Granulocyte/macrophage-colony stimulating factor produced by recombinant avian poxviruses enriches the regional lymph nodes with antigen-presenting cells and acts as an immunoadjuvant.

Recombinant avian poxviruses [fowlpox and canarypox (ALVAC)], restricted for replication in nonavian cell substrates and expressing granulocyte/macrophage-colony stimulating factor (avipox-GM-CSF), were evaluated for their ability to enrich an immunization site with antigen-presenting cells (APCs) and, in turn, function as biological vaccine adjuvants. Avipox-GM-CSF administered as a single s.c. injection significantly enhanced the percentage and absolute number of APCs in the regional lymph nodes that drain the injection site. Both the magnitude and duration of the cellular and phenotypic increases within the lymph nodes induced by the avipox-GM-CSF viruses were significantly (P < 0.05) greater than those measured in mice treated with four daily injections of recombinant GM-CSF protein. Temporal studies revealed that the APC enrichment of regional lymph nodes was sustained for 21-28 days after injection of the recombinant avipox virus expressing GM-CSF and, moreover, three injections of the recombinant virus could be given without any appreciable loss of in vivo bioactivity. Mice expressing human carcinoembryonic antigen (CEA) as a transgene (CEA.Tg) developed CEA-specific humoral and cell-mediated immunity after being immunized with avipox-CEA. The coadministration of recombinant avipox viruses expressing CEA and GM-CSF significantly enhanced CEA-specific host immunity with an accompanying immunotherapeutic response in tumor-bearing CEA.Tg mice. The optimal use of avipox-GM-CSF, in terms of dose and dose schedule, especially when used with different immunogens, remains to be determined. Nonetheless, the present findings demonstrate: (a) the effective delivery of GM-CSF to an immunization site using a recombinant avian poxvirus; (b) the compatibility of delivering an antigen and GM-CSF in replication-defective viruses to enhance antigen-specific immunity; and (c) the combined use of recombinant avipox viruses expressing CEA and GM-CSF to generate antitumor immunity directed at a self tumor antigen.

Identification of NY-ESO-1 peptide analogues capable of improved stimulation of tumor-reactive CTL.

Expression of NY-ESO-1 in a high proportion of different human tumors makes this protein a very attractive vaccine target. NY-ESO-1 peptides, recognized by HLA-A2-restricted CTL, have recently been described. However, it remains unclear how efficiently tumors generate these epitopes, and whether peptide analogues can be used for optimal expansion and activation of NY-ESO-1-specific HLA-A2-restricted CTL. By generating unique CTL clones, we demonstrate that NY-ESO-1-positive tumor cells are efficiently killed by HLA-A2-restricted CTL specific for the peptide epitope NY-ESO-1 157-165. Presentation of this epitope is not affected by the presence or absence of the proteasome subunits low molecular proteins 2 and 7 and is not blocked by proteasome inhibitors, while it is impaired in the TAP-deficient cell line LBL 721.174. NY-ESO-1 157-165 peptide analogues were compared for their antigenicity and immunogenicity using PBL from melanoma patients. Three peptides, containing the carboxyl-terminal cysteine substituted for either valine, isoleucine, or leucine, were recognized at least 100 times more efficiently than the wild-type peptide by specific CTL. Peptide analogues were capable of stimulating the expansion of NY-ESO-1-specific CTL from PBL of melanoma patients much more efficiently than wild-type peptide. These findings define the processing requirements for the generation of the NY-ESO-1 157-165 epitope. Identification of highly antigenic NY-ESO-1 peptide analogues may be important for the development of vaccines capable of expanding NY-ESO-1-specific CTL in cancer patients.

Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations.

Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

Gene gun-based nucleic acid immunization alone or in combination with recombinant vaccinia vectors suppresses virus burden in rhesus macaques challenged with a heterologous SIV.

