approach for dengue vaccine development.     

CCL5-independent helper T lymphocyte responses to immuno-dominant pneumococcal surface protein A epitopes

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several antigens, in addition to the polysaccharide capsule, have been implicated in both the virulence and protective immunity against Streptococcus pneumoniae; one of the best-studied S. pneumoniae antigens is pneumococcal surface protein A (PspA). Recently, it was shown that genetic polymorphisms could diminish CCL5 expression, which results in increased susceptibility to and progression of infectious diseases. We previously showed CCL5 blockade reduced PspA-specific humoral and cellular pneumococcal immunity, during S. pneumoniae strain EF3030-induced carriage, by diminishing IFN-γ and enhancing IL-10 secretion by effector T cells. We also identified immuno-dominant helper T lymphocyte (HTL) epitopes in PspA peptide 19-23 (PspA_199-246), which caused comparatively more cytokine secretion and proliferation responses by splenic and cervical lymph node (CLN) CD4⁺ T cells from mice previously challenged with S. pneumoniae strain EF3030. In this study, we sought to determine if PspA_199-246-specific CD4⁺ T cells responses were resistant to the effect of CCL5 deficiency. In short, T cell responses against these HTL epitopes were resistant to CCL5 inhibition, than compared to cells from control or naïve mice, and unaffected by reduced co-stimulatory molecule expression caused by CCL5 blockade. CCL5 deficiency also corresponded with a higher number of IL-10⁺ CD11b⁺ CD11c^Lo and CD11b⁺ CD11c^Hi cells and lower IFN-γ expression by similar cells, than compared to controls. These data confirm CCL5 is an essential factor for optimal pneumococcal adaptive immunity and show CD4⁺ T cell responses to PspA_199-246 are largely resistant to CCL5 deficiency.     

An adenoviral vector expressing lipoprotein A,a major antigen of Mycoplasma mycoides subspecies mycoides,elicits robust immune responses in mice

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC), is a devastating respiratory disease of cattle. In sub-Saharan Africa, where CBPP is enzootic, live attenuated vaccines are deployed but afford only short-lived protection. In cattle, recovery from experimental MmmSC infection has been associated with the presence of CD4⁺ T lymphocytes that secrete interferon gamma in response to MmmSC, and in particular to the lipoprotein A (LppA) antigen. In an effort to develop a better vaccine against CBPP, a viral vector (Ad5-LppA) that expressed LppA was generated from human adenovirus type 5. The LppA-specific immune responses elicited by the Ad5-LppA vector were evaluated in mice, and compared to those elicited by recombinant LppA formulated with a potent adjuvant. Notably, a single administration of Ad5-LppA, but not recombinant protein, sufficed to elicit a robust LppA-specific humoral response. After a booster administration, both vector and recombinant protein elicited strong LppA-specific humoral and cell-mediated responses. Ex vivo stimulation of splenocytes induced extensive proliferation of CD4⁺ T cells for mice immunized with vector or protein, and secretion of T helper 1-associated and proinflammatory cytokines for mice immunized with Ad5-LppA. Our study – by demonstrating the potential of a viral-vectored prototypic vaccine to elicit prompt and robust immune responses against a major antigen of MmmSC – represents a first step in developing a recombinant vaccine against CBPP.     

Enhancement of in vivo and in vitro immune functions by a conformationally biased,response-selective agonist of human C5a: Implications for a novel adjuvant in vaccine design

A conformationally biased, agonist of human C5a_65–74 (EP67) was assessed for its adjuvant activities in vitro and in vivo. EP67 induced the release of the inflammatory (Th1) type cytokines from C5a receptor (CD88)-bearing antigen presenting cells (APC). EP67 did not induce the release of these cytokines from splenic APCs obtained from C5a receptor knockouts (CD88^−/−). Serum from mice immunized with EP67–ovalbumin (OVA) contained high OVA-specific antibody (Ab) titers [IgG1, IgG2a (IGg2c), IgG2b]. Mice receiving OVA alone produced only IgG1 Abs, indicating the ability of EP67 to induce a Th1-like Ab class switch. Spleen cell cultures from wild type mice but not CD88^−/− mice showed an enhanced OVA-specific proliferative response in vitro. These results indicate the ability of EP67 to drive a Th1-mediated immune response and its potential use as a unique adjuvant.     

