Sequence-based epitope mapping of high pathogenicity avian influenza H5 clade 2.3.4.4b in Latin America.

High Pathogenicity Avian Influenza (HPAI) poses a significant threat to public and animal health. Clade 2.3.4.4b recently emerged from the Eastern hemisphere and disseminated globally, reaching the Latin American (LATAM) region in late 2022 for the first time. HPAI in LATAM has resulted in massive mortalities and culling of poultry and wild birds, causing infection in mammals and humans. Despite its meaningful impact in the region, only occasional evidence about the genetic and epitope characteristics of the introduced HPAI is reported. Hence, this study seeks to phylogenetically characterize the molecular features and the source of HPAI in LATAM by evaluating potential antigenic variations. For such a purpose, we analyzed 302 whole genome sequences. All Latin American viruses are descendants of the 2.3.4.4b clade of the European H5N1 subtype. According to genomic constellations deriving from European and American reassortments, the identification of three major subtypes and eight sub-genotypes was achievable. Based on the variation of antigenic motifs at the HA protein in LATAM, we detected three potential antigenic variants, indicating the HA-C group as the dominant variant. This study decidedly contributes to unraveling the origin of the 2.3.4.4b clade in LATAM, its geographic dissemination, and evolutionary dynamics within Latin American countries. These findings offer useful information for public health interventions and surveillance initiatives planned to prevent and manage the transmission of avian influenza viruses.

Sequence-Based Antigenic Analyses of H1 Swine Influenza A Viruses from Colombia (2008-2021) Reveals Temporal and Geographical Antigenic Variations.

Swine influenza is a respiratory disease that affects the pork industry and is a public health threat. It is caused by type A influenza virus (FLUAV), which continuously undergoes genetic and antigenic variations. A large amount of information regarding FLUAV in pigs is available worldwide, but it is limited in Latin America. The HA sequences of H1 subtype FLUAV-positive samples obtained from pigs in Colombia between 2008-2021 were analyzed using sequence-based antigenic cartography and N-Glycosylation analyses. Of the 12 predicted global antigenic groups, Colombia contained five: four corresponding to pandemic strains and one to the classical swine H1N1 clade. Circulation of these clusters was observed in some regions during specific years. Ca2 was the immunodominant epitope among Colombian viruses. The counts of N-Glycosylation motifs were associated with the antigenic cluster ranging from three to five. The results show for the first time the existence of antigenic diversity of FLUAV in Colombia and highlight the impact of spatial and temporal factors on this diversity. This study provides information about FLUAV variability in pigs under natural conditions in the absence of vaccination and emphasizes the need for surveillance of its phylogenetic and antigenic characteristics.

New Insights into Avian Infectious Bronchitis Virus in Colombia from Whole-Genome Analysis.

Infectious Bronchitis (IB) is a respiratory disease caused by a highly variable Gammacoronavirus, which generates a negative impact on poultry health worldwide. GI-11 and GI-16 lineages have been identified in South America based on Infectious Bronchitis virus (IBV) partial S1 sequences. However, full genome sequence information is limited. In this study we report, for the first time, the whole-genome sequence of IBV from Colombia. Seven IBV isolates obtained during 2012 and 2013 from farms with respiratory disease compatible with IB were selected and the complete genome sequence was obtained by NGS. According to S1 sequence phylogenetic analysis, six isolates belong to lineage GI-1 and one to lineage GVI-1. When whole genome was analyzed, five isolates were related to the vaccine strain Ma5 2016 and two showed mosaic genomes. Results from complete S1 sequence analysis provides further support for the hypothesis that GVI-1, considered a geographically confined lineage in Asia, could have originated in Colombia. Complete genome information reported in this research allow a deeper understanding of the phylogenetic evolution of variants and the recombination events between strains that are circulating worldwide, contributing to the knowledge of coronavirus in Latin America and the world.

Zooming in on the molecular characteristics of swine influenza virus circulating in Colombia before and after the H1N1pdm09 virus.