Gene gun-based DNA immunization alone or in combination with recombinant vaccinia vectors was evaluated for the ability to elicit protective immune responses in rhesus macaques challenged with a pathogenic, heterologous simian immunodeficiency virus (SIV). Six monkeys primed with seven consecutive doses of DNA encoding SIVmac239 gp120 and gp160 (DNA + DNA) were divided into two groups. Three of these animals received another DNA booster immunization and the remaining three received a booster immunization containing a homologous, live recombinant vaccinia virus expressing SIVmac251 gp160 (DNA + VAC). In addition, a group of 15 animals primed with recombinant vaccinia vectors were divided into two groups. One group of six monkeys received another immunization of vaccinia (VAC + VAC) and the other nine animals received a DNA (mac239) booster immunization (VAC + DNA). Geometric mean end-point IgG titres in the DNA + VAC and VAC + DNA groups were substantially higher than the responses seen in the VAC + VAC and DNA + DNA groups, demonstrating a synergistic relationship between DNA-based vaccines and recombinant vaccinia virus-based vaccines. All vaccinates and five naive controls were challenged 19 weeks after the final booster immunization with 10 animal infectious doses of SIVDelta/B670. The vaccines did not prevent infection. However, all vaccine groups showed significant virus load reductions from seven to 56 days post challenge when compared to controls. Although the DNA + DNA group developed the lowest prechallenge antibody responses, the most significant reduction (200-fold) in virus load was associated with this group. In addition, a significant delay in CD4+ T cell loss relative to controls was observed in the DNA + DNA group. These results demonstrate that a gene gun-based DNA vaccine provided some attenuation of infection and CD4+ T cell loss after a heterologous challenge.

Human immunodeficiency virus type 1-specific cytotoxic T lymphocytes (CTL), virus load, and CD4 T cell loss: evidence supporting a protective role for CTL in vivo.

The relationships between primary human immunodeficiency virus type 1 (HIV-1) Gag-specific cytotoxic T lymphocyte (CTL) frequency, virus load, and CD4 T cell loss were evaluated in a group of 46 HIV-1-infected persons with hemophilia. Freshly isolated peripheral blood mononuclear cells in limiting dilution assays were used to measure HIV-1 Gag-specific CTL frequencies. Concurrent measurements of virus load and lymphocyte surface markers were obtained. No correlation between Gag-specific CTL frequency and concurrent CD4 cell count was observed. A significant inverse relationship was observed between HIV-1 Gag-specific CTL frequency and provirus load as measured by polymerase chain reaction. Subjects with higher CTL frequencies were found to have more stable CD4 cell counts over time. These results provide additional evidence to support the concept that the predominant role of this virus-specific cellular immune response is to limit viral replication and CD4 cell loss in HIV-1 infection.

Virus burden in long-term survivors of human immunodeficiency virus (HIV) infection is a determinant of anti-HIV CD8+ lymphocyte activity.

Persons infected with human immunodeficiency virus (HIV) for > 8 years were studied to delineate virologic and immunologic attributes of long-term survival. Whereas those with 300-700 CD4+ cells/microL often had circulating cytotoxic T lymphocytes (CTL) against HIV antigens, those with > 1000 CD4+ cells/microL did not. The subjects with > 1000 CD4+ cells/microL had low virus burden, low levels of Gag-specific CTL precursors, and minimal CD8+ cell activation. Overall, elevated levels of CD8+ cells, CD38 antigen expression on CD8+ cells, and anti-HIV functions were correlated with increased virus burden, provirus load, and HIV plasma RNA levels. A factor that suppressed HIV replication was spontaneously secreted from CD8+ cells of most subjects but not from those with high CD4+ cell counts. CD8+ cell activities, therefore, may reflect chronic viral stimulation of the immune system. Long-term survivors with high levels of CD4+ cells maintained control of viral replication but lacked the CD8+ cell activities.

HIV-1-specific cytotoxic T lymphocyte responses in the first year of life.

