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1.  Benefit risk assessment and update on the use of docetaxel in the management of breast cancer 
The objective of this paper is to review the data supporting the use of docetaxel in the treatment of breast cancer, focusing on pharmacokinetics, efficacy in adjuvant and metastatic trials alone and in combination with chemotherapeutic and targeted agents, and the toxicity of docetaxel in comparison to paclitaxel. Docetaxel is a semisynthetic product derived from the European yew tree Taxus baccata L. It promotes the assembly of microtubules, stabilizes them, and thereby prevents their depolymerization. Docetaxel has been incorporated into neo-adjuvant chemotherapy regimens, both with and without anthracyclines. The inclusion of taxanes such as docetaxel in polychemotherapy regimens in early breast cancer is associated with a statistically significant reduction in mortality. As a single agent, docetaxel is highly active in the treatment of metastatic breast cancer. In first-line treatment of metastatic breast cancer, the combination of docetaxel and capecitabine was associated with an improvement in overall survival; however, toxicity was higher. The toxicity profile of docetaxel has been well documented and is predictable; the most frequent adverse effects are neutropenia and febrile neutropenia. Taxane-specific adverse effects, such as peripheral neuropathy, are also expected but are manageable with appropriate dosing and scheduling.
doi:10.2147/CMAR.S49321
PMCID: PMC3798099  PMID: 24143122
taxanes; docetaxel; clinical trial; adverse effects; peripheral neuropathy; neutropenia
2.  Combination of Docetaxel and Recombinant Vaccine Enhances T-Cell Responses and Antitumor Activity 
Purpose
Taxanes comprise some of the most widely used cancer chemotherapeutic agents. Members of this drug family, including docetaxel, are commonly used to treat breast, prostate, and lung cancers, among others. This study was designed to determine if this taxane has the ability to modulate components of the immune system independent of antitumor activity and to investigate the potential synergistic activities of the combination of docetaxel and vaccine therapy.
Experimental Design
We examined the in vivo effects of docetaxel on immune-cell subsets and on the function of CD4+, CD8+, and regulatory T cell (Treg) populations in response to antigen-specific vaccination. We also examined the antitumor effects of the combination of docetaxel and vaccine in a preclinical model in which docetaxel has no observable effect on tumor growth.
Results
These studies demonstrate for the first time that (a) docetaxel modulates CD4+, CD8+, CD19+, natural killer cell, and Treg populations in nontumor-bearing mice; (b) unlike cyclophosphamide, docetaxel does not inhibit the function of Tregs; (c) docetaxel enhances CD8+ but not CD4+ response to CD3 crosslinking; (d) docetaxel administered after vaccination provides optimal enhancement of immune response to recombinant viral vaccines; (e) docetaxel combined with recombinant viral vaccine is superior to either agent alone at reducing tumor burden; and (f) docetaxel plus vaccine increases antigen-specific T-cell responses to antigen in the vaccine as well as to cascade antigens derived from the tumor.
Conclusions
These findings suggest potential clinical benefit for the combined use of docetaxel and recombinant cancer vaccines.
doi:10.1158/1078-0432.CCR-07-4025
PMCID: PMC2682419  PMID: 18519787
Docetaxel; chemoimmunotherapy; vaccine; T-cell response; antitumor
3.  A Randomized Phase II Study of Concurrent Docetaxel Plus Vaccine Versus Vaccine Alone in Metastatic Androgen Independent Prostate Cancer 
Purpose: Docetaxel has activity against androgen insensitive prostate cancer (AIPC) and preclinical studies have demonstrated that taxane-based chemotherapy can enhance antitumor response of vaccines. The primary objective of this study was to determine if concurrent docetaxel (with dexamethasone) had any effect on generating an immune response to the vaccine. Secondary endpoints were whether vaccine could be given safely with docetaxel and the clinical outcome of the treatment regimen.
Experimental Design: The vaccination regimen was composed of (1) recombinant vaccinia virus (rV) that expresses the prostate-specific antigen gene (rV-PSA) admixed with (2) rV that expresses the B7.1 costimulatory gene (rV-B7.1), and (3) sequential booster vaccinations with recombinant fowlpox virus (rF-) containing the PSA gene (rF- PSA). Patients received GM-CSF with each vaccination. Twenty-eight patients with metastatic AIPC were randomized to receive either vaccine and weekly docetaxel or vaccine alone. Patients on the vaccine alone arm were allowed to cross over to receive docetaxel alone at time of disease progression. The ELISPOT assay was used to monitor immune responses for PSA-specific T cells.
Results: The median increase in these T-cell precursors to PSA was 3.33-fold in both arms following 3 months of therapy. In addition, immune responses to other prostate cancer associated tumor antigens were also detected post-vaccination. Eleven patients who progressed on vaccine alone crossed over to receive docetaxel at time of progression. Median PFS on docetaxel was 6.1 months after receiving vaccine compared with 3.7 months with the same regimen in a historical control.
Conclusion: This is the first clinical trial to demonstrate that docetaxel can be administered safely with immunotherapy without inhibiting vaccine specific T-cell responses. Furthermore, patients previously vaccinated with an anticancer vaccine may respond longer to docetaxel compared with a historical control of patients receiving docetaxel alone. Larger prospective clinical studies will be required to validate these findings.
doi:10.1158/1078-0432.CCR-05-2059
PMCID: PMC1526707  PMID: 16489082
Genitourinary cancers: prostate; Phase I-III clinical trials; Cancer vaccines; Immune responses to cancer
4.  Cationic Lipid/DNA Complex-Adjuvanted Influenza A Virus Vaccination Induces Robust Cross-Protective Immunity▿  
Journal of Virology  2010;84(24):12691-12702.
Influenza A virus is a negative-strand segmented RNA virus in which antigenically distinct viral subtypes are defined by the hemagglutinin (HA) and neuraminidase (NA) major viral surface proteins. An ideal inactivated vaccine for influenza A virus would induce not only highly robust strain-specific humoral and T-cell immune responses but also cross-protective immunity in which an immune response to antigens from a particular viral subtype (e.g., H3N2) would protect against other viral subtypes (e.g., H1N1). Cross-protective immunity would help limit outbreaks from newly emerging antigenically novel strains. Here, we show in mice that the addition of cationic lipid/noncoding DNA complexes (CLDC) as adjuvant to whole inactivated influenza A virus vaccine induces significantly more robust adaptive immune responses both in quantity and quality than aluminum hydroxide (alum), which is currently the most widely used adjuvant in clinical human vaccination. CLDC-adjuvanted vaccine induced higher total influenza virus-specific IgG, particularly for the IgG2a/c subclass. Higher levels of multicytokine-producing influenza virus-specific CD4 and CD8 T cells were induced by CLDC-adjuvanted vaccine than with alum-adjuvanted vaccine. Importantly, CLDC-adjuvanted vaccine provided significant cross-protection from either a sublethal or lethal influenza A viral challenge with a different subtype than that used for vaccination. This superior cross-protection afforded by the CLDC adjuvant required CD8 T-cell recognition of viral peptides presented by classical major histocompatibility complex class I proteins. Together, these results suggest that CLDC has particular promise for vaccine strategies in which T cells play an important role and may offer new opportunities for more effective control of human influenza epidemics and pandemics by inactivated influenza virus vaccine.
doi:10.1128/JVI.00769-10
PMCID: PMC3004296  PMID: 20943978
5.  Intranasal Vaccination Promotes Detrimental Th17-Mediated Immunity against Influenza Infection 
PLoS Pathogens  2014;10(1):e1003875.
