Journal of Immune Based Therapies and Vaccines - Latest Articles

Evaluation of recombinant invasive, non-pathogenic Eschericia coli as a vaccine vector against the intracellular pathogen, Brucella

Jerome Harms — 2009-01-06T00:00:00Z

Background: There is no safe, effective human vaccine against brucellosis. Live attenuated Brucella strains are widely used to vaccinate animals. However these live Brucella vaccines can cause disease and are unsafe for humans. Killed Brucella or subunit vaccines are not effective in eliciting long term protection. In this study, we evaluate an approach using a live, non-pathogenic bacteria (E. coli) genetically engineered to mimic the brucellae pathway of infection and present antigens for an appropriate cytolitic T cell response. Methods: E. coli was modified to express invasin of Yersinia and listerialysin O (LLO) of Listeria to impart the necessary infectivity and antigen releasing traits of the intracellular pathogen, Brucella. This modified E. coli was considered our vaccine delivery system and was engineered to express Green Fluorescent Protein (GFP) or Brucella antigens for in vitro and in vivo immunological studies including cytokine profiling and cytotoxicity assays. Results: The E. coli vaccine vector was able to infect all cells tested and efficiently deliver therapeutics to the host cell. Using GFP as antigen, we demonstrate that the E. coli vaccine vector elicits a Th1 cytokine profile in both primary and secondary immune responses. Additionally, using this vector to deliver a Brucella antigen, we demonstrate the ability of the E. coli vaccine vector to induce specific Cytotoxic T Lymphocytes (CTLs). Conclusion: Protection against most intracellular bacterial pathogens can be obtained mostly through cell mediated immunity. Data presented here suggest modified E. coli can be used as a vaccine vector for delivery of antigens and therapeutics mimicking the infection of the pathogen and inducing cell mediated immunity to that pathogen.

Vaccine based on a ubiquitous cysteinyl protease and streptococcal pyrogenic exotoxin A protects against Streptococcus pyogenes sepsis and toxic shock

Robert Ulrich — 2008-10-31T00:00:00Z

Background: The gram-positive bacterium Streptococcus pyogenes is a common pathogen of humans that causes invasive infections, toxic-shock syndrome, rheumatic fever, necrotizing fasciitis and other diseases. Detection of antibiotic resistance in clinical isolates has renewed interest in development of new vaccine approaches for control S. pyogenes sepsis. In the study presented, a novel protein vaccine was examined. The vaccine was based on a recombinant protein fusion between streptococcal pyrogenic exotoxin B (SpeB), a cysteinyl protease expressed by all clinical isolates, and streptococcal pyrogenic exotoxin A (SpeA), a superantigen produced by a large subset of isolates. Results: A novel protein was produced by mutating the catalytic site of SpeB and the receptor binding surface of SpeA in a fusion of the two polypeptides. Vaccination of HLA-DQ8 transgenic mice with the SpeA-SpeB fusion protein protected against a challenge with the wild-type SpeA that was lethal to naïve controls, and vaccinated mice were protected from an otherwise lethal S. pyogenes infection. Conclusion: These results suggest that the genetically attenuated SpeA-SpeB fusion protein may be useful for controlling S. pyogenes infections. Vaccination with the SpeA-SpeB fusion protein described in this study may potentially result in protective immunity against multiple isolates of S. pyogenes due to the extensive antibody cross-reactivity previously observed among all sequence variants of SpeB and the high frequency of SpeA-producing strains.

Effects of recombinant human growth hormone on HIV-1-specific T-cell responses, thymic output and proviral DNA in patients on HAART: 48-week follow-up