Influenza is one of the most critical viral agents involved in the respiratory disease complex affecting swine production systems worldwide. Despite the absence of vaccination against swine influenza virus in Colombia, the serologic reactivity to classic H1N1 and H3N2 subtypes reported since 1971 indicates the virus has been circulating in the country's swine population for several decades. However, successful isolation and sequencing of field virus from pigs was nonexistent until 2008, when H1N1 classical influenza virus was identified. One year later, due to the emergence of the influenza A (H1N1) pdm09 virus, responsible for the first global flu pandemic of the 21st century, it was introduced in the country. Therefore, to understand the impact of the introduction of the H1N1pdm09 virus in Colombia on the complexity and dynamics of influenza viruses previously present in the swine population, we carried out a study aiming to characterize circulating viruses genetically and establish possible reassortment events that might have happened between endemic influenza viruses before and after the introduction of the pandemic virus. A phylogenetic analysis of ten swine influenza virus isolates from porcine samples obtained between 2008 and 2015 was conducted. As a result, a displacement of the classical swine influenza virus with the pdmH1N1 virus in the swine population was confirmed. Once established, the pandemic subtype exhibited phylogenetic segregation based on a geographic pattern in all the evaluated segments. The evidence presents reassortment events with classic viruses in one of the first H1N1pdm09 isolates. Thus, this study demonstrates complex competition dynamics and variations in Colombian swine viruses through Drift and Shift.

Characterization and evaluation of Colombian propolis on the intestinal integrity of broilers.

Nutritional additives such as propolis seek to improve intestinal health as an alternative to the global ban on in-feed antibiotics used as growth promoters (AGP). The objective of this study was to evaluate the effect of propolis supplementation in diet of broilers. Four hundred and fifty straight-run Ross 308 AP broilers were fed with a basal diet (BD) throughout the whole experimental period. Birds were randomly distributed into 5 groups at d 14: negative control without antibiotics nor propolis (AGP-), positive control 500 ppm of Zinc Bacitracin as growth promoter (AGP+), and 3 groups supplemented with 150, 300, and 450 ppm of propolis. Every group included 6 replicates of 15 birds each. Propolis concentration was increased from d 22 to 42, in experimental groups to 300, 600, and 900 ppm of propolis, and 10% of raw soybean was included as a challenge in all groups during the same period. Analysis of productive parameters, intestinal morphometry, and relative quantification of genes associated with epithelial integrity by qPCR were performed at 21 and 42 d. The groups with the greatest weights were those that consumed diets including 150 (21 d) and 900 ppm (42 d) of propolis compared with all treatments. The lowest score of ISI was found at 300 (21 d) and 600 ppm (42 d). A lower degree of injury in digestive system was seen with the inclusion of 300 ppm (21 d) and 900 ppm (42 d). Up-regulation of zonula occludens-1 (ZO-1) was observed in jejunum of broilers supplemented with 150 and 300 ppm at 21 d. Up-regulation of ZO-1 and TGF-ß was also evidenced in ileum at all propolis inclusion levels at 42-day-old compared to AGP+ and AGP-. The beneficial effects were evidenced at inclusion levels of 150 ppm in the starter and 900 ppm in the finisher. According to the results, the Colombian propolis inclusion can improve productive performance, physiological parameters, and gene expression associated with intestinal integrity.

Rhabdoid melanoma in a harpy eagle (Harpia harpyja).

A 28-year-old male harpy eagle (Harpia harpyja) with a history of anorexia, hyporexia, lethargy, and progressive weight loss was found dead and submitted for post-mortem examination. Gross findings include dark brown discolouration of testes and lungs; the testes were bilaterally enlarged, glistening brown-grey to blackish in appearance, firm, smooth, and multilobulated. The lungs contained a mass with similar features to the testicles, irregularly shaped with multiple nodules. Histology of testis showed round, polygonal and pleomorphic cells, containing melanin pigments and a typical eosinophilic vacuole in their cytoplasm and with severe pleomorphism. An immunohistochemistry panel with Melan-A, vimentin, CK AE1/AE/3, MUM-1 and CD-68 were performed, yielding a positive reaction for Melan-A and vimentin. The morphology of the tumour cells, the presence of melanin pigment and the immunoreactivity for Melan-A and vimentin by the cells led to a diagnosis of rhabdoid melanoma. This is the first case of this pathology in the testis with lung metastasis in a harpy eagle.