Influenza disease is a global health issue that causes significant morbidity and mortality through seasonal epidemics. Currently, inactivated influenza virus vaccines given intramuscularly or live attenuated influenza virus vaccines administered intranasally are the only approved options for vaccination against influenza virus in humans. We evaluated the efficacy of a synthetic toll-like receptor 4 agonist CRX-601 as an adjuvant for enhancing vaccine-induced protection against influenza infection. Intranasal administration of CRX-601 adjuvant combined with detergent split-influenza antigen (A/Uruguay/716/2007 (H3N2)) generated strong local and systemic immunity against co-administered influenza antigens while exhibiting high efficacy against two heterotypic influenza challenges. Intranasal vaccination with CRX-601 adjuvanted vaccines promoted antigen-specific IgG and IgA antibody responses and the generation of polyfunctional antigen-specific Th17 cells (CD4+IL-17A+TNFα+). Following challenge with influenza virus, vaccinated mice transiently exhibited increased weight loss and morbidity during early stages of disease but eventually controlled infection. This disease exacerbation following influenza infection in vaccinated mice was dependent on both the route of vaccination and the addition of the adjuvant. Neutralization of IL-17A confirmed a detrimental role for this cytokine during influenza infection. The expansion of vaccine-primed Th17 cells during influenza infection was also accompanied by an augmented lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it highlights the importance of both route of vaccination and adjuvant selection in vaccine development
Author Summary
Influenza virus remains a global health risk causing significant morbidity and mortality each year, with the elderly (>65 years) and the very young particularly prone to severe respiratory disease. Scientists are working to develop highly efficacious vaccines capable of eliciting broad cross-clade protection from influenza infection. Adjuvants as well as the route of immunization are known to modulate the type, quality and breadth of immune responses to vaccines. In this study, we demonstrated intranasal vaccination with influenza antigens, and a novel synthetic TLR4-based adjuvant system provided protection against a lethal heterologous viral challenge. Immunization stimulated mucosal influenza-specific IgA antibody responses together with systemic IgG antibodies. While intranasal immunization stimulated the production of protective antibodies, vaccination via this route also promoted the generation of influenza-specific Th17 CD4+ T cells. These vaccine-induced Th17 cells increased inflammation and morbidity without contributing to viral clearance following challenge. Antibody neutralization of IL-17A during influenza infection significantly reduced the enhanced lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it demonstrates the importance of both route of immunization and adjuvant selection in vaccine development.
doi:10.1371/journal.ppat.1003875
PMCID: PMC3900655  PMID: 24465206
6.  Update on taxane development: new analogs and new formulations 
The taxanes (paclitaxel and docetaxel) represent an important class of antineoplastic agents that interfere with microtubule function leading to altered mitosis and cellular death. Paclitaxel (Taxol®) was originally extracted from a yew tree (Taxus spp., Taxaceae) a small slow-growing evergreen, coniferous tree. Due to the initial scarcity of paclitaxel, docetaxel (Taxotere®) a semisynthetic analog of paclitaxel produced from the needles of European yew tree, Taxus baccata was developed. Docetaxel differs from paclitaxel in two positions in its chemical structure and this small alteration makes it more water soluble. Today, paclitaxel and docetaxel are widely prescribed antineoplastic agents for a broad range of malignancies including lung cancer, breast cancer, prostate cancer, Kaposi’s sarcoma, squamous cell carcinoma of the head and neck, gastric cancer, esophageal cancer, bladder cancer, and other carcinomas. Although very active clinically, paclitaxel and docetaxel have several clinical problems including poor drug solubility, serious dose-limiting toxicities such as myelosuppression, peripheral sensory neuropathy, allergic reactions, and eventual development of drug resistance. A number of these side effects have been associated with the solvents used for dilution of these antineoplastic agents: Cremophor EL for paclitaxel and polysorbate 80 for docetaxel. In addition, reports have linked these solvents to the alterations in paclitaxel and docetaxel pharmacokinetic profiles. In this review, we provide preclinical and clinical data on several novel taxanes formulations and analogs which are currently US Food and Drug Administration (FDA)-approved or in clinical development in various solid tumor malignancies. Of the new taxanes nab-paclitaxel and cabazitaxel have enjoyed clinical success and are FDA-approved; while many of the other compounds described in this review are unlikely to be further developed for clinical use in daily practice. Furthermore, the successful clinical emergence of novel nontaxane microtubule-targeting chemotherapy agents such as epothilones and eribulin is liable to further restrict the development of novel taxanes.
doi:10.2147/DDDT.S28997
PMCID: PMC3523563  PMID: 23251087
taxane(s); novel taxanes; taxane analogs; new taxane formulations; new antimicrotubule agents
7.  Effects of Different Adjuvants in the Context of Intramuscular and Intranasal Routes on Humoral and Cellular Immune Responses Induced by Detergent-Split A/H3N2 Influenza Vaccines in Mice 
Influenza A/H3N2 viruses have caused the most severe epidemics since 1968 despite current immunization programs with inactivated vaccines. We undertook a side-by-side preclinical evaluation of different adjuvants (Alum, AS03, and Protollin) and routes of administration (intramuscular [i.m.] and intranasal [i.n.]) for assessing their effect on the immunogenicity and cross-reactivity of inactivated split vaccines (A/H3N2/New York/55/2004). Humoral and T cell-mediated immune responses against the homologous virus and a heterologous drifted strain (A/H3N2/Wisconsin/67/2005) were measured in BALB/c mice at 2, 6, and 19 weeks postboost. The AS03- and Alum-adjuvanted i.m. vaccines induced at least an 8-fold increase over the nonadjuvanted vaccine in functional antibody titers against both the homotypic and heterotypic strains and low IgG2a and high IgG1 levels, suggesting a mixed Th1/Th2 response with a Th2 trend. The Protollin-adjuvanted i.n. vaccine induced the lowest IgG1/IgG2a ratio, which is indicative of a mixed Th1/Th2-type profile with a Th1 trend. This adjuvanted vaccine was the only vaccine to stimulate a mucosal IgA response. Whatever the timing after the boost, both hemagglutination inhibition (HAI) and microneutralization (MN) titers were higher with the AS03-adjuvanted i.m. vaccine than with the protollin-adjuvanted i.n. vaccine. Finally, the Alum-adjuvanted i.m. vaccine and the lower-dose Protollin-adjuvanted i.n. vaccine elicited significantly higher CD4+ Th1 and Th2 responses and more gamma interferon (IFN-γ)-producing CD8+ T cells than the nonadjuvanted vaccine. Our data indicate that the adjuvanted vaccines tested in this study can elicit stronger, more persistent, and broader immune responses against A/H3N2 strains than nonadjuvanted inactivated influenza vaccines.
doi:10.1128/CVI.05441-11
PMCID: PMC3272927  PMID: 22190392
8.  Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination 
Background
Influenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus.