CD4 T-cell responses among adults and young children in response to Streptococcus pneumoniae and Haemophilus influenzae vaccine candidate protein antigens

We characterized cytokine profiles of CD4⁺ T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4⁺ T cells producing IL-2, (p = 0.004). Vaccine antigen-specific CD4⁺ T-cell populations in adults were largely of effector (T_EM) and/or central memory (T_CM) phenotypes as defined by CD45RA⁻CCR7⁺ or CD45RA⁻CCR7⁻ respectively; however among young children antigen-specific IL-2 producing CD4⁺ T cells demonstrated CD45RA⁺ expression (non-memory cells). We conclude that adults have circulating memory CD4⁺ T cells (CD45RA⁻) that can be stimulated by all the tested Spn and NTHi protein vaccine candidate antigens, whereas young children have a more limited response.     

Complete protection against a H5N2 avian influenza virus by a DNA vaccine expressing a fusion protein of H1N1 HA and M2e

Most influenza vaccines target hemagglutinin (HA) in order to protect the host against infection. However, theses vaccines are strain-specific due to major antigenic variations of HA. Since it is difficult to predict epidemic and pandemic strains of influenza virus, the development of effective vaccines against divergent influenza viruses is urgently needed. Although M2e-based vaccines are associated with weaker protection than HA-based vaccines that induce neutralizing antibodies against challenge virus matched-strain, the extracellular domain of Matrix 2 protein (M2e) is one of a potential broad-spectrum immunogen because it contains highly conserved sequences among influenza A viruses. In this study, M2e sequence was fused to H1N1 HA DNA (M2e-HA) and the immunogenicity and antiviral efficacy of this DNA vaccine was evaluated in response to challenge with a heterosubtypic H5N2 avian influenza virus. Compared to vaccination with HA or M2e DNA alone, vaccination with M2e-HA DNA or combination of M2e DNA and HA DNA (M2e DNA + HA DNA) induced a broad immunity without evidence of immune interference. In addition, HA-specific CD8⁺ and M2e-specific T cell responses elicited by M2e-HA DNA vaccination were significantly higher than those of HA or M2e DNA vaccine alone, respectively. Following challenge with a heterosubtypic influenza virus infection, vaccination with M2e-HA DNA conferred complete protection against mortality. In combination, these results suggest that DNA vaccines expressing a fusion protein, M2e-HA, may provide an attractive approach for the development of broad-spectrum influenza vaccines.     

Reciprocal changes in CD11c+CD11b+ and CD11c+CD8α+ dendritic cell subsets determine protective or permissive immune response in murine experimental VL

CD11c⁺CD8α⁺ and CD11c⁺CD11b⁺ dendritic cells are two major subsets of murine splenic CD11c⁺ DCs which play a crucial role in T cell priming and shaping Th1/Th2 responses, but their role in the context of experimental visceral leishmaniasis (VL) is poorly understood. Herein, we showed that L. donovani infection in Balb/c mice preferentially decreased the population abundance of CD11c⁺CD11b⁺ DCs and increased relative abundance of splenic CD11c⁺CD8α ⁺DCs. During infection, splenic CD11c⁺CD11b⁺ DCs induced Th1 differentiation whereas CD11c⁺CD8α⁺ DCs promoted Th2 differentiation. Additionally, treatment of infected mice with miltefosine as experimental control exhibited host defense allowing the restoration of CD11c⁺CD11b⁺ population and decrease in CD11c⁺CD8α⁺ subset. Furthermore, reciprocal regulation of immune accessory surface molecules, Sema4A and OX40L critically determined Th1/Th2 response induced by these DC subsets during VL. L. donovani infection significantly induced OX40L expression and slightly downregulated SEMA 4A expression in CD11c⁺CD8α⁺ DCs whereas miltefosine treatment significantly downregulated OX40L expression along with pronounced upregulation of SEMA 4A expression in CD11c⁺CD11b⁺ DCs. SiRNA mediated knockdown of SEMA 4A markedly reduced CD11c⁺CD11b⁺ driven IFN-γ, TNF-α and IL-12 synthesis in miltefosine treated mice whereas functional blocking of OX40L decreased CD11c⁺CD8α⁺ induced IL-10, IL-4 and TGF-β synthesis in L. donovani infected group. Vaccination of Balb/c mice with antigen-pulsed + CpG-ODN-activated DC subsets revealed that only antigen-pulsed CD11c⁺CD11b⁺ DCs eliminated parasite load in visceral organ and restored protective Th1 cytokine response. Collectively, our results suggest that differential regulation of splenic CD11c⁺ subsets by L. donovani is essential for disease progression and specific subtypes may be exploited as prophylactic measures against visceral leishmaniasis.     