Key Factors That Enable the Pandemic Potential of RNA Viruses and Inter-Species Transmission: A Systematic Review.

Viruses play a primary role as etiological agents of pandemics worldwide. Although there has been progress in identifying the molecular features of both viruses and hosts, the extent of the impact these and other factors have that contribute to interspecies transmission and their relationship with the emergence of diseases are poorly understood. The objective of this review was to analyze the factors related to the characteristics inherent to RNA viruses accountable for pandemics in the last 20 years which facilitate infection, promote interspecies jump, and assist in the generation of zoonotic infections with pandemic potential. The search resulted in 48 research articles that met the inclusion criteria. Changes adopted by RNA viruses are influenced by environmental and host-related factors, which define their ability to adapt. Population density, host distribution, migration patterns, and the loss of natural habitats, among others, have been associated as factors in the virus-host interaction. This review also included a critical analysis of the Latin American context, considering its diverse and unique social, cultural, and biodiversity characteristics. The scarcity of scientific information is striking, thus, a call to local institutions and governments to invest more resources and efforts to the study of these factors in the region is key.

Role of vaccine therapy in cancer: biology and practice.

Vaccines constitute a potential new therapeutic approach for a range of human cancers. Unlike other therapeutics, vaccines initiate a dynamic process in the host immune system that can be exploited with subsequent therapies. Indeed, recent preclinical and clinical studies with cancer vaccines have provided evidence that this unique therapeutic modality should lead to consideration of new paradigms in both clinical trial design and endpoints and in combination therapies. The present article reviews and sets out a rationale for these new paradigms, with a focus on prostate cancer.

A randomized phase II trial of docetaxel (taxotere) plus thalidomide in androgen-independent prostate cancer.

New therapeutic alternatives are needed to improve outcomes in patients with androgen-independent prostate cancer (AIPC). For several years, researchers at the National Cancer Institute have been interested in elucidating the importance of angiogenesis in the pathogenesis of prostate cancer and in identifying inhibitors of this process. Thalidomide has been shown to inhibit the ability of tumors to recruit new blood vessels. In a recent phase II trial of thalidomide in AIPC, 28% of patients achieved a prostate-specific antigen (PSA) decrease of >40%. The taxane docetaxel also produces PSA and measurable disease responses when used as monotherapy or as a component of combination chemotherapy for AIPC. Thus, based on the single-agent activity of thalidomide and docetaxel, we initiated a randomized phase II study of weekly docetaxel with or without thalidomide, 200 mg at bedtime, in patients with chemotherapy-naive metastatic AIPC. Docetaxel, 30 mg/m(2) intravenously, was administered every 7 days for 3 weeks, followed by a 1-week rest period. Both regimens have been well tolerated among the first 59 treated patients, with a near absence of grade (3/4) myelosuppression. Fatigue, hyperglycemia, and pulmonary toxicity were seen in both groups. Thrombotic events have been seen in the combination arm. Thirty-five percent (6 of 17) of the patients receiving docetaxel alone and 53% (19 of 36) of those receiving docetaxel and thalidomide have had a PSA decrease of at least 50%. Combining a cytotoxic agent with an angiogenesis inhibitor is a promising area of investigation for prostate cancer management.

The infection of human dendritic cells with recombinant avipox vectors expressing a costimulatory molecule transgene (CD80) to enhance the activation of antigen-specific cytolytic T cells.