Results
Both H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses.
Conclusion
Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.
doi:10.1186/1423-0127-20-19
PMCID: PMC3615951  PMID: 23517052
S-OIV; H1N1; H5N1; Adjuvant; Alum; MPLA
9.  Combined TLR7/8 and TLR9 Ligands Potentiate the Activity of a Schistosoma japonicum DNA Vaccine 
Background
Toll-like receptor (TLR) ligands have been explored as vaccine adjuvants for tumor and virus immunotherapy, but few TLR ligands affecting schistosoma vaccines have been characterized. Previously, we developed a partially protective DNA vaccine encoding the 26-kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST).
Methodology/Principal Findings
In this study, we evaluated a TLR7/8 ligand (R848) and a TLR9 ligand (CpG oligodeoxynucleotides, or CpG) as adjuvants for pVAX1-Sj26GST and assessed their effects on the immune system and protection against S. japonicum. We show that combining CpG and R848 with pVAX1-Sj26GST immunization significantly increases splenocyte proliferation and IgG and IgG2a levels, decreases CD4+CD25+Foxp3+ regulatory T cells (Treg) frequency in vivo, and enhances protection against S. japonicum. CpG and R848 inhibited Treg-mediated immunosuppression, upregulated the production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10, IL-2, and IL-6, and decreased Foxp3 expression in vitro, which may contribute to prevent Treg suppression and conversion during vaccination and allow expansion of antigen-specific T cells against pathogens.
Conclusions
Our data shows that selective TLR ligands can increase the protective efficacy of DNA vaccines against schistosomiasis, potentially through combined antagonism of Treg-mediated immunosuppression and conversion.
Author Summary
There is evidence that TLR activation can block Treg cell responses and thereby break tolerance to self-antigens. It is expected that the use of TLR ligands as vaccine adjuvants will induce potent anti-pathogen immune responses and simultaneously overcome immune inhibition mediated by Tregs. However, the impact of TLR ligands on schistosomiasis vaccines is unclear. Here, we demonstrate that the use of a TLR7/8 ligand (R848) and a TLR9 ligand (CpG) as adjuvants in combination with the S. japonicum vaccine pVAX1-Sj26GST improves disease protection. The combination of CpG and R848 administered after vaccination causes an immune response marked by an upregulation of splenocyte proliferation and IgG and IgG2a levels that also coincides with a decreased proportion of CD4+CD25+ Tregs in mice. We also show that combined adjuvant use of CpG and R848 may impair Treg development and function by promoting the secretion of proinflammatory cytokines and reducing Foxp3 expression. Our findings suggest that in combination with the vaccine, TLR ligands may protect the effector response from Treg-mediated suppression, thereby eliciting the appropriate immune response to improve vaccine efficacy. Immunization combined with the TLR ligands CpG and R848 thus represents a promising new approach for the design of schistosoma vaccines.
doi:10.1371/journal.pntd.0002164
PMCID: PMC3617091  PMID: 23593527
10.  Cross-Reactive Neuraminidase Antibodies Afford Partial Protection against H5N1 in Mice and Are Present in Unexposed Humans 
PLoS Medicine  2007;4(2):e59.
Background
A pandemic H5N1 influenza outbreak would be facilitated by an absence of immunity to the avian-derived virus in the human population. Although this condition is likely in regard to hemagglutinin-mediated immunity, the neuraminidase (NA) of H5N1 viruses (avN1) and of endemic human H1N1 viruses (huN1) are classified in the same serotype. We hypothesized that an immune response to huN1 could mediate cross-protection against H5N1 influenza virus infection.
Methods and Findings
Mice were immunized against the NA of a contemporary human H1N1 strain by DNA vaccination. They were challenged with recombinant A/Puerto Rico/8/34 (PR8) viruses bearing huN1 (PR8-huN1) or avN1 (PR8-avN1) or with H5N1 virus A/Vietnam/1203/04. Additional naïve mice were injected with sera from vaccinated mice prior to H5N1 challenge. Also, serum specimens from humans were analyzed for reactivity with avN1. Immunization elicited a serum IgG response to huN1 and robust protection against the homologous challenge virus. Immunized mice were partially protected from lethal challenge with H5N1 virus or recombinant PR8-avN1. Sera transferred from immunized mice to naïve animals conferred similar protection against H5N1 mortality. Analysis of human sera showed that antibodies able to inhibit the sialidase activity of avN1 exist in some individuals.
Conclusions
These data reveal that humoral immunity elicited by huN1 can partially protect against H5N1 infection in a mammalian host. Our results suggest that a portion of the human population could have some degree of resistance to H5N1 influenza, with the possibility that this could be induced or enhanced through immunization with seasonal influenza vaccines.
Humoral immunity against endemic human H1N1 influenza viruses can partially protect mice against H5N1 challenge, raising the possibility that a portion of the human population could have some degree of resistance against avian flu.
Editors' Summary
Background.
Every winter, millions of people catch influenza—a viral infection of the airways. Most recover quickly but influenza can kill infants, elderly people, and chronically ill individuals. To minimize these deaths, the World Health Organization recommends that vulnerable people be vaccinated against influenza every autumn. Annual vaccination is necessary because flu viruses continually make small changes to the viral proteins (antigens) that the immune system recognizes. Each year's vaccine contains disabled versions of the circulating strains of influenza A type H1N1 and H3N2 viruses, and of influenza B virus. The H and N refer to the major influenza A antigens (hemagglutinin and neuraminidase), and the numbers refer to the type of each antigen; different H1N1 and H3N2 virus strains contain small variations in their respective hemagglutinin and neuraminidase type. Vaccines provide protection against seasonal influenza outbreaks, but sometimes flu viruses emerge that contain major antigenic changes, such as a different hemagglutinin type. These viruses can start pandemics (global outbreaks) because populations have little immunity to them. Many scientists believe that avian (bird) H5N1 influenza virus (which has caused about 250 confirmed cases of human flu and 150 deaths) could trigger the next human pandemic.
Why Was This Study Done?
Avian influenza H5N1 virus has not started a human pandemic yet because it cannot move easily between people. If it acquires this property, it could kill millions before an effective vaccine could be developed, so researchers are looking for other ways to provide protection against avian H5N1. One possibility is that an immune response to the human type 1 neuraminidase (huN1) in circulating H1N1 influenza virus strains and vaccines could provide some protection against avian H5N1 influenza virus, which contains the closely related avian type 1 neuraminidase (avN1). In this study, the researchers have investigated this possibility in mice and in a small human study.
What Did the Researchers Do and Find?
