Therapy of established B16-F10 melanoma tumors by a single vaccination of CTL/T helper peptides in VacciMax(®)

BACKGROUND: Melanoma tumors are known to express antigens that usually induce weak immune responses of short duration. Expression of both tumor-associated antigens p53 and TRP2 by melanoma cells raises the possibility of simultaneously targeting more than one antigen in a therapeutic vaccine. In this report, we show that VacciMax(® )(VM), a novel liposome-based vaccine delivery platform, can increase the immunogenicity of melanoma associated antigens, resulting in tumor elimination. METHODS: C57BL/6 mice bearing B16-F10 melanoma tumors were vaccinated subcutaneously 6 days post tumor implantation with a mixture of synthetic peptides (modified p53: 232–240, TRP-2: 181–188 and PADRE) and CpG. Tumor growth was monitored and antigen-specific splenocyte responses were assayed by ELISPOT. RESULTS: Vaccine formulated in VM increased the number of both TRP2- and p53-specific IFN-γ producing splenocytes following a single vaccination. Vaccine formulated without VM resulted only in enhanced IFN-γ producing splenocytes to one CTL epitopes (TRP2:180–188), suggesting that VM overcomes antigen dominance and enhances immunogenicity of multiple epitopes. Vaccination of mice bearing 6-day old B16-F10 tumors with both TRP2 and p53-peptides formulated in VM successfully eradicated tumors in all mice. A control vaccine which contained all ingredients except liposomes resulted in eradication of tumors in no more than 20% of mice. CONCLUSION: A single administration of VM is capable of inducing an effective CTL response to multiple tumor-associated antigens. The responses generated were able to reject 6-day old B16-F10 tumors

Evaluation of the protective potential of antibody and T cell responses elicited by a novel preventative vaccine towards respiratory syncytial virus small hydrophobic protein

The small hydrophobic (SH) glycoprotein of human respiratory syncytial virus (RSV) is a transmembrane protein that is poorly accessible by antibodies on the virion but has an ectodomain (SHe) that is accessible and expressed on infected cells. The SHe from RSV strain A has been formulated in DPX, a unique delivery platform containing an adjuvant, and is being evaluated as an RSV vaccine candidate. The proposed mechanism of protection is the immune-mediated clearance of infected cells rather than neutralization of the virion. Our phase I clinical trial data dearly showed that vaccination resulted in robust antibody responses, but it was unclear if these immune responses have any correlation to immune responses to natural infection with RSV. Therefore, we embarked on this study to examine these immune responses in older adults with confirmed RSV infection. We compared vaccine-induced (DPX-RSV(A)) immune responses from participants in a Phase 1 clinical trial to paired acute and convalescent titers from older adults with symptomatic laboratory-confirmed RSV infection. Serum samples were tested for anti-SHe IgG titers and the isotypes determined. T cell responses were evaluated by IFN-gamma ELISPOT. Anti-SHe titers were detected in 8 of 42 (19%) in the acute phase and 16 of 42 (38%) of convalescent serum samples. IgG1, IgG3, and IgA were the prevalent isotypes generated by both vaccination and infection. Antigen-specific T cell responses were detected in 9 of 16 (56%) of vaccinated participants. Depletion of CD4(+) but not CD8(+) T cells abrogated the IFN-gamma ELISPOT response supporting the involvement of CD4(+) T cells in the immune response to vaccination. The data showed that an immune response like that induced by DPX-RSV(A) could be seen in a subset of participants with confirmed RSV infection. These findings show that older adults with clinically significant infection as well as vaccinated adults generate a humoral response to SHe. The induction of both SHe-specific antibody and cellular responses support further clinical development of the DPX-RSV(A) vaccine

A respiratory syncytial virus vaccine based on the small hydrophobic protein ectodomain presented with a novel lipid-based formulation is highly immunogenic and safe in adults : a first-in-humans study