HIV-1-specific CTL responses were prospectively evaluated in infants born to HIV-1-seropositive women to assess the capability of the young infant to generate HIV-1-specific CTL and to examine the potential role of HIV-1-specific CTL in the pathogenesis of vertical infection. Our results indicate that some young infants, and even the fetus, seem to be capable of generating virus-specific CTL responses. The detection of HIV-1-specific CTL responses varied among infants, however, with respect to timing, HIV-1 gene product recognition, and the magnitude of detectable responses; HIV-1-specific CTL responses were uncommonly detected in the first few months of life. The less consistent detection of HIV-specific CTL in early infancy contrasts with reports of the detection of HIV-1-specific CTL soon after primary infection in adults. HIV-1-specific CTL were not detected in any uninfected infants born to HIV-1-seropositive women. This description of HIV-1-specific CTL in infants may have important implications for understanding the pathogenesis of vertical HIV-1 infection and for the development of a vaccine to interrupt vertical infection.

High-titer immune responses elicited by recombinant vaccinia virus priming and particle boosting are ineffective in preventing virulent SIV infection.

Eighteen rhesus monkeys were vaccinated with recombinant vaccinia viruses expressing SIVmac antigens in 3 separate rounds of experiments. Twelve of the monkeys were primed with a trivalent vaccinia virus recombinant expressing Gag, Pol, and Env polypeptides that can assemble into SIV pseudovirion particles and boosted with SIV particles in adjuvant. Four of the monkeys were primed with different vaccinia virus recombinants expressing env or gag+env followed by SIV particle boosts; two received vaccinia virus recombinants alone (env or env+gag). Despite the induction of vigorous immune responses, 17 of 18 rhesus monkeys became infected on challenge with a low dose of virulent SIVmac. The single protected animal was one of three challenged with homologous cloned SIV exactly matched to the clone used for construction of trivalent vaccinia virus recombinant and particles. Vaccination may have diminished SIV burdens and rates of CD4+ cell declines in some of the animals, but vaccinated/challenge/infected animals eventually developed fatal disease similar to control animals. These results highlight the extreme difficulty in achieving vaccine protection against virulent SIVmac infection even under idealized laboratory conditions.

Induction of antigen-specific killer T lymphocyte responses using subunit SIVmac251 gag and env vaccines containing QS-21 saponin adjuvant.

Subunit vaccines based on recombinant proteins have proved useful for inducing antibody responses and they are safe for widespread use because they do not contain any live components. Unfortunately, they do not typically induce the types of cell-mediated immune responses required to control viral pathogens; specifically, they do not induce CD8+ cytotoxic T lymphocyte (CTL) responses. To increase the immunogenicity of recombinant proteins, we have used the QS-21 saponin adjuvant in subunit vaccine formulations. In the current study, experimental subunit vaccine formulations containing recombinant p55gag or gp120env proteins from the mac251 strain of the simian immunodeficiency virus (SIVmac251) and the QS-21 adjuvant were used to immunize rhesus macaques. These formulations induced SIV gag- or env-specific cellular immunity that was detectable in vitro and included killer cell activity. The induction of killer cells required prior vaccination and the responses were antigen specific for the immunogens contained in the vaccine formulations. Autologous target cells were required to detect these responses, suggesting genetic restriction, and effector cells appeared to be present in both the CD4+ and CD8+ T lymphocyte subpopulations. These data suggest that the vaccine-induced killer cell activity that was detected was mediated by both CD4+ and CD8+ lymphocytes. Despite the presence of these killer cells, all of the animals became infected with the SIVmac251 on experimental challenge. These findings demonstrated that antigen-specific killer cell responses could be induced by a subunit vaccine formulated with the QS-21 saponin adjuvant. The characteristics of the responses suggested that the effector cells were T lymphocytes, expressing either CD4 or CD8.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of cytotoxic T lymphocyte responses to SIV proteins in SIV-infected macaques using antigen-specific stimulation with recombinant vaccinia and fowl poxviruses.