The researchers immunized mice with DNA encoding the huN1 present in a circulating H1N1 virus. They then examined the immune response of the mice to this huN1 and to avN1 from an avian H5N1 virus isolated from a human patient (A/Vietnam/1203/04). Most of the mice made antibodies (proteins that recognize antigens) against huN1; a few also made detectable levels of antibodies against avN1. All the vaccinated mice survived infection with a man-made flu virus containing huN1, and half also survived infection with low doses of a man-made virus containing avN1 or A/Vietnam/1203/04. To test whether the antibodies made by the vaccinated mice were responsible for this partial protection, the researchers collected serum (the liquid part of blood that contains the antibodies) from them and injected it into unvaccinated mice. Again, about half of the mice survived infection with the H5N1 virus, which indicates that the huN1-induced immunity against H5N1 is largely mediated by antibodies. Finally, the researchers tested serum samples from 38 human volunteers for their ability to inhibit neuraminidase from an H1N1 virus and two H5N1 viruses (antibodies to neuraminidase reduce viral replication and disease severity by inhibiting neuraminidase activity). Most of the sera inhibited the enzyme from the H1N1 virus; and seven also inhibited the enzyme from both H5N1 viruses.
What Do These Findings Mean?
These findings indicate that a vaccine containing huN1 induces the production of antibodies in mice that partly protect them against H5N1 infection. In addition, the human study suggests that some people may have some degree of resistance to H5N1 influenza because of exposure to H1N1 viruses or routine influenza vaccination. These results, while intriguing, don't show that there is actual protection, but it seems well worth doing additional work to address this question. The researchers also suggest that many more people might have been infected already with H5N1 but their strong H1N1 immunity meant they had only mild symptoms, and this hypothesis also deserves further investigation. Overall, these findings raise the possibility that seasonal influenza vaccination may provide some protection against pandemic H5N1. It is worth discussing whether, even while further studies are underway, seasonal vaccination should be increased, especially in areas where H5N1 is present in birds.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040059.
A related PLoS Medicine Perspective article by Laura Gillim-Ross and Kanta Subbarao is available
US Centers for Disease Control and Prevention provides information about influenza for patients and professionals, including key facts about avian influenza and vaccination
US National Institute of Allergy and Infectious Disease has a feature on seasonal, avian and pandemic flu
World Health Organization has fact sheets on influenza and influenza vaccines, and information on avian influenza
UK Health Protection Agency provides information on seasonal, avian, and pandemic influenza
doi:10.1371/journal.pmed.0040059
PMCID: PMC1796909  PMID: 17298168
11.  Evaluation of recombinant Onchocerca volvulus activation associated protein-1 (ASP-1) as a potent Th1-biased adjuvant with a panel of protein or peptide-based antigens and commercial inactivated vaccines 
Vaccine  2008;26(39):5022-5029.
Alum, the only adjuvant approved for clinical applications, can induce strong humoral (Th2) but weak cellular (Th1) immune responses. It is necessary to develop safe and effective adjuvants capable of inducing both humoral and cellular immune responses. We previously showed that activation-associated protein-1 (ASP-1) derived from Onchocerca volvulus has potent adjuvant activity. In this study, we have further evaluated the adjuvanticity of recombinant ASP-1 using a panel of recombinant proteins or synthetic peptide-based antigens, including ovalbumin (OVA), synthetic HIV peptide (HIV-p), recombinant HIV gp41 (rgp41) and HBV HBsAg, as well as three commercially available inactivated vaccines against haemorrhagic fever with renal syndrome (HFRS), Influenza and Rabies. Our results indicate that ASP-1 induced significantly higher IgG1 (Th2-associated) and IgG2a (Th1-associated) responses than alum adjuvant against OVA antigen, HIV-p, and rgp41. Consistently, it induced similar level of IgG1 responses as alum but higher level of IgG2a and IFN-γ-producing T cell responses than alum adjuvant against HBsAg. Further, ASP-1 improved both IgG1 and IgG2a responses to three commercial inactivated vaccines when used separately or in combination. In conclusion, the recombinant ASP-1, unlike alum adjuvant, is able to induce both Th1 and Th2-associated humoral responses and Th1 cellular responses, suggesting that it can be further developed as a promising adjuvant for subunit-based and inactivated vaccines.
doi:10.1016/j.vaccine.2008.07.028
PMCID: PMC2597511  PMID: 18675867
Adjuvant; Onchocerca volvulus; Activation-associated protein-1; Vaccine
12.  Focal Adhesion Kinase Silencing Augments Docetaxel-Mediated Apoptosis in Ovarian Cancer Cells 
Objective
Docetaxel causes cell death through induction of apoptosis; however, cell death characteristics for docetaxel have not yet been fully elucidated. We examined the role of focal adhesion kinase (FAK) cleavage in docetaxel-mediated apoptosis.
Methods
FAK degradation after treatment with docetaxel was determined in both taxane-sensitive (HeyA8 and SKOV3) and taxane-resistant (HeyA8-MDRand SKOV3-TR) ovarian cancer celllines by Westernblot analysis. Cell growth was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. FAK-targeting small interfering RNA (siRNA) was used to decrease FAK expression. Apoptosis and caspase activity were determined using commercially available kits.
Results
SKOV3 and HeyA8 celllines were both sensitive to docetaxel (IC50 levels,1–6.2 nmol/L), whereas the SKOV3-TR and HeyA8-MDR cells were resistant (IC50 ≥250 nmol/L for both). Docetaxel induced high rates of apoptosis in SKOV3 and HeyA8 cells (84% and 66% apoptosis, respectively) but minimal apoptosis (5–8%) in SKOV3-TR and HeyA8-MDR cells. Similarly, FAK was cleaved in SKOV3 and HeyA8 cells in response to docetaxel treatment but unchanged in the resistant cells. Caspase-3 and caspase-8 activity also increased significantly in docetaxel-treated SKOV3 and HeyA8 cells but not in the taxane-resistant cells. DEVD-fmk (caspase-3 blocker) was able to block both FAK cleavage and apoptosis mediated by docetaxel in SKOV3 and HeyA8 cells. FAK siRNA transfection resulted in 70% to 90% decrease in FAK levels in all cell lines within 72 hours. FAK silencing augmented docetaxel-mediated growth inhibition (5- to 8-fold increase) and apoptosis in both of the taxane-sensitive and taxane-resistant cell lines.
Conclusions
Docetaxel induces FAK cleavage, mediated through activation of caspase-3, in taxane-sensitive ovarian cancer cells but not in taxane-resistant cells. The absence of FAK degradation may contribute to cell survival in taxane-resistant cells. FAK silencing promotes the in vitro efficacy of docetaxel in both taxane-sensitive and taxane-resistant cell lines and may serve as a novel therapeutic approach.
doi:10.1158/1078-0432.CCR-05-1728
PMCID: PMC3144933  PMID: 16361572
13.  Optimizing the Dose of Pre-Pandemic Influenza Vaccines to Reduce the Infection Attack Rate 
PLoS Medicine  2007;4(6):e218.
Background
The recent spread of avian influenza in wild birds and poultry may be a precursor to the emergence of a 1918-like human pandemic. Therefore, stockpiles of human pre-pandemic vaccine (targeted at avian strains) are being considered. For many countries, the principal constraint for these vaccine stockpiles will be the total mass of antigen maintained. We tested the hypothesis that lower individual doses (i.e., less than the recommended dose for maximum protection) may provide substantial extra community-level benefits because they would permit wider vaccine coverage for a given total size of antigen stockpile.