Human dendritic cells (DCs) express MHC class I and II molecules and several T-cell costimulatory molecules that contribute to their efficiency as antigen-presenting cells (APCs). Whereas most human DC populations uniformly express some costimulatory molecules such as B7-2 (CD86), previous studies have shown a wide variation in the expression of B7-1 (CD80) among different human DC preparations. In the studies reported here, we demonstrate that replication-defective avipox vectors expressing B7-1 can be used to rapidly and efficiently infect human DCs and can enhance the efficacy of human DCs to activate specific human T-cell populations. This has been demonstrated both in systems using peptide as a source of signal 1 and in systems using recombinant avipox vector to deliver signal 1. The antigen used in these studies was the tumor-associated human carcinoembryonic antigen (CEA). An immunodominant 9-mer CTL epitope for CEA (designated CAP-1) has been previously characterized (K. Y. Tsang et al., J. Natl. Cancer Inst. (Bethesda), 87: 982-990, 1995). The source of signal 1 used in these studies was (a) the CAP-1 peptide; (b) recombinant avipox-CEA; or (c) the dual transgene recombinant avipox-CEA/B7-1. These studies demonstrate that CEA-specific T cells are more efficiently activated using as APCs peptide-pulsed DCs infected with avipox-B7-1, as compared with peptide-pulsed DCs infected with wild-type vector, or with uninfected peptide-pulsed DCs. Greater activation of CEA-specific T cells was also obtained using as APCs DCs that were infected with avipox-CEA/B7-1 as compared with the use of DCs infected with avipox-CEA. A CEA tetramer was also used to isolate high- and low-tetramer-binding CEA-specific T-cell populations. Although both high- and low-tetramer-binding T cells had the ability to lyse CEA peptide-pulsed targets, only the high-tetramer-binding T cells had the ability to lyse colon carcinoma cells expressing CEA, which suggests the existence of tetramer-binding populations with different T-cell receptor (TCR) affinities. The demonstrated safety of recombinant avipox vectors in humans and the previously demonstrated ability to administer them multiple times without host immune response limitations indicate that these vectors expressing B7-1 have a potential use in enhancing the efficacy of human DC immunotherapy protocols using either peptide or recombinant vector to deliver signal 1.

The influence of granulocyte macrophage colony-stimulating factor and prior chemotherapy on the immunological response to a vaccine (ALVAC-CEA B7.1) in patients with metastatic carcinoma.

Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to be an effective vaccine adjuvant because it enhances antigen processing and presentation by dendritic cells. ALVAC-CEA B7.1 is a canarypox virus encoding the gene for the tumor-associated antigen carcinoembryonic antigen (CEA) and for a T-cell costimulatory molecule, B7.1. After an initial dose escalation phase, this study evaluated vaccination with 4.5 x 10(8) plaque-forming units ALVAC-CEA B7.1 alone (n = 30) or with GM-CSF (n = 30) in patients with advanced CEA-expressing tumors to determine whether the addition of the adjuvant GM-CSF enhances induction of CEA-specific T-cells. Patients were vaccinated with vaccine intradermally every other week for 8 weeks. GM-CSF was given s.c. for 5 days beginning 2 days before vaccination. Patients with stable or responding disease after four immunizations received monthly boost injections alone or with GM-CSF. Biopsies of vaccine sites were obtained 48 h after vaccination to evaluate leukocytic infiltration and CEA expression. Induction of peripheral blood CEA-specific T-cell precursors was assessed in HLA-A2 positive patients by an ELISPOT assay looking for the production of IFN-gamma. Therapy was well tolerated. All of the patients had evidence of leukocytic infiltration and CEA expression in vaccine biopsy sites. In the patients receiving GM-CSF, leukocytic infiltrates were greater in cell number but were less likely to have a predominant lymphocytic infiltrate compared with patients receiving vaccine in the absence of the cytokine adjuvant. After four vaccinations, CEA-specific T-cell precursors were statistically increased in HLA-A2 positive patients who received vaccine alone. However, the GM-CSF plus vaccine cohort of HLA-A2 positive did not demonstrate a statistically significant increase in their CEA-specific T-cell precursor frequencies compared with baseline results. The number of prior chemotherapy regimens was negatively correlated with the generation of a T-cell response, whereas there was a positive correlation between the number of months from the last chemotherapy regimen and the T-cell response. ALVAC-CEA B7.1 is safe in patients with advanced, recurrent adenocarcinomas that express CEA, is associated with the induction of a CEA-specific T-cell response in patients treated with vaccine alone but not with vaccine and GM-CSF, and can lead to disease stabilization for up to 13 months.