Methods to analyze CD8+ CTL responses to simian immunodeficiency virus (SIV)-encoded proteins are essential to understand lentivirus immunopathogenesis and protective immune responses. Recombinant infectious shuttle vectors are useful for analyzing CTL responses to many viruses, including HIV. Therefore, CTL responses in SIV-infected Macaca fascicularis to SIV env and SIV gag/pol were evaluated using specific antigen stimulation with recombinant vaccinia (rVV) and fowl poxviruses (rFPV) containing SIV genes. Peripheral blood mononuclear cells from SIV-infected animals were stimulated with autologous cells infected with rVV expressing SIV env/gag/pol, and CTLs specific for SIV env and for SIV gag/pol were detected by testing for lytic activity in target cells expressing these genes separately. Lymphocyte subset purifications from the effector population demonstrated that the CTL response was mediated by CD8+ cells, and the use of brefeldin A to selectively block antigen presentation in association with MHC class I products affirmed this cytolytic activity was class I restricted. The use of rVV to analyze responses to SIV genes is potentially problematic in hosts immunized to vaccinia. Fowl poxvirus is an alternative virus that has many of the molecular advantages of vaccinia virus but is genomically distinct. Therefore, the ability of rFPV to expand and detect SIV-specific CTLs was evaluated. Although there was no cytopathic effect following infection with rFPV, macaque cells infected with this vector did express rFPV gene products, and could be used as stimulator and target cells to detect SIV-specific CD8+ CTLs. The results suggest that these recombinant viral vectors can be used to specifically stimulate CD8+, MHC class I-restricted CTLs reactive to SIV proteins, and should facilitate evaluating CTL responses in both SIV-infected animals and animals vaccinated against SIV.

Immunization with lentivirus-like particles elicits a potent SIV-specific recall cytotoxic T-lymphocyte response in rhesus monkeys.

An effective acquired immunodeficiency syndrome (AIDS) vaccine should be capable of eliciting human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) responses. We have explored the use of lentivirus-like particles produced in mammalian cells infected with a recombinant vaccinia virus to immunize for the induction of CTL in the simian immunodeficiency virus (SIV)/rhesus monkey model for AIDS. SIV-like particles in a threonyl-MDP-based adjuvant did not elicit a high frequency of SIV gag-specific effector cells in naive rhesus monkeys. However, immunization with these particles elicited a potent recall CTL response in monkeys previously vaccinated with a recombinant vaccinia-SIV virus. These observations suggest that poxvirus-produced virus-like particles may represent a safe immunogen for use in periodic boosting to maintain viable cell-mediated immunity to the AIDS virus.

Formation of lentivirus particles by mammalian cells infected with recombinant fowlpox virus.

Recombinant fowlpox viruses (FPV) containing the env or gag-pol genes of simian immunodeficiency virus from macaques (SIVmac) were constructed. The env, gag, and pol-encoded polypeptides were efficiently expressed and processed in avian cells productively infected with FPV as well as in mammalian cells, in which FPV infection is abortive. In addition, the recombinant FPV expressing the gag-pol genes directed the formation of defective, lentivirus-like particles which were released into the culture medium of infected cells. Coinfection of cells with the env and gag-pol recombinant viruses resulted in the generation of particles containing SIVmac envelope glycoprotein. The applications of this system to vaccine development are discussed.

HIV-1 gag-specific cytotoxic T lymphocytes recognize multiple highly conserved epitopes. Fine specificity of the gag-specific response defined by using unstimulated peripheral blood mononuclear cells and cloned effector cells.