Methods and Findings
We used a mathematical model to predict infection attack rates under different policies. The model incorporated both an individual's response to vaccination at different doses and the process of person-to-person transmission of pandemic influenza. We found that substantial reductions in the attack rate are likely if vaccines are given to more people at lower doses. These results are applicable to all three vaccine candidates for which data are available. As a guide to the magnitude of the effect, we simulated epidemics based on historical studies of immunogenicity. For example, for one of the vaccines for which data are available, the attack rate would drop from 67.6% to 58.7% if 160 out of the total US population of 300 million were given an optimal dose rather than 20 out of 300 million given the maximally protective dose (as promulgated in the US National Pandemic Preparedness Plan). Our results are conservative with respect to a number of alternative assumptions about the precise nature of vaccine protection. We also considered a model variant that includes a single high-risk subgroup representing children. For smaller stockpile sizes that allow vaccine to be offered only to the high-risk group at the optimal dose, the predicted benefits of using the homogenous model formed a lower bound in the presence of a risk group, even when the high-risk group was twice as infective and twice as susceptible.
Conclusions
In addition to individual-level protection (i.e., vaccine efficacy), the population-level implications of pre-pandemic vaccine programs should be considered when deciding on stockpile size and dose. Our results suggest that a lower vaccine dose may be justified in order to increase population coverage, thereby reducing the infection attack rate overall.
Steven Riley and colleagues examine the potential benefits of "stretching" a limited supply of vaccine and suggest that substantial reductions in the attack rate are possible if vaccines are given to more people at lower doses.
Editors' Summary
Background.
Every winter, millions of people catch influenza, a viral infection of the nose, throat, and airways. Most recover quickly, but the disease can be deadly. In the US, seasonal influenza outbreaks (epidemics) cause 36,000 excess deaths annually. And now there are fears that an avian (bird) influenza virus might trigger a human influenza pandemic—a global epidemic that could kill millions. Seasonal epidemics occur because flu viruses continually make small changes to their hemagglutinin and neuraminidase molecules, the viral proteins (antigens) that the immune system recognizes. Because of this “antigenic drift,” an immune system response (which can be induced by catching flu or by vaccination with disabled circulating influenza strains) that combats flu one year may provide only partial protection the next year. “Antigenic shift” (large changes in flu antigens) can cause pandemics because communities have no immunity to the changed virus.
Why Was This Study Done?
Although avian influenza virus, which contains a hemagglutinin type that differs from currently circulating human flu viruses, has caused a few cases of human influenza, it has not started a human pandemic yet because it cannot move easily between people. If it acquires this property, which will probably involve further small antigenic changes, it could kill millions of people before scientists can develop an effective vaccine against it. To provide some interim protection, many countries are preparing stockpiles of “pre-pandemic” vaccines targeted against the avian virus. The US, for example, plans to store enough pre-pandemic vaccine to provide maximum protection to 20 million people (including key health workers) out of its population of 300 million. But, given a limited stockpile of pre-pandemic vaccine, might giving more people a lower dose of vaccine, which might reduce the number of people susceptible to infection and induce herd immunity by preventing efficient transmission of the flu virus, be a better way to limit the spread of pandemic influenza? In this study, the researchers have used mathematical modeling to investigate this question.
What Did the Researchers Do and Find?
To predict the infection rates associated with different vaccination policies, the researchers developed a mathematical model that incorporates data on human immune responses induced with three experimental vaccines against the avian virus and historical data on the person–person transmission of previous pandemic influenza viruses. For all the vaccines, the model predicts that giving more people a low dose of the vaccine would limit the spread of influenza better than giving fewer people the high dose needed for full individual protection. For example, the researchers estimate that dividing the planned US stockpile of one experimental vaccine equally between 160 million people instead of giving it at the fully protective dose to 20 million people might avert about 27 million influenza cases in less than year. However, giving the maximally protective dose to the 9 million US health-care workers and using the remaining vaccine at a lower dose to optimize protection within the general population might avert only 14 million infections.
What Do These Findings Mean?
These findings suggest that, given a limited stockpile of pre-pandemic vaccine, increasing the population coverage of vaccination by using low doses of vaccine might reduce the overall influenza infection rate more effectively than vaccinating fewer people with fully protective doses of vaccine. However, because the researchers' model includes many assumptions, it can only give an indication of how different strategies might perform, not firm numbers for how many influenza cases each strategy is likely to avert. Before public-health officials use this or a similar model to help them decide the best way to use pre-pandemic vaccines to control a human influenza pandemic, they will need more information about the efficacy of these vaccines and about transmission rates of currently circulating viruses. They will also need to know whether pre-pandemic vaccines actually provide good protection against the pandemic virus, as assumed in this study, before they can recommend mass immunization with low doses of pre-pandemic vaccine, selective vaccination with high doses, or a mixed strategy.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040218.
US Centers for Disease Control and Prevention provide information on influenza and influenza vaccination for patients and health professionals (in English, Spanish, Filipino, Chinese, and Vietnamese)
The World Health Organization has a fact sheet on influenza and on the global response to avian influenza (in English, Spanish, French, Russian, Arabic, and Chinese)
The MedlinePlus online encyclopedia devotes a page to flu (in English and Spanish)
The UK Health Protection Agency information on avian, pandemic, and seasonal influenza
The US National Institute of Allergy and Infectious Diseases has a comprehensive feature called “focus on the flu”
doi:10.1371/journal.pmed.0040218
PMCID: PMC1892041  PMID: 17579511
14.  Immune changes in patients with advanced breast cancer undergoing chemotherapy with taxanes 
British Journal of Cancer  2002;87(1):21-27.
Besides cytotoxicity, taxanes induce other biological effects, especially in the immune system. Taxanes have demonstrated immunostimulatory effects against neoplasms, supporting the idea that these agents suppress cancer through several mechanisms and not solely through inhibiting cell division. The purpose of the present study was to evaluate the effect of taxanes (paclitaxel and docetaxel) and investigate their ability in alterating important immunological parameters in breast cancer patients. Thirty women with advanced breast cancer undergoing chemotherapy were randomly assigned into two groups treated with either single agent Paclitaxel or Docetaxel. Sera from patients before the first and after the last treatment cycle and from normal donors were assayed by ELISA for IL-2, IL-1β, IFN-γ, GM-CSF, IL-6, TNF-α, and PGE2 levels. In these same blood samples, NK and LAK cell activity was tested in the total PBMC population against NK-sensitive K562 tumour targets, respectively, and autologous mixed lymphocyte reaction was tested by 3H-thymidine proliferation assays. All patients in both groups responded to therapy. Significant differences were observed in the following immune parameters between the control group of healthy blood donors and the pretreatment values of both taxane groups; IL-2, GM-CSF, IFN-γ levels and NK and LAK cell cytotoxicity were depressed, whereas TNF-α and IL-6 levels were raised in breast cancer patients before treatment compared to controls. There were no significant differences between the two treatment groups regarding any of the parameters studied. Both drugs led to increases in MLR values, NK and LAK cell cytotoxicity, and IL-6, GM-CSF, IFN-γ levels, and decreases for IL-1, TNF, and PGE2 levels. The percentage of these differences was greater for docetaxel in comparison to paclitaxel (P<0.0001). More specifically, docetaxel demonstrated a more pronounced effect on enhancing MLR, NK, LAK activity and IFN-γ, IL-2, IL-6, and GM-CSF levels, as well as caused more potent reduction in IL-1 and TNF-α levels when compared to paclitaxel. The present study indicates that patients responded to treatment of advanced breast cancer with single-agent paclitaxel or docetaxel leads to an increase in serum IFN-γ, IL-2, IL-6, GM-CSF cytokine levels and enhancement of PBMC NK and LAK cell activity, while they both lead to a decrease of acute phase serum cytokine levels of IL-1 and TNF-α. Moreover, the effects of docetaxel are in all the above parameters more pronounced than those of paclitaxel.