Human antibody and memory B and T-cell responses after primary and booster immunisation against Neisseria meningitidis B

Vaccination against disease aims at the induction of long-lasting cellular and humoral immune responses. Few studies have addressed the mechanisms by which meningococcal vaccines generate and sustain immunological memory. The goal of this study was to investigate the development of long-term humoral and cellular memory to Neisseria meningitidis serogroup B (MenB) in health subjects after immunisation with the Cuban outer membrane protein (OMP) vaccine (VA-MENGOC-BC^®). The results showed that three doses of vaccine were necessary to induce a detectable memory B-cell response (mean of 0.46%) which became undetectable 6 months later. After boosting, only 2 of 5 individuals responded with an increase in memory B-cell frequencies (values of 0.15% and 0.34%). Bactericidal and opsonic antibody levels were higher after primary immunisation (log₂ mean and median of 4.7 and 1212, respectively) when compared with post-booster response (log₂ mean of 2.6 and median of 285, respectively). Together, these data suggest a failure of vaccine to induce long-term memory B-cell and serological memory in adults. However, we observed a significant and functional memory T-cell response specially after boosting, with a predominance of activated (CD69⁺) central memory T-cell (CD4⁺CD45⁻CCR7⁺) response. Therefore, this study suggests that vaccination with the MenB vaccine induced the generation and activation of memory T-cells but failed to maintain the memory B-cell population at a stable size and/or function.     

Heterologous human/rat HER2-specific exosome-targeted T cell vaccine stimulates potent humoral and CTL responses leading to enhanced circumvention of HER2 tolerance in double transgenic HLA-A2/HER2 mice

DNA vaccines composed of heterologous human HER2 and rat neu sequences induce stronger antibody response and protective antitumor immunity than either HER2 or neu DNA vaccines in transgenic mice. We previously developed HER2-specific exosome-targeted T-cell vaccine HER2-T_EXO capable of stimulating HER2-specific CD8⁺ T-cell responses, but only leading to partial protective immunity in double-transgenic HLA-A2/HER2 mice with self-immune tolerance to HER2. Here, we constructed an adenoviral vector AdV_HuRt expressing HuRt fusion protein composed of NH₂-HER2_1-407 (Hu) and COOH-neu_408-690 (Rt) fragments, and developed a heterologous human/rat HER2-specific exosome-targeted T-cell vaccine HuRt-T_EXO using polyclonal CD4⁺ T-cells uptaking exosomes released by AdV_HuRt-transfected dendritic cells. We found that the HuRt-T_EXO vaccine stimulates enhanced CD4⁺ T-cell responses leading to increased induction of HER2-specific antibody (∼70 µg/ml) compared to that (∼40 µg/ml) triggered by the homologous HER2-T_EXO vaccine. By using PE-H-2K^d/HER2_23-71 tetramer, we determined that HuRt-T_EXO stimulates stronger HER2-specific CD8⁺ T-cell responses eradicating 90% of HER2-specific target cells, while HER2-T_EXO-induced CD8⁺ T-cell responses only eliminating 53% targets. Furthermore, HuRt-T_EXO, but not HER2-T_EXO vaccination, is capable of suppressing early stage-established HER2-expressing 4T1_HER2 breast cancer in its lung metastasis or subcutaneous form in BALB/c mice, and of completely protecting transgenic HLA-A2/HER2 mice from growth of HLA-A2/HER2-expressing BL6-10_A2/HER2 melanoma. HuRt-T_EXO-stimulated HER2-specific CD8⁺ T-cells not only are cytolytic to trastuzumab-resistant HLA-A2/HER2-expressing BT474/A2 breast tumor cells in vitro but also eradicates pre-established BT474/A2 tumors in athymic nude mice. Therefore, our novel heterologous human/rat HER2-specific T-cell vaccine HuRt-T_EXO, circumventing HER2 tolerance, may provide a new therapeutic alternative for patients with trastuzumab-resistant HER2⁺ breast tumor.     