Anna Herasimtschuk — 2008-10-31T00:00:00Z

Background: Efficacious immune-based therapy in treated chronic HIV-1 infection requires the induction of virus-specific CD4+ T cells and subsequent maturation and maintenance of specific memory CD8+ T cells. Concomitant daily administration of recombinant human growth hormone (rhGH) with highly active antiretroviral therapy (HAART) was used in chronically infected patients with lipodystrophy in an attempt to reconstitute these virus-specific T-cell responses. Methods: Individuals with chronic HIV-1 infection on HAART were enrolled on a randomized, double-blinded, placebo-controlled study to receive rhGH therapy. We assessed HIV-1-specific proliferative CD4+ and interferon-gamma (IFN-γ)-producing CD8+ T-cell responses, quantified thymic output and proviral HIV-1 DNA at the following time points: baseline; after 12 weeks of rhGH therapy; at 24 weeks, after randomization into three groups [placebo weeks 12–24 (Group A), alternate-day dosing weeks 12–24 (Group B), and twice-per-week dosing weeks 12–24 (Group C)]; and at 48 weeks after all patients had received HAART alone for the final 24 weeks. Results: We found significant increases in both proliferative CD4+ and IFN-γ-producing CD8+ HIV-1-specific T-cell responses after daily administration of rhGH. This increase was focused on HIV-1 Gag-specific T-cell responses. Following subsequent randomisation into different dosing regimens, HIV-1-specific proliferative CD4+ T-cell responses declined in patients receiving less frequent dosing of rhGH, while virus-specific IFN-γ-producing CD8+ T-cell responses were maintained for longer periods of time. There was no significant change in thymic output and the cell-associated HIV-1 DNA remained stable in most patients. An increased anti-HIV-1 Nef-specific CD4+ T-cell proliferative response was correlated to a decrease in proviral load, and an increased HIV-1 Gag-specific IFN-γ-producing CD8+ T-cell response correlated with an increase in proviral load. Conclusion: The implication of these data is that daily dosing of rhGH with HAART, in addition to improving HIV-1-associated lipodystrophy, may reverse some of the T-lymphocyte dysfunction seen in most treated HIV-1-positive patients, in a dose-dependent manner. Such immune-based therapeutic strategies used in treated, chronic HIV-1 infection may enable the induction of virus-specific CD4+ T cells essential for the subsequent 'kick-start' and expansion of virus-specific CD8+ T cells.Trial registrationGH in Lipoatrophy IMP22350.

CXCL10 blockade protects mice from cyclophosphamide-induced cystitis

Senthilkumar Sakthivel — 2008-10-28T00:00:00Z

Background: Alterations in serum CXCR3 ligand levels were examined in interstitial cystitis (IC) patients; similar expression patterns in serum as well as CXCR3, CXCR3 ligands, and cytokines expressed by peripheral and local leukocyte subpopulations were characterized during cyclophosphamide (CYP)-induced acute cystitis in mice. Results: Serum levels of monokine-induced by interferon-γ (IFN-γ) (MIG/CXCL9), IFN-γ-inducible protein-10 (IP-10/CXCL10), and IFN-γ-inducible T cell α chemoattractant (I-TAC/CXCL11) were elevated in patients with IC. These clinical features closely correlated with CYP-induced cystitis in mice. Serum levels of these CXCR3 ligands and local T helper type 1 (Th1) cytokines were also increased. We demonstrate that CXCR3 as well as CXCL9, CXCL10 and CXCL11 mRNA were significantly expressed by urinary bladder lymphocytes, while CXCR3 and CXCL9 transcripts were significantly expressed by iliac lymph node leukocytes following CYP treatment. We also show that the number of CD4+ T cells, mast cells, natural killer (NK) cells, and NKT cells were increased at systemic (spleen) and mucosal (urinary bladder and iliac lymph nodes) sites, following CYP-induced cystitis in mice. Importantly, CXCL10 blockade attenuated these increases caused by CYP. Conclusion: Antibody (Ab)-mediated inhibition of the most abundant serum CXCR3 ligand, CXCL10, in mice decreased the local production of CXCR3 ligands as well as Th1 cytokines expressed by local leukocytes, and lowered corresponding serum levels to reduce the severity of CYP-induced cystitis. The present study is among the first to demonstrate some of the cellular and molecular mechanisms of chemokines in cystitis and may represent new drug target for this disease.