British Journal of Cancer (2002) 87, 21–27. doi:10.1038/sj.bjc.6600347 www.bjcancer.com
© 2002 Cancer Research UK
doi:10.1038/sj.bjc.6600347
PMCID: PMC2364288  PMID: 12085250
breast cancer; cytokines; immune; paclitaxel; docetaxel
15.  The Adjuvanticity of an O. volvulus-Derived rOv-ASP-1 Protein in Mice Using Sequential Vaccinations and in Non-Human Primates 
PLoS ONE  2012;7(5):e37019.
Adjuvants potentiate antigen-specific protective immune responses and can be key elements promoting vaccine effectiveness. We previously reported that the Onchocerca volvulus recombinant protein rOv-ASP-1 can induce activation and maturation of naïve human DCs and therefore could be used as an innate adjuvant to promote balanced Th1 and Th2 responses to bystander vaccine antigens in mice. With a few vaccine antigens, it also promoted a Th1-biased response based on pronounced induction of Th1-associated IgG2a and IgG2b antibody responses and the upregulated production of Th1 cytokines, including IL-2, IFN-γ, TNF-α and IL-6. However, because it is a protein, the rOv-ASP-1 adjuvant may also induce anti-self-antibodies. Therefore, it was important to verify that the host responses to self will not affect the adjuvanticity of rOv-ASP-1 when it is used in subsequent vaccinations with the same or different vaccine antigens. In this study, we have established rOv-ASP-1's adjuvanticity in mice during the course of two sequential vaccinations using two vaccine model systems: the receptor-binding domain (RBD) of SARS-CoV spike protein and a commercial influenza virus hemagglutinin (HA) vaccine comprised of three virus strains. Moreover, the adjuvanticity of rOv-ASP-1 was retained with an efficacy similar to that obtained when it was used for a first vaccination, even though a high level of anti-rOv-ASP-1 antibodies was present in the sera of mice before the administration of the second vaccine. To further demonstrate its utility as an adjuvant for human use, we also immunized non-human primates (NHPs) with RBD plus rOv-ASP-1 and showed that rOv-ASP-1 could induce high titres of functional and protective anti-RBD antibody responses in NHPs. Notably, the rOv-ASP-1 adjuvant did not induce high titer antibodies against self in NHPs. Thus, the present study provided a sound scientific foundation for future strategies in the development of this novel protein adjuvant.
doi:10.1371/journal.pone.0037019
PMCID: PMC3355165  PMID: 22615877
16.  Effect of Adjuvants on Responses to Skin Immunization by Microneedles Coated with Influenza Subunit Vaccine 
PLoS ONE  2012;7(7):e41501.
Recent studies have demonstrated the effectiveness of vaccine delivery to the skin by vaccine-coated microneedles; however there is little information on the effects of adjuvants using this approach for vaccination. Here we investigate the use of TLR ligands as adjuvants with skin-based delivery of influenza subunit vaccine. BALB/c mice received 1 µg of monovalent H1N1 subunit vaccine alone or with 1 µg of imiquimod or poly(I:C) individually or in combination via coated microneedle patches inserted into the skin. Poly(I:C) adjuvanted subunit influenza vaccine induced similar antigen-specific immune responses compared to vaccine alone when delivered to the skin by microneedles. However, imiquimod-adjuvanted vaccine elicited higher levels of serum IgG2a antibodies and increased hemagglutination inhibition titers compared to vaccine alone, suggesting enhanced induction of functional antibodies. In addition, imiquimod-adjuvanted vaccine induced a robust IFN-γ cellular response. These responses correlated with improved protection compared to influenza subunit vaccine alone, as well as reduced viral replication and production of pro-inflammatory cytokines in the lungs. The finding that microneedle delivery of imiquimod with influenza subunit vaccine induces improved immune responses compared to vaccine alone supports the use of TLR7 ligands as adjuvants for skin-based influenza vaccines.
doi:10.1371/journal.pone.0041501
PMCID: PMC3405087  PMID: 22848514
17.  Pre-Clinical Evaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine 
PLoS ONE  2008;3(8):e2954.
Background
Hepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations.
Methodology and Principal Findings
Physical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/−17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached ≥106 titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-γ and TNF-α cytokine production and elevated levels of IgG2 subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4°C, 6 months at 25°C and 6 weeks at 40°C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models.
Conclusions
Our results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy.
doi:10.1371/journal.pone.0002954
PMCID: PMC2496893  PMID: 18698426
18.  Alterations in tumor necrosis factor signaling pathways are associated with cytotoxicity and resistance to taxanes: a study in isogenic resistant tumor cells 
Introduction
The taxanes paclitaxel and docetaxel are widely used in the treatment of breast, ovarian, and other cancers. Although their cytotoxicity has been attributed to cell-cycle arrest through stabilization of microtubules, the mechanisms by which tumor cells die remains unclear. Paclitaxel has been shown to induce soluble tumor necrosis factor alpha (sTNF-α) production in macrophages, but the involvement of TNF production in taxane cytotoxicity or resistance in tumor cells has not been established. Our study aimed to correlate alterations in the TNF pathway with taxane cytotoxicity and the acquisition of taxane resistance.
Methods
MCF-7 cells or isogenic drug-resistant variants (developed by selection for surviving cells in increasing concentrations of paclitaxel or docetaxel) were assessed for sTNF-α production in the absence or presence of taxanes by enzyme-linked immunosorbent assay (ELISA) and for sensitivity to docetaxel or sTNF-α by using a clonogenic assay (in the absence or presence of TNFR1 or TNFR2 neutralizing antibodies). Nuclear factor (NF)-κB activity was also measured with ELISA, whereas gene-expression changes associated with docetaxel resistance in MCF-7 and A2780 cells were determined with microarray analysis and quantitative reverse transcription polymerase chain reaction (RTqPCR).
Results
MCF-7 and A2780 cells increased production of sTNF-α in the presence of taxanes, whereas docetaxel-resistant variants of MCF-7 produced high levels of sTNF-α, although only within a particular drug-concentration threshold (between 3 and 45 nM). Increased production of sTNF-α was NF-κB dependent and correlated with decreased sensitivity to sTNF-α, decreased levels of TNFR1, and increased survival through TNFR2 and NF-κB activation. The NF-κB inhibitor SN-50 reestablished sensitivity to docetaxel in docetaxel-resistant MCF-7 cells. Gene-expression analysis of wild-type and docetaxel-resistant MCF-7, MDA-MB-231, and A2780 cells identified changes in the expression of TNF-α-related genes consistent with reduced TNF-induced cytotoxicity and activation of NF-κB survival pathways.
