An algorithm for evaluating human cytotoxic T lymphocyte responses to candidate AIDS vaccines.

Development of an effective vaccine against HIV-1 will likely require the induction of a broad array of immune responses, including virus-specific CTLs and neutralizing antibodies. One promising vaccine approach involves live recombinant canarypox (CP)-based vectors (ALVAC) containing multiple HIV-1 genes. In phase I clinical trials in HIV-1-seronegative volunteers, the cumulative rate of detection of HIV-1-specific CTLs has been as high as 60-70%. In the present study, the factors associated with CTL responsiveness were evaluated in a subset of vaccinees immunized with a CP vector expressing portions of the gag, pro, and env genes of HIV-1 (ALVAC-HIV). CTL responses were detected in one of seven examined. While the responding individual had both CD4+ and CD8+ CTLs directed at multiple HIV-1 antigens, this response was not detectable 1 year after the last vaccination. In-depth characterization of "CTL nonresponders" showed that nonresponsiveness was not associated with defects in antigen processing or presentation. A generalized defect in CTL responsiveness was ruled out by parallel assays to detect CMV-specific CTLs from these same volunteers. Furthermore, HIV-1-specific memory CTLs were not detectable by peptide stimulation or by a novel technique for flow cytometric visualization of Gag epitope-specific T lymphocytes while HIV-1-seropositive donors frequently had 0.1-3% of CD8+ cells stain positively for this epitope (SLYNTVATL). Taken together, these results suggest that the lack of detectable HIV-1 CTLs in these volunteers was not due to classic MHC-linked nonresponsiveness.

Induction of human immunodeficiency virus type 1 (HIV-1)-specific cytolytic T lymphocyte responses in seronegative adults by a nonreplicating, host-range-restricted canarypox vector (ALVAC) carrying the HIV-1MN env gene.

CD8+ cytolytic T lymphocytes (CTL) are likely to be an important component of effective vaccines against human immunodeficiency virus type 1 (HIV-1). CTL can be induced most effectively with live virus vectors. However, because of concerns about the safety of such vectors, a nonreplicating canarypox vector (ALVAC) capable of expressing foreign genes in mammalian cells has been developed. This study evaluated the capacity of an ALVAC vector expressing the HIV-1MN envelope (env) glycoprotein to induce HIV-1-specific CTL in seronegative volunteers. Protocols were designed to determine whether immunization with ALVAC alone or in combination with subunit boosting could induce CTL in vaccinia-immune and -naive volunteers. A simple method for antigen-specific in vitro stimulation was used to detect CTL responses in HIV-1-seronegative vaccine recipients. The results indicate that low doses of a nonreplicating virus vector alone can elicit both CD4+ and CD8+ HIV-1-specific CTL in a subset of seronegative volunteers.

Studies of the mechanism of cytolysis by HIV-1-specific CD4+ human CTL clones induced by candidate AIDS vaccines.

Vaccine-induced, virus-specific CTLs may rapidly eliminate the host cells that first become infected after virus exposure, thereby preventing disseminated infection. Thus, there is much interest in the ability of candidate AIDS vaccines to elicit CTLs. All HIV-1 envelope (env) protein-based vaccines tested to date in seronegative humans induce CTLs from the CD4+ subset. Because the mechanism of cytolysis by CD4+ CTLs is controversial, a detailed study of the cytolytic reactions mediated by vaccine-induced, HIV-1-specific human CD4+ CTL clones was conducted. CD4+ CTL clones induced rapid destruction of Ag-pulsed target cells. Lysis was readily detectable within 15 min. Lysis was not a result of syncytium formation between CD4+ effector cells and env-expressing targets. Target cell destruction was not dependent upon de novo RNA or protein synthesis in either the effector or the target cell. Expression of perforin mRNA was detected by Northern blotting and reverse-transcriptase-PCR in CD4+ CTL clones but not in autologous B lymphoblastoid cell lines. Immunohistochemical studies demonstrated perforin protein in cytoplasmic granules in CD4+ CTL clones. Lysis by CD4+ CTLs was strictly dependent upon extracellular Ca2+ and was highly specific, with no lysis of innocent bystander cells. DNA fragmentation was detectable in target cells, but did not precede 51Cr release. Taken together, these results provide a dramatically different view of cytolysis by human CD4+ CTLs. Target cells are lysed by a rapid and efficient mechanism that involves a preformed mediator and that is functionally similar to the mechanism used by CD8+ CTLs.