Background: Respiratory syncytial virus infection can cause lower respiratory tract infection in older adults comparable to influenza, but no vaccines are available. Methods: This was a randomized, observer-blinded, first-in-humans study of a novel synthetic RSV antigen based on the ectodomain of the small hydrophobic glycoprotein (SHe) of RSV subgroup A, formulated with either the lipid and oil-based vaccine platform DepoVax (DPX-RSV[A]) or alum (RSV[A]-Alum), in healthy, 50-64-year-old individuals. Two dose levels (10 or 25 mu g) of SHe with each formulation were compared to placebo. A booster dose was administered on day 56. Results: There was no indication that the vaccine was unsafe. Mild pain, drowsiness, and muscles aches were the most common solicited adverse events (AEs), and the frequencies of the AEs did not increase after dose 2. Robust anti-SHe-specific immune responses were demonstrated in the DPX-RSV(A) 10-mu g and 25-mu g groups (geometric mean titer, approximately 10-fold and 100-fold greater than that of placebo at days 56 and 236, respectively), and responses were sustained in the DPX-RSV(A) 25-mu g group at day 421. Responses to the RSV(A)-Alum vaccines were very low. Conclusions: A novel antigen from the SH protein of RSV, formulated in a lipid and oil-based vaccine platform, was highly immunogenic, with sustained antigen-specific antibody responses, and had an acceptable safety profile

Rejection of large HPV-16 expressing tumors in aged mice by a single immunization of VacciMax® encapsulated CTL/T helper peptides

The incidence of cancer increases significantly in later life, yet few pre-clinical studies of cancer immunotherapy use mice of advanced age. A novel vaccine delivery platform (VacciMax®,VM) is described that encapsulates antigens and adjuvants in multilamellar liposomes in a water-in-oil emulsion. The therapeutic potential of VM-based vaccines administered as a single dose was tested in HLA-A2 transgenic mice of advanced age (48–58 weeks old) bearing large palpable TC1/A2 tumors. The VM-based vaccines contained one or more peptides having human CTL epitopes derived from HPV 16 E6 and E7. VM formulations contained a single peptide, a mixture of four peptides or the same four peptides linked together in a single long peptide. All VM formulations contained PADRE and CpG as adjuvants and ISA51 as the hydrophobic component of the water-in-oil emulsion. VM-formulated vaccines containing the four peptides as a mixture or linked together in one long peptide eradicated 19-day old established tumors within 21 days of immunization. Peptide-specific cytotoxic cellular responses were confirmed by ELISPOT and intracellular staining for IFN-γ producing CD8+ T cells. Mice rendered tumor-free by vaccination were re-challenged in the opposite flank with 10 million HLF-16 tumor cells, another HLA-A2/E6/E7 expressing tumor cell line. None of these mice developed tumors following the re-challenge. In summary, this report describes a VM-formulated therapeutic vaccine with the following unprecedented outcome: a) eradication of large tumors (> 700 mm3) b) in mice of advanced age c) in less than three weeks post-immunization d) following a single vaccination

First-in-man application of a novel therapeutic cancer vaccine formulation with the capacity to induce multi-functional T cell responses in ovarian, breast and prostate cancer patients

BACKGROUND: DepoVax(TM) is a novel non-emulsion depot-forming vaccine platform with the capacity to significantly enhance the immunogenicity of peptide cancer antigens. Naturally processed HLA-A2 restricted peptides presented by breast, ovarian and prostate cancer cells were used as antigens to create a therapeutic cancer vaccine, DPX-0907. METHODS: A phase I clinical study was designed to examine the safety and immune activating potential of DPX-0907 in advanced stage breast, ovarian and prostate cancer patients. A total of 23 late stage cancer patients were recruited and were divided into two dose/volume cohorts in a three immunization protocol. RESULTS: DPX-0907 was shown to be safe with injection site reactions being the most commonly reported adverse event. All breast cancer patients (3/3), most of ovarian (5/6) and one third of prostate (3/9) cancer patients exhibited detectable immune responses, resulting in a 61% immunological response rate. Immune responses were generally observed in patients with better disease control after their last prior treatment. Antigen-specific responses were detected in 73% of immune responders (44% of evaluable patients) after the first vaccination. In 83% of immune responders (50% of evaluable patients), peptide-specific T cell responses were detected at ≥2 time points post vaccination with 64% of the responders (39% of evaluable patients) showing evidence of immune persistence. Immune monitoring also demonstrated the generation of antigen-specific T cell memory with the ability to secrete multiple Type 1 cytokines. CONCLUSIONS: The novel DepoVax formulation promotes multifunctional effector memory responses to peptide-based tumor associated antigens. The data supports the capacity of DPX-0907 to elicit Type-1 biased immune responses, warranting further clinical development of the vaccine. This study underscores the importance of applying vaccines in clinical settings in which patients are more likely to be immune competent. TRIAL REGISTRATION: ClinicalTrials.gov NCT0109584

Genome-wide analysis identifies 12 loci influencing human reproductive behavior.