An alternative approach to combination vaccines: intradermal administration of isolated components for control of anthrax, botulism, plague and staphylococcal toxic shock

Garry Morefield — 2008-09-03T00:00:00Z

Background: Combination vaccines reduce the total number of injections required for each component administered separately and generally provide the same level of disease protection. Yet, physical, chemical, and biological interactions between vaccine components are often detrimental to vaccine safety or efficacy. Methods: As a possible alternative to combination vaccines, we used specially designed microneedles to inject rhesus macaques with four separate recombinant protein vaccines for anthrax, botulism, plague and staphylococcal toxic shock next to each other just below the surface of the skin, thus avoiding potentially incompatible vaccine mixtures. Results: The intradermally-administered vaccines retained potent antibody responses and were well- tolerated by rhesus macaques. Based on tracking of the adjuvant, the vaccines were transported from the dermis to draining lymph nodes by antigen-presenting cells. Vaccinated primates were completely protected from an otherwise lethal aerosol challenge by Bacillus anthracis spores, botulinum neurotoxin A, or staphylococcal enterotoxin B. Conclusion: Our results demonstrated that the physical separation of vaccines both in the syringe and at the site of administration did not adversely affect the biological activity of each component.The vaccination method we describe may be scalable to include a greater number of antigens, while avoiding the physical and chemical incompatibilities encountered by combining multiple vaccines together in one product.

CpG increases vaccine antigen-specific cell-mediated immunity when administered with hepatitis B vaccine in HIV infection.

Jonathan Angel — 2008-08-12T00:00:00Z

Background: Lack of adequate adjuvancy is a possible explanation for lack of vaccine immunogenecity. Immunostimulatory CpGs are potent vaccine adjuvants and may be an important component of the development vaccines, particularly those for which a cellular immune response is required for protection. We have previously demonstrated that CpG ODN co-administration with hepatitis B vaccine results in earlier, stronger and more sustained antibody responses to hepatitis B surface antigen in HIV infected individuals, and wished to determine if, in this population, helper T-cell responses were also enhanced. Methods: We conducted a double-blind, placebo-controlled trial in hepatitis B susceptible, effectively treated HIV-seropositive individuals. Participants received hepatitis B vaccine, with either placebo or CPG 7909 1.0 mg at week 0, 4 and 8. To determine the impact of CpG on cellular immune responses, lymphoproliferative responses (LPR) were evaluated by [3H]-thymidine incorporation at baseline and weeks 4, 8, 12, 24, and 48. Immunophenotyping of lymphocyte subsets was also determined at these time points. Results: Of 36 patients enrolled, 18 received hepatitis B vaccine alone, and 18 received hepatitis B vaccine with CpG. Inclusion of CPG 7909 was associated with a greater proliferative response to HBsAg at all time points following initial vaccination. This increase was statistically significant at 8 weeks (p = 0.042) and 48 weeks (p = 0.024). Similar results were observed when LPR were evaluated as stimulation indices (SI). No differences in proliferative responses to HIV p24 Ag were observed, nor were there any differences in lymphocyte subsets. Conclusion: In addition to enhancing humoral responses to vaccination, we describe for the first time that CPG 7909 enhances cellular immunity to vaccine antigen in a typically hyporesponsive population. This adjuvancy may be important in the development of an effective vaccine for which a cellular immune response is required for protection.

Immunostimulatory effects of three classes of CpG oligodeoxynucleotides on PBMC from HCV chronic carriers

Curtis Cooper — 2008-06-09T00:00:00Z

Background: Chronic hepatitis C virus (HCV) infection results from weak or absent T cell responses. Pegylated-interferon-alpha (IFN-α) and ribavirin, the standard of care for chronic HCV, have numerous immune effects but are not potent T cell activators. A potent immune activator such as TLR9 agonist CpG oligodeoxynucleotide (CpG) may complement current treatment approaches. Methods: Peripheral blood mononuclear cells (PBMC) obtained from HCV chronic carriers who failed previous treatment and from healthy donors were incubated in vitro with the three main CpG classes (A, B or C), recombinant IFN-α-2b (IntronA) and/or ribavirin. Proliferation and cytokine secretion (IFN-α, IL-10 and IP-10) were evaluated. Results: CpG induced proliferation and cytokine secretion in patterns expected for each CpG class with similar group means for HCV and healthy donors. IntronA and ribavirin, alone or together, had no detectable effects. IntronA and C-Class CpG together induced more IFN-α than CpG alone in most subjects. IFN-α secretion was proportional to the number of plasmacytoid dendritic cells in PBMC from healthy donors but not HCV donors in whom responses were highly heterogeneous. Conclusion: The strong immune stimulatory effect of CpG on PBMC isolated from treatment-failed HCV patients suggests possible utility alone or in combination with current HCV antiviral treatment.