Enhanced activation of human T cells via avipox vector-mediated hyperexpression of a triad of costimulatory molecules in human dendritic cells.

T-cell activation usually requires at least two signals. The first signal is antigen-specific, and the second signal(s) involves the interaction of a T-cell costimulatory molecule(s) on the antigen-presenting cell (APC) with its ligand on the T cell. Dendritic cells (DCs) are the most potent APCs, attributable, in part, to their expression of several T-cell costimulatory molecules. Human DCs generated in vitro, however, will vary in methods of generation and maturation and in terms of expression of different phenotypic markers-including costimulatory molecules-among different donors. We report here that a recombinant avipox (fowlpox, rF) vector has been constructed that can efficiently express the transgenes for three human T-cell costimulatory molecules (B7-1, ICAM-1, and LFA-3) as a result of individual early avipox promoters driving the expression of each transgene. This triad of costimulatory molecules (designated TRICOM) was selected because each has an individual ligand on T cells and each has been shown previously to prime a unique signaling pathway in T cells. We report here that rF-TRICOM can efficiently infect human DCs of different states of maturity and hyperexpress each of the three costimulatory molecules on the DC surface without affecting other DC phenotypic markers. Infection of influenza or human papilloma virus 9-mer peptide-pulsed DCs from different individuals, or at different stages of maturity with rF-TRICOM, resulted in enhanced activation of T cells from peripheral blood mononuclear cells of autologous donors after 24 h of incubation with DCS: This enhanced activation was analyzed by both titrating the peptide and differing the DC:effector cell ratios. No effect was observed using the control wild-type avipox vector. No increase in apoptosis was observed in T cells hyperstimulated with the TRICOM vector, and no decrease in interleukin-12 production was seen in lipopolysaccharide-stimulated DCs infected with rF-TRICOM. Antibody-blocking experiments demonstrated that enhanced T-cell activation by TRICOM was attributed to each of the three costimulatory molecules. Peptide-pulsed, rF-TRICOM-infected DCs were also shown to be more effective than peptide-pulsed DCs in activating T cells to 9-mer peptides derived from two relatively weak "self" immunogens, i.e., human prostate-specific antigen and human carcinoembryonic antigen. These studies thus demonstrate for the first time that a vector that can simultaneously hyperexpress three costimulatory molecules can be used to efficiently infect human DCs, leading to enhanced peptide-specific T-cell activation. The use of this approach for in vitro studies and clinical applications in immunotherapy is discussed.

Phase I study in advanced cancer patients of a diversified prime-and-boost vaccination protocol using recombinant vaccinia virus and recombinant nonreplicating avipox virus to elicit anti-carcinoembryonic antigen immune responses.

PURPOSE: This trial sought to determine, for the first time, the validity in human vaccinations of using two different recombinant vaccines in diversified prime-and-boost regimens to enhance T-cell responses to a tumor antigen. PATIENTS AND METHODS: Eighteen patients with advanced tumors expressing carcinoembryonic antigen (CEA) were randomized to receive either recombinant vaccinia (rV)-CEA followed by three avipox-CEA vaccinations, or avipox-CEA (three times) followed by one rV-CEA vaccination. Subsequent vaccinations in both cohorts were with avipox-CEA. Immunologic monitoring was performed using a CEA peptide and the enzyme-linked immunospot assay for interferon gamma production. RESULTS: rV-CEA followed by avipox-CEA was superior to the reverse order in the generation of CEA-specific T-cell responses. Further increases in CEA-specific T-cell precursors were seen when local granulocyte-macrophage colony-stimulating factor (GM-CSF) and low-dose interleukin (IL)-2 were given with subsequent vaccinations. The treatment was extremely well tolerated. Limited clinical activity was seen using vaccines alone in this patient population. Antibody production against CEA was also observed in some of the treated patients. CONCLUSION: rV-CEA was more effective in its role as a primer of the immune system; avipox-CEA could be given up to eight times with continued increases in CEA T-cell precursors. Future trials should use rV-CEA first followed by avipox-CEA. Vaccines specific to CEA are able to generate CEA-specific T-cell responses in patients without significant toxicity. T-cell responses using vaccines alone may be inadequate to generate significant anticancer objective responses in patients with advanced disease. Cytokines such as GM-CSF and IL-2 may play a key role in generating such responses.