Conclusions
We report for the first time that taxanes can promote dose-dependent sTNF-α production in tumor cells at clinically relevant concentrations, which can contribute to their cytotoxicity. Defects in the TNF cytotoxicity pathway or activation of TNF-dependent NF-κB survival genes may, in contrast, contribute to taxane resistance in tumor cells. These findings may be of strong clinical significance.
doi:10.1186/bcr3083
PMCID: PMC3496117  PMID: 22225778
19.  miR-337-3p and Its Targets STAT3 and RAP1A Modulate Taxane Sensitivity in Non-Small Cell Lung Cancers 
PLoS ONE  2012;7(6):e39167.
NSCLC (non-small cell lung cancer) often exhibits resistance to paclitaxel treatment. Identifying the elements regulating paclitaxel response will advance efforts to overcome such resistance in NSCLC therapy. Using in vitro approaches, we demonstrated that over-expression of the microRNA miR-337-3p sensitizes NCI-H1155 cells to paclitaxel, and that miR-337-3p mimic has a general effect on paclitaxel response in NSCLC cell lines, which may provide a novel adjuvant strategy to paclitaxel in the treatment of lung cancer. By combining in vitro and in silico approaches, we identified STAT3 and RAP1A as direct targets that mediate the effect of miR-337-3p on paclitaxel sensitivity. Further investigation showed that miR-337-3p mimic also sensitizes cells to docetaxel, another member of the taxane family, and that STAT3 levels are significantly correlated with taxane resistance in lung cancer cell lines, suggesting that endogenous STAT3 expression is a determinant of intrinsic taxane resistance in lung cancer. The identification of a miR-337-3p as a modulator of cellular response to taxanes, and STAT3 and RAP1A as regulatory targets which mediate that response, defines a novel regulatory pathway modulating paclitaxel sensitivity in lung cancer cells, which may provide novel adjuvant strategies along with paclitaxel in the treatment of lung cancer and may also provide biomarkers for predicting paclitaxel response in NSCLC.
doi:10.1371/journal.pone.0039167
PMCID: PMC3377607  PMID: 22723956
20.  Intranasal Immunization with a Formalin-Inactivated Human Influenza A Virus Whole-Virion Vaccine Alone and Intranasal Immunization with a Split-Virion Vaccine with Mucosal Adjuvants Show Similar Levels of Cross-Protection 
The antigenicity of seasonal human influenza virus changes continuously; thus, a cross-protective influenza vaccine design needs to be established. Intranasal immunization with an influenza split-virion (SV) vaccine and a mucosal adjuvant induces cross-protection; however, no mucosal adjuvant has been assessed clinically. Formalin-inactivated intact human and avian viruses alone (without adjuvant) induce cross-protection against the highly pathogenic H5N1 avian influenza virus. However, it is unknown whether seasonal human influenza formalin-inactivated whole-virion (WV) vaccine alone induces cross-protection against strains within a subtype or in a different subtype of human influenza virus. Furthermore, there are few reports comparing the cross-protective efficacy of the WV vaccine and SV vaccine-mucosal adjuvant mixtures. Here, we found that the intranasal human influenza WV vaccine alone induced both the innate immune response and acquired immune response, resulting in cross-protection against drift variants within a subtype of human influenza virus. The cross-protective efficacy conferred by the WV vaccine in intranasally immunized mice was almost the same as that conferred by a mixture of SV vaccine and adjuvants. The level of cross-protective efficacy was correlated with the cross-reactive neutralizing antibody titer in the nasal wash and bronchoalveolar fluids. However, neither the SV vaccine with adjuvant nor the WV vaccine induced cross-reactive virus-specific cytotoxic T-lymphocyte activity. These results suggest that the intranasal human WV vaccine injection alone is effective against variants within a virus subtype, mainly through a humoral immune response, and that the cross-protection elicited by the WV vaccine and the SV vaccine plus mucosal adjuvants is similar.
doi:10.1128/CVI.00016-12
PMCID: PMC3393367  PMID: 22552600
21.  Enhanced Immunogenicity, Mortality Protection, and Reduced Viral Brain Invasion by Alum Adjuvant with an H5N1 Split-Virion Vaccine in the Ferret 
PLoS ONE  2011;6(6):e20641.
Background
Pre-pandemic development of an inactivated, split-virion avian influenza vaccine is challenged by the lack of pre-existing immunity and the reduced immunogenicity of some H5 hemagglutinins compared to that of seasonal influenza vaccines. Identification of an acceptable effective adjuvant is needed to improve immunogenicity of a split-virion avian influenza vaccine.
Methods and Findings
Ferrets (N = 118) were vaccinated twice with a split-virion vaccine preparation of A/Vietnam/1203/2004 or saline either 21 days apart (unadjuvanted: 1.9 µg, 7.5 µg, 30 µg, or saline), or 28 days apart (unadjuvanted: 22.5 µg, or alum-adjuvanted: 22.5 or 7.5 µg). Vaccinated animals were challenged intranasally 21 or 28 days later with 106 EID50 of the homologous strain. Immunogenicity was measured by hemagglutination inhibition and neutralization assays. Morbidity was assessed by observed behavior, weight loss, temperature, cytopenias, histopathology, and viral load.
No serum antibodies were detected after vaccination with unadjuvanted vaccine, whereas alum-adjuvanted vaccination induced a robust antibody response. Survival after unadjuvanted dose regimens of 30 µg, 7.5 µg and 1.9 µg (21-day intervals) was 64%, 43%, and 43%, respectively, yet survivors experienced weight loss, fever and thrombocytopenia. Survival after unadjuvanted dose regimen of 22.5 µg (28-day intervals) was 0%, suggesting important differences in intervals in this model. In contrast to unadjuvanted survivors, either dose of alum-adjuvanted vaccine resulted in 93% survival with minimal morbidity and without fever or weight loss. The rarity of brain inflammation in alum-adjuvanted survivors, compared to high levels in unadjuvanted vaccine survivors, suggested that improved protection associated with the alum adjuvant was due to markedly reduced early viral invasion of the ferret brain.
Conclusion
Alum adjuvant significantly improves efficacy of an H5N1 split-virion vaccine in the ferret model as measured by immunogenicity, mortality, morbidity, and brain invasion.
doi:10.1371/journal.pone.0020641
PMCID: PMC3110201  PMID: 21687736
22.  Functional p53 Determines Docetaxel Sensitivity in Prostate Cancer Cells 
The Prostate  2012;73(4):418-427.
BACKGROUND
Docetaxel is the first line treatment for castration resistant prostate cancer (CRPC). However, docetaxel resistance rapidly develops. Identifying the critical mechanisms giving rise to docetaxel resistance is the major challenge in advanced prostate cancer.
METHODS
The effects of docetaxel on human DU145, PC3, LNCaP, and C4-2 prostate cancer cells were examined in cell culture, and p53 expression were analyzed by Western blot analysis. The potential role of p53 in docetaxel sensitivity in prostate cancer cells was tested by either p53 silencing using shRNA or p53 overexpression by introducing wild-type p53.