An HIV-1 envelope protein vaccine elicits a functionally complex human CD4+ T cell response that includes cytolytic T lymphocytes.

T cell responses play a critical role in host defense against viral infection. Therefore, the functional properties of HIV-1-specific human T cells induced by an experimental AIDS vaccine were analyzed in detail at the clonal level. Seronegative human volunteers were immunized with a purified recombinant form of the HIV-1 envelope glycoprotein gp160 in a phase I vaccine trial. In a subset of gp160 recipients, this vaccine was shown to elicit a virus-specific CTL response. Antibody blocking and single cell cloning experiments demonstrated that the vaccine-induced cytolytic activity was mediated by CD4+, MHC class II-restricted T cells. Because little is known about the regulation of CD4+ CTL in any system, a detailed analysis of CTL responses in vaccinees was carried out. Longitudinal and cross-sectional studies revealed that the CD4+ CTL response was regulated in a complex manner and was not clearly correlated with MHC class II genotype, Ag dose, or number of immunizations. Cloning studies were carried out to determine what fraction of the vaccine-induced T cells were cytolytic and to examine patterns of cytokine production by vaccine-induced T cells. These experiments demonstrated that, for some vaccinees, CD4+ CTL dominated the in vitro T cell response to gp160 at certain time points. The level of cytolytic activity, which was a stable property of individual clones, varied among clones over a wide and continuous range. Analysis of cytokine secretion by gp160-specific CD4+ T cell clones revealed Th0-, Th1-, and Th2-like patterns, with CD4+ CTL clones showing Th0- or T'1-like patterns. Interestingly, many Th0- and Th1-like CTL clones produced very little IL-2, a finding that may explain the complicated regulation of this response. These results illustrate the complex nature of the human T cell response to subunit vaccines consisting of purified recombinant viral proteins.

Chromatin substructure: an electron microscopic study of thin-sectioned chromatin subjected to sequential protein extraction and water swelling procedures.

Electron microscopic observations and measurements were made on thin-sectioned chromatin fibers and fibrils obtained from nuclei of mature chicken erythrocytes. The nuclei were isolated in low ionic strength gum arabic and octanol then extracted sequentially with (1) 0.14 M NaCl, (2) 0.25 N HCl, (3) buffer saturated phenol, (4) hot 5% SDS and 0.14 M 2-mercaptoethanol and, (5) 0.4 N NaOH. The amount of nuclear protein removed at each of the first four extraction steps was 1, 86, 3 and 11% of the total, respectively. Each extract was characterized by electrophoretic profiles. At each extraction the chromatin was fixed by adding large quantities of a mixture of equal volumes of sodium cacodylate buffered 8% (w/v) glutaraldehyde (pH 6.8) and 2% OsO4 (w/v), directly into (1) an aliquot of the chromatin in extraction fluid, and (2) an aliquot of the chromatin after water washing and swelling. Three size classes of chromatin structure were seen in thin sections prepared for high resolution transmission electron microscopy and stained with uranyl acetate and lead citrate. A thick fiber of about 25 + nm diameter was the predominant large fiber seen in freshly isolated nuclei or in nuclei after salt extraction. This 25 + nm fiber has a substructure consisting of 3.2-5.2 nm diameter fibrils. After water swelling of such freshly isolated or salt extracted nuclei a fiber of about 10 nm diameter was the predominant large fiber instead of the 25 nm diameter fiber. The HCl extraction step which is known to remove histones, caused the disappearance of both the 25 nm and the 10 nm fibers. High magnification (600,000 x) micrographs of the chromatin at all procedural steps, except the last NaOH step, reveal the fibril to be omnipresent. This fibril tends to decrease somewhat in diameter during the protein extraction steps to a 2.5 nm diameter fibril after the hot SDS extraction. A fibril of 2.5 nm diameter is expected of naked double helical DNA stained with a positive stain. The NaOH, which is known to denature DNA, completely destroyed the remaining fibril. We inerpret our results to indicate that the larger chromatin fiber seen in micrographs of thin-sectioned chromatin has a fibrillar substructure which probably represents a double coil of native DNA which may have a thin protein coating of its own. The latter fibril may in turn be wrapped around a hydrophobic histone domain, perhaps reflected in the 10 nm diameter fiber which is seen upon swelling of the chromatin. This 10 nm diameter fiber is thought to be further packaged by folding into the 25 + nm diameter chromatin fiber most frequently reported in thin sections of eukaryotic cell nuclei in situ.