Use of ultraviolet light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes

Charles Gullo — 2008-04-28T00:00:00Z

Background: As the eradication of tumor cells in vivo is most efficiently performed by cytolytic T lymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described. Methods: Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity. Results: By day 10 of cultures, PBMCs primed using UV-irradiated tumor cells demonstrated a higher percentage of activated CD8+/CD4- T lymphocytes than non-primed PBMCs or PBMCs primed using mitomycin-treated MM cells. Cytotoxicity assays revealed that primed PBMCs were markedly more effective (p < 0.01) than non-primed PBMCs in killing RPMI 8226 MM cells. Surface expression of glucose regulated protein 94 (Grp94/Gp96) and Grp78 were both found to be induced in UV-treated MM cells. Conclusion: Since, HSP-associated peptides are known to mediate tumor rejection; these data suggest that immune-mediated eradication of MM cells could be elicited via a UV-induced HSP process. The finding that the addition of 17-allylamide-17-demethoxygeldanamycin (17AAG, an inhibitor of HSP 90-peptide interactions) resulted in decreased CTL-induced cytotoxicity supported this hypothesis. Our study, therefore, provides the framework for the development of anti-tumor CTL cellular vaccines for treating MM using UV-irradiated tumor cells as immunogens.

A new approach for the large-scale generation of mature dendritic cells from adherent PBMC using roller bottle technology

Ryan Campbell-Anson — 2008-03-06T00:00:00Z

Background: Human monocyte-derived DC (mDC) loaded with peptides, protein, tumor cell lysates, or tumor cell RNA, are being tested as vaccines against multiple human malignancies and viral infection with great promise. One of the factors that has limited more widespread use of these vaccines is the need to generate mDC in large scale. Current methods for the large-scale cultivation of mDC in static culture vessels are labor- and time- intensive, and also require many culture vessels. Here, we describe a new method for the large-scale generation of human mDC from human PBMC from leukopheresis or buffy coat products using roller bottles, never attempted before for mDC generation. We have tested this technology using 850 cm2 roller bottles compared to conventional T-175 flat-bottom static culture flasks. Methods: DC were generated from adherent human PBMC from buffy coats or leukopherisis products using GM-CSF and IL-4 in T-175 static flasks or 850 cm2 roller bottles. The cells were matured over two days, harvested and analyzed for cell yield and mature DC phenotype by flow cytometry, and then functionally analyzed for their ability to activate allogeneic T-cell or recall antigen peptide-specific T-cell responses. Results: Monocytes were found to adhere inside roller bottles to the same extent as in static culture flasks. The phenotype and function of the mDC harvested after maturation from both type of culture systems were similar. The yield of mDC from input PBMC in the roller bottle system was similar as in the static flask system. However, each 850 cm2 roller bottle could be seeded with 4–5 times more input PBMC and could yield 4–5 times as many mDC per culture vessel than the static flasks as a result. Conclusion: Our results indicate that the roller bottle technology can generate similar numbers of mDC from adherent PBMC as traditional static flask methods, but with having to use fewer culture vessels. Thus, this may be a more practical method to generate mDC in large-scale cutting down on the amount of laboratory manipulations, and can save both time and labor costs.

An HIV/AIDS Prophylactic vaccine is possible

Qiu Zhong — 2007-12-19T00:00:00Z

One needs to think outside of the box, as one of us (Ronald B Luftig) learned from many years as a mathematician, and a biophysicist.In this short Review, the need to focus on producing high levels of neutralizing antibodies (NAbs) to incoming and conformationally altered virus after it has bound to CD4+ cells is essential.Increasing the number of gp120 molecules on the surface of L-2 particles, could allow for an enhanced number of NAbs.The attempt at increasing CD8+ T cell responses in recent vaccine trials has not worked perhaps because it may have allowed HIV to enter into remote sanctuaries. Our approach focuses on increasing NAbs, before high levels of CD8+ T cells are produced.