RESULTS
We found that DU145 (mutant p53) and PC3 (p53 null) cells were less sensitive than LNCaP and C4-2 cells expressing functional p53 in response to docetaxel. Docetaxel treatment induces considerably higher apoptosis in LNCaP and C4-2 cells than in DU145 and PC3 cells in a dose dependent manner. Docetaxel increases the levels of ser15 phosphorylation of p53 in a dose dependent manner in both LNCaP and C4-2 cells, while has no effect on the levels of ser15 phosphorylation of p53 in DU145 cells. These results suggest that p53 phosphorylation is associated with docetaxel sensitivity in prostate cancer cells. To further confirm whether p53 activation can induce cell sensitivity to docetaxel treatment, we used p53 shRNA to knock down p53 expression in C4-2 cells and determined the cells response to docetaxel treatment. Knockdown of p53 significantly down regulated p53 phosphorylation and blocked docetaxel induced apoptotic cell death compared to the vector control. To further confirm this observation, we established a stable knock out p53 in C4-2 cells. Down regulation of p53 in the stable p53 knock out C4-2 cells significantly inhibited docetaxel induced apoptotic cell death. We also used wild-type (WT) p53 to over express p53 in DU145 cells, and found that expression of WT-p53 in DU145 cells increased their sensitivity to docetaxel.
CONCLUSIONS
These results demonstrate that docetaxel induces p53 phosphorylation and that p53 status is a crucial determinant of docetaxel sensitivity in prostate cancer cells.
doi:10.1002/pros.22583
PMCID: PMC3938015  PMID: 22996738
prostate cancer; docetaxel; p53
23.  Disparate adjuvant properties among three formulations of “alum” 
Vaccine  2012;31(4):653-660.
Aluminum adjuvants, commonly referred to as “alum,” are the most widespread immunostimulants in human vaccines. Although the mechanisms that promote humoral responses to alum-adsorbed antigens are still enigmatic, alum is thought to form antigen depots and induce inflammatory signals that, in turn, promote antibody production. It was recently noted that Imject® alum, a commercial aluminum-containing adjuvant commonly used in animal studies, is not the physicochemical equivalent of aluminum adjuvant present in human vaccines. This difference raises concerns about the use of Imject® alum in animal research as a model for approved aluminum adjuvants. Here, we compared the capacity of Imject® alum, Alhydrogel®, and a traditional alum-antigen precipitate to induce humoral responses in mice to the hapten-carrier antigen, NP-CGG [(4-hydroxy-3-nitrophenyl)acetyl-chicken γ-globulin]. The magnitude of humoral responses elicited by Alhydrogel® and precipitated alum was significantly greater than that induced by Imject® alum. The strength of the humoral responses elicited by different alum formulations was correlated with the quantity of pro-inflammatory cytokines induced and the numbers of inflammatory cells at the site of immunization. Moreover, Imject® exhibited a severely reduced capacity to adsorb protein antigens compared to Alhydrogel® and precipitated alum. These findings reveal substantial differences in the immunostimulatory properties of distinct alum preparations, an important point of consideration for the evaluation of novel adjuvants, the assessment of new alum-based vaccines, and in mechanistic studies of adjuvanticity.
doi:10.1016/j.vaccine.2012.11.044
PMCID: PMC3541451  PMID: 23200935
alum; adjuvant; antibody; inflammation
24.  THE CONTRIBUTION OF TYPE I INTERFERON SIGNALING TO IMMUNITY INDUCED BY ALPHAVIRUS REPLICON VACCINES 
Vaccine  2008;26(39):4998-5003.
The type I interferon (IFN) system is critical for protecting the mammalian host from numerous virus infections and plays a key role in shaping the anti-viral adaptive immune response. In this report, the importance of type I IFN signaling was assessed in a mouse model of alphavirus-induced humoral immune induction. Venezuelan equine encephalitis virus replicon particles (VRP) expressing the hemagglutinin (HA) gene from influenza virus (HA-VRP) were used to vaccinate both wildtype (wt) and IFN α/β receptor knockout (RKO) mice. HA-VRP vaccination induced equivalent levels of flu-specific systemic IgG, mucosal IgG, and systemic IgA antibodies in both wt and IFN RKO mice. In contrast, HA-VRP vaccination of IFN RKO mice failed to induce significant levels of flu-specific mucosal IgA antibodies at multiple mucosal surfaces. In the VRP adjuvant system, co-delivery of null VRP with ovalbumin (OVA) protein significantly increased the levels of OVA-specific serum IgG, fecal IgG, and fecal IgA antibodies in both wt and RKO mice, suggesting that type I IFN signaling plays a less significant role in the VRP adjuvant effect. Taken together, these results suggest that, 1) at least in regard to IFN signaling, the mechanisms which regulate VRP-induced immunity differ when VRP are utilized as expression vectors as opposed to adjuvants, and 2) type I IFN signaling is required for the induction of mucosal IgA antibodies directed against VRP-expressed antigen. These results potentially shed new light on the regulatory networks which promote immune induction, and specifically mucosal immune induction, with alphavirus vaccine vectors.
doi:10.1016/j.vaccine.2008.07.011
PMCID: PMC3595171  PMID: 18656518
viral immunity; Type I IFN; mucosal IgA; adjuvant activity
25.  An adjuvanted inactivated murine cytomegalovirus (MCMV) vaccine induces potent and long-term protective immunity against a lethal challenge with virulent MCMV 
BMC Infectious Diseases  2014;14:195.
Background
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes serious problems in immunocompromised or immunologically immature hosts. Vaccination is the preferred approach for prevention of HCMV infection, but so far no approved HCMV vaccine is available. In this study, we assessed the immunogenicity and protective immunity of a formalin-inactivated murine cytomegalovirus vaccine (FI-MCMV) in a mouse model in combination with adjuvants MF59, alum, or chitosan.
Methods
Specific-pathogen-free BALB/c mice aged 6–8 weeks were immunized twice, 3 weeks apart, with various doses of FI-MCMV (0.25 μg, 1 μg, 4 μg) with or without adjuvant. Mice were challenged with a lethal dose (5 × LD50) of a more virulent mouse salivary gland-passaged MCMV 3 weeks after the second immunization. The protective immunity of the vaccine was evaluated by determining the survival rates, residual spleen and salivary gland viral loads, body weight changes, and serum anti-MCMV IgG titers.
Results
Immunization with FI-MCMV vaccine induced a high level of specific antibody response. Antigen sparing was achieved by the addition of an adjuvant, which significantly enhanced the humoral response to vaccine antigens with a wide range of doses. The level of live virus detected in the spleen on day 5 and in the salivary glands on day 21 after the lethal challenge was significantly lower in adjuvant-treated groups than in controls. Survival rates in adjuvant-treated groups also increased significantly. Furthermore, these protective immune responses were sustained for at least 6 months following immunization.
Conclusions
These results show that inactivated MCMV vaccine is effective, and that the adjuvanted FI-MCMV vaccine provides more effective and longer-term protection than the adjuvant-free vaccine.
doi:10.1186/1471-2334-14-195
PMCID: PMC4005462  PMID: 24720840
Cytomegalovirus; Inactivated vaccine; Adjuvant; Long-term protective immunity

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