Pneumococcal polysaccharide vaccination induces polysaccharide-specific B cells in adult peripheral blood expressing CD19CD20CD3CD70CD27IgMCD43CD5

Pneumococcal polysaccharide vaccines have been used to elicit a protective anti-pneumococcal polysaccharide antibody response against Streptococcus pneumoniae in healthy individuals. Identifying human B cells which respond to T-cell independent type-2 antigens, such as pneumococcal polysaccharides, has been challenging. We employed pneumococcal polysaccharides directly conjugated to fluorophores in conjunction with flow cytometry to identify the phenotype of B cells that respond to pneumococcal polysaccharide vaccination. We have previously identified that the majority of pneumococcal polysaccharide-selected cells responding to vaccination are CD27⁺IgM⁺ (IgM⁺ memory) cells. In this study, we further characterized pneumococcal polysaccharide-selected cells in the peripheral blood to better identify how the various B cell phenotypes responded 7 and 30 days post-immunization. We show that 7 days post-immunization the majority of pneumococcal polysaccharide-selected IgM⁺ memory cells (PPS14⁺ 56.5%, PPS23F⁺ 63.8%) were CD19⁺CD20⁺CD27⁺IgM⁺CD43⁺CD5^+/−CD70⁻, which was significantly increased compared to pre-immunization levels. This phenotype is in alignment with recent publications describing human B-1 cells. PPS-responsive B cells receded to pre-immunization levels by day-30. These findings suggest that this B-1 like cell population plays an important role in early responses to S. pneumoniae infection and possibly other T-cell independent type-2 antigens in humans     

A tumor-specific neoepitope expressed in homologous/self or heterologous/viral antigens induced comparable effector CD8+ T-cell responses by DNA vaccination

Somatic mutations in tumors often generate neoproteins that contain MHC-I-binding neoepitopes. Little is known if and how efficient tumor-specific neoantigens activate CD8⁺ T cells. Here, we asked whether a de novo generated neoepitope, encoded either within an otherwise conserved and ubiquitously expressed self-antigen or in a chimeric HBV core antigen expression platform, providing heterologous helper functions, induces CD8⁺ T cells in C57Bl/6J mice by DNA immunization. For it, we chose an established D^b/Sp_{244-252/R251H} neoepitope generated in the murine Endophilin-B2/SH3GLB2 (EndoB2-Sp) protein by a single amino acid exchange. We showed that a single injection of EndoB2-Sp expression vectors efficiently primed dimer/pentamer⁺, IFN-γ⁺ and cytolytic D^b/Sp_{244-252/R251H}-specific effector CD8⁺ T cells in C57Bl/6J mice. Priming of D^b/Sp_{244-252/R251H}-specific CD8⁺ T cells proceeded independent from CD4⁺ T-cell help in MHC-II-deficient Aα^-/- mice. As compared to the homologous EndoB2-Sp vaccine, the selective expression of the D^b/Sp_{244-252/R251H} neoepitope in chimeric particle-forming and assembly-deficient HBV core antigens induced comparable frequencies D^b/Sp_{244-252/R251H}-specific CD8⁺ T cells with the same cytolytic effector phenotype. The homologous EndoB2 carrier, but not the nine-residue neoepitope presented on chimeric HBV core particles, induced EndoB2-specific IgG antibody responses. The HBV core expression platform is thus an attractive option to selectively induce neoepitope-specific effector CD8⁺ T cells by DNA vaccination. These novel findings have practical implications for the design of heterologous/self and heterologous/viral cancer vaccines that prime and/or activate neoepitope-specific CD8⁺ T cells.