The genetic architecture of human reproductive behavior-age at first birth (AFB) and number of children ever born (NEB)-has a strong relationship with fitness, human development, infertility and risk of neuropsychiatric disorders. However, very few genetic loci have been identified, and the underlying mechanisms of AFB and NEB are poorly understood. We report a large genome-wide association study of both sexes including 251,151 individuals for AFB and 343,072 individuals for NEB. We identified 12 independent loci that are significantly associated with AFB and/or NEB in a SNP-based genome-wide association study and 4 additional loci associated in a gene-based effort. These loci harbor genes that are likely to have a role, either directly or by affecting non-local gene expression, in human reproduction and infertility, thereby increasing understanding of these complex traits

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Anti-PD-1 increases the clonality and activity of tumor infiltrating antigen specific T cells induced by a potent immune therapy consisting of vaccine and metronomic cyclophosphamide

Abstract Background Future cancer immunotherapies will combine multiple treatments to generate functional immune responses to cancer antigens through synergistic, multi-modal mechanisms. In this study we explored the combination of three distinct immunotherapies: a class I restricted peptide-based cancer vaccine, metronomic cyclophosphamide (mCPA) and anti-PD-1 treatment in a murine tumor model expressing HPV16 E7 (C3). Methods Mice were implanted with C3 tumors subcutaneously. Tumor bearing mice were treated with mCPA (20 mg/kg/day PO) for seven continuous days on alternating weeks, vaccinated with HPV16 E749-57 peptide antigen formulated in the DepoVax (DPX) adjuvanting platform every second week, and administered anti-PD-1 (200 μg/dose IP) after each vaccination. Efficacy was measured by following tumor growth and survival. Immunogenicity was measured by IFN-γ ELISpot of spleen, vaccine draining lymph nodes and tumor draining lymph nodes. Tumor infiltration was measured by flow cytometry for CD8α+ peptide-specific T cells and RT-qPCR for cytotoxic proteins. The clonality of tumor infiltrating T cells was measured by TCRβ sequencing using genomic DNA. Results Untreated C3 tumors had low expression of PD-L1 in vivo and anti-PD-1 therapy alone provided no protection from tumor growth. Treatment with DPX/mCPA could delay tumor growth, and tri-therapy with DPX/mCPA/anti-PD-1 provided long-term control of tumors. We found that treatment with DPX/mCPA/anti-PD-1 enhanced systemic antigen-specific immune responses detected in the spleen as determined by IFN-γ ELISpot compared to those in the DPX/mCPA group, but immune responses in tumor-draining lymph nodes were not increased. Although no increases in antigen-specific CD8α+ TILs could be detected, there was a trend for increased expression of cytotoxic genes within the tumor microenvironment as well as an increase in clonality in mice treated with DPX/mCPA/anti-PD-1 compared to those with anti-PD-1 alone or DPX/mCPA. Using a library of antigen-specific CD8α+ T cell clones, we found that antigen-specific clones were more frequently expanded in the DPX/mCPA/anti-PD-1 treated group. Conclusions These results demonstrate how the efficacy of anti-PD-1 may be improved by combination with a potent and targeted T cell activating immune therapy

Using MRI to evaluate and predict therapeutic success from depot-based cancer vaccines

In the preclinical development of immunotherapy candidates, understanding the mechanism of action and determining biomarkers that accurately characterize the induced host immune responses is critical to improving their clinical interpretation. Magnetic resonance imaging (MRI) was used to evaluate in vivo changes in lymph node size in response to a peptide-based cancer vaccine therapy, formulated using DepoVax (DPX). DPX is a novel adjuvant lipid-in-oilâbased formulation that facilitates enhanced immune responses by retaining antigens at the injection site for extended latencies, promoting increased potentiation of immune cells. C57BL/6 mice were implanted with C3 (HPV) tumor cells and received either DPX or control treatments, 5 days post-implantation. Complete tumor eradication occurred in DPX-vaccinated animals and large volumetric increases were observed in the vaccine-draining right inguinal lymph node (VRILN) in DPX mice, likely corresponding to increased localized immune response to the vaccine. Upon evaluating the relative measure of vaccine-potentiated immune activation to tumor-induced immune response (VRILN/VLILN), receiver-operating characteristic (ROC) curves revealed an area under the curve (AUC) of 0.90 (±0.07), indicating high specificity and sensitivity as a predictive biomarker of vaccine efficacy. We have determined that for this tumor model, early MRI lymph node volumetric changes are predictive of depot immunotherapeutic success