Tumor-host responses to various nutritional feeding procedures in rats.

Parenteral and enteral nutrition are being used as adjuncts to cancer therapy. A liquid diet formulation containing a 27% solution of glucose and 3.9% crystalline amino acids with electrolytes and vitamins was given continuously for a week via parenteral (iv), and via intragastric (ig) routes and also was given ad libitum via the oral or per os (po) route to groups of Buffalo rats with and without a Morris No. 7777 transplantable hepatoma to find out how these feeding procedures affect tumor-host interactions. Other groups of rats with and without the hepatoma were given solid food ad libitum. The following parameters were examined: mortality, carcass and organ weights, body and tumor growth, nitrogen balance, energy intake, fluid balance, urinalysis, hematology values, and serum protein levels. The results are considered with respect to the influence of the tumor on the host and the influence of the feeding procedure on the animal with and without a tumor. The presence of the hepatoma was associated with: higher mortality, a decrease in carcass mass, leucocytosis, anemia, a decrease in serum IgG, transferrin and albumin, and an increase in serum alpha fetoprotein. The iv and ig feeding procedures alone resulted in some mortality which was exacerbated by the presence of the tumor. Mortality was especially high in the tumorous rats on the ig feeding procedure. The degree of positive nitrogen balance and carcass mass was similar in non-tumorous rats fed the same liquid diet formula when given iv, ig, or po. Tumorous rats fed the liquid diet ad libitum showed anorexia and a significantly lower nitrogen balance. The iv and ig feeding of tumorous rats at a level which was well above those of the tumorous rats given solid or liquid diet ad libitum maintained the same degree of positive nitrogen balance as non-tumorous rats. Even though the iv feeding of tumorous rats maintained about the same degree of positive nitrogen balance as non-tumorous rats, these tumorous rats still suffered loss of carcass mass. It appears that the large rapidly growing hepatoma has priority for available nutrition over the host. It is further suggested that the rapidly growing hepatoma places an ever increasing demand on the available nutrients. Thus, a point is eventually reached where even supplemental nutritional support can no longer meet the needs of the growing hepatoma and the host.

Stimulation of growth of a transplantable hepatoma in rats by parenteral nutrition.

Young BUF rats of similar ages were inoculated with the transplantable Morris hepatoma No. 7777. At 4 weeks after inoculation, 1 group was given total iv (parenteral) feeding at high caloric and nutritional levels for 2 weeks. Such total iv feeding (hyperalimentation) of rats stimulated a more rapid tumor growth in the host. In addition, the tumors from rats fed parenterally for 2 weeks had higher mitotic activity and larger areas of necrosis, which indicate that iv feeding caused the tumor to undergo faster cell turnover with greater cell production and cell loss. Analysis of organ weights showed that parenteral feeding caused atrophy of the intestines, whereas spleen weights of the hepatoma-bearing rats fed iv were greater than those of the orally fed hepatoma-bearing rats.