CTL directed at the highly conserved HIV-1 gag protein have been described in HIV-1 seropositive persons and may be an important host defense against this retrovirus. Presently only limited data are available regarding the specific epitopes recognized by these CTL. In this study, we have performed a detailed examination of the gag-specific CTL response in three HIV-1 seropositive subjects, using both unstimulated PBMC and cloned CTL. Lysis of gag-expressing targets was found to be mediated by CD3+CD8+ lymphocytes and restricted by class I Ag. Multiple class I Ag were found to restrict gag epitopes in each subject studied, with as many as three of these Ag involved in presenting gag CTL epitopes in a single subject. The majority of gag-specific CTL activity was found to be directed against epitopes in the p24 subunit of the gag protein, with at least seven different HLA class I-restricted CTL p24 epitopes identified in these three subjects. Less CTL activity was directed against p17 subunit of gag and two CTL epitopes were identified in this protein. Although as many as four different epitopes in gag were recognized using CTL from a single subject, none of the epitopes was recognized by CTL from more than one subject. Analysis of gag epitope recognition using cloned CTL demonstrated heterogeneity and specificity not appreciated using unstimulated PBMC. The identification of multiple relatively conserved epitopes in the HIV-1 gag protein and the heterogeneity of CTL responses to this protein may have important implications for vaccine development and our understanding of AIDS pathogenesis.

Importance of the nef gene for maintenance of high virus loads and for development of AIDS.

When rhesus monkeys were infected with a form of cloned SIVmac239 having a premature stop signal at the 93rd codon of nef, revertants with a coding codon at this position quickly and universally came to predominate in the infected animals. This suggests that there are strong selective forces for open functional forms of nef in vivo. Although deletion of nef sequences had no detectable effect on virus replication in cultured cells, deletion of nef sequences dramatically altered the properties of virus in infected rhesus monkeys. Our results indicate that nef is required for maintaining high virus loads during the course of persistent infection in vivo and for full pathologic potential. Thus, nef should become a target for antiviral drug development. Furthermore, the properties of virus with a deletion in nef suggest a means for making live-attenuated strains of virus for experimental vaccine testing.

Recombinant virus vaccine-induced SIV-specific CD8+ cytotoxic T lymphocytes.

Evidence indicates that cytotoxic T lymphocytes (CTLs) may be important in containing the spread of the human immunodeficiency virus (HIV) in the infected host. Although the use of recombinant viruses has been proposed as an approach to elicit protective immunity against HIV, the ability of recombinant viral constructs to elicit CD8+ CTL responses in higher primates has never been demonstrated. A live recombinant virus, vaccinia-simian immunodeficiency virus of macaques (SIVmac), was used to determine whether such a genetically restricted, T lymphocyte-mediated antiviral response could be generated in a primate. Vaccinia-SIVmac vaccination elicited an SIVmac Gag-specific, CD8+ CTL response in rhesus monkeys. These CTLs recognized a peptide fragment that spans residues 171 to 195 of the Gag protein. The rhesus monkey major histocompatibility complex (MHC) class I gene product restricting this CTL response was defined. Both the vaccinated and SIVmac-infected monkeys that shared this MHC class I gene product developed CTLs with the same Gag epitope specificity. These findings support the use of recombinant virus vaccines for the prevention of HIV infections in humans.

Two distinct lymphocyte populations mediate simian immunodeficiency virus envelope-specific target cell lysis.

The relative contributions of the effector lymphocyte responses to the AIDS virus envelope glycoprotein (env) were explored in simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys. CD8+, MHC class I-restricted, env-specific CTL were cloned from PBL of SIVmac-infected monkeys, indicating that such cells constitute a component of the env-specific effector lymphocyte response. A limiting dilution 51Cr release assay was then established for quantitating the frequency of SIVmac-specific effector lymphocytes in PBL of rhesus monkeys. Using this assay we demonstrate that SIVmac env-specific effector lymphocytes are comprised of both CD16-, MHC class I-restricted and CD16+, MHC class I-unrestricted cells. We also demonstrate that the env-specific response is the predominant SIVmac-specific effector lymphocyte response in rhesus monkeys. These studies document the complexity of the effector lymphocyte response to the AIDS virus envelope glycoprotein and establish the role played by two distinct effector cell populations in this response.

Studies of cloned simian immunodeficiency virus-specific T lymphocytes. gag-specific cytotoxic T lymphocytes exhibit a restricted epitope specificity.