The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines.

An enzyme-linked immunosorbent spot (ELISPOT) assay for interferon gamma production has been used to analyze specific T cell responses to a Flu 9-mer peptide, and a 9-mer peptide of carcinoembryonic antigen (CEA). Assays were performed on peripheral blood mononuclear cells (PBMC) of HLA-A2-positive patients with CEA-expressing carcinomas, both before and after vaccination with CEA-based vaccines, and from HLA-A2-positive healthy blood donors. The ELISPOT assay utilized aliquots of frozen PBMC, and assays were performed after 24 h in culture with peptide to rule out any artifacts due to long-term in vitro stimulation cycles. An internal standard was used for each assay to define reproducibility of the assay, and all samples from a given patient (pre- and post-vaccination, with both the Flu and CEA peptides) were analyzed simultaneously. The results indicated a trend towards healthy blood donors having higher levels of Flu-specific T cell precursors than do colon carcinoma patients, but these results were not statistically significant (P = 0.06). On the other hand, slightly higher CEA-specific T cell responses were observed in cancer patients with CEA-expressing carcinomas than in healthy blood donors. PBMC from two CEA-based vaccine clinical trials were analyzed for T cell responses to the same CEA peptide and to the Flu control peptide. The first trial consisted of three monthly vaccinations of CEA peptide (designated PPP) in adjuvant. The second trial consisted of cohorts receiving three monthly vaccinations of avipox-CEA recombinant (designated AAA) or cohorts receiving a primary vaccination with recombinant vaccinia-CEA followed by two monthly vaccinations with avipox-CEA (designated VAA). Few, if any, CEA-specific T cell responses were seen in the PPP vaccinations, while the majority of patients receiving the poxvirus CEA recombinants demonstrated increases in CEA-specific T cell responses and no increases in Flu-specific responses. CEA-specific IgG responses were also demonstrated in patients following recombinant CEA poxvirus vaccinations. Statistical analyses of the T cell responses to the same CEA peptide demonstrated a P value of 0.028 for the recombinant poxvirus vaccines, as compared with the peptide vaccine. There were no differences seen (P = 0.37) in Flu-specific responses after these two types of CEA vaccination. These results thus provide the first evidence that poxvirus recombinant-based vaccines are more potent in the initiation of tumor-antigen-specific T cell responses than vaccines employing peptide in adjuvant, when assays are conducted in an identical manner, and in defining responses to the same peptide. These results also demonstrate for the first time that an ELISPOT assay, performed over a 24-h period and without in vitro sensitization, can be successfully used to monitor immune responses to a tumor-associated antigen in cancer patients.

Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas.

Coordinated presentation of antigen and costimulatory molecules has been shown to result in the induction of an antigen-specific T-cell response rather than the development of anergy. This study evaluated the vaccine ALVAC-CEA B7.1, a canary pox virus that has been engineered to encode the gene for the tumor-associated antigen carcinoembryonic antigen (CEA) and B7.1, a T-cell costimulatory molecule. Patients with CEA-expressing tumors were immunized with 2.5 x 10(7) (n = 3), 1.0 x 10(8) (n = 6), and 4.5 x 10(8) (n = 30) plaque-forming units intradermally every other week for 8 weeks. Patients with stable or responding disease received monthly boost injections. Biopsies of vaccine sites were obtained 48 h after vaccination to evaluate leukocytic infiltration and CEA expression. Induction of CEA-specific T-cell precursors was assessed by an ELISPOT assay looking for the production of IFN-gamma. Therapy was well tolerated, without significant toxicity attributable to vaccine. All patients had evidence of leukocytic infiltration and CEA expression in vaccine biopsy sites. Six patients with elevated serum CEA values at baseline had declines in their levels lasting 4-12 weeks. These patients all had stable disease after four vaccinations. After four vaccinations, patients who were HLA-A-2-positive demonstrated increases in their CEA-specific T-cell precursor frequencies to a CEA-A2-binding peptide from baseline. The number of prior chemotherapy regimens was inversely correlated with the ability to generate a T-cell response. ALVAC-CEA B7.1 is safe in patients with advanced, recurrent adenocarcinomas that express CEA, and it is associated with the induction of a CEA-specific T-cell response.