CD8+ CTL inhibit the replication of HIV and simian immunodeficiency virus of macaques (SIVmac) in PBL and, therefore, are likely to play an important role in containing the spread of the AIDS virus in infected individuals. We have generated a series of gag-specific lytic T lymphocyte clones from PBL: of an SIVmac-infected rhesus monkey. These T cell clones are CD3+CD8+ and are MHC class I-restricted in their target specificity. They are, therefore, CTL. Interestingly, all gag-specific CTL clones, as well as the gag-specific lytic activity of PBL of this monkey, demonstrated specificity for a single 25 amino acid fragment of the SIVmac gag protein. Moreover, they were restricted in their lytic function by a single MHC class I allele. These findings illustrate a powerful method for cloning AIDS virus-specific T lymphocytes and demonstrate a remarkably restricted epitope specificity of this AIDS virus-specific CTL response.

Analysis of c-erbB-2 expression in breast carcinomas with clinical follow-up.

Various monoclonal antibodies reactive with protooncogene products or tumor-associated antigens have been utilized to investigate breast carcinoma biology or antigen expression with potential prognostic relevance. Murine monoclonal antibody TA1, generated by immunization of BALB/c mice with whole c-erbB-2 (neu) transformed NIH/3T3 cells, recognizes the extracellular domain of the c-erbB-2 protein and binds a Mr 185,000 protein by immunoprecipitation. Using avidin-biotin-peroxidase techniques and monoclonal antibody TA1, 313 archival primary adenocarcinomas of the breast were evaluated for c-erbB-2 overexpression; 290 of these were used for multiparametric statistical analysis. Historical, clinical (age, laterality), histological (nuclear grade, tumor size, lymph node status, lymphatic or blood invasion), and hormone receptor data as well as clinical outcome (minimal follow-up, 6 years; median follow-up, 8.5 years) were compared to TA1 staining. For these 290 patients Cox regression multivariate analysis showed the strongest correlation between lymph node status or estrogen receptor status and overall survival (P = 0.0001 and 0.049, respectively). TA1 staining did not significantly correlate with survival (P = 0.395). However, univariate analysis of certain patient subpopulations showed a significant correlation if the examined tumors were subdivided into negative or focally reactive and those with greater than or equal to 40% cellular reactivity. For T3, T4 patients, strong TA1 immunoreactivity correlated with a shortened disease-free survival (log rank P = 0.0018; Wilcoxon p = 0.0078) and overall survival (log rank P = 0.0002; Wilcoxon P = 0.0013). For these patients the overall survival at 6 years was markedly different between the strongly reactive tumors (0%) and the negative to weakly reactive tumors (55%). In lymph node-positive patients a trend between high TA1 reactivity and a worse overall survival was also noted (log rank P = 0.128; Wilcoxon P = 0.054), with a 6-year survival of 42% in the strongly reactive tumors (n = 16) and 65% in the negative to weakly reactive carcinomas (n = 105). No correlation between TA1 immunoreactivity and other historical, clinical, and histological features were noted. c-erbB-2 overexpression as measured by immunohistochemical techniques, therefore, may have clinical significance in certain patient subpopulations.

Generation and characterization of monoclonal antibodies specific for the human neu oncogene product, p185.

A series of monoclonal antibodies specific for the extracellular domain of the human neu gene product (p185) have been produced. The generation of these monoclonal antibodies, and their biochemical and immunological characterization is described. The immunization protocol utilized a series of injections of NIH3T3 cells, cyclophosphamide, and a neu transfected NIH3T3 cell line (designated 18-3-7) which expressed the full length human neu-encoded protein. This immunization regimen induced an immune response to the extracellular portion of p185 on the 18-3-7 cells. A panel of ten hybridomas were identified which secreted monoclonal antibodies with a variety of epitope specificities, and reacted with p185 in a number of different experimental formats. As the neu gene product has been associated with human breast cancers, a series of monoclonal antibodies such as these could prove useful in the diagnosis, prognosis and/or treatment of these human malignancies.