A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in advanced prostate cancer.

A recombinant vaccinia virus encoding human prostate-specific antigen (rV-PSA) was administered as three consecutive monthly doses to 33 men with rising PSA levels after radical prostatectomy, radiation therapy, both, or metastatic disease at presentation. Dose levels were 2.65 x 10(6), 2.65 x 10(7), and 2.65 x 10(8) plaque forming units. Ten patients who received the highest dose also received 250 microg/m2 granulocyte-macrophage colony-stimulating factor (GM-CSF) as an immunostimulatory adjunct. No patient experienced any virus-related effects beyond grade I cutaneous toxicity. Pustule formation and/or erythema occurred after the first dose in all 27 men who received > or =2.65 x 10(7) plaque forming units. GM-CSF administration was associated with fevers and myalgias of grade 2 or lower in 9 of 10 patients. PSA levels in 14 of 33 men treated with rV-PSA with or without GM-CSF were stable for at least 6 months after primary immunization. Nine patients remained stable for 11-25 months; six of these remain progression free with stable PSA levels. Immunological studies demonstrated a specific T-cell response to PSA-3, a 9-mer peptide derived from PSA. rV-PSA is safe and can elicit clinical and immune responses, and certain patients remain without evidence of clinical progression for up to 21 months or longer.

Agonist peptide from a cytotoxic t-lymphocyte epitope of human carcinoembryonic antigen stimulates production of tc1-type cytokines and increases tyrosine phosphorylation more efficiently than cognate peptide.

The identification of an agonist peptide (YLSGADLNL, designated CAP1-6D) to an immunodominant cytotoxic T-lymphocyte (CTL) epitope (designated CAP1) of human carcinoembryonic antigen (CEA) has previously been reported. The agonist peptide harbors a single amino acid substitution at a non-MHC anchor residue and is proposed to exert its effects at the level of the T-cell receptor (TCR). The type and magnitude of cytokines produced by CAP1-reactive CTL upon stimulation with the agonist peptide, CAP1-6D, were compared to those obtained upon stimulation with the cognate CAP1 peptide. In addition, early events in the TCR signaling pathway were examined for differences in tyrosine phosphorylation. Upon stimulation with the agonist peptide CAP1-6D, several different CEA-specific CTL lines exhibited a marked shift in the peptide dose response, which resulted in as much as a 1,000-fold increase in the levels of GM-CSF and gamma-IFN produced as compared with the use of the CAP1 peptide. However, levels of IL-4 and IL-10, which are associated with anti-inflammatory effects, were very low or non-existent. The cytokine profile of CAP1- and CAP1-6D-specific CTL is consistent with a Tc1-type CTL. Consistent with these findings, CEA-specific CTL showed increased tyrosine phosphorylation of TCR signaling proteins ZAP-70 and TCR zeta chains in response to both peptides. However, when CAP1-6D was compared with the wild-type peptide, the increase in ZAP-70 phosphorylation was greater than the increase in zeta phosphorylation. CTL generated with the CAP1-6D agonist were shown capable of lysis of human carcinoma cells expressing native CEA. The ability to upregulate the production of GM-CSF, gamma-IFN, TNFalpha and IL-2 with the agonist peptide, as compared with CAP1, may help in initiating or sustaining anti-tumor immune responses and thus potentially prove to be useful in the treatment of CEA-positive tumors.

Multiple myeloma after treatment of essential thrombocythemia.

An 83-year-old black woman with previously diagnosed essential thrombocythemia was treated with thiotepa to control the excessive number of platelets. Approximately 10 years later, multiple myeloma developed. This appears to be the first case of multiple myeloma occurring after alkylating agent therapy for essential thrombocythemia. Although the mechanism of the development of multiple myeloma has yet to be defined, continuing progress in cell biology may prove a strong association between multiple myeloma and other hematologic malignancies.